Synthesis and analysis of activity of a potential anti-melanoma prodrug with a hydrazine linker

Article abstract of DOI:10.1016/j.ejmech.2013.10.080

A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl) amino]benzoyl}-N′-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1- yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.

Full text of DOI:10.1016/j.ejmech.2013.10.080

European Journal of Medicinal Chemistry 71 (2014) 98e104
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and analysis of activity of a potential anti-melanoma
prodrug with a hydrazine linker
a
a
a
_
Bozena Fra˛ckowiak-Wojtasek , Beata Ga˛sowska-Bajger , Magdalena Mazurek ,
Agnieszka Raniszewska a, Marieke Logghe a , Ryszard Smolarczyk , Tomasz Cichon ,
,
1,
2
b
ꢀ b
Stanis1aw Szala b, Hubert Wojtasek a
,
*
a Faculty of Chemistry, Opole University, Ul. Oleska 48, 45-052 Opole, Poland
b Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice
_
Branch, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen
mustard effector e (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine) has been
synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a
rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were
formed. However, during the reaction one of the products was converted into the other. The stable
product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-
indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically
generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should
not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector
release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for
B16eF10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice
it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic
cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure
resembling tyrosine, occasionally used in the treatment of melanoma.
Received 1 July 2013
Received in revised form
8 October 2013
Accepted 31 October 2013
Available online 9 November 2013
Keywords:
Anti-melanoma prodrug
Nitrogen mustard
Hydrazine
Tyrosinase
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
improvement in the treatment of this notoriously drug-resistant
tumor in patients with mutations in this protein. Monoclonal an-
Malignant melanoma is the most aggressive skin tumor result-
ing from neoplastic transformation of melanocytes, which shows a
steadily increasing incidence [1]. For decades little progress in the
therapy of this tumor has been made with dacarbazine (DTIC)
remaining the major drug used in metastatic disease [2]. Recently,
however, breakthroughs have been made with the discovery of
selective BRAF inhibitors (vemurafenib, GSK2118436), which have
been evaluated in clinical trials and demonstrated exceptional
tibodies against CTLA-4 (ipilimumab) also show significantly
improved effectiveness compared to immunotherapy with inter-
feron and interleukin 2 [3,4]. However, the respective drugs (Yer-
voy, Zelboraf) are effective in a limited subsets of patients. They are
also extremely expensive and in many countries are not refunded
by the national healthcare systems. Therefore, pathways for
discovering new therapeutic agents for this tumor with better ef-
ficacy and lower side effects still remain open.
In mammals the metabolic pathway unique to melanocytes is
melanogenesis, which offers the possibility of developing a tar-
geted chemotherapy specific to this tumor. Tyrosinase (EC 1.14.18.1)
is the key enzyme in this pathway catalyzing the two initial and rate
Abbreviations: DTIC, dacarbazine (5-(3,3-dimethyl-1-triazenyl)imidazole-4-
carboxamide); MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; PBS, phosphate buffered saline.
limiting steps: hydroxylation of L-tyrosine to L-Dopa and its sub-
* Corresponding author. Tel.: þ48 77 452 7122; fax: þ48 77 452 7101.
sequent oxidation to dopaquinone, which undergoes a series of
non-enzymatic and enzymatic reactions leading to melanins [5].
Tyrosinase can also convert other monophenols and o-diphenols to
o-quinones, which are inherently cytotoxic. Tyrosinase activity in
melanocytes seems to be correlated with their malignant
E-mail address: Hubert.Wojtasek@uni.opole.pl (H. Wojtasek).
1
Permanent address: Katholieke Hogeschool Sint-Lieven, Gebroeders de Smet-
straat 1, 9000 Gent, Belgium.
