Synthesis of labelled 1-azafagomine

Article abstract of DOI:10.1039/a907365e

(3,4-trans-4,5-trans)-4,5-Dihydroxy-3-(hydroxymethyl)hexahydro-(3- ¹³C)pyridazine (azafagomine, 1a) was synthesised in 7 steps starting from (2-¹³C) malonic acid 5a through conversion to pentadienoic acid and Diels-Alder reaction with 4-phenyl-1,2,4-triazole-3,5-dione, reduction, epoxidation, epoxide hydrolysis and deprotection. The Royal Society of Chemistry 1999.

Full text of DOI:10.1039/a907365e

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Synthesis of labelled 1-azafagomine
Steen Uldall Hansen and Mikael Bols*
Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark
Received (in Lund, Sweden) 3rd September 1999, Accepted and transferred from
Acta Chem. Scand. 28th September, 1999
(3,4-trans-4,5-trans)-4,5-Dihydroxy-3-(hydroxymethyl)hexahydro-(3-13C)pyridazine (azafagomine, 1a) was
synthesised in 7 steps starting from (2-13C) malonic acid 5a through conversion to pentadienoic acid and Diels–Alder
reaction with 4-phenyl-1,2,4-triazole-3,5-dione, reduction, epoxidation, epoxide hydrolysis and deprotection.
Introduction
Biomolecules labelled with stable isotopes are useful probes in
NMR studies of intermolecular interactions.1 By labelling with
NMR-active nuclei that occur at low natural abundance it is
possible to tremendously enhance signals that are otherwise
difficult to observe. This may allow the gathering of new inform-
ation about electronic or conformational properties during
host–guest binding, particularly when the host is a protein.
Recently we discovered the glycosidase inhibitor 1-azafago-
mine 1.2 This compound inhibits both α- and β-glucosidase
Fig. 1 The chemical structure of 1-azafagomine and its anticipated
interaction with a glycosidase.
potently and was expected to be a transition state analogue.
Scheme 1 Previously published synthesis of ( )-1.2
While 1 was designed to mimic, when protonated, an oxo-
carbenium ion and anomeric carbocation at the same time, the
observation that 1 was a very weak base (pKa 3.9)2 cast some
doubt on whether 1 could be protonated by the enzyme inside
its active site. It therefore became interesting to try to elucidate
whether 1 would become protonated when being bound to α- or
β-glucosidase. This we anticipated might be possible with 13C-
labelled 1 containing the label in a position that would be
affected by protonation of the nitrogens. These positions could
be C-3 or C-6 since the chemical shifts of these differed con-
siderably.2 In this paper we report a synthesis of ( )-1 contain-
ing 13C at C-3.
synthesis and delay the reduction of the carboxylic acid. In the
previous work, we had carried out the Diels–Alder reaction
with pentadienoic acid, but never solved the problem of reduc-
tion of the adduct to 2.
2-(13C)-Labelled pentadienoic acid 6a was synthesised in
55% yield by reaction3 of 2-(13C)malonic acid 5a with acrolein
(acrylaldehyde) in excess (Scheme 2). The acid 6a, a relatively
unstable compound, was promptly treated with 4-phenyl-1,2,4-
triazole-3,5-dione, which was obtained in situ by preoxidation
of 4-phenylurazole with tert-butyl hypochlorite. This reaction
gave the labelled Diels–Alder adduct 7a in 76% yield.2
A
sequence of reactions was found that could reduce the acid: 7a
was converted (SOCl2) to the acid chloride, which was treated
with NaBH4 in 1,4-dioxane–water4 to give the labelled alcohol
2a. Carrying out the reduction of the acid chloride in DMF was
also possible, but direct reduction of the acid 7a with LiAlH4
gave a low yield. Epoxidation of 2a was carried out as in the
unlabelled case2 using methyl(trifluoromethyl)dioxirane giving
labelled trans-epoxide 3a in 58% yield. The hydrolysis of 3a to
triol 4a was also carried out essentially as with 3, but in some
runs an interesting, previously undetected, by-product 8a was
observed in occasionally up to 40% yield. Compound 8a was
Results and discussion
Though our previous synthesis of ( )-1 (Scheme 1) became the
basis for the preparation of 3-(13C)-1, it was clear that the syn-
thesis had to undergo some adjustments before it could be used.
