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Thin-layer chromatography of some 5-nitroimidazoles of pharmaceutical interest
Nate
Thin-layer interest
chrom&agrz@y
ENRICO
GATTAVECCHIA
Ciid
IkrlituA? “G.
ad
Ctic~~*,
of some
DOMENICA Uniwrsity
S-nitroimicf2zafes
of pkarm2ceutical
T0NEJ.B
of Bo!~z.q
Vi5 Seln;i 2, aI26
EbKogm (Itaiyj
(Recciwd Dacmber ?7th, 1979)
Nitroimidazoles are effective against anaerobeP and their ability to increase the efkt of radiation on hy7o.k cells is we11 know~P_ Given the possible clinical usefulness of these compounds, we tried to develop separations for se%?eral.5-nitroimidazoles that have recently been tested for their potential radiosensitizing action’. We report here a systematic study of the thin-layer chromatographic (TJX) properties of 14 Enitroimidazoks on thin layers of silica gel.
Samples
and reagents The DA nitroimidazoles shown in Fig. 1 were supplied by the &tit&o De
Angeli (Milan, Italy), MY 4Oj20 by ,MIDI (Milan, Italy), metronidazole by Farmitalia @Wan, Italy); and 2-metbyI_Y>5)-nitroimidazole by Aldrich Europe (Beer+ Belgium). AU of the chemicals used (Merck, Darmstadt, G.F.R.) were of analyticalreagent _-de.
Thiu-layer plates and chrontatograpi~ic procedures TLC wx performed on 0.25 mm pre-coated layers of silica gel G-60 Fx (Merck) using the solvents listed in Table I. The plates were pre-washed wSth the eluent mixrure to be used. Aliquots (5 ~1) of ethanolic solutions containing from IOea to 10S3 M of nitroimidazok were spotted. DA 3831 was chromatographed as the ammonium salt prepared accordin,= to Braun and Geenen6. After spotting, the plates were developed in a chromatographic tank lined with filter-paper at 30 “C using the ascending technique; the run of the developing eluent was 10 cmThe developed layers were air dried and the spots located by ffuorescence quenching under 255run radiation_ All of the chromato_erams were then sprayed with 1.5% titanium(II1) chloride in 10% acetic acid and heated at 80 “C for 20 min to reduce the nitro group. The plates were then sprayed with one of the following chromo-
genic reagents: (1) 0.5% p-dimethykminobenzaldehyde in ethanol-concxx&rated hyc?rocbioric acid (7595); (2) diazotized sulphanilic acid (prepared according to Grimmett uld Richards’; (3) 0.2 % ninhycirinin acetone. These reasnts were prepared immediately before use.
DA 3829
DA
3as1
DA
3831
DA
39lS
DA 3839
.
CH2CH20H
iI c&J---N
DA
MY 40/20
3354
‘:
ma
w
DA 3604
2 --metlryl 4(S) ni?roimidozole
Fig. 1. Structuresof nitroimidazoles.
RESULTS The RF x 100 values given in Table I represent the average of a minimum of differed by kss than three separate chromatographic runs. The RF x 100 values & 3 from run to run. The solvent systems used allowed the effkctive chromatographic separation of all of the nitroimidazoles examined. For example, althou& DA
NOTES
342 TABLE
I
& x 100 VALUES AND SOLVENT SYsliEMs -_ COltZgQZZZ
COLOUR
REACTIONS
OF S-NITRO~MOLES
IN VARIOUS cozout reacti-cKs=f
Ehiq?s&au A
3
C
D
E
I
2
3
66 53 58 10
80 80
71 70
52 65
ii
&tl)
ii
65 63 0
57 77 6
82 85 87
as 85 4
DA 3539 DA 3854
34 1
30 0
cs bg bz
cs
74 77
52 87 3 (tl)
m Ir-yl tn
tn
D-A 3532 DA 3551 DA 393 1
bl gr s s
br
MY 4Q20 DA 3837
43 38 41 4
DA Ia DA 3329 DA 3853
13 (tl) 14 (d) ;o(tI)
4(tl)
66
600 37
2.3 (tl)
45
--
2-_MztbvC1(S~roimidawk
%tr&~k
DA 3333 DA 39i5
66 lP(ti)
yls ~1s
61 60
ii& s
33 (tl) 72 H
br
81 82
11 (tl) H(tl) 66
PI-y1 tn ix
gr k-g!r
m m
73
60
47
it-gr
m
br br gr-gy m
g-br cs V
r-v Pk
br br
0 a lt-gr m r 2 31 (tl) 14 w ____._~._~~ - Solvent systems used: A = chlorofotitbyl ether-metEmnol-dich!orometbane(6:212:1); amrnonia~thanol (1:9); D = aatom B = &yl aatzte+nethanol~-hesane (7~.‘-1); C = 3% Zsy: a~nmonia (59:l); E = acetm*hyI acetate-2S% ammonia (65:15:4). tl = lailiag. ** For chronrogenic reqents I-3. set text. Colours: bg = bcigc; bl = blue; br = bro&n; ____
cs=coIourkrs;g=green;~=~;It=Light;or=oian~;pk=p~;pl=~e;r=red; tn = tan; Y = viokt ; yi = yellow. 3304 and DX 3829 are tailed and poorly resolved in solvent systems A and B, they could be succcsFuily resolved by using solvent system C.
