The synthesis of N-glycosyl-N′-pyrazolylmethylene aminothioureas under a 4A molecular sieve

Article abstract of DOI:10.1002/jccs.200800056

Aseries of novel N-glycosyl-N′-pyrazolylmethylene aminothioureas (4a-4e, 5a-5e) were synthesized from N-glycosyl-N′-aminothioureas (2a-2d) and 4-formylpyrazole (3a-3e). Activated 4A molecular sieves were adopted for dehydrated reagent to improve the reaction rate and yield. The structures of the new compounds were identified on the basis of IR, ¹H NMR and MS spectra. Simultaneously, the compounds were detected by fluorescence spectrophotometer and had preferable fluorescence activity, so they can be selected as a kind of novel fluorescence labeled derivative of sugar.

Full text of DOI:10.1002/jccs.200800056

Journal of the Chinese Chemical Society, 2008, 55, 385-389
385
The Synthesis of N-Glycosyl-N¢-pyrazolylmethylene Aminothioureas under
a 4Å Molecular Sieve
Yan-Wu Zhao (
College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi Xinjiang 830046, P. R. China
) and Ling-Hua Cao* (
)
A series of novel N-glycosyl-N¢-pyrazolylmethylene aminothioureas (4a-4e, 5a-5e) were synthesized
from N-glycosyl-N¢-aminothioureas (2a-2d) and 4-formylpyrazole (3a-3e). Activated 4Å molecular
sieves were adopted for dehydrated reagent to improve the reaction rate and yield. The structures of the
new compounds were identified on the basis of IR, 1H NMR and MS spectra. Simultaneously, the com-
pounds were detected by fluorescence spectrophotometer and had preferable fluorescence activity, so they
can be selected as a kind of novel fluorescence labeled derivative of sugar.
Keywords: Glycosylisothiocyanate; Thiourea; 4-Formylpyrazole; 4Å Molecular sieve; Fluores-
cence characteristic.
INTRODUCTION
Saccharide compounds are the main source of all or-
ods of glycosyl compunds, the fluorescence derivative
method is the primary means for its excellent sensitivity,
outstanding selectivity and small quantity of sample used
and so on.12 Thus, if glucide compounds are made into ap-
propriate fluorescence-labeled derivatives, it will be a very
significant development in analyzing and determining
glycosyl compounds.
ganisms which maintain energy that life activities require.
In fact, as the endogenesis substance of life, glucide com-
pounds participate almost in all of the biological courses of
organisms.1 In the field of carbohydrates, glycosyl isothio-
cyanates are versatile synthetic intermediates that have
been widely used in the synthesis of carbohydrate deriva-
tives, mostly having thiourea structure, of synthetic, bio-
logical and pharmaceutical interest.2 The thioureas and
their derivatives have antifungal and antibacterial activity
and can also modulate the plant growth.3,4 Their broad bio-
logical activities have attracted a considerable amount of
attention in recent years.5,6 Pyrazole derivatives are a sig-
nificant member in the heterocyclic family for their impor-
tance in the production of medicine and agricultural chemi-
cals.7 Furthermore, a number of pyrazole derivatives have
been reported to exhibit strong physiological and pharma-
cologic activities, such as antiviral, antipyretie, analgestic,
and anti-inflammatory activities.8-11 We designed and syn-
thesized several new thiourea derivatives by incorporating
glycosyl moieties with pyrazole fragments. These deriva-
tives might have biologic activities.
In this study, we characterized the synthesis and fluo-
rescence characteristics of new glycosyl thioureas and ca-
talysis of a 4Å molecular sieve in the reaction. Correspond-
ing glycosyl aminothioureas were prepared from anhy-
drous hydrazine and glycosyl isothiocyanate, then the title
compounds were synthesized in terms of the following syn-
thesis route (Scheme I).
