University of Cambridge, Department of Materials Science and Metallurgy, Pembroke Street, Cambridge CB2 3QZ, United Kingdom, Tel.: +44 1223 334 356, Fax: +44 1223 334 567
1Silesian University of Technology, Department of Organic Chemistry, Biochemistry and Biotechnology, Krzywoustego 4, 44-100 Gliwice, Poland, Tel.: +48 32 237 1792, Fax: +48 32 237 2094
2University of Cambridge, Department of Materials Science and Metallurgy, Pembroke Street, Cambridge CB2 3QZ, United Kingdom, Tel.: +44 1223 334 356, Fax: +44 1223 334 567
Corresponding author email
Associate Editor: S. Flitsch Beilstein J. Org. Chem.2010,6, No. 34.https://doi.org/10.3762/bjoc.6.34 Received 20 Jan 2010,
Accepted 23 Mar 2010,
Published 12 Apr 2010
We present the synthesis and selected physicochemical properties of several novel symmetrical and unsymmetrical α,ω-nucleobase mono- and bis-amide conjugated systems containing aliphatic, aromatic or saccharidic linkages. The final stage of the synthesis involves condensation of a subunit bearing carboxylic group with an amine subunit. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was found to be a particularly effective condensing agent. The subunits containing carboxylic groups were obtained by acidic hydrolysis of N-1 Michael adducts of uracils or N-9 Michael adducts of 6-chloropurine with methyl acrylate. The amines used were aliphatic/aromatic diamines, adenine, 5-substituted 1-(ω-aminoalkyl)uracils and 5′-amino-2′,5′-dideoxythymidine. The title compounds may find application as antiprotozoal agents. Moreover, preliminary microscopy TEM studies of supramolecular behaviour showed that target molecules with bolaamphiphilic structures were capable of forming highly ordered assemblies, mainly nanofibres.
There are numerous reports on the synthesis and application of symmetrical and unsymmetrical α,ω-nucleobase amide-conjugated systems (Figure 1) [1-6]. These molecules are constructed usually via amide bond(s) from two subunits containing nucleobases or their analogues (B1, B2) bearing various linkages (A1, A2).
We have previously reported compounds containing 5-substituted uracil-1-yl subunits bound via short (I, II) or extra-short (III) amide-linkages [1]. These compounds exhibited medium antiprotozoal activity; thus I and III exhibited growth inhibition of parasities – Leishmania donovani and Plasmodium falciparum – at levels of 21.4 and 30.6%, respectively [2]. Compound II reduced the Plasmodium falciparum population by only 17.4%, although it was the most active derivative against it within the group tested. Very recently, Accetta et al. reported remarkable symmetrical amide-conjugated bis-α,ω-uracil based systems (IV). These compounds exhibited antiproliferative and erythroid differentiation induction properties towards human chronic myelogenous leukaemia K562 cells [3]. Moreover, α,ω-nucleobase amide-conjugated molecules possess the tendency to form meta-stable complexes with DNA and thus can perturb the cell replication. For example, amide-linked heterodimer synthons consisting of acyclic nucleoside units and 5′-amino-2′,5′-dideoxythymidine are PNA/DNA chimeras (V) [4]. Furthermore, amide-conjugated systems of solely biologically active units may be utilised in combined anti-HIV therapy, e.g. 2′,3′-dideoxycytidine and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine conjugate (ddC-HEPT) (VI) [5].
In addition, Shimizu et al. in 2001 reported structures, which are formally α,ω-nucleobase bolaamphiphiles (VII), capable of forming extraordinary nanotopologies [6].
In order to synthesise a new group of modified, mainly pyrimidinic nucleosides, we employed a simple and effective synthetic pathway based on the catalytic aminolysis of carboxylic acids. Our synthetic approach surpasses all the procedures previously reported, not only in terms of higher yields, even although earlier reports of the synthesis of symmetrical α,ω-nucleobase amide-conjugated systems were based on a similar strategy. These methods involved two-step procedures such as catalytic condensation, e.g. in the presence of a suitable coupling agent – N,N′-dicyclohexylcarbodiimide (DCC) [7], or acylation of appropriate amines with acyl halides [3] or “active esters” [6]. Monoacylation of aliphatic diamines, either in the presence of MgCl2[8,9] or 9-borabicyclo[3.3.1]nonane (9-BBN) [10], has been used for the synthesis of unsymmetrical derivatives. Interestingly, considering the physicochemical aspect of our work, two of the compounds synthesised containing aliphatic long-chain linkages revealed a tendency to self-assemble in the initial TEM studies.
