The search for cytotoxic coordination compounds of biogenic metals, which have lower
overall toxicity compared to clinically used platinum-containing drugs, has attracted
significant interest in recent years.[1 ]
[2 ] Among the studied compounds of biogenic transition metals (Co, Cu, Zn, Fe), the
most promising as anticancer agents may be copper coordination compounds, which can
exhibit high cytotoxic activity by various mechanisms.[3–10 ] Copper-containing complexes with 2-thio-imidazol-4-ones are among the classes of
such compounds with proven antitumor activity,[11 ]
[12 ] and ability to inhibit telomerase activity.[13 ]
It is known that coordination compounds containing Cu(I) can effectively penetrate
tumor cells.[14 ] Improving drug penetration into cells allows a reduction of their therapeutic dose
and, therefore, general toxicity. One strategy to stabilize Cu(I) in molecules is
to use an organic ligand with a redox potential close to Е
0
Сu(II)/Cu(I) . In this way, we have previously shown that the copper-containing coordination compounds
of 2-thioimidazol-4-ones with ferrocene-containing organic ligands had cytotoxicity
comparable to clinically used drugs.[15 ] To find the an optimal redox-active fragment to achieve maximum cytotoxicity, we
synthesized three novel 2-thioimidazol-4-ones containing organic ligands possessing
the 2-methylnaphthalene-1,4-dione (menadione) fragment, instead of the previously
utilized bulky ferrocene moiety.
Menadione (2-methyl naphthoquinone) is available from natural sources, in particular
from the leaves and nuts of Juglans nigra and Juglans regia .[16 ] It has near to the Е
0
Сu(II)/Cu(I) potential of quinone/hydroquinone redox transition (Е
0 = +0.2V)[17 ] and can potentially facilitate the intercalation into tumor cell DNA due to its
planar π-system. In addition, a recent study of 1,4-naphthaquinone derivatives has
shown that these compounds are selective natural inhibitors of Cdc25 and MKK7, which
are key enzymes in the cell cycle and oncogenesis, with inhibition constants in the
order of 100 nM,[18 ] which could lead to an additional enhancement of the antiproliferative effect of
menadione conjugates on cancer cells.
To our surprise, examination of the published methods for incorporating an amine fragment
into the menadione substrate revealed that there are no protocols for the direct amination
of this quinone with primary amines under mild conditions. Menadione reactions with
methylamine in a wide range of solvents have been reported, but the yields do not
exceed 30–40%. In particular, the simplest protocol (dichloromethane, 28 °C, 24 hours)
leads to the adduct of menadione and methylamine in a yield of 40%.[19 ] Use of ethanol as solvent and a ten-fold excess of amine in the presence of sodium
hydroxide for 24 hours gave an increased yield, but complicated the isolation and
purification of the product.[20 ] Further attempts to increase the yield of the amination product required in use
of harsher conditions, including reaction in HClO4 medium without organic solvent.[21 ] However, such conditions are incompatible with amines possessing labile functional
groups.
Two- and three-stage syntheses have also been developed, involving the halogenation
of menadione followed by the replacement of the halogen atom with an amine; however,
these approaches also resulted in low yields and led to mixtures of halogen substitution,
methyl substitution, and polyamination products, even when the simplest amine, methylamine,
was used.[22 ] In the case of more complex amines the problem of side reactions increased and isolation
of the target products was difficult.[23 ]
However, some methods are known for the amination of menadione and other quinones
with simple amines (propyl, butylamine, etc.), as well as branched non-functional
amines (tert -butylamine, neopentylamine, etc.).[24 ] Using propargylamine as a simple functionalized amine, it was shown that direct
amination in alcohol media could result in yields up to 80%. However, the authors
of this article also did not extend their finding to more complex and functional amines.[25 ]
To obtain the desired target compounds, we have developed a two-step synthesis including
the direct amination of menadione with functionalized primary alkyl amines under mild
conditions, followed by conjugation of the obtained menadione derivatives with substituted
5-pyrididylmethylene-2-thioimidazol-4-ones.
We began our investigation of the menadione amination at room temperature with propargylamine
as a simple functionalized primary amine (Table [1 ]). A range of polar and nonpolar solvents were tested (DCM, DCM/EtOH mixture, EtOH,
DMF) and it was found that the yields in polar media were somewhat higher than in
nonpolar ones, and the highest yield was observed when 95% ethanol was used as a solvent.
The optimal reaction time was 9 to 12 hours. With a further increase in time, the
yield of the target product increased slightly, but significant amounts of polymeric
material were formed.
Table 1 Optimization of the Reaction Conditionsa
Entry
Solvent
Time (h)
Yield (%)b
1
DCM
3
24
2
DCM/EtOH 10:1
3
28
3
DCM/EtOH 1:1
3
35
4
EtOH
3
45
5
DMF
3
30
6
EtOH 95%
6
63
7
EtOH 95%
9
75
8
EtOH 95%
12
80
9
EtOH 95%
24
84
10
EtOH (argon atm.)
12
77
11
EtOH 95%(sunlight)
12
49
a All reactions were performed using menadione (0.3 mmol) and propargylamine (0.9 mmol)
in solvent (10 mL) in the absence of light.
b Yields calculated based on purified product.
Carrying out the reaction under an inert atmosphere did not lead to an increase in
the yield or to less darkening of the reaction mixture. In contrast, carrying out
the reaction in the dark is important, although this was not mentioned in any previous
reports on the amination of menadione and its derivatives. Apparently, due to the
photo-oxidation of menadione,[26 ] higher yields can be obtained in the absence of light.