2
Present address: Universiteit Gent, Campus Schoonmeersen, Valentin Vaer-
wyckweg 1, 9000 Gent, Belgium.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.080

B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
99
transformation [6,7]. Application of tyrosinase as a melanocyte-
specific enzyme for activation of anti-melanoma prodrugs was
therefore considered long time ago [8]. The concept of selectively
releasing cytotoxic agents in melanocytes from tyrosinase-
activated prodrugs was developed more than a decade ago and
named Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT)
[9]. Initially, the cytotoxic agents were attached to the primary
amino group of tyrosinase substrates (tyrosine, dopamine and
related compounds) by carbamate or urea linkers [9e11]. However,
later studies on such tyramine and dopamine derivatives showed
that conversion of the amino group to amide, carbamate or urea
derivatives made the nitrogen atom insufficiently nucleophilic for
cyclization of the corresponding o-quinones to dihydroxydihy-
droindoles and therefore the release of the active group was un-
likely [12]. In the meantime, new derivatives were developed,
where the effector part was connected to the aromatic amino group
of 4-aminophenol or 6-aminodopamine via a urea or thiourea
linker [13]. Recently, tyramine and dopamine derivatives of tri-
azenes (DTIC analogs) have been prepared as potential tyrosinase-
activated anti-melanoma prodrugs [14]. Again, however, reduced
nucleophilicity of the modified amine nitrogen hindered the
release of the effector after enzymatic activation.
We have recently shown that the hydrazine group in amino acid
phenylhydrazides [15] and in the antitumor drug procarbazine [16]
can be oxidized by o-quinones and therefore indirectly by tyrosi-
nase. Based on these results we have postulated that this redox
exchange reaction can be utilized in activation of anti-melanoma
prodrugs with a hydrazine linker (Scheme 1). Before designing
target compounds we have first tested the concept with carbidopa,
an approved drug containing both the catechol and hydrazine
moieties. Detection of 6,7-dihydroxy-3-methylcinnoline as one of
the major products of oxidation of his compound by tyrosinase
demonstrated that the nucleophilic attack of the hydrazine group
in the side-chain on the generated o-quinones, which led to cycli-
zation, competed with the intramolecular redox exchange reaction
between these two moieties [17]. This cyclization reaction is un-
desired from the point of view of designing anti-melanoma pro-
drugs, because it may reduce the yield of effector release. Therefore,
after initial attempts, we have given up work on alkyl and dialkyl
hydrazines and concentrated on acylated hydrazine derivatives.
Here we report the synthesis of an aniline mustard prodrug with a
hydrazine linker and a phenolic activator oxidizable by tyrosinase,
analysis of its enzymatic activation and its effect on murine mela-
noma in vitro and in vivo.
2. Results and discussion
2.1. Synthesis
The synthesis of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-
(4-hydroxybenzyl)hydrazine was accomplished in a 7-step proce-
dure. First, tert-butyl 4-[bis-(2-hydroxyethyl)amino]benzoate was
prepared from tert-butyl 4-aminobenzoate and ethylene oxide [18].
The hydroxyl groups were then replaced with chlorine atoms using
methanesulfonyl chloride. The carboxylic group was deprotected
and converted to a hydrazide in a reaction with tert-butox-
ycarbonylhydrazine. After removal of the protecting group, the
hydrazide was coupled with 4-hydroxybenzaldehyde yielding a
hydrazone, which was then reduced with hydrogen gas on a
palladium catalyst (Scheme 2).
2.2. Analysis of the activation of the prodrug by tyrosinase
Spectrophotometric analysis of the oxidation of the synthesized
compound by tyrosinase demonstrated that it served as a substrate
of the enzyme e rapid changes in the UVeVis spectrum occurred
with a new maximum of absorption at 337 nm (Fig. 1). This spec-
trum did not correspond to o-quinones and suggested rather that
an additional electron donating group was attached to the aromatic
ring (i.e. intramolecular or intermolecular nucleophilic attack).
HPLC analysis showed the presence of 2 major products (Fig. 2).
Their proportions differed depending on the reaction conditions
(time of the reaction, concentration of the substrate, proportion of
the substrate to enzyme concentrations). Time-course analysis
demonstrated that one of the products, eluting just behind the
substrate, was eventually converted into the second product,
whose UVeVis spectrum closely resembled the spectrum of the
reaction mixture, with a maximum of absorption at 337 nm. This
compound was isolated by extraction and column chromatography
and analyzed by NMR and high resolution mass spectrometry. Re-
sults of this analysis allowed its unequivocal identification as 5,6-
dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate
e a cyclization product formed by a nucleophilic attack of the hy-
drazide nitrogen atom on the enzyme-generated o-quinone
O
H
O
H
N
N
N
N
H
tyrosinase
H
Cl
Cl
N
N
Cl
Cl
O
OH
O
O
+ H2O
- N2
O
OH
N
Cl
N
+
N
Cl
Cl
N
OH
Cl
OH
OH
OH
Scheme 1. The concept of tyrosinase-activated anti-melanoma prodrugs with a hydrazine linker e postulated reactions occurring during oxidation of N-{4-[bis-(2-chloroethyl)
amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine.

