Synthesis and Physicochemical Characterization of Fluorescent (E)-2-Stilbenyloxyalkylthiouracils and Isomer Differentiation using EIMS

Article abstract of DOI:10.1002/jhet.5570410207

Twelve new fluorescent (E)-2-stilbenyloxyalkylthiouracils and 6-methyluracils 5a-5I were prepared. EI induced mass spectral fragmentation of these compounds was investigated. Fragmentation pathways are proposed on the basis of accurate mass and metastable

Full text of DOI:10.1002/jhet.5570410207

Mar-Apr 2004
177
Synthesis and Physicochemical Characterization of Fluorescent (E)-2-
Stilbenyloxyalkylthiouracils and Isomer Differentiation using EIMS
·
Elzbieta Wyrzykiewicz *, Monika Wendzonka
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland
Received July 1, 2003
Twelve new fluorescent (E)-2-stilbenyloxyalkylthiouracils and 6-methyluracils 5a-5l were prepared. EI
induced mass spectral fragmentation of these compounds was investigated. Fragmentation pathways are pro-
posed on the basis of accurate mass and metastable transition measurements. Correlation between the inten-
+.
sities of the M and the selected fragment ions of these compounds is discussed. The data obtained permit a
1
13
distinction of the metamers. The H and C NMR spectra of these compounds were assigned unambigu-
ously using a combination of heteronuclear (HETCOR) spectra and the chemical shifts. The data derived
from these spectra can be used to differentiate the isomers.
J. Heterocyclic Chem., 41, 177 (2004).
Introduction.
Results and Discussion.
Thio derivatives of pyrimidine bases are of interest
because of their biological and pharmacological activities
[1-7]. Many of the modified nucleobases and nucleosides
both with additional five- and six-membered rings are flu-
orescent [8-11] and offer a possibility of gaining informa-
tion concerning DNA and RNA structure and dynamic
[12-14]. Many have been shown to enter biochemical path-
ways and have given indications about their binding to dif-
ferent enzymes [15-16]. The fluorescent derivatives of
cytosine and cytidine have been widely reported in the lit-
erature [11,17-20]. However, to the best of our knowledge,
very little work has been published on the synthesis and
physicochemical properties of fluorescent derivatives of 2-
thiouracils. This fact has stimulated us to prepare a series
of (E)-2-stilbenyloxyalkylthiouracils (5 a,5 c,5 e,5g,5 i,5 k)
and (E) - 2 - s t i l b e n y l o x y a l k y l t h i o - 6 - m e t h y l u r a c i l s
(5 b,5 d,5f,5 h,5j,5 l) with the idea that these compounds
bearing on pyrimidine ring (E)-stilbenyloxyalkylthio sub-
stituents with strongly fluorescent (E)-stilbene moiety,
would certainly be fluorescent [21,22].
A few series of twelve new fluorescent (E)-2-stilbeny-
loxyalkylthiouracils (5 a,5c,5e,5 g,5i ,5k) and (E) - 2 - s t i l-
benyloxyalkylthio-6-methyluracils (5b,5d,5f,5h ,5j ,5l)
have been synthesized in the reaction of 2-thiouracil (or 2-
thio-6-methyluracil) with corresponding (E) - b r o-
moalkoxystilbenes 3a-3f. Treatment of 2-thiouracil (or 2-
thio-6-methyluracil) with 3a-3f in 0.3 M solution of NaOH
in methanol at room temperatures afforded 5 a -5 l
(Scheme 1). A series of new 3a-3f have been synthesized
in the reaction of (E)-stilbenol-4, [(E)-4'-chlorostilbenol-4,
(E) 4'-nitrostilbenol-4)] with 1,2-dibromoethane (1,3-
dibromopropane; 1,4-dibromobutane; 1,5-dibromopen-
tane; 1,6-dibromohexane) in 0.3 M NaOH solution of
DMSO at room temperature. The structures of all com-
pounds obtained were determined by examining their
UV/VIS, IR, 1H NMR and 13C NMR spectra as well as on
the basis of elemental analyses (Tables 1-7).
The 1H and 13C NMR data of 3a-3f and 5 a-5l are given in
Tables 3-7. Assignments of the 1H NMR and 13C NMR res-
onances of these compounds were deduced on the basis of
signal multiplicities, and by the concerted application of the
two–dimensional NMR technique HETCOR. The HET-
COR results allow unequivocal assignment of the 13C NMR
spectra proposed on the basis of chemical shifts theory,
additivity rules and by comparing the measured and calcu-
lated chemical shifts [31]. (E) configuration in the stilbene
part of the molecules of 3 a-3f and 5 a-5l was determined on
the basis of their UV/VIS and IR spectra. It has been pointed
out that in the UV/VIS spectra of 3 a-3f λmax are in the range
320-378 nm (Table 3), as well as 5 a-5 l 318.5-362.5 nm
(Table 4) respectively. According to the literature [26-28]
(E)-stilbenes exhibited the values of λmax in range 290-360
nm and for (Z)-stilbenes values of λmax fall in the range 260-
280 nm. The infrared spectra of 3 a-3f and 5 a-5l show a
strong band in the range 960-987 cm-1 which according to
the literature [29-30] can be attributed to the out-of-plane
deformation vibration of the C-H bond of the (E)-ethylene
bridge of the stilbene skeletone (Tables 3,4).
