Method for producing halogenated N-arylpyrazoles

Abstract

The present invention relates to a process for preparing compounds of the formula (I) ##STR00001##
by halogenating compounds of the formula (II), ##STR00002##
where in R.sup.1, R.sup.2, R.sup.3 and X are defined according to the invention.

Claims

1. A process for preparing a compound of formula (I) ##STR00008## wherein X is chlorine, bromine or iodine; R.sup.1 is hydrogen, cyano, halogen, C.sub.1-C.sub.4-alkyl optionally substituted by halogen or CN, or C.sub.1-C.sub.4-alkoxy optionally substituted by halogen, R.sup.2 is trifluoromethylsulfonyl, trifluoromethylsulfinyl, trifluoromethylsulfanyl, halogen, C.sub.1-C.sub.4-alkyl optionally substituted by halogen, or C.sub.1-C.sub.4-alkoxy optionally substituted by halogen; and R.sup.3 is hydrogen, cyano, halogen, C.sub.1-C.sub.4-alkyl optionally substituted by halogen or CN, or C.sub.1-C.sub.4-alkoxy optionally substituted by halogen, comprising halogenating a compound of formula (II) ##STR00009## wherein R.sup.1, R.sup.2 and R.sup.3 are as defined above, with an organic halogenating compound with addition of ≥0.0001 equivalent and <0.3 equivalent, based on the total molar amount of compound of the formula (II) used, of at least one acid selected from the group consisting of mineral acids, sulfonic acids, carboxylic acids and Lewis acids.

2. The process according to claim 1, wherein the organic halogenating compound is selected from the N-halosuccinimides, the 1,3-dihalo-5,5-dimethylhydantoins or the halocyanuric acids.

3. The process according to claim 1, wherein the halogenating compound is selected from N-bromosuccinimide (NBS), N-iodosuccinimide (NIS), 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), 1,3-diiodo-5,5-dimethylhydantoin (DIDMH), 1,3,5-tribromo-1,3,5-triazine-2,4,6-trione or 1,3-dibromo-1,3,5-triazine-2,4,6-trione.

4. The process according to claim 1, wherein the acid is used in a proportion of ≥0.001 equivalent and ≤0.15 equivalent, based on the total molar amount of compound (II) used.

5. The process according to claim 1, wherein the at least one acid is a carboxylic acid.

6. The process according to claim 1, wherein the acid is selected from HF, HCl, HBr, HI, H.sub.2SO.sub.4, HNO.sub.3 and H.sub.3PO.sub.4, optionally substituted arylsulfonic and alkylsulfonic acids, optionally substituted alkylcarboxylic and arylcarboxylic acids, optionally substituted alkyldicarboxylic and aryldicarboxylic acids, and anhydrous or hydrated fluoride, chloride or bromide salts, nitrates, acetates, sulfates or trifluoromethanesulfonates (OTf) of lithium or of the alkaline earth metals, of the boron-group metals and of the transition metals.

7. The process according to claim 1, wherein the acid is selected from HCl, HF, HNO.sub.3, H.sub.2SO.sub.4, H.sub.3PO.sub.4, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, para-toluenesulfonic acid, acetic acid, propionic acid, trifluoroacetic acid, trichloroacetic acid and anhydrous or hydrated salts selected from the fluoride, chloride or bromide salts, nitrates or trifluoromethanesulfonates (OTf) of the metals B or Al, from the nitrates or trifluoromethanesulfonates (OTf) of the alkaline earth metals Mg or Ca, or the nitrates or trifluoromethanesulfonates (OTf) of the transition metals Fe, Zn, Cu or Sc.

8. The process according to claim 1, wherein the acid is selected from the group consisting of HNO.sub.3, HF, H.sub.2SO.sub.4, methanesulfonic acid, para-toluenesulfonic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, Mg(NO.sub.3).sub.2, Ca(NO.sub.3).sub.2, Fe.sub.2(NO.sub.3).sub.3, Zn(NO.sub.3).sub.2, Zn(OTf).sub.2, Cu(NO.sub.3).sub.2, Sc(NO.sub.3).sub.3, Ca(OTf).sub.2, Mg(OTf).sub.2, Cu(OTf).sub.2, BBr.sub.3, BCl.sub.3, BF.sub.3*OEt.sub.2, Al(NO.sub.3).sub.3, Al(OTf).sub.3, Fe(OTf).sub.3, Cu(OTf).sub.2 and Sc(OTf).sub.3.