100
B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
O
O
O
O
O
a
b
O
HO
HO
Cl
N
N
H2N
Cl
c
O
O
O
H
N
NH2
N
H
N
H
Boc
OH
e
d
Cl
Cl
Cl
N
N
N
Cl
Cl
Cl
f
OH
OH
O
O
H
N
N
N
H
N
H
g
Cl
Cl
N
N
Cl
Cl
Scheme 2. Synthesis of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine: a) ethylene oxide, AcOH; b) MsCl, Py; c) 90% TFA in DCM; d) EDC, BtOH, Boc-
NHNH2; e) 1.25 M HCl in MeOH; f) 4-hydroxybenzaldehyde, EtOH; g) H2, Pd/C, MeOH.
(Michael addition). This result was surprising, since we expected
that the acylation of the nitrogen atom of the hydrazine linker
would sufficiently lower its nucleophilicity to prevent cyclization.
This reaction is, of course, undesired, since it prevents the effector
release from the enzyme-oxidized prodrug. In fact, we were not
able to detect the released mustard drug (4-[bis-(2-chloroethyl)
amino]benzoic cid) in any reaction mixture by TLC, HPLC or MS
analysis. The intermediate, eluting slightly behind the substrate in
the HPLC analysis, was also isolated. Its mass and NMR spectra
matched the spectra of the hydrazone e the last intermediate in the
synthetic procedure, (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-
(4-hydroxybenzylidene)hydrazine).
Based on these results we can propose the reactions occurring
during the oxidation of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-
N0-(4-hydroxybenzyl)hydrazine by tyrosinase. The enzyme oxi-
dizes the phenolic group of the activator to o-quinone. The hy-
drazide nitrogen can then attack the o-quinone generating a cyclic
product, which is further oxidized undergoing aromatization e
spontaneous or tyrosinase-catalyzed (Scheme 3). Alternatively the
o-quinone can undergo a redox exchange reaction with a substrate
molecule, generating the detected hydrazone. It is rather unlikely
that this product is generated directly by the intermolecular redox
exchange reaction. We therefore postulate that the diazene is
formed first, which then undergoes tautomerization. A similar re-
action sequence has been detected before during the metabolism of
procarbazine [19]. We have also observed this azo-hydrazone tau-
tomerization after oxidation of procarbazine by o-quinones (indi-
rectly by tyrosinase), although in that case the azo compound was
stable enough to be identified as the major product [16]. Benzyla-
zoalkanes are generally considered less stable than their isomeric
alkylhydrazones [19]. Studies of the degradation of the hydrazine-
containing antidepressant isocarboxazid (N0-benzyl-5-methyl-1,2-
oxazole-3-carbohydrazide) in vivo identified benzoic acid as one
of the major metabolites, presumably formed via the diazene,
hydrazone, and benzaldehyde intermediates [19]. In vitro oxidation
of a model compound, N-(4-chlorobenzyl)-N0-benzoylhydrazine, by
rat liver microsomes produced the corresponding hydrazone as the
major product. In this case, however, the formation of this product
was attributed mainly to N-hydroxylation catalyzed by cyto-
chromes P450, followed by dehydration [20].
1.2
1
0.8
0.6
0.4
0.2
0
2.3. Effect of the synthesized compound on murine melanoma
The effect of the synthesized prodrug on cells in culture was
tested with B16eF10 murine melanoma cells and HECa10 and
NIH3T3 control cells. It was toxic to all three cell lines (lethal at
250
(Fig. 3). It was then examined on solid tumors generated in mice. At
100 g dose it showed moderate activity in reducing the tumor size,
mM) and showed limited specificity for the melanoma cells
200
300
400
500
m
Wavelength (nm)
only slightly better than the parent mustard drug (4-[bis-(2-
chloroethyl)amino]benzoic acid), but was significantly weaker
than melphalan (Fig. 4). These results are consistent with the
analysis of its enzymatic activation, which demonstrated that its
oxidation by tyrosinase did not lead to effector release.