This paper deals with the synthesis and physicochemi-
1
cal properties of 5a-5 l. The UV/VIS, IR, H NMR and
13C NMR of (E)-bromoalkoxystilbenes (3 a-3 f) have also
been investigated because these compounds are also
unknown in the literature. The analyses of 1H NMR, 13C
NMR and EI mass spectra of 5a-5l have been performed
to check the possibility of differentiation of (E)-2-stil-
benyloxyalkylthiouracils (5 a,5 c,5e,5g,5 i,5k ) and (E)-2-
stilbenyloxyalkylthio-6-methyluracils (5 b,5d,5 f,5h,5 j,5 l).
The investigation of electron-impact induced mass spec-
tra was also undertaken to discover whether it is possible
to distinguish the metamers with molecular formulas
C2 1 H2 0 N2O2S (5 b ,5 c ); C2 2 H2 1 N3O4S (5 f ,5 g ) and
C23H23N3O4S (5 h,5 i) on the basis of differences in val-
ues of µ1 and µ2 i.e. the ratio of the intensity of selected
fragment ion peaks to that of the parent ion peak, and to
compare the data with those previously obtained in our
laboratory [23-25].

178
E. Wyrzykiewicz, M. Wendzonka
Vol. 41
Scheme 1
ring in the molecules of 5a-5l. The determination of the
presence of 6-methyl-uracil ring in the molecules of 5b,
5d, 5f, 5h, 5j and 5l is also possible on the basis of the
presence in the 13C NMR spectra of these compounds the
signals of carbons of C VI -methyl group of uracil ring.
The values of the chemical shifts of the signals of these
carbons fall in the range 18.94-24.23 δ.
A comparison of the number and positions of the C II, C
IV, C VI carbon atom signals in the region 160-170 ppm of
13C NMR spectra of (E)-2-stilbenyloxyalkylthiouracils
(5a,5c,5e,5g,5i,5k ) and (E)-2-stilbenyloxyalkylthio-6-
methyluracils (5 b,5 d,5 f,5 h,5 j,5l) allows a differentiation
between metameric 5b and 5c [C21H20N2O2S], 5f and 5g
[C22H21N3O4S] as well as 5h and 5i [C23H23N3O4S].
This data are given in tabular form below:
5b (E)-2-[stilbenyl-4-oxyethylthio-6-metyluracil];
C II 167.05 ppm
C IV 160.63 ppm
C VI 162.24 ppm
5c (E)-2-[stilbenyl-4-oxypropylthiouracil]
C II 166.40 ppm
C IV 161.56 ppm
C VI 149.76 ppm
1
In the H NMR spectra of (E)-2-stilbenyloxyalkyl-
thiouracils (5 a,5 c,5 e,5g,5 i,5k) are seen two doublets of
CV-H and CVI-H protons of uracil ring. The values of the
chemical shifts of the signals of these protons were estab-
lished in the range 6.52-6.60 δ and 7.45-8.00 δ, respec-
tively (Table 4). In the 1H NMR spectra of (E)-2-stilbenyl-
oxyalkylthio-6-methyluracils (5 b,5 d,5f,5 h,5 j,5 l) are seen
two singlets of CV-H and CVI-CH3 protons of 6-methylu-
racil ring. The values of the chemical shifts of the signals
of these protons fall in the range 6.37-6.83 δ and 2.24-2.49
5f (E)-2-[4'-nitrostilbenyl-4-oxypropylthio-], 6-methyluracil,
C II 165.70 ppm
C IV 164.56 ppm
C VI 160.01 ppm
5g (E)-2-[4'-nitrostilbenyl-4-oxybutylthio-]uracil
C II 161.70 ppm
C IV 160.89 ppm
C VI 146.18 ppm
5h (E)-2-[4'-nitrostilbenyl-4-oxybutylthio-], 6-methyluracil
1
δ, respectively. The presence of these signals in the H
NMR spectra of 2-(E)-stilbenyloxyalkylthiouracils and
2-(E)-stilbenyloxyalkylthio-6-methyluracils allows for the
determination of the presence of uracil or 6-methyluracil
C II 165.70 ppm
C IV 164.56 ppm
C VI 160.30 ppm
Table
1
Chemical and Physical Data of Compounds 3a-f
Comp.
Formula
(mol. weight)
M.p.
[°C]
Yield
%
Rf
TLC
Elemental analysis
Calculated
(Found)
C
H
N
-
3a
3b
3c
3d
3e
3f
C
H
OBr
OBr
123-4
108-9
72-4
72
62
30
16
17
72
0.50
0.52
0.74
0.74
0.72
0.51
63.36
4.95
16 15
303.20
(63.13)
64.35
(63.74)
56.35
(56.15)
57.45
(57.85)
55.88
(5.15)
4.95
(5.23)
4.42
(4.62)
4.79
(4.80)
5.39
C
H
-
17 17
317.22
C
H
NO Br
3.84
(3.60)
3.72
(3.78)
3.43
(3.41)
-
17 16
3
362.23
C
H
NO Br
82-3
18 18
3
376.25
C
H
NO Br
77-8
19 20
3
408.25
(55.65)
60.91
(5.11)
6.69
C
H
ONBrCl
136-8
20 22
394.20
(60.60)
(6.73)

Mar-Apr 2004
Synthesis and Physicochemical Characterization
179
Table
2
Chemical and Physical Data of Compounds 5a-l
Comp.