Results and Discussion
The subunits containing carboxylic groups were obtained by acidic hydrolysis of N-1 Michael adducts of uracils or N-9 Michael adducts of 6-chloropurine with methyl acrylate (Scheme 1A) [1]. Uracil derivatives were obtained via the fully regioselective one-step procedure as previously reported [11,12]. To construct systems containing modified purinic nucleobase, we synthesised the N-9 adduct (1ea) of 6-chloropurine with methyl acrylate in 90% yield using Hünig’s base (diisopropylethylamine) as a deprotonating agent (Scheme 1B) (see Supporting Information File 1).
X-ray analysis confirmed the structure of this regioisomer (Figure 2) [13]. This simple method appears to be the most N-9 regioselective process compared to other methods [14-16].
The N-7 adduct of 6-chloropurine (1eb) was also isolated in 10% yield. Hydrolysis of the N-9 adduct under acidic conditions (5% HClaq) gave 9-(2-carboxyethyl)-6-chloro-9H-purinium-3 chloride (1e). As amine components, readily accessible amines such as aliphatic α,ω-diamines, phenylene diamines, adenine, 5-substituted 1-(ω-aminoalkyl)uracils and 5′-amino-5′-deoxythymidine were chosen.
To accomplish the synthesis of the title compounds, we developed a simple and effective method of coupling the carboxylic subunit with an amino compound. As condensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was found to be very convenient and efficient (Figure 3) [19].
We have previously used this catalyst successfully in our search for unsymmetrical α,ω-nucleobase molecules, which are structurally related to the title compounds, i.e. short linkage amide- [1] and ester-conjugated systems [12].
Symmetrical α,ω-nucleobase amide-conjugated systems
The first set of compounds – symmetrical aliphatic and aromatic α,ω-nucleobase amide-conjugated systems (4–15, Table 1) – was synthesised from 3-(5-substituted-uracil-1-yl)propionic acids 1a–d and the appropriate aliphatic α,ω-diamines 2a–e, and the aromatic diamines 3a–c in DMF (see Supporting Information File 1).
Table 1:
Symmetrical aliphatic and aromatic α,ω-nucleobase amide-conjugated systems.
Acidic subunit
X
Amine subunit
R
Product
Yield [%]
1c
Br
2a
(CH2)2
4
57
1a
H
2b
(CH2)3
5
64
1b
CH3
2b
(CH2)3
6
60
1d
NO2
2b
(CH2)3
7
55
1b
CH3
2c
(CH2)6
8
67
1c
Br
2c
(CH2)6
9
31
1a
H
2d
(CH2)9
10
74
1b
CH3
2e
(CH2)10
11
59
1d
NO2
2e
(CH2)10
12
67
1c
Br
3a
o-phenylene
13
35
1c
Br
3b
m-phenylene
14
37
1a
H
3c
p-phenylene
15
68
The yields vary from moderate to good. The lower yields noted in the table arise as a result of the method of isolation (see Supporting Information File 1). Despite its susceptibility to nucleophilic attack, 5-nitrouracil acid derivative 1d also reacted with aliphatic diamines and gave the expected diamides 7 and 12.
Unsymmetrical α,ω-nucleobase amide-conjugated systems
The second group – unsymmetrical α,ω-nucleobase amide-conjugated systems and their synthetic precursors (Table 2) – was synthesised from monoacid and monoamine derivatives with the exception of mono-amine precursor 16 which was synthesised from 1,6-hexylenediamine.
This synthesis was based on the classical approach of half-conversion of the bifunctional reactant with the monofunctional compound by using an excess of diamine (3 equiv) with respect to the acidic reactant. Nevertheless, this procedure led to problems in the purification step and partial decomposition of DMT-MM during the reaction. This was especially significant when compared to the synthesis of N-Boc monoprotected amine reactant 17 (See Supporting Information File 1).
The compounds in this second group were obtained in yields of 31–95%. The synthesis of nucleosides containing aliphatic linkages (19–22) presented no difficulties on work-up.
Table 2:
Unsymmetrical α,ω-nucleobase amide-conjugated systems based on aliphatic or saccharidic linkages and their exemplary synthetic precursors.
