Sugar-derived di- and tetrahydropyridazinones: Synthesis of new glycosidase inhibitors

Article abstract of DOI:10.1002/1522-2675(20000705)83:7<1599::AID-HLCA1599>3.0.CO;2-0

The N-unsubstituted D-arabino-tetrahydropyridazinone 7 is a micromolar inhibitor of β-glucosidases from sweet almonds (competitive), Caldocellum saccharolyticum (mixed), yeast α-glucosidase (competitive), jack bean α- mannosidase (competitive), and snail β-mannosidase (competitive). The N- substituted tetrahydropyridazinones 22, 24, and 26 are weak inhibitors of these glycosidases, and so are the dihydropyridazinones 8 and 17-19, where the best inhibition was observed for 8 (K(i) = 56 μM for jack bean α- mannosidase). The tetrahydropyridazinones were obtained by reduction of the corresponding dihydropyridazinones with NaCNBH₃, and the dihydropyridazinones were prepared by treatment with hydrazine or substituted hydrazines of the known and readily available D-threo-pent-2-uluronate 11.

Full text of DOI:10.1002/1522-2675(20000705)83:7<1599::AID-HLCA1599>3.0.CO;2-0

Helvetica Chimica Acta ± Vol. 83 (2000)
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Sugar-Derived Di- and Tetrahydropyridazinones: Synthesis of New
Glycosidase Inhibitors
by Chepuri Venkata Ramana and Andrea Vasella*
Laboratorium für Organische Chemie, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich
The N-unsubstituted d-arabino-tetrahydropyridazinone 7 is a micromolar inhibitor of b-glucosidases from
sweet almonds (competitive), Caldocellum saccharolyticum (mixed), yeast a-glucosidase (competitive), jack
bean a-mannosidase (competitive), and snail b-mannosidase (competitive). The N-substituted tetrahydropyr-
idazinones 22, 24, and 26 are weak inhibitors of these glycosidases, and so are the dihydropyridazinones 8 and
17 ± 19, where the best inhibition was observed for 8 (Ki ˆ 56 mm for jack bean a-mannosidase). The
tetrahydropyridazinones were obtained by reduction of the corresponding dihydropyridazinones with
NaCNBH3, and the dihydropyridazinones were prepared by treatment with hydrazine or substituted hydrazines
of the known and readily available d-threo-pent-2-uluronate 11.
Introduction. ± The competitive and selective inhibition of retaining b-d-pyrano-
side-cleaving glycosidases by 1,5-glyconolactones such as 1 has been rationalised by
correlating their structure with that of related oxycarbenium cations [1][2]. The
similarity between glyconolactones and oxycarbenium cations has led to the double
hypothesis that oxycarbenium cations, or closely related species, are reactive
intermediates of the enzymic glycoside hydrolysis, and thus similar to the transition
state, and that glycono-1,5-lactones are transition-state analogues. While it has become
clear that taking oxycarbenium cations as lead compounds for the design of transition-
state analogues neglects the important interaction with the catalytic acid [3][4], the
imitation of glycosyl cations has been fruitful in suggesting valuable modifications of
the structure of the archetypical lactone and piperidine-type inhibitors. Thus, Ganem
and co-workers have combined elements of shape (as dominant in the lactone 1) and
charge (as in deoxynojirimycin 2) in their amidinium cations 3 [5][6]. Bols and co-
workers [7] and later Ichikawa et al. [8] have changed the position of the positively
charged N-centre, resulting in the synthesis of isofagomine 4 and the azaglucose 5 [9].
Bols and co-workers have also combined the basic sites of deoxynojirimycin and
isofagomine in the hexahydropyridazine (piperidazine) 6 [10 ± 12], and Zhang et al.
have prepared 1,4,5,6-tetrahydropyridazine derivatives as inhibitors of influenza
neuraminidase [13]. Additional modulations and combinations of shape- and charge-
related structural motifs are of interest to learn more about the details of the reaction
mechanism of glycosidases.
We wondered about the inhibitory power, selectivity, and pH dependence of
tetrahydro- and dihydropyridazinones of type 7 and 8. These compounds possess two
structurally differentiated inhibitory motifs, viz. a ring N-atom corresponding to the
basic site of nojirimycin, and a lactam group. The lactam CˆO group may function as
H-bond acceptor for the catalytic acid of b- and possibly also of a-glycosidases [14],

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Helvetica Chimica Acta ± Vol. 83 (2000)
while the ring N-atom of 7 corresponding to that of nojirimycin might be protonated
at sufficiently low pH values and lead to a charge/charge interaction with
the catalytic base. Thus, the inhibition of b-glycosidases might give information about
the location of the catalytic acid ± since the CˆO group is shifted from
its position in 1 ± and provide a pH-modulated selectivity of the inhibition of b- and
a-glycosidases.
An established method for the construction of tetrahydropyridazinone derivatives
involves the reduction by catalytic hydrogenation [15][16] or by hydride reagents [17 ±
19] of dihydropyridazinones1). Dihydropyridazinones are accessible by several
methods ([22] and references cited therein), and particularly from g-oxo acids or g-
oxo esters [15 ± 19][23], and hydrazine or substituted hydrazines. The known methyl
1,3,4-tri-O-acetyl-d-threo-pent-2-uluronate (11; Scheme), readily available from 3,4,6-
tri-O-acetyl-d-glucal (10) [24], appeared to be an ideal starting compound for our
needs. Condensation of the oxo ester 11 with hydrazine and mono-substituted
hydrazines, followed by deacetylation should lead to dihydropyridazinones and, hence,
also to the desired tetrahydropyridazinones.
Results and Discussion. ± Oxidative cleavage of triacetylglucal 10 with RuO2/NaIO4
in CCl4/H2O [24], followed by esterification with Me3SiCHN2 in MeOH/PhH [25]
yielded 63% of the oxo ester 11 on a 60-mmol scale. This ester was treated with a slight
excess of hydrazine monohydrate in MeOH [26] to afford the crystalline dihydropyr-
idazinone 12 in a yield of 75% after chromatography. This dihydropyridazinone was
completely deacetylated with aq. NH3 in MeOH, to afford the triol 8 (80%). In a
similar fashion, 11 was treated with methyl-, (2-hydroxyethyl)-, or (2-phenethyl)hy-
drazine [27]. Methylhydrazine yielded predominantly the diacetyl N-methyldihydro-
1
)
Meng and Hesse have reported the synthesis of the protected tetrahydropyridazinone 9 [20]. This derivative
has not been deprotected, and the implication that deprotection may not be trivial has been confirmed by
exploratory experiments with a related derivative [21].

Helvetica Chimica Acta ± Vol. 83 (2000)
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Scheme
a) RuO2 ´ x H2O, NaIO4, CCl4/H2O, then Me3SiCHN2, MeOH/PhH; 63%. b) i) Hydrazine monohydrate,
MeOH; 75% (12). ii) Methylhydrazine, EtOH; 82% (13/14, 40 :1). iii) (2-Hydroxyethyl)hydrazine, EtOH;
77% (15). iv) (2-Phenethyl)hydrazine, EtOH; 65% (16). c) Aq. NH3, MeOH; 80% (8); 90% (17); 85% (18);
70% (19 from 10, with(2-phenethyl)hydrazine, EtOH and then aq. NH3). d) NaCNBH3, MeOH, AcOH; 58%
(7/21 2 :3); 75% (22/23 1:1); 87% (24/25 1:1); 89% (26/27 3 :2). e) 5% Pd/C, 5 bar of H2, MeOH/AcOH, then
aq. NH3, MeOH; 63%.
pyridazinone 13 (80%) besides a small amount of the triacetate 14 (2%). Deacetylation
of 13 provided the N-methyldihydropyridazinone 17 in 90% yield. The deprotected N-
(2-hydroxyethyl)dihydropyridazinone 18 was obtained in an overall yield of 65% from
11. (2-Phenethyl)hydrazine reacted only slowly with 11. Purification of the resulting
diacetyl N-(2-phenethyl)dihydropyridazinone 16 (65%) proved cumbersome, while the
deacetylated dihydropyridazinone 19 (70%) was readily purified.