Formula
(mol. weight)
M.p.
[°C]
Yield
%
Rf
TLC
Elemental analysis
Calculated
(Found)
C
H
N
S
5a
5b
5c
5d
5e
5f
C
H
N O S
246-8
213-5
222-3
187-9
93-4
20
53
19
24
73
40
77
65
57
81
16
19
0.73
0.59
0.66
0.57
0.48
0.51
0.54
0.47
0.54
0.57
0.67
0.65
66.85
(66.77)
69.23
(66.92)
69.23
(69.94)
65.18
(65.44)
61.61
(61.72)
62.41
(62.58)
61.11
(61.04)
63.16
(63.12)
63.10
(63.05)
63.86
(63.56)
62.88
(63.00)
64.79
5.01
(4.94)
5.49
(5.56)
5.49
(5.59)
5.43
(5.52)
4.65
(4.62)
4.96
(5.02)
5.09
(5.23)
5.26
(5.36)
5.26
(5.24)
5.54
(5.48)
5.89
(5.56)
5.83
7.79
(7.60)
7.69
(7.40)
7.69
(7.41)
6.91
(6.41)
10.26
(10.05)
9.93
(9.98)
9.72
(9.41)
9.60
(9.47)
9.60
(9.57)
9.31
(9.18)
6.11
(6.23)
6.04
(5.86)
8.91
(8.82)
8.79
(8.70)
8.79
(8.62)
7.90
(7.82)
7.82
(7.51)
7.56
(7.51)
7.56
(7.62)
7.32
(7.39)
7.32
(7.40)
7.09
(7.00)
7.12
(8.06)
6.91
20 18
2 2
350.06 x 1/ H O
2
2
C
H N O S
21 20 2 2
364.04
C
H N O S
21 20 2 2
364.04
C
H N O S
22 22 2 2
405,3 x 11/ H O
2
2
C
H N O S
21 19 3 4
409,41
C
H
N O S
175-7
154-6
132-4
148-50
139-41
189-91
169-71
22 21
3 4
423.44
C H N O S
5g
5h
5i
22 21 3 4
432.44 x 1/ H O
2
2
C
H N O S
23 23 3 4
437.46
C
H N O S
23 23 3 4
437.46
C H N O S
5j
24 25 3 4
451.49
C H N O SCl
5k
5l
24 25 2 2
449.45 x 1/ H O
2
2
C
H N O SCl
25 27 2 2
463.45 x 1/ H O
(64.83)
(5.86)
(6.78)
2
2
5i (E)-2-[4'-nitrostilbenyl-4-oxypentylothiouracil
Scheme 2
C II 165.65 ppm
C IV 164.86 ppm
C VI 146.00 ppm
On the basis of the low and high resolution electron–impact
mass spectra, as well as fragment ion spectra from the first
field–free region, recorded using linked scans at constant B/E
(Tables 8, 9), the principal mass spectral fragmentation routes
of the molecular ions of compounds 5a-5l are interpreted as
shown in Scheme 2 and 3. The stilbenyloxyalkyl substituents
of the molecular ions of 5a-5l might have a tricyclic
dihydrophenanthrenyloxyalkyl structure, as it was postulated
by us earlier in the cases of the mass decompositions of the
molecular ions of N- (E)-stilbenyloxyalkylcarbonyl substi-
tuted hydrazones of arylaldehydes [25,32,33]. The common
features of the mass spectral fragmentation of the molecular
ions of 5a-5l are simple cleavages of Csp3-O bonds in the
thioalkyloxy chain i.e. the elimination of stilbenyloxy radi-
cals. By these simple inductive cleavage the even-electron
fragment ions d are derived. These ions might have a bicyclic
structure with the positive charge probably situated on the
annular nitrogen atom, as was proposed earlier for the mass
fragmentation of alkoxycarbonylalkylthiouracils [34].
It ought to be pointed out that cleavages of Csp2-S and
Csp3-N bonds in these even-electron fragment ions leading
to ejection of neutral alkenethiols molecules to give even-
electron fragment ions situated in the mass spectra of (E)-2-
stilbenyloxyalkylthio-6-methylurails (5 a,5c,5 e,5 g,5 i,5 k) at

180
E. Wyrzykiewicz, M. Wendzonka
Vol. 41
Table 3
1
UV/VIS, IR and H NMR Data of 3a-f
–1
1
Comp.
