Volume 2 number 1 1 November 1 975
Volume 2 number 11
November1975
Nucleic Acids Research
Nucleic Acids Research
A facile synthesis of 5-(perfluoroalkyl)-pyrimidines+
D. Cech, R. Wohlfeil and G. Etzold
Humboldt-University Berlin, Department of Chemistry, 104 Berlin, GDR R eceived 15 October 1975
ABSTRACT In the paper a synthetic two stage procedure is described for the preparation of perfluoroalkylated derivatives of uracil and its nucleosides. Using copper bronze a perfluoroalkyl-copper-complex is formed from perfluoroalkyl iodides in polar aprotic solvents, such as DMSO, and under inert conditions. The reaction of this complex with uracil, uridine and 2'-deoxyuridine leads to the corresponding 5-substituted perfluoroalkyl derivatives. It is shown by mass spectra that the substitution always takes place at the 5-position of the pyrimidine. The chemical and physical properties of the formed compounds are described.
INTRODUCTION Among the well-known antimetabolites of the nucleic acid metabolism 5-trifluoromethyluracil and its 2'-deoxyribofuranoside have proved particularly effective 1). Higher homologues of the series of the 5-perfluoroalkyl pyrimidines, however, have not been described as yet. It is true that PREOPRASHENSKAYA and co-workers 293) described a synthesis of similar derivatives obtained by a transformation of 5-hydroxymethyluracil with tetrafluoroethylenes, but the compounds obtained do not represent true homologues in the series of the perfluoroalkyl derivatives as the perfluoroalkyl group is always separated from the pyrimidine ring by a CH2-group. So we first studied the question to what extent the procedures used for 5-trifluoromethyluracil were also applicable to the synthesis of higher homologues. Whereas methods for the preparation from 5-carboxyuracil and SF 4) or the electrochemical introduction of a perfluoroalkyl group 5) are out of the question from the first, a further
2183
Nucleic Acids Research difficulty arises from the difficult-accessibility of 2 H-perfluoroacetic acid derivatives which are required for the preparation of these compounds by usual procedure of cyclocondensation with urea. There are a number of methods for the direct introduction of perfluoroalkyl groups into aromatic compounds 6,7,8), however, non one was used for the perfluoroalkylation of pyrimidines. RESULTS AND DISCUSSION In view of the difficulties, we considered the use of and COE 10) the method recently described by McLOUGHLIN for the introduction of perfluoroalkyl groups into benzene and its derivatives also for the synthesis of perfluoroalkylated pyrimidines. According to McLOUGHLIN, perfluoroalkyl iodides are transformed with aromatic iodine compounds in the presence of Cu-bronze under inert conditions in polar aprotic solvents. The formation of perfluoroalkyl compounds proceeds via a copper complex according to the following equation : RFJ
+
ArJ
RFAr
Cu
Ar RF
=
=
+
2 CuJ
aryl perfluoroalkyl
As aromates can also be used, we were of the opinion that possibly also pyrimidines can be perfluoroalkylated in spite of the presence of two readily protonisable H-atoms. First, the perfluoroalkyl-copper-complex (RFCuL3 t L = DMSO; R? = CF3 (CF2)n CF2-, n = 2, 4, 6 and 8) was prepared by the transformation of perfluoroalkyl iodides with specially prepared Cu-bronze in DMSO at 110 °C with exclusion of oxygen. If this complex (n=4) is reacted with 5-iodouracil for example, one obtains, apart from non-transformed initial product, only uracil, whereas a transformation of the previously isolated complex with iuracil in DMSO yields a new, uv-absorbing compound. Mass spectrometric examination of the structure of the new compound showed that the sum formula corresponds exactly to that qf 5-(Z-hexyl)-uracil (Ib) 11). The spectrum
2184
Nucleic Acids Research contains
fragments m/e
=
241
and
m/e
=
91
(for
+O;C-CH=CF2)
which prove the lokalisation at the C-5-position. The fragment patterns show that a bond splitting occurs between C-4 and N-3 and between C-5 and C-6 of the uracil 12).Moreover, the site of the UV-maximum in acidic and alkaline media (significant shift of the maximum in alkaline media) confirms that the substitution did not occur at the N-atoms of the pyrimidine.
Figure 1
CF2-(ICF2)n-CF3
HN
O NJ R I II
R R
=
III
R
=
=
a n H; ribofuranoside ;
2, b
n = 4, c n = 6 2, b n = 4, c n d n =8 deoxyribofuranoside a n = 2, b n =
a n
=
=
6,
=
8
The yields of the reaction amount to approximately 10 to 20 per cent in addition to non transformed uracil, with two side products of less importance being formed the structure of which has not been cleared up yet. We believe that the small yields are due to side reactions in the transformation of the complex with uracil which are not yet understood. The spectrometric determination of the formation of the complex showed yields of approx. 60 per cent in relation to the alkyl iodide used 13). It was found in our experiments that a variation of the reaction conditions did not bring about higher yields. For example, when using other solvents, such as pyridine or DMF no reaction was obtained, and the addition of catalysts such as azo-bis-isobutyronitril, which was supposed to accelerate the radical decomposition of the complex, did not yield any remarkable results.