9. The process according to claim 1, wherein the reaction is conducted in a temperature range of −78 to 200° C.

10. The process according to claim 1, wherein R.sup.2 is halogen-substituted C.sub.1-C.sub.4-alkyl or halogen-substituted C.sub.1-C.sub.4-alkoxy.

11. The process according to claim 1, wherein R.sup.2 is fluorine-substituted C.sub.1-C.sub.4-alkyl or fluorine-substituted C.sub.1-C.sub.4-alkoxy.

12. The process according to claim 1, wherein R.sup.1 and R.sup.3 in each case independently of one another are a substituent selected from hydrogen, Cl, Br, F, C.sub.1-C.sub.3-alkyl, halogen-substituted C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy or halogen-substituted C.sub.1-C.sub.3-alkoxy.

13. The process according to claim 1, wherein R.sup.1 and R.sup.3 are not simultaneously hydrogen.

14. The process according to claim 1, wherein R.sup.1 is halogen or (C.sub.1-C.sub.3)-alkyl, R.sup.2 is fluorine-substituted C.sub.1-C.sub.4-alkyl or fluorine-substituted C.sub.1-C.sub.4-alkoxy, and R.sup.3 is halogen, C.sub.1-C.sub.3-alkyl or fluorine-substituted C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy or fluorine-substituted C.sub.1-C.sub.3-alkoxy.

15. The process according to claim 1, wherein X is bromine or iodine.

16. The process according to claim 9, wherein the reaction is conducted in a temperature range from −20 to 100° C.

Description

EXAMPLES

(1) The following examples explain the process according to the invention in more detail without limiting the invention thereto.

1) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(2) 11.1 g (28.0 mmol, 0.5 eq) of 1,3-diiodo-5,5-dimethylhydantoin were initially charged in 25 ml of acetonitrile and admixed over 0.5 h at an internal temperature of 20° C. with a solution of 22.7 g (purity: 93%, 55.6 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 0.28 g (2.8 mmol, 0.05 eq) of 96% sulfuric acid, dissolved in 25 ml of acetonitrile. After addition was complete, the mixture was stirred further for 10 min and subsequently complete conversion to the iodopyrazole was detected by means of HPLC.sup.a). 10 ml of water were then added and the reaction was terminated by adding 5 ml of saturated sodium sulfite solution. The solvent was partially distilled off under reduced pressure and the product was filtered after precipitation with 20 ml of water. The residue was washed twice with 80 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless-to-yellowish solid: yield 29.1 g (98% of theory).

(3) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.84 ppm (s, 1H); 7.71 ppm (s, 2H); 7.65 ppm (s, 1H).

2) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(4) 5.1 g (12.9 mmol, 0.505 eq) of 1,3-diiodo-5,5-dimethylhydantoin were initially charged in 10 ml of acetonitrile and admixed over 15 min at an internal temperature of 20° C. with a solution of 10.0 g (purity: 98%, 25.7 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 80 mg (1.3 mmol, 0.05 eq) of glacial acetic acid, dissolved in 10 ml of acetonitrile. After addition was complete, the reaction was heated to 50° C. and stirred further at this temperature. After 10 h, a 90% conversion to the iodopyrazole was detectable by means of HPLC.sup.a). The reaction was terminated by adding 5 ml of saturated sodium sulfite solution and the product was filtered after precipitation with 100 ml of water. The residue was washed twice with 20 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a pale orange solid: yield 12.1 g (82% of theory).

3) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(5) 51.4 g (129.9 mmol, 0.505 eq) of 1,3-diiodo-5,5-dimethylhydantoin were initially charged in 100 ml of acetonitrile and admixed over 0.5 h at an internal temperature of 20° C. with a solution of 100.0 g (purity: 98%, 257.2 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 26 mg (0.26 mmol, 0.001 eq) of 96% sulfuric acid, dissolved in 100 ml of acetonitrile. After addition was complete, the reaction mixture was stirred at 50° C. After 1 h, complete conversion to the iodopyrazole was detectable by means of HPLC.sup.a). 50 ml of water were then added and the reaction was terminated by adding 50 ml of saturated sodium sulfite solution. The solvent was partially distilled off under reduced pressure and the product was filtered after precipitation with 300 ml of water. The residue was washed twice with 100 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless-to-yellowish solid: yield 129.5 g (94% of theory).

4) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(6) 5.1 g (12.9 mmol, 0.505 eq) of 1,3-diiodo-5,5-dimethylhydantoin were initially charged in 10 ml of acetonitrile and admixed over 15 min at an internal temperature of 20° C. with a solution of 10.0 g (purity: 98%, 25.7 mmol, 1.0eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 134 mg (0.26 mmol, 0.01 eq) of Fe(OTf).sub.3, dissolved in 10 ml of acetonitrile. After addition was complete, the reaction mixture was stirred further at RT. After 2 h, complete conversion to the iodopyrazole was detectable by means of HPLC.sup.a). The reaction was then terminated by adding 5 ml of saturated sodium sulfite solution and the product was filtered after precipitation with 100 ml of water. The residue was washed twice with 100 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless-to-yellowish solid: yield 12.7 g (88% of theory).

5) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(7) 6.3 g (27.0 mmol, 1.05 eq) of N-iodosuccinimide were initially charged in 10 ml of acetonitrile and admixed over 15 min at an internal temperature of 20° C. with a solution of 10.0 g (purity: 98%, 25.7 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 131 mg (1.29 mmol, 0.05 eq) of 96% sulfuric acid, dissolved in 10 ml of acetonitrile. After addition was complete, the reaction mixture was stirred at this temperature. After 1 h, complete conversion to the iodopyrazole was detectable by means of HPLC.sup.a). The reaction was then terminated by adding 5 ml of saturated sodium sulfite solution and the product was filtered after precipitation with 100 ml of water. The residue was washed twice with 100 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless-to-yellowish solid: yield 12.7 g (94% of theory).

6) 1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(8) 10.0 g (purity: 97.4%, 25.5 mmol, 1.0eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole were dissolved in 20 ml of acetonitrile and admixed with 0.26 mg (2.55 μmol, 0.0001 eq) of 96% H.sub.2SO.sub.4. After addition of 1.53 g (3.9 mmol, 0.15 eq) of 1,3-diiodo-5,5-dimethylhydantoin, the solution was heated to 60° C. and stirred at this temperature. After 5 h, a further 1.53 g (3.9 mmol, 0.15 eq) of 1,3-diiodo-5,5-dimethylhydantoin were added and the mixture was stirred further at 60° C., and the addition was repeated after a total of 10 h with a further 2.04 g (5.2 mmol, 0.20 eq) of 1,3-diiodo-5,5-dimethylhydantoin. After a total of 17 h at 60° C., 99% conversion to the iodopyrazole was detectable by means of HPLC.sup.a). The product was not isolated.

7) 4-bromo-1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-1H-pyrazole (I-2

(9) 3.86 g (13.2 mmol, 0.51 eq) of 1,3-dibromo-5,5-dimethylhydantoin were initially charged in 50 ml of acetonitrile and admixed over 0.5 h at an internal temperature of 20° C. with a solution of 10.0 g (purity: 99%, 26.2 mmol, 1.0eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 0.13 g (1.3 mmol, 0.05 eq) of 96% sulfuric acid, dissolved in 50 ml of acetonitrile. After addition was complete, the mixture was stirred further for 10 min and subsequently complete conversion to the bromopyrazole was detected by means of HPLC.sup.a). 10 ml of water were then added and the reaction was terminated by adding 5 ml of saturated sodium sulfite solution. The solvent was partially distilled off under reduced pressure and the product was filtered after precipitation with 20 ml of water. The residue was washed twice with 80 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless solid: yield 11.6 g (96% of theory).