UV/VIS
IR (cm
)
H NMRδ (ppm)
λ max
(lg ε)
ν C=C
HC=CH
trans
νC-O-
3a
3b
3c
3d
3e
3f
240
306
320
240
308
320
259
276
376
258
275
376
258
276
378
240
310
328
(3.95)
(4.43)
(4.42)
(3.97)
(4.42)
(4.41)
(4.14)
(4.12)
(4.30)
(4.08)
(4.05)
(4.32)
(4.12)
(4.09
1606
1595
1511
1603
1588
1511
1604
1588
1511
1604
1589
1512
1604
1589
1510
1605
1573
1511
OCH 4.32 t J= 6Hz
2
BrCH 3.66 t J= 6Hz
2
961
968
969
974
960
968
1255
1246
1253
1254
1249
1253
Ar
6.88-7.51 m
OCH 4.13 t J= 6Hz
2
BrCH 3.61 t J= 6Hz
2
Ar
6.88-7.51 m
OCH 4.33 t J= 6Hz
2
BrCH 3.66 t J= 6Hz
2
Ar
6.88-7.51 m
OCH 4.13 t J= 6Hz
2
BrCH 3.61 t J= 6Hz
2
Ar
6.88-7.51 m
OCH 3.98 t J= 6Hz
2
BrCH 3.48 t J= 6Hz
2
(4.35)
(4.05)
(4.44)
(4.47)
Ar
6.86-7.52 m
OCH 3.98 t J= 6Hz
2
BrCH 3.48 t J= 6Hz
2
Ar
6.86-7.45 m
Scheme 3
5h, 5j and 5l the even-electron fragment ions situated at m/z
109 [C5H5N2O] allows one to identify the presence of the
6-methyl group on the uracil moiety of these compounds.
It was found that the cleavage of the Csp3-O bond in the
thioalkoxy chain of 5 a -5l follows also McLaff e r t y
rearrangement involving the migration of a hydrogen atom
to a double bond of the phenyl ring of the (E)-stilbene skele-
ton. Odd-electron fragment ions c (Scheme 1) or b (Scheme
2) were obtained by this fragmentation pathways. The dif-
ferences in the fragmentation of metameric pairs of (E)-2-
stilbenyloxyalkylthiouracils (5 a,5c,5e,5 g,5 i,5k) and (E)-2-
stilbenyloxyalkylthio-6-methyluracils (5 b,5d,5 f,5h,5 j,5 l)
i.e. 5 b and 5 c; 5f and 5 g;5h and 5 i have been quantified by
comparing the calculated values of the coefficients µ1 and
µ2 i.e. the abundances of the selected fragment ions relative
to the abundances of the molecular ions.
% rel. int. d  +
µ 1 = ------------------
% rel. int. a+.
% rel. int. c  +
µ 2 = -------------------
% rel. int. a+.
It has been established by us previously that the diff e r-
ences in the values of coefficients µ are useful for differenta-
tion of the positional isomers of benzylthiouracils [35-37].
As can be seen from the data in Table 10, the differences
between the values of the coefficients µ1 and µ2 [i.e. the
calculated quotients of the relative intensities of the
selected fragment ions d (µ1) and c (µ2) as well as molecu-
lar ions M+. a] are sufficient to differentiate isomers. The
(E)-2-stilbenyloxyalkylthiouracils (5b, 5f, 5h) are distin-
guished from metameric (E)-2-stilbenyloxyalkylthio-6-
methyluracils (5c, 5g, 5i) on the basis of the lower values
of µ1 and the higher values of µ2.
m/z 95 [C4H3N2O] (ion e, Table 8, Scheme 2; ion g Table 9,
Scheme 3) and in the mass spectra of (E)-2-stilbenyl-
oxyalkylthio-6-methyluracils (5b,5d,5 f,5h,5 j,5 l) at m/z 109
[C5H5N2O] (ion e, Table 8, Scheme 2; ion g Table 9,
Scheme 3). The presence in the mass spectra of 5b, 5d, 5 f,

Mar-Apr 2004
Synthesis and Physicochemical Characterization
181
Table 4
1
UV/VIS, IR and H NMR Data of 5a-l
–1
1
Comp.