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Nucleic Acids Research Under the reaction conditions that proved to be favourable for the transformation of perfluorohexyl copper with uracil, also perfluorobutyl- and perfluorooctyl copper complexes showed a reaction. It was, however, found that the reactivity markedly decreases with the chain length. 5-(F-octyl)-uracil (Ic) could only be obtained in small quantities, and 5-(F-decyl)-uracil can no longer be prepared under the above mentioned conditions. After it had been proved that the introduction of the perfluoroalkyl residual is possible and reaction takes place at the desired position, it appeared to be of special interest to find out whether this reaction was also possible with nucleosides. For this, uridine and 2'-deoxyuridine were suitable substances. It was found that the reaction of uridine with the perfluoroalkyl copper complex in DMSO at 110 .0C gives 5-perfluoroalkylated uridines. In this way it was possible to isolate 5-(F-butyl)-uridine (IIa), 5-(F-hexyl)-uridine (IIb), 5-(f-octyl)-uridine (IIc) and, unlike uracil, also 5-(F-decyl)-uridine (Id). Finally, the synthesis of 5-(F-butyl)-2'-deoxyuridine (IIIa) and 5-(F-decyl)-2'-deoxyuridine (IIIb) from the non-protected 2'-deoxyuridine proved that the method developed by us is generally applicable, both with regard to the chain length of the perfluoroalkyl residue and the existence of a substituent on the N-1 atom. In spite of the sugar component, the perfluoroalkylated nucleosides are still sufficiently volatile and show a mole peak in the mass spectroscopy from which the sum formula can be ascertained in all cases. After splitting off of the sugar component, analogues fragment patterns are found for the remaining amount of uracil. Among the side products formed in the synthesis of the nucleosides, the formation of perfluoroalkylated bases is of special interest; these could be identified both by mass spectroscopy and by the position of the UV-maximum in the alkaline media. In this process it still remains unclear whether the splitting of the glycosidic bond takes place only after perfluoroalkylation, or whether it is brought about by the reaction conditions on the free nucleoside and 2186
Nucleic Acids Research the uracil formed in this way is perfluoroalkylated afterwards. The absence of uracil and the presence of non-transformed uridine in the reaction mixture suggest, however, that under these condition uridine itself is stable. This shows that either the perfluoroalkyl group decreases the stability of the glycosidic bond, or that in the course of the reaction with the copper complex high energy transition stages occur which promote the splitting.
Tab. 1
compound
m.p. (
Ia Ib Ic
Uracil 5-TMU IIa IIb IIc IId IIIa
IIIb
0C)
250 d. 250 d. 250 d. 335 d. 245-248 203-205 220-223 229-234 232-234 182-184 248-250
A max
(pH 7)
(pH 13)
258 258 258
283 283 283 284 270
260 256 263 263 263 263 263 263
263 263 263 263 262 262
pKvalue a)
7,4 8,4
9,5 7,4
Rfvalue b)
0,65 0,66 0,67 0,24 0,38 0,52 0,54 0,55
0,56 0,59
0,62
5-TMU = 5-trifluoromethyluracil; a) determined by UV-spectroscopy; b) solvent: ethylacetate/iso-propanol/water (12:1:6, upper ph.) A summery of some physical properties of the prepared compounds is given in Tab.1. The UV-spectra of the perfluoroalkyl uracils show the same maxima as uracil and are similar to that of thymine and 5-fluorouracil 14). The spectra show also a bathochromic shifting in the alkaline media which is an additional proof to the fact that the substitution on N-1 has not occured. When comparing the pK-values of 5-(F-butyl)-uracil (Ia) and 5-(F-hexyl)-uracil (Ib) with those of uracil and 5-trifluoromethyluracil 15), one will see that the influence of the perfluoroalkyl residue de2187
Nucleic Acids Research creases with increasing chain length-as the inductive effect of the alkyl group in the higher homologues does not signi-
ficantly change in its relation to the CF3-group. The polarity of the compounds show a different behaviour. When drawing conclusions from the Rf-values of the thin-layer chromatography with regard to the polarity, the high values are an expression of a polarity that decreases with increasing length of the chain. This is also reflected in the good solubility of the compounds in non-polar solvents. We do not know the mechanism of the reaction as yet. It may be a homolytic cleavage of the copper complex 16) and the attack of the resulting perfluoroalkyl radicals on the 5-6 - double bond of the pyrimidine. However, further experiments are necessary to corroborate this.