Hydrogenation of the diacetyl N-methyldihydropyridazinone 13 in the presence of
5% Pd/C proceeded with reductive cleavage of the allylic CÀO bond [28][29], and
deacetylation of the reduction product yielded 20 (63%). Hydrogenation of 13 in the
presence of PtO2/Al2O3 [19] provided a complex mixture, while reduction of the
deprotected dihydropyridazinones 8 and 17 ± 19 with NaCNBH3/MeOH [18] in the
presence of AcOH proceeded smoothly to yield a 3 :2 to 1:1 mixture of the
tetrahydropyridazinones 7, 22, 24, and 26 and their respective diastereoisomers 21, 23,
25, and 27.

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The dihydropyridazinone 12 is characterised by a singlet (CˆN) at 154.45 and another singlet at 167.12
(NÀCˆO) ppm in the 13C-NMR spectrum. HÀC(2) resonated at much higher field than to HÀC(3). J(2,3) of
12.5 Hz indicates a diaxial relation. The position of the free OH group is evidenced by the chemical shift of
HÀC(3) and a long-range coupling between HÀC(3) and both HÀC(5) and H'ÀC(5) of 0.6and 0.9 Hz,
respectively, evidences the C(3)ÀOH group. The structure of 12 and of 17 was confirmed by crystal structure
analysis2) (Fig. 1); structural parameters agree with those of known dihydropyridazinone derivatives [30]. Two
molecules of 17 are connected by H2O, resulting in a 1:2 ratio for 17/H2O in the unit cell. The NMR spectra of
the dihydropyridazinone 20 are characterised by a quadruplet for (C(5)) at 18.54 and another quadruplet (NMe)
at 34.91 ppm, a C(4) singlet at 157.16, a C(1) singlet at 167.11 ppm, and a br. singlet (1.94 ppm) for CH3(5) with a
weak long-range coupling to HÀC(3), resonating as a broad dd (4.04 ppm).
Fig. 1. ORTEP Representation of 12 and 17 ´ 2 H2O
The assignment of the d-arabino configuration to 7, 22, 24, and 26, and of the l-xylo-
configuration to 21, 23, 25, and 27 is based on NOE experiments. NOEꢀs of 4.5 to 7.1%
were observed for HÀC(2) and HÀC(4) of 22. No analogous NOE was found for 23,
evidencing that HÀC(2) and HÀC(4) are cis in 22, and trans in 23. The d-arabino-
configuration of 7, 24, and 26 is indicated by the close similarity of their NMR spectra
with the one of 22, whereas the spectra of 21, 25, and 27 resemble that of 23. For 7, 22,
24, and 26, the large J(2,3) value of 9.7 ± 9.0 Hz and the medium J(3,4) value of 6.2 ±
N
5.4 Hz evidence a S2 conformation of the d-arabino-tetrahydropyridazinones. This is
corroborated by Amber* force-field calculations (Macromodel V.6, gas phase) leading
N
to a calculated J(2,3) value of 9.5 and a calculated J(3,4) value of 6.5 Hz for the S2
conformation that is by ca. 2 kcal/mol more stable than the corresponding 3H4
conformer. The epimers 20, 22, 24, and 26 show medium J(2,3) and J(3,4) values of
6.2 ± 5.3 and 5.3 ± 3.9 Hz, respectively, in agreement with a 1SN conformation (calculated
J(2,3) ˆ 6.3 and J(3,4) ˆ 6.0 Hz).
Inhibition Studies. ± The dihydropyridazinones 8, 17, 18, and 19, and the d-arabino-
tetrahydropyridazinones 7, 22, 24, and 26 were tested against a-glucosidase from
brewerꢀs yeast, b-glucosidases from sweet almonds and Caldocellum saccharolyticum,
a-mannosidase from jack beans, and b-mannosidase from snails. Among the four
2
)
The crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as
deposition No. CCDC-142596( 12) and CCDC-142597 (17). Copies of the data can be obtained, free of
charge, on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: ‡44(1223)336033; e-
mail: deposit@ccdc.cam.ac.uk).

Helvetica Chimica Acta ± Vol. 83 (2000)
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dihydropyridazinones 8, 17, 18, and 19, only 8 proved to be a (weak) inhibitor of the b-
glucosidases from sweet almonds (IC50 0.9 mm) and C. saccharolyticum (IC50 0.6m m),
a-mannosidase from jack beans (IC50 142 mm, Ki 46 mm, competitive inhibition), and b-
mannosidase from snails (IC50 4.6m m). A weak inhibition of yeast a-glucosidase was
observed with 19 (IC50 6.5 mm). Among the tetrahydropyridazinones, only the N-
unsubstituted 7 is a good inhibitor of b-glucosidases from almonds (IC50 21 mm, Ki
13 mm, competitive inhibition) and from C. saccharolyticum (IC50 9 mm, Ki 9 mm, mixed
inhibition), but not of yeast a-glucosidase (IC50 4.0 mm). Surprisingly, 7 is also a good
inhibitor of jack bean a-mannosidase (IC50 47 mm, Ki 25 mm, competitive inhibition) and
snail b-mannosidase (IC50 10 mm, Ki 6 mm, competitive inhibition). Among the N-
substituted tetrahydropyridazinones, only 26 was a weak inhibitor (IC50 3.5 mm) of
yeast a-glucosidase. Also, the N-substituted tetrahydropyridazinones 22 (IC50 2.8 mm
for jack bean a-mannosidase), 24, and 26 are poor inhibitors of mannosidases.
The effect of pH on the inhibition by 7 of b-glucosidases from sweet almonds
(pH 4.2 ± 7.4, pH optimum 5.6[31]) and C. saccharolyticum (pH 4.2 ± 7.4, pH optimum
6.2 [32]), a-mannosidase from jack beans (pH 4.0 ± 6.2, pH optimum 4.0 ± 5.0
[33][34]), and b-mannosidase from snails (pH 4.0 ± 5.6, pH optimum 4.5 [35]) was
studied. The pH dependence of the inhibition is represented by the IC50 vs. pH plot in
Fig. 2. Except for the b-glucosidases from sweet almonds, the pH dependence of the
inhibition of 7 is in accordance with the pH dependence of the enzymic activity. The
optimum inhibition of b-glucosidases from sweet almonds at pH 4.2 may not be
informative, as it is a mixture of glycosidases. The results indicate that 7 is not fully
protonated over the pH range examined. Related acylhydrazines possess pKHA values
between 1.5 and 3.4 [36]. Attempts to determine the pKHA value of 7 by titration
showed no inflection of titration curve above pH 3, where the determination of pH is
rather inaccurate. The expected interaction of a-glucosidases with protonated 7 could
not be tested, as the enzyme is almost inactive below pH 4.5, and the pH dependence of
the inhibition shows that realisation of the concept indicated in the Introduction
requires a more strongly basic center corresponding to OÀC(5).
Fig. 2. pHDependence of the inhibition of glycosidases by the tetrahydropyridazinone 7

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The stronger inhibition of b-glucosidases than of yeast a-glucosidase by 7 is in
keeping with the hypothesis of a (bidentate?) interaction between the catalytic acid
and the lactam moiety of 7. This interaction may also explain the inhibition of b-
mannosidases, and denote a flexibility of the catalytic acid, as it may be required in
conjunction with the postulated conformational change of the aglycon (À1) moiety
during enzyme hydrolysis [37][38]. However, the requirement for an unsubstituted
lactam NÀH group, suggesting a H-bond to a suitable acceptor, does not allow us to
exclude a possible cooperative interaction with the catalytic nucleophile. The inhibition
of a-mannosidase from jack beans by 7 and 8 is suprising, considering that only a
moderate inhibition of this enzyme has been observed for deoxynojirimycin 2 [7] and
deoxymannojirimycin [7], isofagomine 4 [7][8], and azafagomine 6 [10], and
considering that 7 is almost inactive against yeast a-glucosidase.