The synthesis relied on the use of penta-2,4-dien-1-ol as an
intermediate, the synthesis of which consisted of esterification
and reduction of pentadienoic acid. Both these reactions were
carried out on relatively sensitive compounds and were also
difficult to carry out on a small scale. These steps were therefore
unattractive in a synthesis with costly materials. We therefore
planned to carry out the Diels–Alder reaction earlier in the
1
identified from changes in the H and 13C NMR spectra as
J. Chem. Soc., Perkin Trans. 1, 1999, 3323–3325
This journal is © The Royal Society of Chemistry 1999
3323

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Fig. 2 Dihydrazino derivative obtained in previous work, resembling
by-product 8a.5
Experimental
General
1
13C NMR and H NMR spectra were recorded on a Varian
Gemini 200 instrument. Mass spectra were obtained on a
Micromass LCT-QTOF instrument. Concentrations were per-
formed on a rotary evaporator at a temperature below 40 ЊC.
(E) (2-13C)-Penta-2,4-dienoic acid 6a
(2-13C)Malonic acid 5a (0.95 g, 9.05 mmol, 99% 13C, Cambridge
Isotope Laboratories, Inc.) was dissolved in 1.4 ml of dry pyrid-
ine and heated to 80 ЊC. Acrolein (0.775 ml, 16.4 mmol) was
added in one portion, and the mixture was stirred for 30 min.
First ice (6 g) and then sulfuric acid 96% (0.8 ml) was added
with stirring, followed by extraction (3 × 15 ml CHCl3), drying
(MgSO4) and evaporation of the solvent. The crude product 6a
was obtained as a yellow solid, which was used immediately
without further purification. Yield: 490 mg (55%); 13C NMR
(d6-DMSO) δ 171.8 (d, C-1, J1,2 293 Hz), 146.4 (d, C-3, J3,2 275
Hz), 133.9 (C-5), 126.2 (d, C-4, J4,2 37 Hz), 120.7 (intensity
100×, C-2); 1H NMR (CDCl3) δ 9.85 (br s, 1H, H-1), 7.28 (ddd,
1H, H-3, J3,2 15.4 Hz, J3,4 11.0 Hz, JH3,C2 2.2 Hz), 6.42 (dddt,
1H, H-4, J4,5c 10.6 Hz, J4,5t 16.8 Hz, JH4,C2 3.7 Hz, J4,2 0.7 Hz),
5.84 (ddd, 1H, H-2, JH2,C2 164 Hz), 5.59 (d, 1H, H-5t), 5.49 (d,
1H, H-5c).
Scheme 2 Synthesis of 13C labelled ( )-1 using a modified synthesis.
Dot (ؒ) depicts 13C.
( )-7,9-Dioxo-8-phenyl-1,6,8-triaza-(2-13C)bicyclo[4.3.0]non-3-
ene-2-carboxylic acid 7a
compared with 4a. In 8a the 13C signals of the ipso, para and
ortho positions of the aromatic group (δC 138.6, 122.4 and
119.7) differ widely from those of 3a or 4a (δC 131.2, 127.4 and
125.7). These new signals fit those of an anilide and resemble
those of a similar compound 9 previously synthesised by us.5
Furthermore, 13C NMR spectroscopy shows two carbonyl
4-Phenylurazole (0.927 g, 5.2 mmol) was suspended in EtOAc
(7 ml) at 0 ЊC and tert-butyl hypochlorite (0.567 g) was added to
give a red homogeneous solution. After 10 min, 6a (0.49 g, 4.95
mmol) was added, and the solution was stirred for 18 h at room
temperature. The precipitated adduct 7a was isolated by fil-
tration, and washed with pentane. Yield: 1.03 g (76%); 13C NMR
(d6-DMSO) δ 168.0 (d, C-2, J2Ј,2 218 Hz), 152.3, 151.5 (C-7,
C-9), 130.9 (Ar ipso), 128.6 (meta), 127.6 (para), 125.4 (ortho),
122.9 (C-4), 119.2 (d, C-3, J3,2 166 Hz), 55.2 (intensity 100×,
C-2), 42.5 (C-5); 1H NMR (DMSO) δ 7.49 (m, 5H, ArH), 6.12
(m, 2H, H-3, H-4), 5.22 (br d, 1H, H-2, JH2,C2 147 Hz), 4.32 (br
d, 1H, H-5eq, J5eq,5ax 16.5 Hz), 4.03 (br d, 1H, H-5 ave.); MS
(ES) m/z 297.0719 (M ϩ Naϩ). Calc. for C1213CH11N3O4 ϩ
Naϩ: m/z, 297.0676.