EXPERIMENTAL Melting points were taken on a heated microscope stage
(Boetius) and are not corrected. Thin-layer chromatography on silica gel was performed on ready-for-use KIESELGEL 60F 254 silica gel plates (Merck, Darmstadt) in the solvent ethylacetate/iso-propanol/water (12:1:6, upper phase). Preparative separations on silica gel were performed on a column of KIESELGEL 60 (particle size 0,063-0,200 mm) in the solvent systems ethylacetate or ethylacetate/iso-propanol/ water (24:1:12, upper phase). The LW-spectra were taken in methanolic or methanolic sodium hydroxide solutions either on a Beckman DK-2 spectrophotometer or on an Unicam SP 800 spectrophotometer. Mass spectra were taken on the MS 902 S (AEI, Manchester) 12) All the procedures were taken in a nitrogen atmosphere, oxygen being absent at all times 17). Preparation of Cu-bronze To a solution of 5 g (0,02 mol) CuSO4*5 H20 in 20 ml water 2 g Zn powder was added. The mixture was then decanted and the residue washed some times with water. The excess of Zn was removed with an aqueous solution of hydrochloric acid (5 %) and the precipitate neutralised with water. For activating, 2 g Cu-bronze was shaken with 20 ml of a 2 % so218
Nucleic Acids Research lution of iodine in acetone. After filtration the residue was washed with 3 portions (15 ml) of a mixture of acetone and concentrated hydrochloric acid (1:1), with pure acetone and dried under diminished pressure.
5-(F-butyl)-uracil (Ia) 1-Iodo-F-butane (3,46 g, 10 mmol), Cu-bronze (1,3 g) and DMS0 (3 ml) were stirred and heated at 110 0C (bath) for 60 min. After standing and cooling the excess of Cu-bronze was filtered, leaving a clear green solution. Uracil (1,12 g, 10 mmol) was then added under exclusion of atmospheric moisture and the mixture were stirred and heated at 110 0C for 60 min. After cooling the yellow precipitate was filtered and the filtrate diluted with 20-30 ml water, filtered,and extracted with ether. The combined ether extracts were dried over sodium sulfate, evaporated, and the residue crystallized from a mixture of methanol and water (7:3) to afford 134 mg of 5-(F-butyl)-uracil (Ia), m.p. 2500C dec. For C8R3F9N202 (330,11) calculated:29,11% C, 0,92% H, 8,49% N; found: 28,86% C, 0,85% H, 9,12% N.
5-(F-hexyl)-uracil (Ib) A mixture of 1-iodo-F-hexane (890 mg, 2 mmol), Cu-bronze (260 mg, 4 mmol) and DMS0 (3 ml) was heated at 110 °C for 60 min. After filtration uracil (112 mg, 1 mmol) was added
under exclusion of atmospheric moisture and the mixture heated at 110 0C for 60 min. The mixture was then processed analogously to the preparation of the compound Ia. Chromatography of the resulting residue on a column of KIESELGEL 60 (100 g) and elution with ethylacetate afforded 90 mg of 5-(F-hexyl)-uracil (Ib), m.p. 250 0C dec. . For C10H3F13N202 (430,13) calculated: 27,92% C, 0,70% H, 6,51% N; found: 27,13% C, 0,65% H, 5,98% N
5- (F-octyl)-uracil (Ic) The perfluorooctyl copper complex was prepared from 1-iodo-F-octane (1,09 g, 2 mmol), Cu-bronze (130 mg, 2 mmol) and 3 ml DM30 analogously to the preparation of the perfluorobutyl-complex. Uracil (112 mg, 1 mmol) was added, the mixture heated at 110 0C for 60 min, and, after cooling, 2189
Nudek Acids Research filtered. The yellow precipitate was then eluted with 0,1 n hydrochloric acid to afford 30 mg of 5-(Frocty1)uracil (Ic), m.p. 250 C dec.. Por 012917N202 (530914) calculated: 27,19% C, 0,57% H, 5,28% N; found: 26,51% C, 0,50% 1H 5,04% N.