Tabfe I also iists the colour reactions of the nitroimidazoles after spraying with the chromogenic reagents_ Opimal colorixtion of the spots occurred within 5 min when solution 2 was used and zfter heating (10 min at SO “C) with reagents 1 and 3. The sensitivity of the colour reactions was found to be at Ieast 0.1 pg_ The coloured Ehriich and Pauii derivatives w-ere very stzble on silica gel znd the chromato_pams couId he preserved for I week or even longer, unlike the colours obtained with ninhydrin, which darkened within 24 h. According to Stambaugh ad Mantheis, a green colour obtained with ninhydrin can be considered tentative proof of a methyl group in the tpositionThis cannot he taken as a general rule for ali 2-methylnitroimidazoles when silica gel is uss as the support for TLC_ In fact, 2-methylA(nitroimidazole and DA 3535, both of which have a methyl group in the Zposi:don, gave a pare yeHow and a deep blue colour, respectively, when sprayed with ninhydrin after reduction wi’Lh titanium(Ii1) chiorideREFERESCES 1 D. Link sd LI. Mukr, Anrhicrub. Ag_ Ciremorke_., 10 (1976) 476. Z P. L. Olin, Snf. J_ Ccnzccr. 40 (1979) 89. 3 G. E_ Adanr. Erir. _Wezi_&ii_, 23 (1973) 48. 4 G_ IC Adams. I. R_ Flo&hart, C. E Smitfren. I. J. Strat’ord, P. Wardmzn Rodhat. I&s_. 67 (1976) 9_ 5 A_ 13icci2. G. Bcnilli and S. Rofiia, h_ J_ I?QG?&.Biol., 36 (1979) 85. 6 D. Iham and I-L Geeoeo, J_ Ck.romztcgr_. 7 (1962) 56. 7 _M_ R. Grimmtxt and E. L. Richards, J. Cimma~~., 20 (1965) 171. S J. E_ Stambaugh zx!d R_ W_ Man&e& J_ Chronza~o~_. 31 (1967) 128.
ad
M_ E
Wa:ts.
Thin-layer interest
chrom&agrz@y
ENRICO
GATTAVECCHIA
Ciid
IkrlituA? “G.
ad
Ctic~~*,
of some
DOMENICA Uniwrsity
S-nitroimicf2zafes
of pkarm2ceutical
T0NEJ.B
of Bo!~z.q
Vi5 Seln;i 2, aI26
EbKogm (Itaiyj
(Recciwd Dacmber ?7th, 1979)
Nitroimidazoles are effective against anaerobeP and their ability to increase the efkt of radiation on hy7o.k cells is we11 know~P_ Given the possible clinical usefulness of these compounds, we tried to develop separations for se%?eral.5-nitroimidazoles that have recently been tested for their potential radiosensitizing action’. We report here a systematic study of the thin-layer chromatographic (TJX) properties of 14 Enitroimidazoks on thin layers of silica gel.
Samples
and reagents The DA nitroimidazoles shown in Fig. 1 were supplied by the &tit&o De
Angeli (Milan, Italy), MY 4Oj20 by ,MIDI (Milan, Italy), metronidazole by Farmitalia @Wan, Italy); and 2-metbyI_Y>5)-nitroimidazole by Aldrich Europe (Beer+ Belgium). AU of the chemicals used (Merck, Darmstadt, G.F.R.) were of analyticalreagent _-de.
Thiu-layer plates and chrontatograpi~ic procedures TLC wx performed on 0.25 mm pre-coated layers of silica gel G-60 Fx (Merck) using the solvents listed in Table I. The plates were pre-washed wSth the eluent mixrure to be used. Aliquots (5 ~1) of ethanolic solutions containing from IOea to 10S3 M of nitroimidazok were spotted. DA 3831 was chromatographed as the ammonium salt prepared accordin,= to Braun and Geenen6. After spotting, the plates were developed in a chromatographic tank lined with filter-paper at 30 “C using the ascending technique; the run of the developing eluent was 10 cmThe developed layers were air dried and the spots located by ffuorescence quenching under 255run radiation_ All of the chromato_erams were then sprayed with 1.5% titanium(II1) chloride in 10% acetic acid and heated at 80 “C for 20 min to reduce the nitro group. The plates were then sprayed with one of the following chromo-
genic reagents: (1) 0.5% p-dimethykminobenzaldehyde in ethanol-concxx&rated hyc?rocbioric acid (7595); (2) diazotized sulphanilic acid (prepared according to Grimmett uld Richards’; (3) 0.2 % ninhycirinin acetone. These reasnts were prepared immediately before use.