All subsequent reactions with substituted amines (Table [2 ]) were carried out under the optimized conditions. It was found that the conditions
were suitable for primary amines with various functional groups (azide, alkyne, hydroxyl,
amine, halogen) and even for glycine. Most reactions proceeded in good yield (up to
82%). Thus, we have developed a convenient method for the addition of N-nucleophiles
to menadione that is suitable for primary amines with various functional groups.
Table 2 Direct Amination of Menadionea
Entry
Product
X
Yield (%)b
m.p. (°C)
1
1
-C≡CH
80
156
2
2
-CH2 -OH
73
98
3
3
-CH2 -N3
c
82
100
4
4
-CH2 -CH2 -N3
c
80
58
5
5
-CH2 -CH2 -NHBoc
57
106
6
6
-CH2 -Cl
78
123
7
7
-CH2 -COOHd
64
174
a All reactions were performed under the optimized conditions.
b Yields calculated based on purified product.
c Azidoamines were prepared by the procedure previously reported for 2-azidoethanamine
and used without purification.[15 ]
d Triethylamine was added to form the free NH2 group from the zwitterion.
It should be noted that 2-azidoethanamine and 3-azidopropan-1-amine are both reasonably
stable and safe to use either in solution or as neat materials when cooled to 0–10 °C,
so they should be added cold. The relatively low yield in the case of product 5 may be explained by increased polymerization in this case.
A comparison of the UV/Vis spectra of target and starting compounds (Figure [1 ]) showed that compounds 1 –6 have an absorption maximum of about 450 nm, which is consistent with their intense
red color, and such adsorption bands are distinct from the absorption band of the
initial menadione (absorption maximum at 332 nm, yellow color). The extinction coefficients
for all amino-menadiones 1 –7 are approximately 2–3 × 103 M–1 сm–1 . The positions of the absorption maxima are slightly dependent on the nature of the
functional group in the amine and on the length of the hydrocarbon spacer between
the functional group and the amino group. The combination of these factors allows
assessment of the progress of the menadione amination to be made visually, by observing
the change in color of the reaction mixture from yellow to bright red.
Thus, UV/Vis spectroscopy may be used for the determination of the product concentrations
in mixtures and in biological tests.
Figure 1 UV/Vis spectra of menadione and compounds 1 –7 (EtOH, 3 × 10–4 M).
Compounds 1 –7 may then potentially be used to incorporate a menadione fragment into the structure
of different organic ligands via linkers of various chemical nature using azide-alkyne
cycloaddition reactions, ester and peptide synthesis, and also by nucleophilic substitution
of a labile halogen (chlorine) atom.
To demonstrate the feasibility of such reactions, the products of the azide-alkyne
cycloaddition (CuACC) of menadione derivatives 1 and 3 with azido- and alkynyl-containing 5-pyridylmethylene-2-thio-imidazolones 8 and 9 were synthesized (Scheme [1 ]). As a result, menadione-substituted derivatives 11 and 12 were obtained in high yields under heterogeneous click reaction conditions.
Scheme 1 CuACC reactions of menadione derivatives 1 and 3 with 5-pyridylmethylene-2-thioimidazolones 8 and 9
For the synthesis of organic ligands by click chemistry, it was important to test
the reactivity of azides and alkynes under the conditions of biphasic catalysis with
Cu(I). Carrying out the reactions in a methylene chloride/water mixture avoided the
loss of catalyst by binding to the chelating part of the ligand and reduced the difficulties
associated with isolating the free product (not bound to copper ions) from the reaction
mixture. Washing the excess copper from the by-product complex with EDTA and other
complexants created additional preparative difficulties due to the low solubility
of the copper complexes of the 2-thiohydantoins in water and the hydrolysis of the
SME fragments. Previously described click reactions of menadione derivatives were
mostly carried out under homogeneous conditions, which were not applicable in our
case.[25 ]
,
[27 ]
[28 ]
[29 ]
[30 ]
[31 ]
It is worthy of note that both aminomenadiones 1 and 3 with propargyl and azide moieties are equally effective under these reaction conditions.
The conjugate 13 , containing menadione and 2-thioimidazole-4-one moieties linked via a biodegradable
ester linker, was synthesized starting from hydroxy-containing quinone 2 and (Z )-2-(5-oxo-4-(pyridin-2-ylmethylidene)-2-thioxoimidazolin-1-yl) acetic acid (10 ; Scheme [2 ]).
Scheme 2 Synthesis of conjugate 13 with an ester linker between menadione- and 2-thioimidazole-4-one-containing fragments
It was shown that the hydroxy-containing menadione derivative 2 is not only capable of being applied to carbodiimide synthesis, but it also gave
satisfactory yields of the target ester under standard reaction conditions and with
standard combinations of activating reagents without additional optimization.
In conclusion, in the framework of this work, various conditions for the direct amination
of menadione have been investigated, optimal conditions were selected for the case
of labile amines, and a series of aminated derivatives was obtained with 60–80% yields.[32 ]
[33 ]
[34 ]
[35 ]
[36 ] The possibility of further conjugation of the obtained menadione functional derivatives
containing propargyl, azide or hydroxyl fragments with substituted 5-pyridylmethylene-2-thioimidazol-4-ones
has been demonstrated. All products were isolated and purified using column chromatography
with reasonable yields. Thus, a convenient synthetic pathway for accessing conjugates
of 2-thioimidazol-4-ones and menadione with various linkers based on a two-step procedure,
which opens up the possibility of obtaining a wide series of promising ligands for
antitumor coordination compounds, has been developed.