5- (F-butyl)-uridine (IIa) 1-Iodo-F-butane (1,38 gs 4 mmol), Cu-bronze (130 mg, 2 mmol) and 3 ml DMSO were stirred and heated at 110 0C for 60 min. After cooling and filtration uridine (245 mg, 1 mmol) was added and the mixture heated at 110 OC for 60 min. 5 ml water was then added and the solution and the precipitate eluted with ether. The combined ether extracts were dried over sodium sulfate and evaporated under diminished pressure. The residue was applied to a column of KIESELGEL 60 (75 g) in the solvent ethylacetate and eluted by the same solvent. The UV-absorbing fractions containing 5-(F-butyl)-uridine (IIa) were evaporated under diminished pressure to afford 16 mg, m.p. 203-205 0C. For C13H1 F9N206 (462,24) calculated: 33,76% C, 2,39% H, 6,06% N; found: 33,05% C, 1,97% H, 5,46% N. 5- (F-hexyl)-uridine (IIb) The compound IIb was processed as given under compound IIa; 1,8 g (4 mmol) 1-iodo-F-hexane, 130 mg (2 mmol) Cubronze and 3 ml DMS0 and 245 mg (1 mmol) uridine. Yield, 35 mg of 5-(F-hexyl)-uridine (IIb), m.p. 220-223 0C. For C15H11F13N206 (562,26) calculated:32,02% C, 1,97% H, 4,98% N; found: 31,26% C, 1,47% H, 4,09% N
5-(F-octyl)-uridine (IIc) 1-Iodo-F-octane (1,1 g, 2 mmol), Cu-bronze (130 mg, and 3 ml DMSO were stirred and heated at 11.0OC (bath) for 60 min. After filtration uridine (245 mg, 1 mmol) was added under exclusion of atmospheric moisture and the mixture was heated at 110 0C. 10 ml ether was then added and the ether/ DMSO-phase was separated and diluted with 20 ml water. The aqueous phase was extracted some times with ether, the combined ether extracts dried over sodium sulfate and evaporated under diminished pressure. The residue was purified on
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Nucleic Acids Research a colulmn of KIESELGEL 60 (110 g, solvent system ethylacetate). The UV-absorbing fractions were evaporated to afford 30 mg of 5-(Z-octyl)-uridine (II), m.p. 229-231 OC. For C17H11,F17N06 (662,28) calculated: 30,81% C, 1,67% H, 4,23% N; found:30,14% C, 1,32% H, 3,86% N
5-(F-decyl)-uridine (IId) Preparation via compound IIc from 245 mg (1 mmol) uridine the perfluorodecyl-copper-complex prepared from 1,3 g (2 mmol) 1-iodo-F-decane, 130 mg (2 mmol) Cu-bronze, and 3 ml DMSO. The resulting 5-(F-decyl)-uridine (IId) was purified by chromatography. Yield, 30 mg, m.p. 232-234 °C. For C19H 1F21tN206 (762,24) calculated: 29992% C, 1,45% H, 3,67% N; found: 291,01 C, 1,57% H, 4,01% N
5-(F-butyl)-2'-deoxyuridine (IIIa) 227 mg (1 mmol) 2'-deoxyuridine and the perfluorobutyl-copper-complex prepared from 1,38 g (4 mmol) 1-iodoF-butane, 260 mg (4 mmol) Cu-bronze and 3 ml DMS0 according to the above mentioned procedure were stirred and heated at 110 C under exclusion of atmospheric moisture for 60 min. After cooling the mixture was diluted with 10 ml water, filtered, and either the solution and the precipitate were extracted with ether. The combined ether extracts were dried over sodium sulfate, evaporated, and the residue chromatographed on a column of KIESELGEL 60 (130 g). Elution with the solvent system ethylacetate/iso-propanol/ water (24:1:12, upper phase) afforded 80 mg chromatographically pure 5-(F-butyl)-2'-deoxyuridine (IIIa), m.p. 1821;84 OC. For C13H11F9N205 (446,08) calculated: 34,97% C, 2,48% H, 6,27% N; found: 34,51%C-, 2,11% H, 5,87% N o
5-(F-decyl)-2'._deoxyuridine (IIIb) The compound IIIb was processed as given under compound IIc; 1,3 g (2 mmol) 1-iodo-F-decane, 130 mg (2 mmol) Cubronze, 3 ml DMS0, and 227 mg (1 mmol) 2'-deoxyuridine. Yield, 23 mg of chromatographically pure 5-(F-decyl)-2'-deoxyuridine (IIIb), m.p. 248-250 0C. For C19H11F-21N205 (746,03) calculated: 30,56% C, 1,48% H, 3,75% N; found: 31,12% C, 1,56% H, 3,76% N
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Nucleic Acids Research +
++
A part of this paper was presented at the 3 rd Symposium on the Chemistry of Nucleic Acid Components, October 8 - 12, 1975, Liblice Castle, Czechoslovakia adress: Academy of Sciences of G.D.R., Central Institute of Molecular Biology, 1115 Berlin-Buch, G.D.R.