1
groups are present in 8a, and the H chemical NMR spectrum
in DMSO shows only 2 OH groups are present, in addition to
an NH group. For comparison, 3 OH signals are clearly seen in
the DMSO spectrum of 4a.
By-product 8a was formed in amounts varying from 0 to
40%. Its formation appears to depend on the reaction time,
because when pure triol 4a was subjected to hydrolysis con-
ditions 8a was formed. Interestingly, no hydrolysis of the
phenylurazole moiety has been observed in this reaction, so
apparently intramolecular attack on the carbonyl group by
the hydroxy group is much more favorable than attack by
water. Thus the by-product is formed from the product, and
to avoid its formation it is important not to prolong the
reaction time.
( )-2-Hydroxymethyl-8-phenyl-1,6,8-triaza-(2-13C)bicyclo-
[4.3.0]non-3-ene-7,9-dione 2a
To freshly distilled SOCl2 (5 ml) was added 7a (1.00 g, 3.65
mmol) and the suspension was refluxed for 90 min, giving a
homogeneous solution. The remaining SOCl2 was removed by
evaporation and the crude acid chloride dried under reduced
pressure. The acid chloride was then dissolved in dry 1,4-
dioxane (12 ml) and added over 30 min to a solution of
water–1,4-dioxane (5 ml:5 ml) containing NaBH4 (400 mg) and
keeping the temperature at 10 ЊC. After stirring the mixture for
2 h, water (20 ml) was added. Extraction with CH2Cl2 (4 × 25
ml), drying (MgSO4) and evaporation gave a crude product
(0.938 g). This was purified by flash chromatography, using
EtOAc as the eluent, giving 2a (0.696 g, 73%); 13C NMR
(DMSO) δ 151.9, 150.2 (C-7, C-9), 131.1 (Ar ipso), 128.4
Nevertheless, both 4a and 8a can be converted to labelled
azafagomine. Hydrazinolysis of 4a, by the method described
for 4,2 gave 3-labelled 1-azafagomine 1a in 65% yield. Similarly,
hydrazinolysis of 8a gave 1a in 75% yield, so it is not necessary
to separate mixtures of 4a and 8a before hydrazinolysis.
Conclusions
We have described a succesful synthesis of the first 13C-labelled
derivative of a 1-azasugar. NMR studies of the inhibitor–
enzyme complexes are underway. The synthesis described is also
an improvement on the previous synthesis of ( )-1, since it
reduces the work with sensitive dienes.
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(meta), 127.4 (para), 125.6 (ortho), 123.0 (d, C-3, J3,2 175 Hz),
121.2 (C-4), 60.5 (d, C-2, J2Ј,2 149 Hz), 54.6 (intensity 100×,
C-2), 42.8 (C-5); 1H NMR (DMSO) δ 7.47 (m, 5H, ArH), 6.03
(m, 2H, H-3, H-4), 5.06 (br s, 1H, 2Ј-OH), 4.46 (br d, 1H, H-2,
JH2,C2 144 Hz), 4.18 (dd, 1H, H-5ax, J5ax,5eq 16.5 Hz, J5ax,4 2.2
Hz), 4.03 (d, 1H, H-5eq), 3.68 (d, 2H, H2-2Ј, J 4.8 Hz); MS (ES)
m/z 283.0927 (M ϩ Naϩ). Calc. for C1213CH13N3O3 ϩ Naϩ: m/z,
283.0884.
