Nine of 16 stereoisomeric polyhydroxylated proline amides are potent β-N-acetylhexosaminidase inhibitors

Article abstract of DOI:10.1021/jo500157p

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of β-N- acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. β-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

Full text of DOI:10.1021/jo500157p

Article
pubs.acs.org/joc
Nine of 16 Stereoisomeric Polyhydroxylated Proline Amides Are
Potent β‑N‑Acetylhexosaminidase Inhibitors
Benjamin J. Ayers,*,† Andreas F. G. Glawar,†,‡ R. Fernando Martínez,† Nigel Ngo,† Zilei Liu,†
George W. J. Fleet,*,† Terry D. Butters,‡ Robert J. Nash,§ Chu-Yi Yu,∥ Mark R. Wormald,‡
Shinpei Nakagawa,⊥ Isao Adachi,⊥ Atsushi Kato,*,⊥ and Sarah F. Jenkinson†
†Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
‡Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
§Phytoquest Limited, IBERS, Plas Gogerddan, Ceredigion, Aberystwyth, SY23 3EB, U.K.
∥CAS Key Laboratory of Molecular Recognition and Function, Institute of Chemistry, Chinese Academy of Science, Beijing 100190,
China.
⊥Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
S
* Supporting Information
ABSTRACT: All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-
3,4-dihydroxyproline amides have been synthesized from lactones
accessible from the enantiomers of glucuronolactone. Nine
stereoisomers, including all eight with a (3R)-hydroxyl config-
uration, are low to submicromolar inhibitors of β-N-acetylhex-
osaminidases. A structural correlation between the proline amides is
found with the ADMDP-acetamide analogues bearing an
acetamidomethylpyrrolidine motif. The proline amides are generally
more potent than their ADMDP-acetamide equivalents. β-N-
Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide
analogue is compared to an azetidine carboxylic acid amide. None of
the amides are good α-N-acetylgalactosaminidase inhibitors.
INTRODUCTION
■
In humans, deficiencies in the levels of β-N-acetylhexosamini-
dases (HexNAcases [EC 3.2.1.52]) can lead to lysosomal storage
disorders (LSDs) such as Tay−Sachs and Sandhoff1−3 and to
Alzheimer’s disease.4 In particular, control of O-GlcNAc levels
provides opportunities for intervention in many pathogenic
conditions,5,6 including diabetes,7,8 cancer,9,10 Parkinson’s
disease,11 and osteoarthritis.12 In addition, specific inhibition of
α-N-acetylgalactosaminidases (α-GalNAcases [EC 3.2.1.49]) by
chemotherapeutic agents has potential in the treatment of the
Schindler−Kanzaki LSD13 and of cancer by prevention of the
degradation of the macrophage-activating factor.14,15
Although about 250 naturally occurring iminosugars have
been isolated from plants, none of them are good HexNAcase
inhibitors. Three microbial natural products, pochonicine 1,16−18
siastatin B 2,19−21 and nagstatin 3,22,23 are nanomolar inhibitors
of HexNAcases (Figure 1). There are many synthetic examples of
micromolar HexNAcase inhibitors that possess an acetamide
acetamide (NHAc) group.
Figure 1. Examples of potent HexNAcase inhibitors containing an
(NHAc) group which include the pyrrolidines ADMDP-
acetamide [(N-(((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxy-
methyl)pyrrolidin-2-yl)methyl)acetamide] 412,24 and LABNAc
5;2 the piperidines PUGNAc 6,25,26 NGT (N-acetyl-D-glucos-
amine-thiazoline) 7,27,28 DNJNAc 8,29 and DGJNAc 9;29 and
Received: January 23, 2014
Published: March 18, 2014
azepanes including 10.30,31
© 2014 American Chemical Society
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azetidine 12L33 carboxylic acids as potent inhibitors of
HexNAcases, which do not contain an NHAc group (Figure
2). An SAR investigation into the inhibitory activity of these
amides toward HexNAcases has not been possible as the
syntheses of amides 11 and 12L do not readily permit
preparation of their stereoisomers. In contrast, we have recently
published a divergent pathway to the synthesis of all the
stereoisomers of pyrrolidine-containing DMDP34 [(2R,3R,4R,-
5R)-2,5-bis(hydroxymethyl)pyrrolidine-3,4-diol] from the enan-
tiomers of glucuronolactone35 via lactone intermediates. We
report herein the extension of this divergent methodology, and
the utilization of these lactone intermediates, to provide access to
all 16 stereoisomeric N-methyl proline amides 13, which are five-
membered analogues of N-methyl pipecolic 11 and azetidine
amides 12L. This permitted an SAR investigation of their activity
toward HexNAcases which is also described. In addition, an SAR
comparison is made between the N-methyl proline amides and
the corresponding isosteric ADMDP-acetamides.
Figure 2. N-Methylamides of piperidine, pyrrolidine, and azetidine
carboxylic acids and the azetidine-acetamide (-D and -L numbering
suffixes are used where both enantiomers of a compound are discussed).
The N-methyl amide moiety in these targets is isosteric to the
NHAc of inhibitors shown in Figure 1; with this is mind, we
propose that the azetidine-acetamide 14, the novel isosteric
analogue of azetidine amide 12L, would be an inhibitor of
HexNAcases. The synthesis and biological evaluation of 14 is also