B1A1–COOH
H2N–A2B2
Pro-
duct
B1A1−CONH−A2B2
Yield [%]
1c
2c
16
33
1b
2ba
17
99
1b
2f
18
68
1c
2g
19
90
1c
2h
20
83
1e
2i
21
95
1e
2j
22
44
1f
2h
23
85
1g
2k
24
31
1c
2l
25
92
Two different acidic and three different amine subunits – synthesised according to known procedures – were utilised in the synthesis of 19, 20 and 23–25. The acidic subunits used were (2S,3S,5R)-3-hydroxy-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydrofuran-2-carboxylic acid (1f) [21] and 4-({(2R,3S,5R)-3-acetoxy-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydro-furan-2-yl}methoxy)-4-oxobutanoic acid (1g) [22]. The amine subunits employed were 3-[5-nitro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]propan-1-aminium trifluoroacetate (2g) [23], 6-[5-nitro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]hexan-1-aminium trifluoroacetate (2h) [23] and 1-[(2R,4S,5R)-5-(aminomethyl)-4-hydroxytetrahydrofuran-2-yl]-5-methylpyrimidine-2,4-(1H,3H)-dione (2l) [24]. The synthesis of the amine subunit 2j was carried out according to the known procedure [25]. The unprotected hydroxyl group in sugar moieties, as expected, was entirely unreactive towards the carboxylic group when compared to the amine functionality, and consequently nucleosides with a saccharide moiety (23, 25) were successfully synthesised.
Self-assembly of aliphatic long-chain symmetrical α,ω-nucleobase amide-conjugated systems
For the microscopic studies, we selected the two model compounds 11 and 12. The supramolecular behaviour of conjugated nucleobase-based bolaamphiphiles was examined by TEM. A JEOL 200 FX, operating at 200 kV, was used to obtain the typical images as shown in Figure 4. For the microscopic studies, saturated ethanolic solutions of 11 and 12 prepared by ultrasonication were subjected to slow vaporisation at −20 °C. Microcrystalline assemblies were grown directly on copper grids for TEM analyses. This procedure of growth accompanied by careful investigations revealed that thymine- (A, B) and 5-nitrouracil-based (C) bolaamphiphiles derived from 1,10-diaminodecane may form nanofibres of 80–300 nm diameter for thymine and 15–100 nm for 5-nitrouracil derivatives, respectively.
The electron diffraction patterns in the case of 11 indicate single crystal fibre diffraction (Figure 4A). A simplified model for the formation of multilayered nanofibres is shown in Figure 5.
A proposal of simplified models of micro- and nanofibres is based on the π-stacking and hydrogen bonding interactions between thymine units. Amide bonds, on the other hand, may be crucial in the inter-layer hydrogen bonding. A linear system of hydrogen bonding appears as a thermodynamically favourable (A). Nevertheless, a less favourable “bent” (or “wobble”) system could also be involved [26]. This fact is reflected in the presence of residual electron diffraction patterns, which are in particular visible in the case of so-called “twin fibres” (B) growing out from the microcrystalline phase – “lizard tail”. A complete model of the nanofibre is presented in Figure 5C along with its longitudinal cross-section (D).
Conclusion
We have synthesised a novel group of twelve symmetrical and ten unsymmetrical α,ω-nucleobase amide-conjugated systems using DMT-MM as the condensing agent. These two groups of compounds will be investigated for antiprotozoal activity within the framework of the Drugs for Neglected Diseases initiative (DNDi). These investigations are currently in progress.
Additionally, we have undertaken preliminary TEM studies on the self-assembly of two symmetrical aliphatic long-chain compounds. Their supramolecular nature was revealed since they form nanofibres during low temperature crystal growth. These experiments were reproducible, but further studies are required.
Supporting Information
Experimental Section
Supporting Information File 1:
The data provides general remarks, procedures, physical properties and 1H and 13C NMR spectra of all newly synthesised compounds.
The authors would like to thank the Institute of Tropical Diseases in Geneva, Switzerland for performing the initial tests of antiprotozoal activity for the unsymmetrical short-linkage α,ω-nucleobase amide-conjugates. These investigations were conducted within the framework of the Drugs for Neglected Diseases initiative (DNDi).
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Crystallographic data of this compound was deposited in The Cambridge Crystallographic Data Centre under the reference number CCDC-728242. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif
Unpublished data. Biological activity was tested in Institute of Tropical Diseases within a framework of Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland.
Zare, A.; Hasaninejad, A.; Beyzavi, M. H.; Parhami, A.; Zare, A. R. M.; Khalafi-Nezhad, A.; Sharghi, H. Can. J. Chem.2008,86, 317–324. doi:10.1139/V08-022
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