8a: 13C NMR (DMSO) δ 155.7 (C-9), 153.7 (C-2Ј), 138.6 (Ar
ipso), 128.0 (meta), 122.4 (para), 119.7 (ortho), 71.6 (d, C-5, J5,6
152 Hz), 67.3 (C-4), 64.4 (d, C-7, J6,7 135 Hz), 56.4 (intensity
100×, C-6), 46.0 (C-3); 1H NMR (DMSO) δ 9.21 (s, 1H,
NH), 7.54 (d, 2H, o-ArH, Jo,m 8.1 Hz), 7.30 (t, 2H, m-ArH,
Jm,o = Jm,p = 7.7 Hz), 7.02 (t, 1H, p-ArH), 5.64 (br s, 1H, 4-OH),
5.40 (s br, 1H, 5-OH), 4.41 (m, 3H, H-4, H-5, H-7a), 3.55 (ddd,
1H, H-6, J6,C6 128 Hz, J6,7a 6.5 Hz, J6,7b 9.0 Hz), 3.21 (m, 3H,
H2-3, H7b); MS (ES) m/z 317.0951 (M ϩ Naϩ). Calc. for
C1213CH15N3O5 ϩ Naϩ: m/z, 317.0939.
The by-product 8a was also obtained from 4a (40 mg), when
treated with HClO4 (75%, 100 µl) in 4 ml of water for 50 h at
100 ЊC. After neutralisation (KHCO3) and concentration a
residue containing a 2:1 mixture of 4a and 8a was obtained.
(2,3-trans-3,4-cis)-3,4-Epoxy-2-hydroxymethyl-8-phenyl-1,6,8-
triaza(2-13C)bicyclo[4.3.0]non-3-ene-7,9-dione 3a
Alcohol 2a (0.50 g, 1.92 mmol) was dissolved in a mixture of
MeCN (15 ml) and water (10 ml). The solution was cooled to
0 ЊC, and 1,1,1-trifluoroacetone (2 ml) and NaHCO3 (1.3 g)
were added, followed by Oxone (6.15 g) in small portions over
a period of 10 min. The mixture was stirred 15 h at room tem-
perature. Another charge of NaHCO3 (0.65 g), 1,1,1-trifluoro-
acetone (1 ml) and Oxone (3 g) was added, and after 2 h the
reaction mixture was worked up by the addition of water (100
ml) and extraction with CHCl3 (5 × 50 ml). The combined
organic layers were dried (MgSO4) and concentrated to give a
solid mixture of trans and cis epoxide (468 mg) in a 3:1
ratio. On addition of CHCl3 (18 ml) pure 3a crystallized out
(308 mg, 58%); 13C NMR (DMSO) δ 150.1 (C-7, C-9), 131.0
(Ar ipso), 128.4 (meta), 127.5 (para), 125.6 (ortho), 59.0 (d,
C-2Ј, J2Ј,2 152 Hz), 53.5 (intensity 100×, C-2), 49.6 (d, C-3, J3,2
(3,4-trans-4,5-trans)-4,5-Dihydroxy-3-(hydroxymethyl)hexa-
hydropyridazine 1a
Triol 4a (83 mg, 0.28 mmol) was dissolved in hydrazine hydrate
(2 ml) and heated at 100 ЊC for 18 h. The solution was concen-
trated to give a syrup. Flash chromatography in EtOH–NH4OH
(25%) 9:1 gave pure 1a (27 mg, 65%); 13C NMR (D2O) δ 70.2 (d,
C-4, J4,3 155 Hz), 69.7 (C-5), 61.4 (intensity 100×, C-3), 58.0 (d,
C-3Ј, J3Ј,3 160 Hz), 49.9 (C-6); 1H NMR (D2O) δ 3.58 (ddd, 1H,
H-3Јa, J3Јa,3Јb 12.1 Hz, J3Јa,3 2.9 Hz, J3Јa,C3 1.8 Hz), 3.42 (ddd, 1H,
H-3Јb, J3Јb,3 5.9 Hz, J3Јb,C3 2.2 Hz), 3.32 (dd, 1H, H-5, J5,6ax 10.3
Hz, J5,6eq 5.1 Hz), 3.09 (dt, 1H, H-4, J4,3,5 9.9 Hz, J4,C3 2.6
Hz), 2.97 (dd, 1H, H-6eq, J6eq,6ax 12.8 Hz, J6eq,5 5.1 Hz), 2.47
(dddd, 1H, H-3, J3,C3 137 Hz, J3,4 9.9 Hz); MS (ES) m/z
172.0776 (M ϩ Naϩ). Calc. for C413CH12N2O3 ϩ Naϩ: m/z
172.0775.