Scheme I
Additionally, nowadays, the research into saccha-
rides is one of the most popular fields in chemistry and bi-
ology. As a result, as far as saccharide compounds are con-
cerned, the means of analyzing and determining the struc-
ture and quality contained in the substance have become
more and more important. Among all of the analytic meth-
1a, 2a, 4a-4e: gly=2,3,4-tri-O-acetyl-b-D-xylopyranosyl;
1b, 2b, 5a-5e: gly=2,3,4-tri-O-acetyl-a-L-arabinopyranosyl;
3a-3e, 4a-4e, 5a-5e: a: R=H; b: R=OCH ; c: R=Cl; d: R=Br; e: R=NO
3
2

386 J. Chin. Chem. Soc., Vol. 55, No. 2, 2008
Zhao and Cao
RESULTS AND DISCUSSION
Table 1. The effect of molecular sieve amount on yield of
product and reaction time
In the process of the synthesis of compounds 3a-3e, it
was extremely difficult to directly form 4-formylpyrazole
by the formylation of pyrazole, but hydrazone reacted with
Vilsmeier-Haak reagent very easily to produce 4-formyl-
pyrazole in the ratio of 1 to 2. Furthermore, the virtue of
this reaction was mild conditions and high yield.
Molecular sieve Method 1
Method 2
amount/g
time/h
yield/%
time/h
yield/%
0.5
1.0
1.5
2.0
4
4
4
4
65
65
65
65
3
3
2
2
75
82
85
83
A glycosyl ring is easily opened in acidic conditions,
therefore the best condition of the reaction is approxi-
mately neutral or weakly alkalescent. We selected sugar
isothiocyanates (1a-1d) of high reaction activity and anhy-
drous hydrazine and let them react in mild conditions.
N-glycosyl-N¢-aminothioureas (2a-2d) were obtained, then
reacted with 4-formylpyrazole (3a-3e) to give a series of
novel N-glycosyl-N¢-(1-phenyl-3-arylpyrazole-4-yl)meth-
ylene aminothioureas (4a-4e, 5a-5e).
there were doublet-peaks of N-H at about d 8.20~8.40;
however, owing to the deshielding effect of the larger con-
jugated system formed by vicinal methyleneamino and cy-
cle-pyrazole, the proton of N¢-H shifted toward a relatively
downfield position of d 9.10~9.30. The proton of NH-CS-
NH shifted toward upfield in comparison with compounds
2a-2b.
Regarding the synthesis of glycosylaminothiourea,
we took anhydrous hydrazine to react with isothiocyanates
in order to avoid the hydrolyzation of isothiocyanates. Be-
cause isothiocyanates also reacted with ethanol, the tem-
perature of the reaction was controlled below 5 °C to pre-
vent a side reaction.
The IR spectra of compounds 4a-4e and 5a-5e ex-
hibited broad bands at 3400-3200 cm-1, assigned to their
NH. The strong bands appearing at 1400-1350 cm-1 were
attributed to character absorption of NH-CS-NH. The
signals at 1745 cm-1 were identified as CO of acetyl in
the sugar ring.
The LC-MS (ESI) displayed that all the compounds
had quasi-molecular ion peaks and they appeared accord-
ing to the base peak, which revealed these target compounds
were comparatively stable. Simultaneously, glycosyl frag-
ment peaks and an isotopic peak cluster were exhibited in
the MS.
In this paper, we attempted to use activated a 4Å mo-
lecular sieve which resulted in shorter reaction time (2-3 h)
and higher yield (75%-85%). A 4Å molecular sieve must
be triturated, and then activated under 500 °C before use.
We show the action of an 4Å molecular sieve in the reaction
of 4a (Table 1).
From the table, we could conclude the optimum con-
ditions of the reaction were using a 1.5 g activated 4Å mo-
lecular sieve, and in this way, the title compounds could be
synthesized within only two hours to achieve high yield.
In the 1H NMR spectra of compounds 2a-2b, 4a-4e,
5a-5e, a few single peaks appearing at about d 2.10 were at-
tributed to hydrogen atoms of acetyl in a sugar ring; how-
ever, multiple peaks appearing at d 3.40~5.50 were attrib-
uted to hydrogen atoms of the sugar ring. In addition, the
signals of the sugar ring C1-H displayed at about d 5.60 and
revealed a triplet-peak for coupling with both C2-H and
N-H and their coupling constants in the range of 8.8~9.6
Hz. To D-xylosyl thiourea derivatives and L-arabinosyl
thiourea derivatives, the configuration of their anomeric
carbon was identical. The proton (C1-H) shifted toward
downfield in contrast with other protons in the sugar ring.