REFERENCES 1 Reges,P.RO and Heidelberger,C. (1965) Biochim. Biophys. Acta 103, 177 2 German,L.C., Polutshik,B.P., Preobrazhenskaya,M.N., Melnik,C.A. and Kurzhena,L.M. (1974) Khim.Geterotsiklich. Soedin. 6, 859 3 Melnik, C.A. ,Preobrazhenskaya,M.N., Bachmetova,A.J., German,L.C. and Polutshik,B.P. (1975) Zhur.org.Khim. 11, 190 4 Mertes,M.P. and Saheb,S.E. (1963) J.Pharm.Sci. 52, 508 5 Hein,L., Cech,D. and Liebenthal,C. G.D.R.-Pat. WPC0 7d/ 185.491, 17.4.1975 6 Tiers,G.V.D. (1960) J.Am.Chem.Soc. 82, 5513 7 Knunyants,I.L.,, Shokind,V.V., Krasuskaya,,M.P. and Khulakyan,S.P. (1967) Izvest.Akad.Nauk S.S.S.R., Otdel, Khim.Nauk 1520 8 Brace,N.0. (1966) U.S.-Pat, 3,271,441 ; CA 66, 2388 9 McLouphlin,V.C.R. and Thrower,J. (1969) Tetrahedron 25, 5921 10 Coe,P.L. and Milner,N.E. (1973) J.Fluorine Chem. 2, 167 11 nomenclature of the perfluoroalkylated compounds see: Young,J.A. (1974) J.Chem.Documentation 14, 98 12 Otto,A., Etzold,G., Wohlfeil,R. and Cech,D. , in preparation 13 Lange,B. (1956) in Kolorimetrische Analyse, p. 1080 Springer Verlag, Berlin 14 Wempen,I. and Fox,J.J. (1964) J.Am.Chem.Soc. 86, 2474 15 Nestler,H.J. and Garrett,E.R. (1968) J.Pharm.Sci. 57, 1117 16 Bacon,R.G.R. and Hill,H.A.O. (1965) Quart.Rev. 19, 95 17
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Herzog,,S.
and Dehnert,J.
(1964)
Z.Chem.
4,
1
Volume 2 number 11
November1975
Nucleic Acids Research
Nucleic Acids Research
A facile synthesis of 5-(perfluoroalkyl)-pyrimidines+
D. Cech, R. Wohlfeil and G. Etzold
Humboldt-University Berlin, Department of Chemistry, 104 Berlin, GDR R eceived 15 October 1975
ABSTRACT In the paper a synthetic two stage procedure is described for the preparation of perfluoroalkylated derivatives of uracil and its nucleosides. Using copper bronze a perfluoroalkyl-copper-complex is formed from perfluoroalkyl iodides in polar aprotic solvents, such as DMSO, and under inert conditions. The reaction of this complex with uracil, uridine and 2'-deoxyuridine leads to the corresponding 5-substituted perfluoroalkyl derivatives. It is shown by mass spectra that the substitution always takes place at the 5-position of the pyrimidine. The chemical and physical properties of the formed compounds are described.
INTRODUCTION Among the well-known antimetabolites of the nucleic acid metabolism 5-trifluoromethyluracil and its 2'-deoxyribofuranoside have proved particularly effective 1). Higher homologues of the series of the 5-perfluoroalkyl pyrimidines, however, have not been described as yet. It is true that PREOPRASHENSKAYA and co-workers 293) described a synthesis of similar derivatives obtained by a transformation of 5-hydroxymethyluracil with tetrafluoroethylenes, but the compounds obtained do not represent true homologues in the series of the perfluoroalkyl derivatives as the perfluoroalkyl group is always separated from the pyrimidine ring by a CH2-group. So we first studied the question to what extent the procedures used for 5-trifluoromethyluracil were also applicable to the synthesis of higher homologues. Whereas methods for the preparation from 5-carboxyuracil and SF 4) or the electrochemical introduction of a perfluoroalkyl group 5) are out of the question from the first, a further
2183
Nucleic Acids Research difficulty arises from the difficult-accessibility of 2 H-perfluoroacetic acid derivatives which are required for the preparation of these compounds by usual procedure of cyclocondensation with urea. There are a number of methods for the direct introduction of perfluoroalkyl groups into aromatic compounds 6,7,8), however, non one was used for the perfluoroalkylation of pyrimidines. RESULTS AND DISCUSSION In view of the difficulties, we considered the use of and COE 10) the method recently described by McLOUGHLIN for the introduction of perfluoroalkyl groups into benzene and its derivatives also for the synthesis of perfluoroalkylated pyrimidines. According to McLOUGHLIN, perfluoroalkyl iodides are transformed with aromatic iodine compounds in the presence of Cu-bronze under inert conditions in polar aprotic solvents. The formation of perfluoroalkyl compounds proceeds via a copper complex according to the following equation : RFJ
+
ArJ
RFAr
Cu
Ar RF
=
=
+
2 CuJ
aryl perfluoroalkyl
As aromates can also be used, we were of the opinion that possibly also pyrimidines can be perfluoroalkylated in spite of the presence of two readily protonisable H-atoms. First, the perfluoroalkyl-copper-complex (RFCuL3 t L = DMSO; R? = CF3 (CF2)n CF2-, n = 2, 4, 6 and 8) was prepared by the transformation of perfluoroalkyl iodides with specially prepared Cu-bronze in DMSO at 110 °C with exclusion of oxygen. If this complex (n=4) is reacted with 5-iodouracil for example, one obtains, apart from non-transformed initial product, only uracil, whereas a transformation of the previously isolated complex with iuracil in DMSO yields a new, uv-absorbing compound. Mass spectrometric examination of the structure of the new compound showed that the sum formula corresponds exactly to that qf 5-(Z-hexyl)-uracil (Ib) 11). The spectrum
2184
Nucleic Acids Research contains
fragments m/e
=
241
and
m/e
=
91
(for
+O;C-CH=CF2)
which prove the lokalisation at the C-5-position. The fragment patterns show that a bond splitting occurs between C-4 and N-3 and between C-5 and C-6 of the uracil 12).Moreover, the site of the UV-maximum in acidic and alkaline media (significant shift of the maximum in alkaline media) confirms that the substitution did not occur at the N-atoms of the pyrimidine.