We thank Dr. B. Bernet for valuable analytical contributions, Dr. B. Schweizer for the crystal-structure
determinations, K. Piens (University of Gent, Belgium) and M. Böhm for the enzymic tests, T. Mäder for HPLC
purifications, B. Brandenberg for NOE experiments, and the Swiss National Science Foundation and F.
Hoffmann La Roche AG, Basel, for generous support.
Experimental Part
General. Solvents were distilled before use. TLC: Merck silica gel 60F-254 plates. HPTLC: Macherey-Nagel
Nano-Sil NH2/UV254 plates; detection by heating with mostain (400 ml of 10% H2SO4 soln., 20 g of
(NH4)6Mo7O24 ´ 6 HO, 0.4 g of Ce(SO4)2). Flash chromatography (FC): silica gel Fluka 60 (0.04 ± 0.063 mm).
2
HPLC: Merck LiChrosorb RP-18 (7 mm) 250 Â 25 nm. M.p. uncorrected. Optical rotations: 1-dm cell. UV
Spectra (lmax in nm (log e)). IR spectra: KBr, neat or 2% CHCl3 soln. 1H- (300 MHz) and 13C-NMR (75 MHz):
chemical shifts d in ppm and coupling constants J in Hz. CI- and FAB-MS: 3-Nitrobenzyl alcohol and NH3 as
matrix. a-Glucosidase (maltase, 3.2.1.20, Type VI, G-4634, as lyophilised powder) from brewerꢀs yeast, b-
glucosidase (3.2.1.21, as lyophilised powder) from C. saccharolyticum, a-mannosidase (3.2.1.24, as suspension in
3.0m (NH4)2SO4 and 0.1 mm ZnCl2, pH 7.5) from jack beans, b-mannosidase (3.2.1.24, as suspension in 3.0m
(NH4)2SO4 and 10 mm AcONa, pH around 4.0) from snails and all nitrophenyl glycosides were purchased from
Sigma and used without any further purification. b-Glucosidase (3.2.1.21, as lyophilised powder) from sweet
almonds was purchased from Fluka and used without any further purification.
Methyl 1,3,4-Tri-O-acetyl-d-threo-pent-2-uluronate (11) [24]. At r.t., a vigorously stirred biphasic mixture of
CCl4/H2O 1:1 (1.2 l) was treated with 10 (16.2 g, 60 mmol), sodium (meta)periodate (78.12 g, 365 mmol), and
ruthenium(IV) dioxide ´ hydrate (240 mg, 60% Ru, 2.4 mol-%), whereupon the mixture became bright yellow.
After stirring for 2 d, the dark purple CCl4 layer was separated and discarded. The black aq. layer was diluted
with H2O (300 ml) and extracted with AcOEt (3 Â 400 ml). The combined org. extracts were washed with brine,
treated with charcoal and anh. Na2SO4, and filtered through Celite (washed previously with AcOEt). After
evaporation, the crude oxo acid (colourless oil, 14.0 g) was dissolved in MeOH/C6H6 1:3 (400 ml), cooled in an
ice-water bath, treated with a 2m soln. of Me3SiCHN2 in hexane (ca. 37 ml) until persistence of the yellow colour,
and stirred for 15 min. Evaporation and FC (AcOEt/hexane 1:4) afforded 11 as pale yellow oil (13 g).
Crystallization from Et2O/hexane gave 11 as colourless needles (11.6g, 63%). Rf(AcOEt/hexane 3 :1) 0.83. M.p.
61 ± 628 (Et2O/hexane) ([24]: 57 ± 588). [a]D25 ˆ À17.2 (c ˆ 1, CHCl3) ([24]: [a]D17 ˆ À10.2 (c ˆ 6, CHCl3)). IR
(KBr): 2940m, 1767s, 1750s, 1445m, 1430m, 1404m, 1379s, 1257m, 1224s, 1200s, 1162m, 1123m, 1075s, 1011m,
957m, 939m, 901w, 871w, 794w. 1H-NMR (CDCl3): 2.16, 2.18, 2.20 (3s, 3 AcO); 3.77 (s, MeO); 4.75 (d, J ˆ 17.4,
HÀC(1)); 4.94 (d, J ˆ 17.4, H'ÀC(1)); 5.62, 5.77 (2d, J ˆ 2.5, HÀC(3), HÀC(4)). 13C-NMR (CDCl3): 20.36
(q, Me); 20.39 (q, 2 Me); 53.13 (q, MeO); 66.71 (t, C(1)); 70.67, 75.43 (2d, C(3), C(4)); 166.94 (s, CˆO); 169.90
‡
(s, 2 CˆO); 170.13 (s, CˆO); 197.50 (s, C(2)). CI-MS: 305 (30, [M ‡ H] ), 292 (5), 275 (21), 262 (18), 245 (59),
203 (89), 43 (100). Anal. calc. for C12H16O9 (304.25): C 47.37, H 5.30; found: C 47.52, H 5.53.
2,5-Di-O-acetyl-11,41-anhydro-4-deoxy-4-(hydroxyimino)-d-threo-pentonamide (12). At 08, a soln. of 11
(1.218 g, 4 mmol) in MeOH (80 ml) was treated with a soln. of 1m hydrazine monohydrate in EtOH (5.6ml,
5.6mmol), and stirred at 0 8 for 2 h, and then at 238 for 8 h. Evaporation and FC (AcOEt/hexane 1:1) gave 12
(710 mg, 75%). Colourless solid. Rf(AcOEt/hexane 3 :1) 0.47. M.p. 160 ± 1618 (MeOH/CHCl3). UV (MeOH):

Helvetica Chimica Acta ± Vol. 83 (2000)
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253 (3.8). [a]2D5 ˆ À282 (c ˆ 1, MeOH). IR (KBr). 3313s, 3253s, 2988s, 1747s, 1704s, 1646m, 1439m, 1398s,
1366m, 1331s, 1150s, 1255m, 1199m, 1119m, 1100m, 1066m, 1053s, 1017w, 926m, 904w, 831w. 1H-NMR
(CD3OD): 2.07, 2.18 (2s, 2 AcO); 4.37 (br. d, J ˆ 12.5, HÀC(3)); 4.76( dd, J ꢀ 14, 0.6, HÀC(5)); 4.96( dd, J ˆ
13.7, 0.9, H'ÀC(5)); 5.44 (d, J ˆ 12.5, HÀC(2)). 13C-NMR (CD3OD): 20.50 (q, 2 Me); 63.35 (t, C(5)); 68.02,
71.63 (2d, C(2), C(3)); 154.45 (s, C(4)); 167.12 (s, C(1)); 171.87, 172.37 (2s, 2 CˆO). ESI-MS: 511 (45, [2M ‡
‡
‡
‡
‡
Na] ), 299 (45), 267 (65, [M ‡ Na] ), 262 (100, [M ‡ NH4] ), 245 (8, [M ‡ H] ). Anal. calc. for C9H12N2O6 ´
0.25 H2O (248.71): C 43.46, H 5.07, N 11.26; found: C 43.43, H 4.95, N 11.11.
X-Ray Crystal-Structure Analysis of 12. Monoclinic P21; a ˆ 6.0370(4) Š, b ˆ 10.059(3) Š, c ˆ 9.7125(7) Š;
a ˆ 908, b ˆ 101.783(5)8, g ˆ 908. Vˆ 577.4(2) Š3, Z ˆ 2; Dcalc ˆ 1.405 Mg/m3. From a crystal of size 0.30  0.10 Â
0.10 mm, 1067 reflexions were measured on an Enraf-Nonius CAD-4 diffractometer [39], with CuKa radiation
(graphite monochromator, l ˆ 1.54184 Š) at 293 K. R ˆ 0.0462, Rw ˆ 0.1344. The structure was solved by direct
methods with SHELXS-96[40]. The non-H-atoms were refined anisotropically with SHELXL-97 [41]. H-
Atoms at O(13) and N(2) were obtained from a difference Fourier map and refined with isotropic displacement
parameters. The rest of the H-atom positions were calculated and included in the structure factor calculation.