The reason was due to the deshielding effect of the oxygen
of the hemiacetal. In the compounds of 4a-4e and 5a-5e,
Solid-state fluorescence spectra of compounds 4a-4e
and 5a-5e were determined with solid powder on a quartz
round plate with excitation and emission slits of 2.5/2.5 nm
by spectrophotometry. The optimum excitation wavelength
of compounds 4a-4e and 5a-5e was 350 nm. The fluores-
cence spectra of compounds 4 and 5 excited at optimum ex-
citation wavelength of each compound respectively; at the
same time, the emission wavelengths of compounds 4 and 5
were measured by the optimum excitation wavelength. The
emission wavelengths of compounds 4a-4e were 406, 453,
412, 404, 526 nm; meanwhile, the emission wavelengths of
5a-5e were 468, 423, 437, 403, 529 nm. As shown in Fig. 1
and Fig. 2, the fluorescence intensity of compounds 4e and
5e was comparatively weak. The reason was mainly due to
the nitro group in these compounds, which was a strong
electron-withdrawing group and could make fluorescence
highly quenched. Compounds 4a-4d and 5a-5d displayed

Synthesis of Glycosyl Pyrazolylmethylene Aminothiourea
J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 387
relatively good fluorescence activity. Furthermore, we
could find that the fluorescence intensity of xylosyl thio-
urea derivatives was stronger than that of arabinosyl thio-
urea derivatives from Fig. 1 and Fig. 2, which might be re-
lated to the structure of the molecules. In addition, a larger
conjugated system formed by a pyrazole ring and 1,3-phen-
yl resulted in an fluorescence property of the compounds.
So, several compounds of good fluorescence activity could
be utilized as labeled derivatives of sugar analysis.
1. Synthesis of glycosyl isothiocyanates (1a, 1b) ac-
cording to the literature13
1a: yield: 41%, m.p. 88-90 °C, (lit 12: m.p. 91-92 °C);
1b: yield: 55%, syrup.
2. Preparation of N-amino-N¢-glycosylthiourea (2a,
2b)
Anhydrous hydrazine (1.2 mL, 5 mmol) was added to
80 mL of ethanol below 5 °C by using an ice-water bath.
The mixture was stirred. Then a solution of 5 mmol of
glycosylisothiocyanates 1 or 2 in ethanol (30 mL) was
added dropwise with maganetic stirring. The reaction mix-
ture was then continuously stirred below 5 °C for at least 10
min. The precipitate was filtered off and crystallized from
petroleum ether and ethyl acetate.
EXPERIMENTAL
General Method
The 1H NMR spectra were recorded on an Inova-400
(using TMS as internal standard, DMSO-d6 as solvent). MS
were determined with a HP 1100 (LC-MS). The IR spectra
were obtained on a Bruker Equinox 55 FT-IR apparatus by
a pressed KBr pellet. Elemental analyses were performed
on a Thermo Flash EA-1112 analyzer. Melting points were
taken on a Yanaco MP-S3 micromelting point apparatus.
Corrected fluorescence spectra were taken on a Hitachi
F-4500 fluorescence spectrophotometer. All reagents were
analytical grade chemicals, which could not be utilized be-
fore not being disposed, except for special introduction.
The boiling point range of petroleum ether was 60-90 °C.
4Å molecular sieves were activated under high tempera-
ture. The TLC was performed by GF254 and 0.5% CMC.
Detection made use of UV light; the mobile phase was pe-
troleum ether and ethyl acetate (1:2).
2a: yield: 75%. m.p. 170-172 °C. (lit 12: m.p. 170-172
°C);
2b: yield: 73%. m.p. 128-130 °C.
3. Preparation of 1-phenyl-3-aryl-4-formylpyrazole
(3a-3e) in terms of the literature14
4. Preparation of N-glycosyl-N¢-(1-phenyl-3-arylpyra-
zole-4-yl)methylene aminothioureas (4a-4e, 5a-5e)
Method 1: N-amino-N¢-glycosylthioureas (2a, 2b) (4
mmol) and 1-phenyl-3-aryl-4-formylpyrazoles (3a-3e) (4
mmol) were added to THF (30 mL) and refluxed for about 4
h. The end of the reaction was detected by TLC. The reac-
tion mixture was concentrated. The pure production was
obtained by recrystallization from ethyl acetate.