DA 3829
DA
3as1
DA
3831
DA
39lS
DA 3839
.
CH2CH20H
iI c&J---N
DA
MY 40/20
3354
‘:
ma
w
DA 3604
2 --metlryl 4(S) ni?roimidozole
Fig. 1. Structuresof nitroimidazoles.
RESULTS The RF x 100 values given in Table I represent the average of a minimum of differed by kss than three separate chromatographic runs. The RF x 100 values & 3 from run to run. The solvent systems used allowed the effkctive chromatographic separation of all of the nitroimidazoles examined. For example, althou& DA
NOTES
342 TABLE
I
& x 100 VALUES AND SOLVENT SYsliEMs -_ COltZgQZZZ
COLOUR
REACTIONS
OF S-NITRO~MOLES
IN VARIOUS cozout reacti-cKs=f
Ehiq?s&au A
3
C
D
E
I
2
3
66 53 58 10
80 80
71 70
52 65
ii
&tl)
ii
65 63 0
57 77 6
82 85 87
as 85 4
DA 3539 DA 3854
34 1
30 0
cs bg bz
cs
74 77
52 87 3 (tl)
m Ir-yl tn
tn
D-A 3532 DA 3551 DA 393 1
bl gr s s
br
MY 4Q20 DA 3837
43 38 41 4
DA Ia DA 3329 DA 3853
13 (tl) 14 (d) ;o(tI)
4(tl)
66
600 37
2.3 (tl)
45
--
2-_MztbvC1(S~roimidawk
%tr&~k
DA 3333 DA 39i5
66 lP(ti)
yls ~1s
61 60
ii& s
33 (tl) 72 H
br
81 82
11 (tl) H(tl) 66
PI-y1 tn ix
gr k-g!r
m m
73
60
47
it-gr
m
br br gr-gy m
g-br cs V
r-v Pk
br br
0 a lt-gr m r 2 31 (tl) 14 w ____._~._~~ - Solvent systems used: A = chlorofotitbyl ether-metEmnol-dich!orometbane(6:212:1); amrnonia~thanol (1:9); D = aatom B = &yl aatzte+nethanol~-hesane (7~.‘-1); C = 3% Zsy: a~nmonia (59:l); E = acetm*hyI acetate-2S% ammonia (65:15:4). tl = lailiag. ** For chronrogenic reqents I-3. set text. Colours: bg = bcigc; bl = blue; br = bro&n; ____
cs=coIourkrs;g=green;~=~;It=Light;or=oian~;pk=p~;pl=~e;r=red; tn = tan; Y = viokt ; yi = yellow. 3304 and DX 3829 are tailed and poorly resolved in solvent systems A and B, they could be succcsFuily resolved by using solvent system C.
Tabfe I also iists the colour reactions of the nitroimidazoles after spraying with the chromogenic reagents_ Opimal colorixtion of the spots occurred within 5 min when solution 2 was used and zfter heating (10 min at SO “C) with reagents 1 and 3. The sensitivity of the colour reactions was found to be at Ieast 0.1 pg_ The coloured Ehriich and Pauii derivatives w-ere very stzble on silica gel znd the chromato_pams couId he preserved for I week or even longer, unlike the colours obtained with ninhydrin, which darkened within 24 h. According to Stambaugh ad Mantheis, a green colour obtained with ninhydrin can be considered tentative proof of a methyl group in the tpositionThis cannot he taken as a general rule for ali 2-methylnitroimidazoles when silica gel is uss as the support for TLC_ In fact, 2-methylA(nitroimidazole and DA 3535, both of which have a methyl group in the Zposi:don, gave a pare yeHow and a deep blue colour, respectively, when sprayed with ninhydrin after reduction wi’Lh titanium(Ii1) chiorideREFERESCES 1 D. Link sd LI. Mukr, Anrhicrub. Ag_ Ciremorke_., 10 (1976) 476. Z P. L. Olin, Snf. J_ Ccnzccr. 40 (1979) 89. 3 G. E_ Adanr. Erir. _Wezi_&ii_, 23 (1973) 48. 4 G_ IC Adams. I. R_ Flo&hart, C. E Smitfren. I. J. Strat’ord, P. Wardmzn Rodhat. I&s_. 67 (1976) 9_ 5 A_ 13icci2. G. Bcnilli and S. Rofiia, h_ J_ I?QG?&.Biol., 36 (1979) 85. 6 D. Iham and I-L Geeoeo, J_ Ck.romztcgr_. 7 (1962) 56. 7 _M_ R. Grimmtxt and E. L. Richards, J. Cimma~~., 20 (1965) 171. S J. E_ Stambaugh zx!d R_ W_ Man&e& J_ Chronza~o~_. 31 (1967) 128.
ad
M_ E
Wa:ts.