Drawings of the molecule were done with PLUTO [42] and ORTEP [43].
Treatment of 11 with Methylhydrazine. At 08, a soln. of 11 (415 mg, 1.36mmol) in EtOH (28 ml) was treated
with 1m methylhydrazine in EtOH (1.9 ml, 1.9 mmol) and stirred at 08 for 2 h and at 238 for 2 h. Evaporation and
FC (AcOEt/hexane 1:3 ! 1:1) gave 13 (283 mg, 80%) and 14 (7 mg, 2%).
2,5-Di-O-acetyl-11,41-anhydro-4-deoxy-4-(hydroxyimino)-1-N-methyl-d-threo-pentonamide (13). Rf(AcOEt/
hexane 3 :1) 0.47. M.p. 123 ± 1248 (CHCl3/hexane). UV (CHCl3): 264 (3.8). [a]2D5 ˆ À415.4 (c ˆ 1, CHCl3). IR
(CHCl3): 3399w, 3038w, 2947w, 1749s, 1693s, 1602w, 1426m, 1378s, 1341w, 1248s, 1123w, 1064s, 1032m, 970w,
930w, 825w. 1H-NMR (CDCl3): 2.13, 2.23 (2s, 2 AcO); 3.34 (s, MeN); 3.50 (d, J ˆ 5.9, exchanged with D2O,
HOÀC(3)); 4.68 (br. dd, J ꢀ 12.9, 5.8, irrad. at 3.50 ! br. d, J ꢀ 12.8, HÀC(3)); 4.75 (dd, J ˆ 13.1, 0.6, irrad. at
4.68 ! d, J ˆ 13.1, HÀC(5)); 5.0 (dd, J ˆ 13.1, 0.9, irrad. at 4.68 ! d, J ˆ 13.1, H'ÀC(5)); 5.45 (d, J ˆ 12.8,
HÀC(2)). 13C-NMR (CHCl3): 20.66, 20.72 (2q, 2 Me); 36.59 (q, MeN); 62.18 (t, C(5)); 67.62, 70.24 (2d, C(2),
‡
C(3)); 151.87 (s, C(4)); 163.23 (s, C(1)); 170.71, 171.50 (2s, 2 CˆO). CI-MS: 259 (24, [M ‡ H] ), 241 (24), 216
(26), 199 (43), 156 (100). Anal. calc. for C10H14N2O6 (258.23): C 46.51, H 5.46, N 10.85; found: C 46.49, H 5.44,
N 10.78.
2,3,5-Tri-O-acetyl-11,41-anhydro-4-deoxy-4-(hydroxyimino)-1-N-methyl-d-threo-pentonamide (14). Colour-
less oil. Rf(AcOEt/hexane 1 :1) 0.53. 1H-NMR (CDCl3): 2.06, 2.11, 2.16 (3s, 3 AcO); 3.36( s, MeN); 4.62
(d, J ˆ 13.1, HÀC(5)); 4.92 (dd, J ˆ 13.1, 0.9, H'ÀC(5)); 5.51 (br. d, J ꢀ 12.1, HÀC(3)); 6.00 (d, J ˆ 12.4,
HÀC(2)). 13C-NMR (CHCl3): 20.49 (q, Me); 20.53 (q, 2 Me); 36.76 (q, MeN); 62.39 (t, C(5)); 66.93, 68.15 (2d,
C(2), C(3)); 147.42 (s, C(4)); 161.83 (s, C(1)); 169.44, 170.08, 170.36 (3s, 3 CˆO). FAB-MS: 601 (20, [2M ‡
‡
‡
H] ), 447 (78), 301 (100, [M ‡ H] ), 199 (100).
2,5-Di-O-acetyl-11,41-anhydro-4-deoxy-1-N-(2-hydroxyethyl)-4-(hydroxyimino)-d-threo-pentonamide (15).
A soln. of 11 (380 mg, 1.25 mmol) in EtOH (25 ml) was cooled to 08, treated with a 1m soln. of (2-
hydroxyethyl)hydrazine in EtOH (1.75 ml, 1.75 mmol), and stirred at 08 for 2 h and at 238 for 2 h. Evaporation
and FC (AcOEt/hexane 7:3) gave 15 (278 mg, 77%). Colourless solid. Rf(AcOEt/hexane 3 :1) 0.12. M.p. 99 ±
99.58 (CHCl3/hexane). UV (CHCl3): 263 (3.75). [a]2D5 ˆ À326.4 (c ˆ 1, CHCl3). IR (CHCl3): 3467m, 3038w,
2945w, 1749s, 1691s, 1427m, 1382s, 1342w, 1248s, 1144w, 1112w, 1066s, 979w, 930w, 86 0w. 1H-NMR (CDCl3): 2.13,
2.23 (2s, 2 AcO); 2.55 (br. s, exchanged with D2O, CH2OH); 3.70 (d, J ˆ 5.3, exchanged with D2O, HOÀC(3));
3.82 ± 3.87 (m, 2 H, irrad. at 2.55 ! change, CH2OH); 3.90 ± 3.94 (m, CH2N); 4.71 (br. dd, J ꢀ 12.9, 4.2, irrad. at
3.70 ! d, J ˆ 12.8, HÀC(3)); 4.81 (dd, J ˆ 13.7, 0.9, irrad. at 4.71 ! d, J ꢀ 13.7, HÀC(5)); 5.01 (dd, J ˆ 13.7, 0.9,
irrad. at 4.71 ! d, J ˆ 13.7, H'ÀC(5)); 5.47 (d, J ˆ 12.8, HÀC(2)). 13C-NMR (CHCl3): 20.71, 20.75 (2q, 2 Me);
50.90 (t, CH2N); 60.53 (t, CH2OH); 62.25 (t, C(5)); 67.54, 70.27 (2d, C(2), C(3)); 152.68 (s, C(4)); 164.09
‡
‡
(s, C(1)); 170.83, 171.56(2 s, 2 CˆO). FAB-MS: 577 (10, [2M ‡ H] ), 289 (100, [M ‡ H] ), 229 (60). Anal. calc.
for C11H16N2O7 (288.25): C 45.83, H 5.59, N 9.72; found: C 45.66, H 5.44, N 9.63.
2,5-Di-O-acetyl-11,41-anhydro-4-deoxy-4-(hydroxyimino)-1-N-(2-phenethyl)-d-threo-pentonamide (16). A
soln. of 11 (566 mg, 1.86 mmol) in MeOH was cooled to À108, treated with a 1m soln. of (2-phenethyl)hydrazine
(2.6ml, 2.6mmol), and stirred at À108 for 2 h and at 238 for 12 h. Evaporation and FC (AcOEt/hexane 1:3)
gave 16 (414 mg, 64%). Colourless solid. Rf(AcOEt/hexane 3 :1) 0.70. M.p. 104 ± 1058 (CH2Cl2/hexane). UV
(CHCl3): 265 (3.8). [a]2D5 ˆ À335.4 (c ˆ 1, CHCl3). IR (CHCl3): 3399w, 3038w, 2947w, 1749s, 1693s, 1602w,
1
1497w, 1453w, 1433w, 1381m, 1248m, 1146w, 1102m, 1075w, 1031w, 971w, 930w, 826w. H-NMR (CHCl3): 2.14,
2.20 (2s, 2 AcO); 2.93 (br. t, J ꢀ 7.6, irrad. at 3.92 ! s, PhCH2); 3.33 (d, J ˆ 5.6, exchanged with D2O, HOÀC(3));
3.92 (dt, J ꢀ 13.6, 7.5, irrad. at 2.93 ! d, J ˆ 13.1, CHN); 4.02 (dt, J ꢀ 13.6, 7.7, irrad. at 2.93 ! d, J ˆ 13.4, CH'N);
4.58 (br. dd, J ˆ 12.8, 5.6, irrad. at 5.40 ! br. d, J ˆ 4.4, irrad. at 3.33 ! d, J ˆ 12.8, HÀC(3)); 4.72 (dd, J ꢀ 13.0,

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Helvetica Chimica Acta ± Vol. 83 (2000)
0.6, irrad. at 4.58 ! d, J ˆ 12.8, HÀC(5)); 4.98 (dd, J ꢀ 12.9, 0.8 irrad. at 4.58 ! d, J ˆ 12.8, H'ÀC(5)); 5.40
(d, J ˆ 12.8, HÀC(2)); 7.18 ± 7.32 (m, 5 arom. H). 13C-NMR (CHCl3): 20.74, 20.82 (2q, 2 Me); 34.09 (t, PhCH2);
49.95 (t, CH2N); 62.25 (t, C(5)); 67.78, 70.21 (2d, C(2), C(3)); 126.80 (d); 128.71 (d, 2 C); 129.16( d, 2 C); 138.37
‡
(s); 151.52 (s, C(4)); 162.76 (s, C(1)); 171.61, 171.66 (2s, 2 CˆO). ESI-MS: 719 (81, [2M ‡ Na] ), 387 (40), 371
‡
(100, [M ‡ Na] ). Anal. calc. for C17H20N2O6 (348.35): C 58.61, H 5.79, N 8.04; found: C 58.58, H 5.75, N 8.18.