Fig. 1. UVeVIS spectra of
a
reaction mixture containing 0.05 mM N-{4-[bis-(2-
g of tyrosinase
chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine and 2.5
m
in 3 ml of 10 mM sodium phosphate buffer, pH 6.8. The spectra displayed were
recorded for 20 min at 2 min intervals.

B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
101
600
500
400
300
200
100
0
140
a
a
120
100
80
60
40
20
0
0
5
10
15
0
100
200
300
400
500
600
Time (min)
Concentration (µM)
140
120
100
80
150
b
b
100
50
0
60
40
20
0
0
0
0
0
5
10
15
0
100
200
300
400
500
600
Time (min)
Concentration (µM)
Fig. 3. Toxicity of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hy-
drazine against B16eF10 (C), NIH3T3 (:), and HECa10 (-) cells in vitro after 24 h (a)
and 48 h (b).
100
80
60
40
20
0
c
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5
5
5
10
10
10
15
Time (min)
Time (min)
Time (min)
100
80
60
40
20
0
d
6
7
8
9
10 11 12 13 14 15 16 17 18
Time after tumor induction (days)
Fig. 4. Effect on the growth of melanoma tumors in mice: A e control, C e 4-[bis-(2-
chloroethyl)amino]benzoic acid, : e N-{4-[bis-(2-chloroethyl)amino]-benzoyl}-N0-(4-
hydroxybenzyl)hydrazine, - e melphalan. Compounds were applied at 100 mg dose.
15
3. Conclusions
Our results have demonstrated that (N-{4-[bis-(2-chloroethyl)
amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine) is effectively
oxidized by tyrosinase. However, the o-quinone formed by its
enzymatic oxidation does not undergo intramolecular redox ex-
change reaction with the hydrazine linker but a Michael addition of
the acylated hydrazine nitrogen atom or an intermolecular redox
exchange with the hydrazine group of a substrate molecule, which
gives, either directly or indirectly, via a diazene intermediate, the
100
80
60
40
20
0
e
15
mixing the reagents (a), and then after 30 min (b), 60 min (c), 120 min (d) with 250
of the enzyme in 50 ml of buffer, and after 120 min with 500 g of the enzyme (e). The
mg
m
chromatograms displayed were recorded at 280 nm. Signals correspond to the
following compounds identified by mass spectrometry, NMR analysis and/or com-
parison with synthetic standards: 7.7 min e the substrate, 8.5 min e N-{4-[bis-(2-
chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzylidene)hydrazine, 10.5 min
dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate.
Fig. 2. HPLC separation of a reaction mixture obtained after oxidation of 0.1 mM N-{4-
[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine by tyrosinase in
10 mM sodium phosphate buffer, pH 6.8. Analyses were performed immediately after
e 5,6-

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B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
O
O
H
N
H
N
N
H
N
H
tyrosinase
Cl
Cl
Cl
Cl
N
N
N
N
Cl
Cl
Cl
Cl
O
OH
OH
O
O
O
H
N
N
N
N
H
OH
OH
tyrosinase
O
O
H
N
N
N
N
H
Cl
Cl
N
N
Cl
Cl
HOH OH
OH
tyrosinase
tyrosinase?
O
N
N
Cl
N
Cl
OH
HOH
Scheme 3. Reactions occurring during oxidation of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine by tyrosinase based on structures of identified
products.
corresponding hydrazone. Neither pathway, however, leads to
release of the effector part from the prodrug. Application of car-
bazates or semicarbazides would probably not prevent the cycli-
zation reaction. Phenylhydrazine derivatives are most likely not an
option, either, because of their general toxicity and higher sus-
ceptibility to oxidation by a variety of enzymes. It seems therefore
that anti-melanoma prodrugs with such a structure cannot be
selectively activated by tyrosinase.
The mass spectrometer was calibrated with sodium formate
clusters.
4.1.2. tert-Butyl 4-[bis-(2-hydroxyethyl)amino]benzoate
Yield: 83%; m.p.: 89e93 ꢀC;
1H NMR (DMSO)
d
: 1.50 (s, 9H); 3.47 (m, 4H); 3.55 (m, 4H); 4.81
(t, 2H, J ¼ 5.21 Hz); 6.70 (d, 2H, J ¼ 8.0 Hz); 7.66 (d, 2H, J ¼ 8.0 Hz);
13C NMR (DMSO)
: 29.61; 54.68; 59.53; 80.60; 111.92; 118.70;
d
132.34; 152.90; 166.85;
IR: 3308, 3007, 2976, 2943, 2880, 1705, 1604, 1160, 1111, 1068,
1055, 1040, 837, 771;
4. Experimental
HRMS (þESI): m/z calculated for C15H23NNaO4 (M þ Na)þ
304.1519; m/z measured: 304.1546.