Figure 1
CF2-(ICF2)n-CF3
HN
O NJ R I II
R R
=
III
R
=
=
a n H; ribofuranoside ;
2, b
n = 4, c n = 6 2, b n = 4, c n d n =8 deoxyribofuranoside a n = 2, b n =
a n
=
=
6,
=
8
The yields of the reaction amount to approximately 10 to 20 per cent in addition to non transformed uracil, with two side products of less importance being formed the structure of which has not been cleared up yet. We believe that the small yields are due to side reactions in the transformation of the complex with uracil which are not yet understood. The spectrometric determination of the formation of the complex showed yields of approx. 60 per cent in relation to the alkyl iodide used 13). It was found in our experiments that a variation of the reaction conditions did not bring about higher yields. For example, when using other solvents, such as pyridine or DMF no reaction was obtained, and the addition of catalysts such as azo-bis-isobutyronitril, which was supposed to accelerate the radical decomposition of the complex, did not yield any remarkable results.
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Nucleic Acids Research Under the reaction conditions that proved to be favourable for the transformation of perfluorohexyl copper with uracil, also perfluorobutyl- and perfluorooctyl copper complexes showed a reaction. It was, however, found that the reactivity markedly decreases with the chain length. 5-(F-octyl)-uracil (Ic) could only be obtained in small quantities, and 5-(F-decyl)-uracil can no longer be prepared under the above mentioned conditions. After it had been proved that the introduction of the perfluoroalkyl residual is possible and reaction takes place at the desired position, it appeared to be of special interest to find out whether this reaction was also possible with nucleosides. For this, uridine and 2'-deoxyuridine were suitable substances. It was found that the reaction of uridine with the perfluoroalkyl copper complex in DMSO at 110 .0C gives 5-perfluoroalkylated uridines. In this way it was possible to isolate 5-(F-butyl)-uridine (IIa), 5-(F-hexyl)-uridine (IIb), 5-(f-octyl)-uridine (IIc) and, unlike uracil, also 5-(F-decyl)-uridine (Id). Finally, the synthesis of 5-(F-butyl)-2'-deoxyuridine (IIIa) and 5-(F-decyl)-2'-deoxyuridine (IIIb) from the non-protected 2'-deoxyuridine proved that the method developed by us is generally applicable, both with regard to the chain length of the perfluoroalkyl residue and the existence of a substituent on the N-1 atom. In spite of the sugar component, the perfluoroalkylated nucleosides are still sufficiently volatile and show a mole peak in the mass spectroscopy from which the sum formula can be ascertained in all cases. After splitting off of the sugar component, analogues fragment patterns are found for the remaining amount of uracil. Among the side products formed in the synthesis of the nucleosides, the formation of perfluoroalkylated bases is of special interest; these could be identified both by mass spectroscopy and by the position of the UV-maximum in the alkaline media. In this process it still remains unclear whether the splitting of the glycosidic bond takes place only after perfluoroalkylation, or whether it is brought about by the reaction conditions on the free nucleoside and 2186
Nucleic Acids Research the uracil formed in this way is perfluoroalkylated afterwards. The absence of uracil and the presence of non-transformed uridine in the reaction mixture suggest, however, that under these condition uridine itself is stable. This shows that either the perfluoroalkyl group decreases the stability of the glycosidic bond, or that in the course of the reaction with the copper complex high energy transition stages occur which promote the splitting.
Tab. 1
compound
m.p. (
Ia Ib Ic
Uracil 5-TMU IIa IIb IIc IId IIIa
IIIb
0C)
250 d. 250 d. 250 d. 335 d. 245-248 203-205 220-223 229-234 232-234 182-184 248-250
A max
(pH 7)
(pH 13)
258 258 258
283 283 283 284 270
260 256 263 263 263 263 263 263
263 263 263 263 262 262
pKvalue a)
7,4 8,4
9,5 7,4
Rfvalue b)
0,65 0,66 0,67 0,24 0,38 0,52 0,54 0,55
0,56 0,59
0,62
5-TMU = 5-trifluoromethyluracil; a) determined by UV-spectroscopy; b) solvent: ethylacetate/iso-propanol/water (12:1:6, upper ph.) A summery of some physical properties of the prepared compounds is given in Tab.1. The UV-spectra of the perfluoroalkyl uracils show the same maxima as uracil and are similar to that of thymine and 5-fluorouracil 14). The spectra show also a bathochromic shifting in the alkaline media which is an additional proof to the fact that the substitution on N-1 has not occured. When comparing the pK-values of 5-(F-butyl)-uracil (Ia) and 5-(F-hexyl)-uracil (Ib) with those of uracil and 5-trifluoromethyluracil 15), one will see that the influence of the perfluoroalkyl residue de2187
Nucleic Acids Research creases with increasing chain length-as the inductive effect of the alkyl group in the higher homologues does not signi-
ficantly change in its relation to the CF3-group. The polarity of the compounds show a different behaviour. When drawing conclusions from the Rf-values of the thin-layer chromatography with regard to the polarity, the high values are an expression of a polarity that decreases with increasing length of the chain. This is also reflected in the good solubility of the compounds in non-polar solvents. We do not know the mechanism of the reaction as yet. It may be a homolytic cleavage of the copper complex 16) and the attack of the resulting perfluoroalkyl radicals on the 5-6 - double bond of the pyrimidine. However, further experiments are necessary to corroborate this.