11,41-Anhydro-4-deoxy-4-(hydroxyimino)-d-threo-pentonamide (8). A soln. of 12 (210 mg, 0.86mmol) in
MeOH (15 ml) was cooled to À208, treated with aq. NH3 (1 ml), kept at À208 for 12 h, warmed to r.t., and
evaporated. The residue was dissolved in H2O (2 ml). Lyophilisation gave 8 (120 mg, 80%). Colourless solid.
M.p. 163 ± 1648 (dec., MeOH/THF). UV (H2O): 286(2.5). [ a]2D5 ˆ À349 (c ˆ 1.5, H2O). IR (KBr): 3385s, 2886w,
1686s, 1653m, 1474w, 1437w, 1329w, 1281w, 1140m, 1097m, 1074m, 1038m, 96 9w, 850w. 1H-NMR ((D6)DMSO):
3.82 (dd, J ˆ 9.6, 5.0, irrad. at 5.70 ! d, J ˆ 9.6, HÀC(3)); 4.01 (dd, J ˆ 14.6, 5.6, irrad. at 4.76 ! d, J ˆ 14.0,
HÀC(5)); 4.12 (dd, J ˆ 9.9, 5.9, irrad. at 5.70 ! change, HÀC(2); 4.18 (dd, J ˆ 14.0, 6.5, irrad. at 4.76 ! d, J ꢀ
14.0, H'ÀC(5)); 4.76( dd, J ˆ 6.6, 5.4, exchanged with D2O, HOÀC(5)); 5.70 (d, J ꢀ 5.5, exchanged with D2O,
HOÀC(2), HOÀC(3)); 10.62 (br. s, HÀN). 13C-NMR ((D6)DMSO): 61.11 (t, C(5)); 67.91, 69.26, (2d, C(2),
‡
‡
C(3)); 156.83 (s, C(4)); 168.95 (s, C(1)). ESI-MS: 319 (84, [2M À H] ), 249 (25), 219 (65), 159 (100, [M À H] ).
Anal. calc. for C5H8N2O4 (160.13): C 37.50, H 5.04, N 17.49; found: C 37.84, H 5.07, N 17.35.
11,41-Anhydro-4-deoxy-4-(hydroxyimino)-1-N-methyl-d-threo-pentonamide (17). A soln. of 13 (270 mg,
1.05 mmol) in MeOH (15 ml) was cooled to À208, treated with aq. NH3 (3 ml), kept at À208, stirred for 12 h,
warmed to r.t., and evaporated. The residue was dissolved in H2O (4 ml). Lyophilisation gave 17 (158 mg, 90%).
Colourless solid. M.p. 95 ± 968 (MeOH). UV (H2O): 288 (3.03). [a]2D5 ˆ À341.9 (c ˆ 1, H2O). IR (KBr): 3287s,
2972m, 2879m, 1684s, 1471m, 1456m, 1415w, 1384w, 1350s, 1308m, 1264w, 1152s, 1125m, 1072s, 1034m, 86 2m.
1H-NMR ((D6)DMSO): 3.16( s, MeN); 3.85 (br. dd, J ꢀ 9.2, 3.6, irrad. at 5.77 ! br. d, J ˆ 9.3, HÀC(3)); 4.04
(dd, J ˆ 14.6, 3.4, irrad. at 4.86 ! d, J ˆ 14.6, HÀC(5)); 4.14 (dd, J ꢀ 9.2, 3.7, irrad. at 5.77 ! d, J ˆ 9.0, HÀC(2));
4.20 (dd, J ˆ 14.6, 3.6, irrad. at 4.90 ! d, J ˆ 14.6, H'ÀC(5)); 4.86( t, J ꢀ 5.7, exchanged with D2O, HOÀC(5));
5.77 (br. s, 2 H, exchanged with D2O, HOÀC(2), HOÀC(3)). 13C-NMR ((D6)DMSO): 35.20 (q, MeN); 60.40
‡
(t, C(5)); 67.01, 68.64 (2d, C(2), C(3)); 156.81 (s, C(4)); 168.61 (s, C(1)). CI-MS: 175 (100, [M ‡ H] ), 157 (51),
145 (39), 141 (41). Anal. calc. for C6H10N2O4 ´ 2 H2O (208.43): C 34.29, H 6.71, N 13.33; found: C 34.76, H 6.45,
N 13.22.
X-Ray Crystal-Structure Analysis of 17 ´ 2 H2O. Monoclinic P21: a ˆ 4.7022(14) Š, b ˆ 12.026(7) Š, c ˆ
8.286(2) Š; a ˆ 90.00(4)8, b ˆ 103.63(2)8, g ˆ 90.00(4)8. Vˆ 455.4(3) Š3; Z ˆ 2; Dcalc ˆ 1.533 Mg/m3. R ˆ 0.0298,
Rw ˆ 0.0777. From a crystal of size 0.2  0.15  0.05 mm 2709 reflexions were measured on an Enraf Nonius
CAD-4 diffractometer [39] with MoKa radiation (graphite monochromator, l ˆ 0.71073 Š) at 220(2) K. The
structure was solved by direct methods with SIR 97 [44]. The non-H-atoms were refined anisotropically with
SHELXL-97 [40]. H-Atoms were obtained from a difference Fourier map and refined isotropically. Drawings of
the molecule were done with PLUTO [42], ORTEP [43].
11,41-Anhydro-4-deoxy-1-N-(2-hydroxyethyl)-4-(hydroxyimino)-d-threo-pentonamide (18). A soln. of 15
(190 mg, 0.66 mmol) in MeOH (10 ml) at À208 was treated with aq. NH3 (3 ml). The mixture was kept at À208
for 12 h, warmed to r.t., and evaporated. The residue was dissolved in H2O (2 ml). Lyophilisation gave 18
(115 mg, 85%). Colourless solid. M.p. 132 ± 1348 (MeOH). UV (H2O): 287 (2.96). [a]2D5 ˆ À363.3 (c ˆ 1, H2O).