4.1. Synthesis
4.1.1. General procedures for the synthesis of the prodrug
4.1.3. tert-Butyl 4-[bis-(2-chloroethyl)amino]benzoate
Chemicals were purchased from SigmaeAldrich, Merck or
POCH (Gliwice, Poland) and used without further purification. All
chemicals and solvents were of reagent grade, except buffer
components and HPLC solvents, which were of analytical grade.
Analytical TLC was performed on precoated aluminum plates
(Merck silica gel 60 F254) and visualized by UV fluorescence,
potassium permanganate solution or ninhydrin solution. Prepar-
ative column chromatography was carried out on silica gel (60e
120 mesh, Merck). Melting points (uncorrected) were determined
on a Boetius apparatus. IR spectra were recorded on a Nicolet 6700
spectrometer. 1H, 13C, and correlation NMR spectra were obtained
using a Bruker Avance 400 MHz spectrometer. Chemical shifts are
given in ppm referenced to the TMS signal. Coupling constants, J,
are expressed in Hz. High-resolution mass spectra (HRMS) were
obtained on a Bruker Daltonik micrOTOF-Q II mass spectrometer
equipped with an ESI source in the positive or negative ion mode.
Yield: 78%; m.p: 68e70 ꢀC;
1H NMR (DMSO)
d
: 1.51 (s, 9H); 3.78 (m, 8H); 6.80 (d, 2H,
J ¼ 8.0 Hz); 7.72 (d, 2H, J ¼ 8.0 Hz);
13C NMR (DMSO)
: 27.85; 40.76; 51.62; 79.27; 110.89; 118.81;
d
130.86; 149.79; 164.91; IR: 3009, 2968, 2930, 2851, 1697, 1607, 1519,
1318, 1165, 770;
HRMS (þESI): m/z calculated C15H22Cl2NO2 (M þ H)þ 318.1022;
m/z measured: 318.1042.
4.1.4. 4-[Bis-(2-chloroethyl)amino]benzoic acid
Yield: 95%; m.p: 162e167 ꢀC;
1H NMR (DMSO)
d
: 3.79 (m, 8H); 6.81 (d, 2H, J ¼ 8.0 Hz); 7.77 (d,
2H, J ¼ 8.0 Hz); 12.29 (bs, 1H);
13C NMR (DMSO)
: 40.75; 51.65; 110.87; 118.10; 131.19; 149.83;
167.18; IR: 3432, 2968, 1678, 1603, 1523, 1416, 1297, 1183, 767;
d

B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
103
HRMS (-ESI): m/z calculated for C11H12Cl2NO2 (M ꢁ H)ꢁ
260.0240; m/z measured 260.0254.
with four channels. Separations were performed at 30 ꢀC at a flow
rate of 0.2 ml/min. Samples dissolved in methanol were injected into
a 5
mL loop and then eluted with the following program: 30%
4.1.5. N-{4-[Bis-(2-chloroethyl)amino]benzoyl}-N0-tert-
butoxycarbonylhydrazine
acetonitrile in water for 2 min, 30e60% gradient of acetonitrile in
water for 8 min, followed by 90% acetonitrile in water for 4 min.
Chromatograms were recorded at 230, 280, 300, and 335 nm.