UV/VIS
IR (cm
νC=O
)
H NMR δ (ppm)
λ max (lg ε)
νC=C
HC=CH
νSCH
2
trans
1658
5e-j
5a
5b
5c
5d
5e
292.5 (4.43)
318.5 (4.47)
1605
1535
1511
961
962
968
967
970
2443
2453
2449
2445
2445
OCH 4.45 t J= 6Hz
2
SCH 3.74 t J= 6Hz
CVI H 8.00 d J= 7Hz
CV H 6. 52 d J= 7Hz
6.90-7.49 m
OCH 4.45 t J= 6Hz
SCH 3.75 t J= 6Hz
CV H 6.43 s
CVI CH 2.49 s
6.93-7.51 m
OCH 4.50 t J= 6Hz
SCH 3.50 t J= 6Hz
CVI H 8.07 d J= 7Hz
CV H 6. 54 d J= 7Hz
6.93-7.51 m
OCH 4.42 t J= 6Hz
SCH 3.61 t J= 6Hz
CV H 6.86 s
CVI CH 2.39 s
6.93-7.51 m
OCH 4.16 t J= 6Hz
SCH 3.53 t J= 6Hz
CVI H 7.49 d J= 7Hz
CV H 6.53 d J= 7Hz
2
Ar
292.5 (4.45)
318.5 (4.47)
1603
1577
1510
1648
1703
1644
1705
2
2
3
Ar
292.5 (4.45)
318.5 (4.44)
1602
1595
1511
2
2
Ar
292.5 (4.44)
318.5 (4.45)
1604
1592
1511
2
2
3
Ar
265.0 (4.32)
359.5 (4.17)
1603
1587
1511
d
d
d
d
d
8.20 J=9Hz
7.61 J=9Hz
6.95 J=9 Hz
9.92 J=9Hz
6.97 J=16Hz
2
A
B
C
D
E
2
d 7.24 J=16Hz
F
5f
5g
5h
5i
266.0 (3.88)
359.5 (3.72)
1605
1587
1511
1635
1657
1644
1668
1655
968
964
961
987
958
2446
2449
2453
2452
2455
OCH 4.19 t J= 6Hz
d
d
d
d
d
8.20 J=9Hz
7.60 J=9Hz
6.94 J=9 Hz
9.91 J=9Hz
6.99 J=16Hz
2
A
B
C
D
E
SCH 3.43 t J= 6Hz
2
CV H 6.84 s
CVI CH 2.29 s
3
d 7.23 J=16Hz
F
271.0 (4.27)
361.0 (4.08)
1605
1589
1511
OCH 4.11 t J= 6Hz
d
d
d
d
d
8.21 J=9Hz
7.61 J=9Hz
6.95 J=9Hz
6.91 J=9Hz
6.99 J=16Hz
2
A
B
C
D
E
SCH 3.43 t J= 6Hz
2
CVI H 7.49 d J= 7Hz
CV H 6.60 d J= 7Hz
d 7.24 J=16Hz
F
270.0 (4.29)
359.5 (4.09)
1605
1589
1512
OCH 4.06 t J= 6Hz
d
d
d
d
d
8.19 J=9Hz
7.58 J=9Hz
6.94 J=9Hz
6.91 J=9Hz
6.97 J=16Hz
2
A
B
C
D
E
SCH 3.45 t J= 6Hz
2
CVI H 6.49 s
CV CH 2.30 s
3
d 7.23 J=16Hz
F
271.5 (4.30)
362.5 (4.12)
1607
1585
1511
OCH 4.03 t J= 6Hz
d
d
d
d
d
8.21 J=9Hz
7.59 J=9Hz
6.96 J=9Hz
6.91 J=9Hz
6.94 J=16Hz
2
A
B
C
D
E
SCH 3.63 t J= 6Hz
2
CVI H 7.49 d J= 7Hz
CV H 6.53 d J= 7Hz
d 7.23 J=16Hz
F
5j
271.0 (4.28)
360.5 (4.05)
1605
1590
1510
OCH 4.00 t J= 6Hz
d
d
d
d
d
8.21 J=9Hz
7.59 J=9Hz
6.94 J=9Hz
6.91 J=9Hz
6.94 J=16Hz
2
A
B
C
D
E
SCH 3.41 t J= 6Hz
2
CVI H 6.49 s
CV CH 2.24 s
3
d 7.22 J=16Hz
F

182
E. Wyrzykiewicz, M. Wendzonka
Vol. 41
Table 4(continued)
–1
1
Comp.
UV/VIS
IR (cm
νC=O
)
H NMR δ (ppm)
λ max (lg ε)
νC=C
HC=CH
νSCH
2
trans
1659
5e-j
5k
5l
292.5 (4.40)
326.0 (4.46)
1604
1592
1511
968
967
2401
2669
OCH 4.11 t J= 6Hz
SCH 3.38 t J= 6Hz
CVI H 7.45 d J= 7Hz
CV H 6.63 d J= 7Hz
d
d
d
d
d
d
d
d
d
d
d
d
8.07 J=9Hz
7.43 J=9Hz
7.16 J=9H
2
A
B
C
D
E
F
2
6.76 J=9Hz
6.92 J=16Hz
7.29 J=16Hz
7.43 J=9Hz
6.96 J=9Hz
6.90 J=9Hz
6.63 J=9Hz
7.00 J=16Hz
7.64 J=16Hz
293.0 (4.37)
323.0 (4.41)
1604
1575
1511
1648
OCH 4.03 t J= 6Hz
SCH 3.26 t J= 6Hz
CV H 6.37 s
CVI CH 2.46 s
2
A
B
C
D
E
F
2
3
Table 5
13
C NMR Shifts of 3a-3f
Carbon
3a
3b
3c
3d
3e
3f
C-1
137.55
127.10
128.67
127.34
126.31
128.02
130.96
127.81
115.01
157.79
67.94
28.97
-
137.63
126.78
128.64
127.25
126.26
126.16
130.45
127.75
114.76
158.43
65.39
29.91
32.34
-
144.30
128.42
124.92
146.48
124.17
132.90
129.13
128.42
114.92
159.29
68.87
29.83
32.48
-
144.14
128.31
124.04
146.25
123.99
132.76
128.87
126.38
114.73
159.35
66.94
29.47
33.46
27.90
-
144.31
128.43
124.15
146.40
124.03
132.92
128.89
126.48
114.84
159.68
67.66
28.93
33.56
28.34
24.77
-
136.20
128.87
128.76
132.64
127.77
125.14
129.65
127.36
114.72
158.98
67.76
C-2,6
C-3,5
C-4
C-α
C-α'
C-1'
C-2',6'
C-3',5'
C-4'
C-I
C-II
29.02
32.62
27.87
25.24
C-III
C-IV
C-V
-
-
-
-
-
-
-
C-VI
-
33.71
The fluorescence properties of compounds 5a -5 l have
been investigated, the fluorescence emission maxima
and quantum yields are summarized in Table 4. The (E)-
2-stilbenyloxyalkylthiouracils (5 a,5c,5k) and (E)-2-stil-
benyloxyalkylthio-6-methyluracils (5 b,5d,5l) are mod-
erately fluorescent, having emission bands at 370-380
nm with the main excitation peak at 320-360 nm at
dioxane. The (E)-2-[4'-nitrostilbenyl-4-oxyalkylthio]-6-
methyluracils (5 f,5h,5j ) are highly fluorescent, having
emissions bands at 520 nm with the main excitation
peak at 360 nm in dioxane. Clearly, the substitution of
(E)-stilbenylaxyalkyl group at the angular sulfur atom
of 2-thiouracil (or 2-thio-6-methyluracil) serves as a
useful method of converting these compounds into fluo-
rescent derivatives.