IR (KBr): 3442s, 2986m, 2881m, 1631s (sh); 1481w, 1450s, 1420m, 1372m, 1330s, 1316m, 1287m, 1257m, 1124s,
1092s, 1062s, 1038m, 994w, 96 0w, 940w, 86 2w, 842m. 1H-NMR ((D6)DMSO): 3.45 ± 3.52 (m, irrad. at 4.6 !
change, CH2OH); 3.62 ± 3.66 (m, irrad. at 3.48 ! change, NCH2); 3.87 (dd, J ˆ 10.0, 4.7, irrad. at 5.72 ! d, J ˆ
10.0, HÀC(3)); 4.06( dd, J ˆ 14.6, 5.3, irrad. at 4.81 ! d, J ˆ 14.3, HÀC(5)); 4.17 (dd, J ꢀ 9.8, 5.8, irrad. at
5.77 ! d, J ˆ 10.0, HÀC(2)); 4.21 (dd, J ꢀ 14.5, 6.1, irrad. at 4.81 ! d, J ˆ 14.3, H'ÀC(5)); 4.60 (t, J ꢀ 5.8, irrad.
at 3.48 ! s, exchanged with D2O, CH2OH); 4.81 (t, J ꢀ 5.9, exchanged with D2O, HOÀC(5)); 5.72 (d, J ˆ 4.7,
exchanged with D2O, HOÀC(3)); 5.77 (d, J ˆ 5.9, exchanged with D2O, HOÀC(2)). 13C-NMR ((D6)DMSO):
49.19 (t, NCH2); 57.52 (t, CH2OH); 60.30 (t, C(5)); 67.16, 68.73 (2d, C(2), C(3)); 156.93 (s, C(4)); 166.85
‡
(s, C(1)). CI-MS: 205 (100, [M ‡ H] ), 187 (66), 175 (29), 169 (25), 161 (49). Anal. calc. for C7H12N2O5 (204.18):
C 41.18, H 5.92, N 13.72; found: C 41.26, H 5.82, N 13.57.
11,41-Anhydro-4-deoxy-4-(hydroxyimino)-1-N-(2-phenethyl)-d-threo-pentonamide (19).
A soln. of 11
(1.064 mg, 3.5 mmol) in MeOH (80 ml) was cooled to À108, treated with a 1m soln. of (2-phenethyl)hydrazine
(5 ml, 5 mmol), stirred at À108 for 2 h and at 238 for 12 h, treated with aq. NH3 (6ml), and again stirred for 4 h.
After evaporation and FC (AcOEt/hexane 3 :7 ! 3 :2), the product was dissolved in MeOH, treated with
charcoal, and filtered through Celite. Evaporation gave 19 (650 mg, 70% yield). Colourless solid. Rf(MeOH/
AcOEt 1:4) 0.8. M.p. 92 ± 948 (MeOH/CHCl3/hexane). UV (H2O): 286(2.85). [ a]2D5 ˆ À248.4 (c ˆ 0.75, H2O).
IR (KBr): 3382s, 2942w, 2854w, 1654s, 1508w, 1456m, 1400w, 1349m, 1294w, 1277w, 1153m, 1132w, 1105w, 1076m,

Helvetica Chimica Acta ± Vol. 83 (2000)
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1040m, 855m, 6 41m. 1H-NMR (D2O): 2.93 (dt, J ꢀ 14.0, 7.0, PhCH); 3.0 (dt, J ꢀ 13.9, 6.9, PhCH'); 3.88 (dt, J ꢀ
13.5, 6.8, CHN); 4.10 (dt, J ꢀ 14, 6.8, CH'N); 4.20, 4.34 (2d, J ˆ 13.1, HÀC(2), HÀC(3)); 4.44 (br. s, HÀC(5),
H'ÀC(5)); 7.18 ± 7.32 (m, 5 arom. H). 13C-NMR (D2O): 35.80 (t, PhCH2); 51.54 (t, CH2N); 62.78 (t, C(5)); 71.31
(d, C(2), C(3)); 129.36( d); 131.32 (d, 2 C); 131.85 (d, 2 C); 141.30 (s); 162.64 (s, C(4)); 171.00 (s, C(1)). ESI-
‡
‡
MS: 551 (20, [2M ‡ Na] ), 287 (100, [M ‡ Na] ). Anal. calc. for C13H16N2O4 ´ 0.25 H2O (268.79): C 58.09,
H 6.19, N 10.42; found: C 58.14, H 6.18, N 10.14.
11,41-Anhydro-4,5-dideoxy-4-(hydroxyimino)-1-N-methyl-d-threo-pentonamide (20). A soln. of 13 (140 mg,
0.5 mmol) in MeOH/AcOH (5 ml, 95 :5) was hydrogenated at 5 bar for 8 h in the presence of 5% Pd-C (14 mg),
filtered, and concentrated. A soln. of the product in MeOH (5 ml) was treated with aq. NH3 (0.8 ml), stirred for
4 h, and evaporated. FC (AcOEt/hexane 3 :7 ! 3 :2) gave 20 (50 mg, 63%). Colourless waxy solid. UV
1
(MeOH): 286(4.05). [ a]2D5 ˆ À402 (c ˆ 0.6, H2O). H-NMR ((D6)DMSO): 1.94 (br. s, irrad. at 4.04 ! s, Me);
3.12 (s, MeN); 3.84 (dd, J ˆ 11.2, 4.4, irrad. at 5.64 ! d, J ˆ 11.2, HÀC(2)); 4.04 (br. dd, J ꢀ 11.1, 4.8, irrad. at
1.94 ! dd, J ˆ 11.2, 5.0, irrad. at 5.83 ! br. d, J ꢀ 11.4, HÀC(3)); 5.64 (d, J ˆ 4.7, exchanged with D2O,
HOÀC(2)); 5.77 (d, J ˆ 5.9, exchanged with D2O, HOÀC(3)). 13C-NMR ((D6)DMSO): 18.52 (q, C(5)); 34.91
(q, MeN); 68.43, 69.21 (2d, C(2), C(3)); 157.16( s, C(4)); 167.11 (s, C(1)). ESI-MS: 229 (55), 213 (100), 197 (44),
‡
‡
181 (36, [M ‡ Na] ), 176(15), 157 (8, [ M À H] ). Anal. calc. for C6H10N2O3 (158.16): C 45.57, H 6.37, N 17.71;
found: C 45.59, H 6.39, N 17.54.
General Procedure for the Reduction of Dihydropyridazinones 8, and 17 ± 19 with NaCNBH3. A soln. or
suspension of the substrate in MeOH/AcOH (99 :1 to 95 :5) was treated with NaCNBH3 (1.5 or 2 equiv. (for
19)). The mixture was stirred for 24 h, treated with NaCNBH3 (1.5 or 2 (for 19)), and again stirred for 2 d.
Evaporation and FC (AcOEt ! AcOEt/MeOH 5 :1) gave a mixture of diastereoisomers. Their ratio was
determined by 1H-NMR spectroscopy. Pure samples of all diastereoisomers were obtained by prep. HPLC, with
the solvent system as for anal. HPTLC.
Reduction of 8. Treatment of 8 (260 mg, 1.62 mmol) in MeOH/AcOH (10 ml, 98 :2) with NaCNBH3
(300 mg, 4.8 mmol), and FC gave 8 (37 mg) and 7/21 2 :3 (153 mg, 58%).
11,4-Anhydro-d-arabinohydrazide (7). Rf(i-ProH/CHCl3/H2O, 4 :3 :0.3) 0.07. IR (KBr): 3433s, 3255s,
2944m, 2888m, 1677s, 1458m, 1407w, 1355m, 1307w, 1263w, 1151m, 1135w, 1074m, 1057m, 1029w, 996m, 957w,
841m. 1H-NMR (D2O): 3.08 (br. q, J ꢀ 6.2, HÀC(4)); 3.57 (dd, J ˆ 9.3, 6.2, irrad. at 3.08 ! change, irrad. at
4.38 ! d, J ˆ 6.2, HÀC(3)); 3.68 (d, J ˆ 6.2, 2 H, irrad. at 3.08 ! br. s, CH2(5)); 4.38 (d, J ˆ 9.3, HÀC(2)).
13C-NMR (D2O): 63.27 (t, C(5)); 64.65 (d, C(4)); 74.63, 75.45 (2d, C(2), C(3)); 178.68 (s, C(1)). ESI-MS: 217
‡
‡
(100), 185 (44, [M ‡ Na] ), 163 (6, [M ‡ H] ). Anal. calc. for C5H10N2O4 (162.14): C 37.04, H 6.22, N 17.28;
found: C 37.07, H 6.22, N 17.08.