Yield: 96%; m.p: 78e85 ꢀC;
1H NMR (DMSO)
d
: 1.42 (s, 9H); 3.77 (m, 8H); 6.80 (d, 2H,
J ¼ 8.0 Hz); 7.75 (d, 2H, J ¼ 8.0 Hz); 8.77 (bs, 1H); 9.89 (bs, 1H);
13C NMR (DMSO)
: 28.04; 40.90; 51.69; 78.87; 110.82; 120.04;
4.2.3. Isolation of the products of the enzymatic reaction
d
To identify the products of the oxidation of N-{4-[bis-(2-
chloroethyl)amino]benzoyl}-N0-(4-hydroxybenzyl)hydrazine
by
129.07; 148.97; 155.60; 165.52; IR: 3396, 3290, 2979, 2932, 1722,
1655, 1608, 1510, 1368, 1252, 1160, 762;
tyrosinase 30.58 mg of the substrate (0.1 mM) was incubated in
800 ml of 10 mM sodium phosphate buffer, pH 6.8, with 4 mg of
tyrosinase. The reaction mixture was stirred for up to 2 h (condi-
tions were optimized for each of the products) and was then
extracted with 2 ꢂ 400 ml of dichloromethane. Because the phases
separated poorly, the mixture was transferred to 50 ml Falcon tubes
and centrifuged at 7000ꢂg for 5 min at room temperature in an
Eppendorf 5810R centrifuge. The organic layer was recovered and
dehydrated with anhydrous sodium sulfate. The solvent was
HRMS (þESI): m/z calculated for C16H23Cl2N3NaO3 (M þ Na)þ
398.1009; m/z measured: 398.0998.
4.1.6. 4-[Bis-(2-chloroethyl)amino]benzoic acid hydrazide
Yield: 90%; m.p: 123e128 ꢀC;
1H NMR (DMSO)
d
: 3.75 (m, 8H); 4.50 (bs, 2H) 6.76 (d, 2H,
J ¼ 8.0 Hz); 7.71 (d, 2H, J ¼ 8.0 Hz); 9.47 (bs, 1H);
13C NMR (DMSO)
: 40.51; 51.22; 110.65; 117.15; 129.25; 149.55;
d
165.19; IR: 3435, 2951, 2927, 2821, 2666, 1606, 1513, 1328, 831, 758;
HRMS (þESI): m/z calculated for C11H16Cl2N3O (M þ H)þ
276.0665; m/z measured: 276.0659.
evaporated, the residue was dissolved in 400
ml of acetonitrile and
applied to a 10 g octadecyl column (BakerbondÒ Octadecyl 40
mm
Prep LC Packing). The sample was eluted with a stepwise gradient
of acetonitrile in water (30 ml of each: 40%, 45%, 50%, 55%, 60%, 70%,
80%, 90% of acetonitrile in water and then with 100% acetonitrile).
Fractions containing a pure product were pooled, the solvent was
evaporated, the residue was dissolved in DMSO and analyzed by
NMR (1H, 13C, HSQC, HMBC).
Separation on a reverse-phase column was not effective for the
isolation of the second product, migrating in HPLC analysis close to
the substrate. This compound was therefore isolated by column
chromatography of the crude extract of the enzymatic reaction on
silica gel (eluent: 1e3% methanol in dichloromethane).
For HPLC analysis reactions were carried out at 5
(1.91 mg of the substrate in 50 ml of buffer) with 250 or 500
tyrosinase for 15, 30, 60, and 120 min. After extraction with
dichloromethane and evaporation of the solvent, samples were
dissolved in 500 mL of methanol, a small portion was diluted 1:5
4.1.7. N-{4-[Bis-(2-chloroethyl)amino]benzoyl}-N0-(4-
hydroxybenzylidene)hydrazine
Yield: 72%; m.p: 119e122 ꢀC;
1H NMR (DMSO)
d
: 3.80 (m, 8H); 6.83 (d, 2H, J ¼ 8.0 Hz); 6.84 (d,
2H, J ¼ 8.0 Hz) 7.54 (d, 2H, J ¼ 8.0 Hz); 7.81 (d, 2H, J ¼ 8.0 Hz); 8.33
(bs, 1H); 9.90 (bs, 1H); 11.41 (bs, 1H);
13C NMR (DMSO)
d: 41.46; 52.22; 111.40; 116.11; 121.45; 126.03;
129.05; 129.79; 147.28; 149.48; 159.59; 162.88; IR: 3420, 3223,
1603, 1509, 1355, 1276, 1188, 1055, 920, 833, 756, 726;
HRMS (þESI): m/z calculated for C18H20Cl2N3O2 (M þ H)þ
380.0927; m/z measured: 380.0936.
mmol scale
mg of
4.1.8. N-{4-[Bis-(2-chloroethyl)amino]benzoil}-N0-(4-
hydroxybenzyl)hydrazine
Yield: 74%; m.p: 116e122 ꢀC;
1H NMR (DMSO)
d: 3.76 (m, 10H); 5.11 (m, 1H); 6.71 (d, 2H,
with methanol and analyzed as described above. The reaction
mixture before extraction and both phases after extraction were
analyzed by TLC with the substrate and the free mustard drug (4-
[bis-(2-chloroethyl)amino]benzoic acid) as standards.