(10) .sup.1H-NMR (CDCl.sub.3, 400 MHz): δ (ppm)=7.80 ppm (s, 1H); 7.71 ppm (s, 2H); 7.63 ppm (s, 1H)

8) 4-bromo-1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-1H-pyrazole (I-2

(11) 4.81 g (27.2 mmol, 1.05 eq) of N-bromosuccinimide were initially charged in 10 ml of acetonitrile and admixed over 15 min at an internal temperature of 20° C. with a solution of 10.0 g (purity: 99%, 26.2 mmol, 1.0eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 0.13 g (1.3 mmol, 0.05 eq) of 96% sulfuric acid, dissolved in 10 ml of acetonitrile. After addition was complete, the mixture was stirred further at this temperature and, after 1 h, complete conversion to the bromopyrazole was detected by means of HPLC.sup.a). The reaction was then terminated by adding 5 ml of saturated sodium sulfite solution and the product was filtered after precipitation with 100 ml of water. The residue was washed twice with 20 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless solid: yield 11.8 g (96% of theory).

9) 4-bromo-1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-1H-pyrazole (I-2

(12) 2.0 g (7.1 mmol, 0.55 eq) of dibromoisocyanuric acid were initially charged in 10 ml of acetonitrile and admixed at an internal temperature of 20° C. with 5.0 g (purity: 98%, 12.9 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole and 63 mg (0.6 mmol, 0.05 eq) of 96% sulfuric acid. After addition was complete, the mixture was stirred further at this temperature and, after 30 min, complete conversion to the bromopyrazole was detected by means of HPLC.sup.a). The reaction was then terminated by adding 5 ml of saturated sodium sulfite solution and the isocyanuric acid formed was removed by filtration after dilution with 20 ml of acetonitrile. The mother liquor was admixed dropwise with 150 ml of water and the precipitated solid was filtered. The residue was washed twice with 30 ml each time of water and, after drying under reduced pressure at 40° C., the product was obtained as a colourless solid: yield 5.8 g (95% of theory).

(13) Further Experiments with Respect to the Acids:

(14) Table 1) gives an overview of further experiments that were carried out analogously to experiment 1) and the conversions of compound (1-1) achieved, determined by means of HPLC.sup.a).

(15) There was variation of the acid used, the temperature and the reaction time. All other parameters and reactants were kept the same.

(16) TABLE-US-00001 TABLE 1 Acid Temperature (° C.) Time (h) Conversion (%) HCl (37%) 40 24 85 H.sub.3PO.sub.4 40 11 89 trifluoroacetic acid 40 9 91 para-toluenesulfonic 25 1 88 acid Mg(OTf).sub.2 40 6.5 91 Ca(OTf).sub.2 40 6.5 91 methanesulfonic acid 25 2 90 Fe.sub.2(NO.sub.3).sub.3*9H.sub.2O 40 2 91 Mg(NO.sub.3).sub.2*6H.sub.2O 40 7 88 BF.sub.3*EtO.sub.2 25 3 92 MgSO.sub.4*4H.sub.2O 40 12 60

(17) Comparative Example without Addition of Acid:

1-[2,6-dichloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)phenyl]-4-iodo-1H-pyrazole (I-1

(18) 0.5 g (1.3 mmol, 1.0 eq) of 1-[2,6-dichloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]phenyl]pyrazole were initially charged in 10 ml of acetonitrile and admixed with 2.85 g (0.7 mmol, 0.55 eq) of 1,3-diiodo-5,5-dimethylhydantoin. The reaction mixture was heated to 65-70° C. and stirred at this temperature for 15 h. After this time, 54% conversion to the desired iodinated product was detectable by means of HPLC.sup.a). The product was not isolated.