Conclusions.
- It follows from the obtained results that the fluores-
cence of 5a - 5l can be attributed to the introduced flu-
orophore i.e. (E)-stilbenyl moiety.

Mar-Apr 2004
Synthesis and Physicochemical Characterization
183
Table 6
13
C NMR Shifts of 5a-5f
Carbon
5a
5b
5c
5d
5e
5f
C-1
136.06
126.32
128.14
127.61
126.23
128.00
128.65
127.75
114.16
155.62
168.89
161.18
108.67
145.35
67.31
32.63
-
137.50
126.41
128.69
127.37
126.34
127.81
128.74
127.71
114.75
157.76
167.05
160.63
108.86
162.24
67.44
32.54
-
137.44
127.35
127.81
127.35
126.20
127.47
128.71
127.71
114.88
157.60
166.4
161.56
108.85
149.76
65.61
28.51
28.24
-
137.31
126.29
129.72
128.34
126.64
129.23
129.83
128.94
115.75
159.35
169.02
161.70
109.30
162.84
67.50
28.57
29.42
-
146.18
128.34
124.46
146.12
136.50
124.13
129.53
129.16
114.81
160.09
165.65
160.65
109.11
146.00
65.74
28.47
28.34
-
146.49
126.51
124.16
146.30
132.97
124.31
128.57
126.56
115.04
159.92
165.70
164.56
108.42
160.01
65.98
28.87
27.32
-
C-2,6
C-3,5
C-4
C-α
C-α'
C-1'
C-2',6'
C-3',5'
C-4'
C-II
C-IV
C-V
C-VI
C
C
C
C
C
C
C
A
B
C
D
E
F
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
19.17
18.94
24.05
G
- (E)-4'-Nitrostilbenyl-4-oxyalkylthiouracils (5 e,5 g,5 i)
and (E)-4'-nitrostilbenyl-4-oxyalkylthio-6-methylu-
racils (5f,5h,5j) may well be the best fluorescent
derivatives of uracil yet prepared.
- (E)-Configuration in the stilbene part of the molecules
of 5a-5l can be determined on the basis of the analysis
of UV/VIS and IR spectra of these compounds.
- The presence of the 6-methyl substituent in the uracil
ring of (E)-2-stilbenyloxyalkylthio-6-methyluracils
(5 b,5 d,5 f,5 h,5 j,5 l) can be identified on the basis of
- The inductive cleavage of these bonds produces even
electron fragment ions d. The cleavage of these bonds
according to McLafferty rearrangement produces odd-
electron fragment ions c (Schemes 2,3; Tables 9, 10).
- The values of µ1 and µ2 (i.e. the ratio of intensities of d
and c fragment ions peaks to those of the molecular ion
peaks M+.) depend on the structure of (E)-2-stilbeny-
loxyalkylthiouracils (5c,5g,5i) and (E)-stilbeny -
loxyalkylthio-6-methyluracils (5 b,5 f,5 h). The differ-
ences in the values of µ1 and µ2 are useful for differenti-
ation between metamers of these compounds derivatives
of uracil (5 b,5 f,5h) and 6-methyluracil (5c,5g,5i).
1
analysis of H NMR, (Table 4), 13C NMR (Table 6)
and EI mass spectra (Tables 8 and 9) of these com-
pounds.
- The differences in the 13C NMR spectra of 5a-5l in
the number and positions in the range 160-170 ppm of
the C II, C IV and C VI carbon atoms of uracil ring,
allow the differentiation of metamers 5b, 5c and 5f, 5g
as well as 5h, 5i.
- The basic mass fragmentation of the molecular ion of
5 a -5 l is due to cleavages of Csp3–O bonds of
oxyalkylthio chains of (E)-stilbenyloxy-alkylthio -
uracils.
EXPERIMENTAL
The purity of all described compounds was checked by
m.p.'s, TLC and elemental analysis. Melting points (uncor-
rected) were determined on a Böetius microscope hot stage. R
values refer to TLC silica gel F TLC plates (Merck) devel-
254
oped with CHCl – n-hexane 4:1 (3a-3 f) and CHCl - MeOH
5:1 (5 a-5l ) and observed under UV light (λ=254 and 366 nm).