11,4-Anhydro-l-xylonohydrazide (21). Rf(i-PrOH/CHCl3/H2O (4 :3 :0.3) 0.13. 1H-NMR (D2O): 3.31
(ddd, J ꢀ 7.2, 5.0, 4.0, irrad. at 4.07 ! dd, J ˆ 7.2, 5.4, HÀC(4)); 3.74 (dd, J ˆ 12.4, 7.2, irrad. at 3.31 ! change,
HÀC(5)); 3.81 (dd, J ꢀ 12.0, 5.2, irrad. at 3.31 ! change, H'ÀC(5)); 4.07 (br. dd, J ꢀ 5.4, 3.9, irrad. at. 3.31 ! br.
d, J ꢀ 5.7, HÀC(3)); 4.15 (d, J ˆ 5.3, HÀC(2)). 13C-NMR (D2O): 60.53 (d, C(4)); 61.22 (t, C(5)); 73.36, 73.48
‡
(2d, C(2), C(3)); 175.85 (s, C(1)). ESI-MS: 217 (100), 185 (44, [M ‡ Na] ). Anal. calc. for C5H10N2O4 (162.14):
C 37.04, H 6.22, N 17.28; found: C 36.90, H 6.15, N 17.18.
Reduction of 17. Reduction of 17 (330 mg, 1.89 mmol) in MeOH/AcOH (15 ml, 96:4) with NaCNBH 3
(356mg, 5.67 mmol) and FC gave 22/23 1:1 (239 mg, 75%).
11,4-Anhydro-1-N-methyl-d-arabinohydrazide (22). Rf(i-PrOH/CHCl3/H2O 1 :1:0.02) 0.18. UV (H2O): 286
(2.04). [a]2D5 ˆ ‡4.2 (c ˆ 1, H2O). IR (neat): 3375s, 3266s, 2936m, 2866m, 1650s, 1485m, 1445m, 1395m, 1341m,
1237w, 1207w, 1157w, 1102m, 1064s, 96 1w, 882m. 1H-NMR (D2O): 3.11 (s, MeN); 3.12 (ddd, J ꢀ 8.2, 5.6, 5.0,
irrad. at 4.35 ! NOE of 4.5%, HÀC(4)); 3.54 (br. dd, J ˆ 9.3, 5.9, irrad. at 3.08 ! br. dd, J ꢀ 9.0, 1.3, irrad. at
3.08 ! NOE of 2.2%, irrad. at 4.35 ! dd, J ꢀ 5.6,1.6, irrad. at 4.35 ! NOE of 3.9%, HÀC(3)); 3.65 (dd, J ˆ 11.8,
7.8, irrad. at 3.08 ! change, irrad. at 3.08 ! NOE of 2.9%, HÀC(5)); 3.72 (dd, J ˆ 11.8, 5.0, irrad. at 3.08 !
change, irrad. at 3.08 ! NOE of 2.7%, H'ÀC(5)); 4.38 (d, J ˆ 9.3, irrad. at 3.08 ! NOE of 7.1%, HÀC(2)).
13C-NMR (D2O): 39.10 (q, MeN); 63.48 (t, C(5)); 64.58 (d, C(4)); 74.91, 75.63 (2d, C(2), C(3)); 176.07 (s, C(1)).
‡
‡
ESI-MS: 375 (100, [2M ‡ Na] ), 230 (27), 199 (36, [M ‡ Na] ), 182 (16).
11,4-Anhydro-1-N-methyl-l-xylonohydrazide (23). Rf(i-PrOH/CHCl3/H2O, 1:1:0.02) 0.25. UV (H2O): 287
(2.3). [a]2D5 ˆ À137.7 (c ˆ 0.7, H2O). IR (neat): 3361s, 2935w, 1650s, 1451w, 1398m, 1344w, 1229w, 1094s, 1054s,
832w. 1H-NMR (D2O): 3.14 (s, MeN); 3.38 (ddd, J ꢀ 7.2, 5.0, 4.0, irrad. at 4.07 ! dd, J ˆ 7.2, 5.4, irrad. at 3.38 !
NOE of 5%, HÀC(4)); 3.74 (dd, J ˆ 11.8, 7.2, irrad. at 3.38 ! change, irrad. at 3.38 ! NOE of 1.4%, HÀC(5));
3.82 (dd, J ꢀ 12.0, 5.2, irrad. at 3.38 ! change, irrad. at 3.38 ! NOE of 2.1%, H'ÀC(5)); 4.07 (dd, J ꢀ 5.6, 4.3,
irrad. at 3.38 ! d, J ˆ 5.7, HÀC(3)); 4.15 (d, J ˆ 5.6, HÀC(2)). 13C-NMR ((D2O): 38.54 (q, MeN); 60.26
‡
(d, C(4)); 61.44 (t, C(5)); 73.87, 73.98 (2d, C(2), C(3)); 173.57 (s, C(1)). ESI-MS: 351 (66, [2M À H] ), 265 (56),

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‡
211 (47), 175 (100, [M À H] ), 149 (42). Anal. calc. for C6H12N2O4 ´ 0.4 H2O (183.38): C 39.30, H 7.04, N 15.28;
found: C 39.20, H 6.84, N 14.96.
Reduction of 18. Reduction of 18 (110 mg, 0.54 mmol) in MeOH/AcOH (10 ml, 98 :2) with NaCNBH3
(101 mg, 1.6mmol) and FC gave 24/25 1:1 (98 mg, 87%).
11,4-Anhydro-1-N-(2-hydroxyethyl)-d-arabinohydrazide (24). Rf(i-PrOH/CHCl3/H2O 4 :3 :0.2) 0.14. UV
(H2O): 291 (2.2). [a]2D5 ˆ 19.7 (c ˆ 0.63, H2O). IR (neat): 3353s, 3277s, 2941w, 2877m, 1651s, 1453m, 1420m,
1348m, 1273m, 1150m, 1061s, 986w, 875w. 1H-NMR (D2O): 3.09 (ddd, J ꢀ 8.3, 5.4, 4.2, HÀC(4)); 3.53 ± 3.47
(m, irrad. at 3.77 ! change, irrad. at 4.41 ! change, HÀC(3), HÀCN); 3.60 (dd, J ˆ 11.8, 8.7, HÀC(5)); 3.69
(dd, J ˆ 11.8, 4.7, H'ÀC(5)); 3.78 ± 3.70 (m, 3 H); 4.41 (d, J ˆ 9.7, HÀC(2)). 13C-NMR (D2O): 52.96( t, NCH2);
60.94 (t, CH2HO); 63.58 (t, C(5)); 64.61 (d, C(4)); 74.75, 75.60 (2d, C(2), C(3)); 176.67 (s, C(1)). ESI-MS: 435
‡
‡
(100, [2M ‡ Na] ), 408 (18), 360 (28), 261 (36), 229 (89, [M ‡ Na] ). Anal. calc. for C7H14N2O5 ´ 0.33 H2O
(212.15): C 39.77, H 6.91, N 13.18; found: C 39.62, H 6.97, N 13.20.