(19) The following halogenated N-arylpyrazoles of the general formula (I) were preparable analogously to experiments 1) and 7):

4-bromo-1-[2-bromo-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)-6-(trifluoromethoxy)phenyl]-1H-pyrazole (I-3

(20) Conversion by HPLC.sup.a): >99% (r.t., 1 h)

(21) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.92 (d, J 1.9 Hz, 1H), 7.79 (s, 1H), 7.63 (s, 2H).

4-bromo-1-[2-chloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)-6-(trifluoromethoxy)phenyl]-1H-pyrazole (I-4

(22) Conversion by HPLC.sup.a): >99% (r.t., 1 h)

(23) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.80 (d, J 1.8 Hz, 1H), 7.79 (s, 1H), 7.64 (s, 1H), 7.59 (s, 1H).

4-bromo-1-[2-chloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-1H-pyrazole (I-5

(24) Conversion by HPLC.sup.a): >99% (r.t., 1 h)

(25) .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) δ (ppm)=8.48 (br s, 1H), 8.47 (s, 1H), 8.06 (br s, 1H), 8.03 (s, 1H).

4-bromo-1-[2-bromo-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-1H-pyrazole (I-6

(26) Conversion by HPLC.sup.a): >99% (r.t., 4 h)

(27) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=8.17 (br s, 1H), 7.99 (br s, 1H), 7.79 (s, 1H), 7.62 (s, 1H).

4-bromo-1-[2-methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-1H-pyrazole (I-7

(28) Conversion by HPLC.sup.a): >99% (40° C., 1 h)

(29) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.87 (br s, 1H), 7.78 (br s, 1H), 7.7 (s, 1H), 7.59 (s, 1H), 2.13 (s, 3H).

1-[2-bromo-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)-6-(trifluoromethoxy)phenyl]-4-iodo-1H-pyrazole (I-8

(30) Conversion by HPLC.sup.a): >99% (r.t., 0.5 h)

(31) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.92 (d, J 1.8 Hz, 1H), 7.83 (s, 1H), 7.65 (s, 1H), 7.63 (s, 1H).

1-[2-chloro-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)-6-(trifluoromethoxy)phenyl]-4-iodo-1H-pyrazole (I-9

(32) Conversion by HPLC.sup.a): >99% (r.t., 0.5 h)

(33) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.83 (d, J 1.9 Hz, 1H), 7.77 (s, 1H), 7.66 (s, 1H), 7.59 (s, 1H).

1-[2-chloro-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-4-iodo-1H-pyrazole (I-10

(34) Conversion by HPLC.sup.a): >99% (r.t., 0.5 h)

(35) .sup.1H-NMR (DMSO-d.sub.6, 400 MHz) δ (ppm)=8.47 (br s, 1H), 8.38 (s, 1H), 8.05 (br s, 1H), 7.97 (s, 1H).

1-[2-bromo-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-4-iodo-1H-pyrazole (I-11

(36) Conversion by HPLC.sup.a): >99% (r.t., 0.5 h)

(37) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=8.16 (br s, 1H), 7.99 (br s, 1H), 7.83 (s, 1H), 7.64 (s, 1H).

4-iodo-1-[2-methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl]-6-(trifluoromethyl)phenyl]-1H-pyrazole (I-12

(38) Conversion by HPLC.sup.a): >99% (r.t., 0.5 h)

(39) .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm)=7.87 (br s, 1H), 7.81 (s, 1H), 7.78 (br s, 1H), 7.61 (s, 1H), 2.11 (s, 3H).

(40) Methods:

(41) The NMR data of the examples are listed in conventional form (6 values, multiplet splitting, number of hydrogen atoms).

(42) The solvent and the frequency in which the NMR spectrum was recorded are stated in each case.

(43) .sup.a)HPLC (High Performance Liquid Chromatography) on a reversed-phase column (C18), Agilent 1100 LC system; Phenomenex Prodigy 100×4 mm ODS3; eluent A: acetonitrile (0.25 ml/1); eluent B: water (0.25 ml TFA/1); linear gradient from 5% acetonitrile to 95% acetonitrile in 7.00 min, then 95% acetonitrile for a further 1.00 min; oven temperature 40° C.; flow rate: 2.0 ml/min.