UV/VIS spectra were recorded with a Specord UV/VIS spec-
trophotometer in dioxane. IR spectra were recorded with a FT-
f
3
3

184
E. Wyrzykiewicz, M. Wendzonka
Vol. 41
Table 7
13
C NMR Shifts of 5g-5l
Carbon
5g
5h
5i
5j
5k
5l
C-1
144.31
123.83
124.04
146.22
132.83
124.15
129.14
126.47
116.00
160.00
161.70
160.89
109.68
146.18
67.72
31.27
28.56
-
146.49
126.51
124.16
146.30
132.97
124.16
129.14
128.41
114.89
159.41
165.70
164.56
108.42
160.30
65.48
28.47
28.34
-
146.27
126.38
123.98
146.27
132.77
124.05
128.85
124.31
114.77
159.41
165.65
164.86
108.22
146.0
67.26
30.24
28.17
25.87
-
144.18
126.39
123.98
144.18
132.86
124.07
128.83
128.32
114.80
159.92
165.40
163.98
108.33
160.92
67.73
135.89
127.88
128.78
132.92
126.06
128.50
131.17
127.41
115.49
157.38
163.91
161.88
109.45
145.52
69.22
136.20
127.87
130.03
132.79
125.63
128.84
130.35
127.44
115.02
158.60
157.15
160.87
109.03
165.09
68.16
C-2,6
C-3,5
C-4
C-α
C-α'
C-1'
C-2',6'
C-3',5'
C-4'
C-II
C-IV
C-V
C-VI
C
C
C
C
C
C
C
A
B
C
D
E
F
30.57
28.86
25.20
28.75
31.65
28.59
25.04
27.93
27.67
-
31.08
28.82
25.21
28.56
27.99
21.54
-
-
-
-
-
-
-
-
24.05
24.23
G
Table 8
Elemental Composition and Relative Intensities of the Ion Peaks in the spectra of
5a-5d and 5k-l According to High Resolution Data
Ion
m/z
Elemental compositon % Relative intensity
5a
5b
5c
5d
5k
5l
+.
M
b
a
350
364
378
440
454
222
236
312
196
230
155
169
183
211
225
95
C
C
C
C
C
C
C
C
C
C
H
N O S
10
-
-
-
-
7
-
-
27
-
100
-
-
-
-
13
-
-
7
-
-
-
8
-
-
14
-
-
100
-
-
-
-
11
-
9
-
18
-
-
-
-
3
-
14
-
-
100
-
-
-
7
-
-
-
28
-
-
-
6
-
22
-
-
-
-
-
72
-
-
-
4
-
77
-
-
-
-
-
20 18
2 2
H
N O S
21 20
2 2
H
N O S
22 22
2 2
H
N O SCl
24 25
2 2
H
N O SCl
18
-
-
3
-
100
-
-
-
-
27
-
25 27
2 2
H
O
O
OCl
O
16 14
H
17 16
H
20 21
c
H
14 12
H
OCl
14 11
d
C H N OS
6
7 2
C H N OS
-
-
-
7
9 2
C H N OS
100
-
-
-
6
-
8
11
H
H
2
C
C
N OS
100
-
13
-
20
-
10 15
2
N OS
11 17
2
e
f
C H N O
4
3 2
109
70
84
C H N O
5
-
25
5
5 2
C H NO
8
-
7
-
3
4
C H NO
10
4
6
13
and pulse width 15 µs. Heteronuclear 2D C NMR- H NMR
chemical shift correlation experiments were carried out using
HETCOR spectra. The spectra were acquired with 2K data
1
IR Bruker IFS – 113 v spectrophotometer in KBr pellets. The
1
13
H NMR (300 MHz) and C NMR (75 MHz) spectra were
recorded on a Varian Mercury Spectrometer operating at
300.07 MHz (proton or 75.46 MHz (carbon). Data were
obtained from CDCl solutions. The chemical shifts were ref-
13
points, 256 increments and spectral width 19.63 KHz for
1
and 4.97 KHz for H.
C
3
Elemental analyses were performed with a Vector Euro EA
3000 analyzer. Low- and high-resolution mass spectra were
recorded on an AMD - Intectra GmbH-Harpstedt D-27243 Model
402 two - sector mass spectrometer (ionizing voltage 70 eV,
accelerating voltage 8 kV, resolution 10,000). Samples were
introduced by a direct insertion probe at the source temperature
erenced to tetramethylsilane. Chemical shifts are given in the δ
1
scale (ppm) and coupling constants in Hertz. H NMR (300.07)
spectra were recorded with spectral width 9 KHz, acquisition
time 2.0 s, pulse width 6 µs and double precision acquisition.