11,4-Anhydro-1-N-(2-hydroxyethyl)-l-xylonohydrazide (25). Rf(i-PrOH/CHCl3/H2O, 4 :3 :0.2) 0.20. UV
(H2O): 287 (2.3). [a]2D5 ˆ À86.1 (c ˆ 0.8, H2O). IR (neat): 3361s, 2941m, 1651s, 1427m, 1353m, 1248m, 1065s,
865w, 832w. 1H-NMR (D2O): 3.36(br. ddd, J ꢀ 7.5, 5.0, 3.4, irrad. at 4.07 ! dd, J ˆ 7.2, 4.7, HÀC(4)); 3.62 ± 3.66
(m, 2 H); 3.70 ± 3.78 (m, 3 H); 3.82 (dd, J ꢀ 12, 4.6, HÀC(5)); 4.07 (br. dd, J ꢀ 6.2, 5.0, irrad. at 3.36 ! d, J ˆ 6.5,
HÀC(3)); 4.22 (d, J ˆ 6.2, HÀC(2)). 13C-NMR (D2O): 52.62 (t, CH2N); 60.82 (t, CH2OH); 61.24 (t, C(5)); 61.50
‡
(d, C(4)); 74.60, 75.09 (2d, C(2), C(3)); 175.05 (s, C(1)). ESI-MS: 435 (100, [2M ‡ Na] ), 413 (12), 315 (38), 261
‡
‡
(20), 229 (89, [M ‡ Na] ), 207 (40, [M ‡ H] ). Anal. calc. for C7H14N2O5 (206.20): C 40.77, H 6.84, N 13.59;
found: C 40.53, H 6.88, N 13.34.
Reduction of 19. Reduction of 19 (199 mg, 0.75 mmol) in MeOH/AcOH (5 ml, 95 :5) with NaCNBH3
(185 mg, 3 mmol) FC gave 26/27 3 :2 (180 mg, 89%).
11,4-Anhydro-1-N-(2-phenethyl)-d-arabinohydrazide (26). Rf(i-PrOH/CHCl3/H2O 1:1 :0.02) 0.34. UV
(H2O): 290 (1.3). [a]D25 ˆ ‡10.1 (c ˆ 0.83, H2O). IR (neat): 3374s, 3277s, 2938m, 1659s, 1497w, 1454m, 1360w,
1288w, 1199w, 1143w, 1082m, 1038m. 1H-NMR (D2O): 2.94 (t, J ˆ 7.2, PhCH2); 3.04 (ddd, J ꢀ 9.0, 5.9, 4.7,
HÀC(4)); 3.44 (dd, J ˆ 9.3, 5.9, HÀC(3)); 3.49 ± 3.59 (m, HÀC(5), NCH); 3.63 (dd, J ꢀ 12.0, 4.5, H'ÀC(5));
3.90 (dt, J ꢀ 13.7, 7.8, CH'N); 4.29 (d, J ˆ 9.3, HÀC(2)); 7.31 ± 7.44 (m, 5 arom. H). 13C-NMR (D2O): 35.38
(t, PhCH2); 52.87 (t, CH2N); 63.57 (t, C(5)); 64.63 (d, C(4)); 74.86, 75.67 (2d, C(2), C(3)); 129.68 (d); 131.79
‡
(d, 2 C); 131.88 (d, 2 C); 141.82 (s); 176.05 (s, C(1)). ESI-MS: 531 (40, [2M À H] ), 355 (72), 301 (65), 265 (100,
‡
[M À H] ). Anal. calc. for C13H18N2O4 (266.29): C 58.64, H 6.81, N 10.52; found: C 58.63, H 6.94, N 10.51.
11,4-Anhydro-1-N-(2-phenethyl)-l-xylonohydrazide (27): Rf(i-PrOH/CHCl3/H2O 1 :1:0.02) 0.43. UV
(H2O): 286(2). [ a]D25 ˆ À98.4 (c ˆ 1, H2O). IR (neat): 3365s, 3237s, 3026w, 2937w, 1651s, 1497w, 1454m,
1
1426m, 1359w, 1292w, 1236w, 1120m, 1058m, 981w, 958w, 908w. H-NMR (D2O): 2.96( t, J ˆ 7.2, PhCH2); 3.19
(ddd, J ꢀ 7.2, 5.0, HÀC(4)); 3.69 (dd, J ꢀ 12.0, 7.0, irrad. at 3.19 ! change, HÀC(5)); 3.73 (dt, J ꢀ 14.2, 7.2, irrad.
at 2.96 ! change. CHN); 3.77 (dd, J ꢀ 12.0, 5.0, irrad. at 3.19 ! change, H'ÀC(5)); 3.83 (dt, J ꢀ 14.0, 7.0, irrad. at
2.96 ! change, CH'N); 3.97 (dd, J ˆ 6.2, 5.3, HÀC(3)); 4.13 (d, J ˆ 6.2, HÀC(2)); 7.31 ± 7.44 (m, 5 arom. H).
13C-NMR (D2O): 35.17 (t, PhCH2); 52.61 (t, CH2N); 61.24 (d, C(4)); 61.43 (t, C(5)); 74.48, 74.86(2 d, C(2),
‡
C(3)); 129.68 (d); 131.75 (d, 2 C); 131.98 (d, 2 C); 141.87 (s); 174.45 (s, C(1)). ESI-MS: 531 (69, [2M À H] ), 355
‡
(52), 301 (36), 265 (100, [M À H] ). Anal. calc. for C13H18N2O4 ´ 0.25 H2O (270.80): C 57.66, H 6.89, N 10.34;
found: C 57.55, H 6.88, N 10.20.
Inhibition of Glycosidases. IC50 Values were determined at a substrate concentration corresponding to Km
of each enzyme. Determination of the inhibition constants (Ki) were performed at different concentrations of
the inhibitor (usually 4 ± 8 concentrations) bracketing the Ki or IC50 value.
a) Inhibition of Brewerꢀs Yeast a-Glucosidase. IC50 Values were determined at 378 with a 0.08m KH2PO4/
K2HPO4/NaCl buffer (pH 6.8), and 4-nitrophenyl a-d-glucopyranoside as substrate. Measurements were
started by addition of the substrate to pre-incubated cuvets containing inhibitor or H2O, enzyme and buffer. The
increase of absorption per min at 400 nm was taken as velocity for the hydrolysis of the substrate. The increase
was linear during all measurements (3 min). IC50 Values were calculated by plotting the inhibitor concentration
vs. the rate of hydrolysis.
b) Inhibition of Sweet Almond b-Glucosidase. Inhibition studies were carried out at 378 with a 0.08m
KH2PO4/K2HPO4 buffer (pH 6.8), and 4-nitrophenyl b-d-glucopyranoside as substrate. Measurements were
started by addition of the substrate to a pre-incubated cuvets containing inhibitor or H2O, enzyme and buffer.
The increase of absorption per min at 400 nm was taken as velocity for the hydrolysis of the substrate. The
increase was linear during all measurements (3 min). For inhibition studies below pH 6.0, reactions were started
by adding substrate and quenching the reaction after 5 min with 0.2m borate buffer (pH 9.2) and taking the

Helvetica Chimica Acta ± Vol. 83 (2000)
1609
absorption of the soln. at 400 nm. IC50 Values were calculated by plotting the inhibitor concentration vs. the rate
of hydrolysis or absorption. Ki Values were determined from Dixon plots.
c) Inhibition of C. saccharolyticum b-Glucosidase. Inhibition studies were carried out at 558, as mentioned
under b.
d) Inhibition of Jack Bean a-Mannosidase. Inhibition studies were carried out at 378 in 50 mmol acetate
buffer (pH 4.5) with 2 mmol of ZnCl2, and 4-nitrophenyl a-d-mannopyranoside as substrate. Reactions were
initiated by adding the enzyme (75 ml) to a pre-incubated cuvet containing the substrate solution in buffer
(1350 ml) and H2O and/or inhibitor solution (75 ml). The velocity of the substrate hydrolysis was measured by
quenching 500 ml aliquots after 1, 2 and 3 min, with 500 ml of 0.2m borate buffer (pH 9.2) and measuring the
absorption at 400 nm, and the increase of absorption was found to be linear. Ki Values were obtained from
Dixon plots.
e) Inhibition of Snail b-Mannosidase. Ki and IC50 values were determined at 258, with 50 mmol acetate
buffer (pH 4.5) and 4-nitrophenyl b-d-mannopyranoside as substrate. The velocity of substrate hydrolysis was
determined by quenching the reaction after 5 min with 0.2m borate buffer (pH 9.2) and measuring the
absorption. Ki Values were obtained from Dixon plots.
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