Method 2: N-amino-N¢-glycosylthiourea (2a, 2b) (4
Fig. 1. Fluorescence spectra of compounds 4a-4e.
Fig. 2. Fluorescence spectra of compounds 5a-5e.

388 J. Chin. Chem. Soc., Vol. 55, No. 2, 2008
Zhao and Cao
mmol) and 1-phenyl-3-aryl-4-formylpyrazoles (3a-3e) (4
mmol) was added to THF (30 mL). Simultaneously, 0.5~2
g of activated 4Å molecular sieve was also added. The fol-
lowing procedure was the same as method 1.
All physical data are listed in Table 2. The data of 1H
NMR, IR and MS are listed in Table 3.
Table 2. Physical data and elemental analysis data and MS for compounds
Elemental analysis (Calcd.)/%
Yield
(%)
No.
Molecule
m.p./°C
MS, m/z (%)
C
H
N
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
C28H29N5O7S
C29H31N5O8S
C28H28ClN5O7S
C28H28BrN5O7S
C28H28N6O9S
C28H29N5O7S
C29H31N5O8S
C28H28ClN5O7S
C28H28BrN5O7S
C28H28N6O9S
85
78
88
85
82
80
76
90
88
86
226-227
241-242
181-182
169-170
155-156
159-161
149-150
157-158
150-151
164-165
58.12 (58.02) 5.06 (5.04) 12.11 (12.08) 580 ([M+H]+, 100)
57.24 (57.13) 5.11 (5.13) 11.46 (11.49) 610 ([M+H]+, 100)
54.69 (54.77) 4.59 (4.60) 11.43 (11.40) 614 ([M+H]+, 100)
50.15 (51.07) 4.30 (4.29) 10.66 (10.63) 658 ([M+H]+, 100)
53.75 (53.84) 4.53 (4.52) 13.44 (13.45) 625 ([M+H]+, 100)
58.13 (58.02) 5.03 (5.04) 12.10 (12.08) 580 ([M+H]+, 100)
57.23 (57.13) 5.12 (5.13) 11.47 (11.49) 610 ([M+H]+, 100)
54.67 (54.77) 4.60 (4.60) 11.43 (11.40) 614 ([M+H]+, 100)
51.16 (51.07) 4.28 (4.29) 10.66 (10.63) 658 ([M+H]+, 100)
53.95 (53.84) 4.51 (4.52) 13.49 (13.45) 625 ([M+H]+, 100)
Table 3. 1H NMR, IR, spectra data for new compounds
No.
1H NMR d
IR (KBr) n/cm-1
4a
2.05, 2.07, 2.08 (s, 9H, 3 ´ CH3CO), 3.48-5.42 (m, 5H, sugar ring-H), 5.58 (t, J = 3318, 1738, 1371, 911
8.8 Hz, 1H, C1-H), 7.35-7.86 (m, 10H, ArH), 7.87 (s, 1H, CH=N), 8.24 (d, J = 8.8
Hz, 1H, NH), 8.67 (s, 1H, pyrazole-H), 9.35 (br, 1H, N¢H)
4b
2.01, 2.05, 2.09 (s, 9H, 3 ´ CH3CO), 3.43 (t, J = 10.8 Hz, 1H, C5-H), 3.72 (s, 3H, 3341, 1729, 1355, 915
OCH3), 4.15 (t, J = 10.8 Hz, 1H, C5-H), 5.01-5.41 (m, 3H, sugar ring-H), 5.59 (t,
J = 9.2 Hz, 1H, C1-H), 7.33-7.85 (m, 9H, ArH), 7.88 (s, 1H, CH=N), 8.22 (d, J =
9.2 Hz, 1H, NH), 8.65 (s, 1H, pyrazole-H), 9.38 (br, 1H, N¢H)
4c
4d
4e
5a
5b
5c
5d
5e
2.03, 2.06, 2.11 (s, 9H, 3 ´ CH3CO), 3.51-5.49 (m, 5H, sugar ring-H), 5.58 (t, J = 3294, 1743, 1389, 920
8.8 Hz, 1H, C1-H), 7.40-7.90 (m, 9H, ArH), 7.94 (s, 1H, CH=N), 8.28 (d, J = 8.8