13
C NMR (75.460 MHz) spectra were recorded with spectral
width 18.76 KHz, acquisition time 1.0 s, recycle delay 1.0 s

Mar-Apr 2004
Synthesis and Physicochemical Characterization
185
Table 9
of ~150 °C. The elemental composition of the ions were deter-
mined by a peak matching method relative to perfluorokerosene
and using the same instrument. All masses measured were in
agreement with those of the composition given in column 3 of
Tables 8 and 9 to within ± 2 ppm. The B/E linked scan spectra in
the first field - free region were investigated using helium as the
Elemental Composition and Relative Intensities of the Ion Peaks in the
Spectra of5e-5j According to High Resolution Data
Ion
m/z
Elemental
compositon
% Relative intensity
5e 5f
5g
5h
5i
5j
-5
collision gas at a pressure of 1.73 x 10 with the ion source tem-
perature of 180 °C, ionization energy of 70 eV and an accelerat-
+.
M
a
409
423
437
451
211
241
169
183
197
211
165
152
109
95
C
C
C
C
C
C
H
N O S
3
-
-
6
-
-
16
-
-
-
-
-
-
-
21 19
3 4
H
N O S
3 4
ing voltage of 8 kV. The values of µ and µ were calculated as
1
22 21
2
H
N O S
-
4
27
-
20
7
averages of three measurements. Fluorescence corrected spectra
were taken in dioxane (isocratic grade for liquid chromatogra-
phy-Merck) with a Perkin Elmer MPF-3 instrument.
Fluorescence quantum yields (in the same solvent) were calcu-
lated on the basis of the value 0.55 for quinine sulfate. (E)-
Stilbenol-4, (E)-4'-nitostilbenol-4 and E-4'-chlorostilbenol-4
were obtained according to the literature [38].
23 23
3 4
H
N O S
-
-
-
-
24 25
3 4
b
c
d
H NO
2
37 11
30 12
9
7
14
45
-
13
9
H
NO
14
-
9
-
26
-
14 11
3
C H N OS
100
-
7
9 2
C H N OS
-
-
-
100 100
-
-
-
8
11 2
C H N OS
-
-
-
-
100
-
100
-
-
9
13
2
C
C
C
H
N OS
93
24
7
40
-
10 15
2
General Procedure for the Preparation of (E)-Bromoalkoxy-
stilbenes (3a-3f).
e
f
g
H
H
59 25
11 11
22
6
19
3
30
8
13
9
12
8
C H N O
-
12
-
-
16
-
-
5
5 2
A 20 ml solution of (E)-stilbenol-4, (2 mmole) or (E)-4'-nitros-
tilbenol-4, (E)-4'-chloro-stilbenol-4) in of 0.3 M NaOH in DMSO
was stired at room temperature while 4 mmole of dibromoalka-
nes (1,2-dibromoethane; 1,3-dibromopropane; 1,4-dibromobu-
tane; 1,5-dibromo- pentane; 1,6-dibromohexane) was added
dropwise. After stiring for 3 hours precipitated solid was filtered.
The filtrate was then acidified (pH 3) with 6 M HCl and diluted
with 80 ml of cold water. The precipitated solid was collected by
filtration, washed with water and dried at room temperature
under vacuum. Recrystallization from ethanol afforded com-
pounds 3a-3f.
C H N O
13
35
25
4
3 2
Table 10
Values of µ Calculated from the EI Mass Spectra of Metameric 5b, 5c,
5f, 5g,5h, 5i
Comp
5b
5c
5f
5g
5h
5i
µ
µ
0.7
2.0
1.8
0.7
0.6
2.0
1.6
0.8
0.4
2.7
1
2
2.25
1.66
General Procedure for the Preparation of (E)-2-Stilbenyloxy-
alkylthiouracils (5a-5l).
Table 11
UV/VIS and Fluorescence Spectra of 5a-5l
2-Thiouracil (or 2-thio-6-methyluracil) (0.07 mole) in 10 ml of
0.3 M methanolic NaOH was stirred in room temperature while
0.07 mole of corresponding (E)-bromoalkoxystilbene (3a-3f) in
10 ml of acetone solution was added dropwise. The reaction mix-
ture was stirred at room temperature for 8 hours. The obtained
solid was filtered, the filtrate was acidified (pH 3) with 6 M HCl
and diluted with 70 ml of cold water. The precipitated solid was
collected by filtration, washed with water and dried at room tem-
perature under vacuum. Recrystallization from ethanol afforded
compounds 5a-5l.
Comp. UV/VISλ
Fluorescence
Quantum
yields
[φ]
max[nm], (log ε) Excitation wave Emission wave
(dioxane)
[nm] (dioxane)
320
5a
5b
5c
5d
5e
5f
303.0 (4.53)
320.0 (4.49)
304.5 (4.51)
320.0 (4.49)
303.5 (4.53)
320.0 (4.49)
304.5 (4.50)
320.5 (4.48)
265.0 (4.32)
359.0 (4.17)
266.0 (4.32)
359.0 (3.72)
271.0 (4.27)
261.0 (4.08)
270.0 (4.29)
259.5 (4.09)
271.9 (4.30)
362.5 (4.12)
271.9 (4.28)
360.5 (4.05)
308.0 (4.50)
326.5 (4.50)
308.5 (4.53)
326.0 (4.54)
370
370
380
370
520
530
520
520
520
520
380
380
0.01
0.01
320
320
320
360
360
360
360
360
360
326
326
0.01
0.01
REFERENCES AND NOTES
0.002
0.002
0.002
0.003
0.002
0.003
0.01
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5h
5i
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