1
178 Hz), 47.3 (C-4), 40.6 (C-5); H NMR (DMSO) δ 7.46 (m,
5H, Ar), 5.39 (dt, 1H, 2Ј-OH, J2Ј-OH,2Ј 5.9 Hz, J2ЈOH,C2 1.5 Hz),
4.46 (dt, 1H, H-2, JH2,C2 145 Hz, J2,2aЈ 4.0 Hz), 4.13 (d, 1H,
H-5ax, J5ax,5eq 13.9 Hz), 3.92 (dd, 1H, H-5eq, J5eq,4 2.2 Hz), 3.80
(m, 2H, H-2Ј), 3.63 (m, 2H, H-3, H-4). MS (ES): m/z 299.0891
(M ϩ Naϩ). Calc. for C1213CH13N3O4 ϩ Naϩ: m/z, 299.0833.
Alternatively 8a (29 mg) was subjected to hydrazinolysis in
hydrazine hydrate (2 ml) at 100 ЊC for 18 h. After work-up as
above 1a (11 mg, 75%) was obtained.
(2,3-trans-3,4-trans)-3,4-Dihydroxy-2-hydroxymethyl-8-phenyl-
1,6,8-triaza(2-13C)bicyclo[4.3.0]non-3-ene-7,9-dione 4a and (4,5-
trans-5,6-trans)-4,5-dihydroxy-8-oxo-2-(N-phenyl)carbamoyl-
1,2-diaza-8-oxa-bicyclo[4.3.0]nonane 8a
Acknowledgements
We thank the Danish Natural Science Research council for
financial support through the THOR program, and the
Carlsberg foundation for providing support for the Micromass
LCT-QTOF instrument.
Epoxide 3a (0.236 g, 0.855 mmol) was dissolved in water (25 ml)
and HClO4 (70%; 0.59 ml) was added. The solution was heated
to 100 ЊC for 5 h, then neutralised with KHCO3 (0.70 g) and
concentrated. Flash chromatography in EtOAc gave pure 4a
(184 mg, 74%), while 8a was also obtained (44 mg, 18%).
4a: 13C NMR (DMSO) δ 152.9, 150.8 (C-7, C-9), 131.2 (Ar
ipso), 128.4 (meta), 127.4 (para), 125.7 (ortho), 65.9 (C-4), 65.3
(d, C-3, J3,2 155 Hz), 60.3 (intensity 100×, C-2), 57.8 (d, C-2Ј,
References
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1
J2Ј,2 149 Hz), 46.1 (C-5); H NMR (DMSO) δ 7.46 (m, 5H,
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J3-OH,3 = J3-OH,C2 = 3.7 Hz), 4.98 (dt, 1H, 2Ј-OH, J2Ј-OH,2Ј 5.9 Hz,
J2Ј-OH,C2 1.5 Hz), 4.00 (dt, 1H, H-2, J2,C2 144 Hz, J2,2Ј 5.8 Hz),
3.79 (m, 4H, H-2Ј, H-3, H-4), 3.66 (dd, 1H, H-5ax, J5ax,5eq 15.0
hz, J5ax,4 2.6 Hz), 3.51 (dd, 1H, H-5eq, J5eq,4 2.6 Hz); MS (ES)
m/z 317.0988 (M ϩ Naϩ). Calc. for C1213CH15N3O5 ϩ Na: m/z,
317.0939.
Paper 9/07365E
J. Chem. Soc., Perkin Trans. 1, 1999, 3323–3325
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