EXPERIMENTAL Melting points were taken on a heated microscope stage
(Boetius) and are not corrected. Thin-layer chromatography on silica gel was performed on ready-for-use KIESELGEL 60F 254 silica gel plates (Merck, Darmstadt) in the solvent ethylacetate/iso-propanol/water (12:1:6, upper phase). Preparative separations on silica gel were performed on a column of KIESELGEL 60 (particle size 0,063-0,200 mm) in the solvent systems ethylacetate or ethylacetate/iso-propanol/ water (24:1:12, upper phase). The LW-spectra were taken in methanolic or methanolic sodium hydroxide solutions either on a Beckman DK-2 spectrophotometer or on an Unicam SP 800 spectrophotometer. Mass spectra were taken on the MS 902 S (AEI, Manchester) 12) All the procedures were taken in a nitrogen atmosphere, oxygen being absent at all times 17). Preparation of Cu-bronze To a solution of 5 g (0,02 mol) CuSO4*5 H20 in 20 ml water 2 g Zn powder was added. The mixture was then decanted and the residue washed some times with water. The excess of Zn was removed with an aqueous solution of hydrochloric acid (5 %) and the precipitate neutralised with water. For activating, 2 g Cu-bronze was shaken with 20 ml of a 2 % so218
Nucleic Acids Research lution of iodine in acetone. After filtration the residue was washed with 3 portions (15 ml) of a mixture of acetone and concentrated hydrochloric acid (1:1), with pure acetone and dried under diminished pressure.
5-(F-butyl)-uracil (Ia) 1-Iodo-F-butane (3,46 g, 10 mmol), Cu-bronze (1,3 g) and DMS0 (3 ml) were stirred and heated at 110 0C (bath) for 60 min. After standing and cooling the excess of Cu-bronze was filtered, leaving a clear green solution. Uracil (1,12 g, 10 mmol) was then added under exclusion of atmospheric moisture and the mixture were stirred and heated at 110 0C for 60 min. After cooling the yellow precipitate was filtered and the filtrate diluted with 20-30 ml water, filtered,and extracted with ether. The combined ether extracts were dried over sodium sulfate, evaporated, and the residue crystallized from a mixture of methanol and water (7:3) to afford 134 mg of 5-(F-butyl)-uracil (Ia), m.p. 2500C dec. For C8R3F9N202 (330,11) calculated:29,11% C, 0,92% H, 8,49% N; found: 28,86% C, 0,85% H, 9,12% N.
5-(F-hexyl)-uracil (Ib) A mixture of 1-iodo-F-hexane (890 mg, 2 mmol), Cu-bronze (260 mg, 4 mmol) and DMS0 (3 ml) was heated at 110 °C for 60 min. After filtration uracil (112 mg, 1 mmol) was added
under exclusion of atmospheric moisture and the mixture heated at 110 0C for 60 min. The mixture was then processed analogously to the preparation of the compound Ia. Chromatography of the resulting residue on a column of KIESELGEL 60 (100 g) and elution with ethylacetate afforded 90 mg of 5-(F-hexyl)-uracil (Ib), m.p. 250 0C dec. . For C10H3F13N202 (430,13) calculated: 27,92% C, 0,70% H, 6,51% N; found: 27,13% C, 0,65% H, 5,98% N
5- (F-octyl)-uracil (Ic) The perfluorooctyl copper complex was prepared from 1-iodo-F-octane (1,09 g, 2 mmol), Cu-bronze (130 mg, 2 mmol) and 3 ml DM30 analogously to the preparation of the perfluorobutyl-complex. Uracil (112 mg, 1 mmol) was added, the mixture heated at 110 0C for 60 min, and, after cooling, 2189
Nudek Acids Research filtered. The yellow precipitate was then eluted with 0,1 n hydrochloric acid to afford 30 mg of 5-(Frocty1)uracil (Ic), m.p. 250 C dec.. Por 012917N202 (530914) calculated: 27,19% C, 0,57% H, 5,28% N; found: 26,51% C, 0,50% 1H 5,04% N.