Hz, 1H, NH), 8.70 (s, 1H, pyrazole-H), 9.37 (br, 1H, N¢H)
2.00, 2.07, 2.09 (s, 9H, 3 ´ CH3CO), 3.50-5.48 (m, 5H, sugar ring-H), 5.62 (t, J = 3301, 1728, 1357, 909
9.6 Hz, 1H, C1-H), 7.37-7.88 (m, 9H, ArH), 7.91 (s, 1H, CH=N), 8.25 (d, J = 9.6
Hz, 1H, NH), 8.69 (s, 1H, pyrazole-H), 9.41 (br, 1H, N¢H)
2.01, 2.05, 2.08 (s, 9H, 3 ´ CH3CO), 3.45-5.47 (m, 5H, sugar ring-H), 5.60 (t, J = 3310, 1739, 1376, 917
9.2 Hz, 1H, C1-H), 7.35-7.85 (m, 9H, ArH), 7.88 (s, 1H, CH=N), 8.30 (d, J = 9.2
Hz, 1H, NH), 8.66 (s, 1H, pyrazole-H), 9.39 (br, 1H, N¢H)
1.68, 2.01, 2.18 (s, 9H, 3 ´ CH3CO), 3.80-5.43 (s, 5H, sugar ring-H), 5.51 (t, J = 3318, 1760, 1366, 911
8.8 Hz, 1H, C1-H), 7.39-7.88 (m, 10H, ArH), 7.93 (s, 1H, CH=N), 8.39 (d, J = 8.8
Hz, 1H, NH), 8.70 (s, 1H, pyrazole-H), 9.90 (br, 1H, N¢H)
1.70, 1.89, 2.24 (s, 9H, 3 ´ CH3CO), 3.70 (s, 3H, OCH3), 3.88-5.49 (m, 5H, sugar 3309, 1758, 1387, 907
ring-H), 5.60 (t, J = 9.6 Hz, 1H, C1-H), 7.42-7.92 (m, 9H, ArH), 7.98 (s, 1H,
CH=N), 8.37 (d, J = 9.6 Hz, 1H, NH) 8.75 (s, 1H, pyrazole-H), 9.97 (br, 1H, N¢H)
1.65, 1.95, 2.23 (s, 9H, 3 ´ CH3CO), 3.82-5.48 (m, 5H, sugar ring-H), 5.52 (t, J = 3321, 1767, 1374, 919
9.6 Hz, 1H, C1-H), 7.40-7.89 (m, 9H, ArH), 7.99 (s, 1H, CH=N), 8.40 (d, J = 9.6
Hz, 1H, NH), 8.70 (s, 1H, pyrazole-H), 9.91 (br, 1H, N¢H)
1.67, 1.99, 2.19 (s, 9H, 3 ´ CH3CO), 3.85-5.42 (m, 5H, sugar ring-H), 5.55 (t, J = 3330, 1751, 1370, 917
8.8 Hz, 1H, C1-H), 7.37-7.87 (m, 9H, ArH), 7.95 (s, 1H, CH=N), 8.36 (d, J = 8.8
Hz, 1H, NH), 8.72 (s, 1H, pyrazole-H), 9.93 (br, 1H, N¢H)
1.72, 1.92, 2.20 (s, 9H, 3 ´ CH3CO), 3.80-5.41 (m, 5H, sugar ring-H), 5.58 (t, J = 3319, 1764, 1359, 921
8.8 Hz, 1H, C1-H), 7.39-7.88 (m, 9H, ArH), 7.96 (s, 1H, CH=N), 8.41 (d, J = 8.8
Hz, 1H, NH), 8.71 (s, 1H, pyrazole-H), 9.95 (br, 1H, N¢H)

Synthesis of Glycosyl Pyrazolylmethylene Aminothiourea
J. Chin. Chem. Soc., Vol. 55, No. 2, 2008 389
6. Zhang, Z.-Y.; Chen, L.-M.; Feng, X.-M.; Zeng, F.-L. Chin.
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ACKNOWLEDGEMENT
This work was supported by the National Nature Sci-
ence Foundation of China (Grant No. 20462006).
8. Kees, K. L.; Fitzgerald, J. J. Jr.; Steiner, K. E.; Mattes, J. F.;
Mihan, B.; Tosi, T.; Mondoro, D.; McCaleb, M. L. J. Med.
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Received July 9, 2007.
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