J ¼ 8.4 Hz); 6.76 (d, 2H, J ¼ 9.2 Hz); 7.14 (d, 2H, J ¼ 8.4 Hz); 7.69 (d,
2H, J ¼ 9.2 Hz); 9.30 (s, 1H); 9.77 (bs, 1H);
13C NMR (DMSO)
d: 41.42; 52.20; 55.19; 111.30; 115.36; 121.26;
128.99; 129.19; 130.33; 149.13; 156.87; 165.76; IR: 3398, 3281,
2960, 2936, 1608, 1516, 1458, 1280, 830, 759, 730;
HRMS (þESI): m/z calculated for C18H22Cl2N3O2 (M þ H)þ
382.1084; m/z measured 382.1077.
4.2.4. MS analysis of the products of the enzymatic reaction
For MS analysis the product purified by reverse phase chroma-
tography was dissolved in acetonitrileewater (100
ml:50 ml) and
10 l of 1 N sodium hydroxide was added. The second product
m
isolated by normal phase chromatography was dissolve in
acetonitrile-water (the same ratio). These samples were injected
directly into the mass spectrometer.
4.2. Analysis of the enzymatic activation of the prodrug
4.2.1. Spectrophotometric analysis of the oxidation of the prodrug
by tyrosinase
4.2.4.1. 5,6-Dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]
Mushroom tyrosinase was isolated as previously described [21].
A stock solution of N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N0-(4-
hydroxybenzyl)hydrazine was prepared in 40% methanol at 1 mM
concentration. The substrate was then diluted to 0.05 mM in 10 mM
sodium phosphate buffer, pH 6.8 giving 2% final methanol con-
benzoate. 1H NMR (DMSO)
d
: 3.80 (m, 8H); 6.86 (d, 2H, J ¼ 8.0 Hz);
7.09 (s, 1H),7.80 (s, 1H), 8.00 (d, 2H, J ¼ 8.0 Hz); 8.15 (s, 1H); 9.40 (bs,
2H);
13C NMR (DMSO)
d: 41.39; 52.17; 101.01; 104.63; 111.03; 118.83;
120.59; 134.00; 135.14; 139.90; 144.98; 149.46; 150.14; 166.64;
HRMS (-ESI): m/z calculated for C18H16Cl2N3O3 (M ꢁ H)ꢁ
392.0574; m/z measured: 392.0580;
centration, and 10 mg, 5 mg or 2.5 mg of tyrosinase from a 1 mg/ml
stock in the same buffer was added. The final reaction volume was
3 ml. The spectra between 200 and 600 nm were recorded at 1 min
intervals for 60 min in a Jasco V-650 UVeVis spectrophotometer.
4.3. Analysis of anti-melanoma activity
4.2.2. HPLC analysis
Products of the enzymatic reactions were analyzed on an Accu-
4.3.1. Cell culture
core C18 analytical column (100 mm length, 2.1 mm diameter, 2.6
particle size) from Thermo Scientific with a guard column connected
to a Dionex UltiMate 3000 HPLC instrument with a UVeVis detector
m
m
The following cell lines were used: B16eF10, NIH3T3 (all from
ꢀ
ATCC); HECa10 (lymph node-derived and provided by Dr. D. Dus,
Institute of Immunology and Experimental Therapy, Wroc1aw,

104
B. Fra˛ckowiak-Wojtasek et al. / European Journal of Medicinal Chemistry 71 (2014) 98e104
Poland). Cells were cultured using RMPI 1640 media supplemented
with 10% FBS and using NUNCLONÔ Surface (NUNCÔ) culture
vessels. Cultures were kept in a humidified standard incubator
(37 ꢀC and 5% CO2).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.10.
080. These data include MOL files and InChiKeys of the most
important compounds described in this article.
4.3.2. Drug cytotoxicity
Cells were grown in 96-well plates (NUNCLONÔ Surface,
2 ꢂ 103 cells per well, 100
mL of RMPI 1640 supplemented with
10% FBS). After 24 h, the tested compound was added in
quadruplicate to the culture medium using eleven different
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Acknowledgments
This work was supported by a grant from the Polish Ministry of
Science and Higher Education No. 2 P05F 00330.