5- (F-butyl)-uridine (IIa) 1-Iodo-F-butane (1,38 gs 4 mmol), Cu-bronze (130 mg, 2 mmol) and 3 ml DMSO were stirred and heated at 110 0C for 60 min. After cooling and filtration uridine (245 mg, 1 mmol) was added and the mixture heated at 110 OC for 60 min. 5 ml water was then added and the solution and the precipitate eluted with ether. The combined ether extracts were dried over sodium sulfate and evaporated under diminished pressure. The residue was applied to a column of KIESELGEL 60 (75 g) in the solvent ethylacetate and eluted by the same solvent. The UV-absorbing fractions containing 5-(F-butyl)-uridine (IIa) were evaporated under diminished pressure to afford 16 mg, m.p. 203-205 0C. For C13H1 F9N206 (462,24) calculated: 33,76% C, 2,39% H, 6,06% N; found: 33,05% C, 1,97% H, 5,46% N. 5- (F-hexyl)-uridine (IIb) The compound IIb was processed as given under compound IIa; 1,8 g (4 mmol) 1-iodo-F-hexane, 130 mg (2 mmol) Cubronze and 3 ml DMS0 and 245 mg (1 mmol) uridine. Yield, 35 mg of 5-(F-hexyl)-uridine (IIb), m.p. 220-223 0C. For C15H11F13N206 (562,26) calculated:32,02% C, 1,97% H, 4,98% N; found: 31,26% C, 1,47% H, 4,09% N
5-(F-octyl)-uridine (IIc) 1-Iodo-F-octane (1,1 g, 2 mmol), Cu-bronze (130 mg, and 3 ml DMSO were stirred and heated at 11.0OC (bath) for 60 min. After filtration uridine (245 mg, 1 mmol) was added under exclusion of atmospheric moisture and the mixture was heated at 110 0C. 10 ml ether was then added and the ether/ DMSO-phase was separated and diluted with 20 ml water. The aqueous phase was extracted some times with ether, the combined ether extracts dried over sodium sulfate and evaporated under diminished pressure. The residue was purified on
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Nucleic Acids Research a colulmn of KIESELGEL 60 (110 g, solvent system ethylacetate). The UV-absorbing fractions were evaporated to afford 30 mg of 5-(Z-octyl)-uridine (II), m.p. 229-231 OC. For C17H11,F17N06 (662,28) calculated: 30,81% C, 1,67% H, 4,23% N; found:30,14% C, 1,32% H, 3,86% N
5-(F-decyl)-uridine (IId) Preparation via compound IIc from 245 mg (1 mmol) uridine the perfluorodecyl-copper-complex prepared from 1,3 g (2 mmol) 1-iodo-F-decane, 130 mg (2 mmol) Cu-bronze, and 3 ml DMSO. The resulting 5-(F-decyl)-uridine (IId) was purified by chromatography. Yield, 30 mg, m.p. 232-234 °C. For C19H 1F21tN206 (762,24) calculated: 29992% C, 1,45% H, 3,67% N; found: 291,01 C, 1,57% H, 4,01% N
5-(F-butyl)-2'-deoxyuridine (IIIa) 227 mg (1 mmol) 2'-deoxyuridine and the perfluorobutyl-copper-complex prepared from 1,38 g (4 mmol) 1-iodoF-butane, 260 mg (4 mmol) Cu-bronze and 3 ml DMS0 according to the above mentioned procedure were stirred and heated at 110 C under exclusion of atmospheric moisture for 60 min. After cooling the mixture was diluted with 10 ml water, filtered, and either the solution and the precipitate were extracted with ether. The combined ether extracts were dried over sodium sulfate, evaporated, and the residue chromatographed on a column of KIESELGEL 60 (130 g). Elution with the solvent system ethylacetate/iso-propanol/ water (24:1:12, upper phase) afforded 80 mg chromatographically pure 5-(F-butyl)-2'-deoxyuridine (IIIa), m.p. 1821;84 OC. For C13H11F9N205 (446,08) calculated: 34,97% C, 2,48% H, 6,27% N; found: 34,51%C-, 2,11% H, 5,87% N o
5-(F-decyl)-2'._deoxyuridine (IIIb) The compound IIIb was processed as given under compound IIc; 1,3 g (2 mmol) 1-iodo-F-decane, 130 mg (2 mmol) Cubronze, 3 ml DMS0, and 227 mg (1 mmol) 2'-deoxyuridine. Yield, 23 mg of chromatographically pure 5-(F-decyl)-2'-deoxyuridine (IIIb), m.p. 248-250 0C. For C19H11F-21N205 (746,03) calculated: 30,56% C, 1,48% H, 3,75% N; found: 31,12% C, 1,56% H, 3,76% N
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A part of this paper was presented at the 3 rd Symposium on the Chemistry of Nucleic Acid Components, October 8 - 12, 1975, Liblice Castle, Czechoslovakia adress: Academy of Sciences of G.D.R., Central Institute of Molecular Biology, 1115 Berlin-Buch, G.D.R.
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