reported.
Scheme 1. Divergent Access to 10 Pyrrolidines from the
Enantiomers of Glucuronolactone34 and Proposed Access to
Proline Amide Targets
RESULTS AND DISCUSSION
■
Synthesis. 1. N-Methyl 5-(Hydroxymethyl)-3,4-dihydrox-
yproline Amides. D-Glucuronolactone 15D, and its readily
available enantiomer 15L,35 are well-established chirons.36−45
We have previously reported the synthesis of all 10 stereo-
isomeric pyrrolidines 17 divergently from the enantiomers of
glucuronolactone via the 16 lactones 16 (Scheme 1).34 The
proline amides 13 can be accessed from the lactones 16 by
reaction with methylamine.
The lactones 16 are the key intermediates in the synthesis of
the target proline amides 13. They were accessed in excellent
yield, divergently from the enantiomers of glucuronolactone 15D
and 15L.34 In the synthesis of L-gulo proline amide 22L (Scheme
2), the lactone 18L was utilized. Activation of the C2-hydroxyl by
Our group has described the synthesis and inhibitory activity
of the N-methylamides of the hydroxylated pipecolic 1132 and
a
Scheme 2. Synthesis of Gulo- and Ido-Configured Proline Amides
a
Reagents and conditions: (i) Tf2O, pyr, DCM −40 °C, 1 h, 95%;34 (ii) 10% Pd−C, H2, 1,4-dioxane, rt, 1.5 h; (iii) MeNH2 (33% in EtOH), 16 h, rt;
(iv) 10% Pd−C, H2, 1,4-dioxane−2 M HCl, rt, 16 h, 66% over three steps; (v) MsCl, pyr, 0 °C−rt, 2 h, 93%;34 (vi) MeNH2 (33% EtOH), 1,4-
dioxane, rt, 16 h, 99%; (vii) 10% Pd−C, H2, NaOAc, 1,4-dioxane, rt, 3 h, then Boc2O, rt, 16 h, 86%; (viii) 10% Pd−C, H2, 1,4-dioxane−2 M HCl, rt,
16 h, from 27L, 77%, from 28L, 75%.
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esterification with trifluoromethanesulfonic (triflic) anhydride in
pyridine afforded the triflate 19L (95%).34 Hydrogenation of the
azide moiety with palladium (10% on carbon) led initially to the
strained bicycle 20L, which upon addition of methylamine
underwent aminolytic ring opening to form the dibenzyl
intermediate 21L. Further reduction under acidic conditions
afforded the proline amide 22L in 66% yield over three steps. The
enantiomeric D-gulo proline amide 22D was similarly formed
from the lactone 18D in a comparable yield. In order to access
the C2-epimeric L-ido proline amide 29L, the C2-epimeric
lactone 23L was employed. The C2-hydroxyl of the lactone 23L
was activated by esterification with methanesulfonyl (mesyl)
chloride in pyridine (93%) to form the mesylate 24L.34 Here,
aminolytic ring-opening was performed prior to reductive
iminocyclization as a bicyclic lactone could not be formed.
Treatment of the mesylate 24L with methylamine afforded an
inseparable mixture of the epimeric open-chain amides 25L and
26L (99%). Azide hydrogenation and subsequent Boc-
protection of the iminocyclized prolines gave the separable,
protected L-gulo 27L (42%) and L-ido (44%) amides 28L. Acidic
hydrogenation of these amides afforded the L-gulo 22L (77%)
and L-ido 29L (75%) proline amides, respectively. The D-ido
target 29D was accessed with the enantiomeric route in a
comparable yield.
Previous work34 had shown that the galacto-system was not
amenable to forming a bicyclic intermediate; the methodology
utilized for accessing ido-configured targets was repeated here.
The lactone 30L was activated as the mesylate 31L by
esterification with mesyl chloride (95%, Scheme 3).34 Treatment
with methylamine led to the formation of two separable, epimeric
open-chain amides 34L and 35L (95%, 7.6:1). Acidic hydro-
genation of 34L and 35L led to the target proline amides L-
galacto 36L (94%) and L-talo 37L (90%), respectively. In the talo-
route, the lactone 32L was activated as the mesylate 33L in the
usual manner (84%).34 Methylamine-induced aminolysis gave an
18.2:1 mixture of the open-chain amides 35L and 34L (96%).
These open-chain amides formed the target proline amides as
described above. The enantiomeric proline amides D-galacto 36D
and D-talo 37D were prepared via the same route.
Scheme 3. Synthesis of Galacto- and Talo-Configured Proline
a
Amides
In contrast to the galacto route, the manno route has previously
been shown to form a stable bicyclic intermediate.45 The triflate
39D was formed from the lactone 38D, which with subsequent
hydrogenation gave the intermediate bicycle 40D (Scheme
4).34,45 Aminolytic ring-opening and acidic hydrogenation led to
the D-manno target 41D (99% over three steps). For the synthesis
of the D-gluco proline amide 46D, the lactone 42D was activated
as the mesylate 43D34 (99%) which with methylamine formed
the open-chain amide 44D (92%). In contrast to previous ido,
galacto, and talo routes that gave a mixture of epimers, retention
of the C2 stereochemistry under the reaction conditions was
observed. However, unlike these previous routes, azide hydro-
genation gave a complex mixture. Boc-protection of the mixture
allowed separation of the protected proline amide intermediate
45D in an acceptable 52% yield. Further attempts at improving
this step were not successful. Acidic hydrogenation of Boc-
protected 45D led to formation of the D-gluco proline amide 46D
a
Reagents and conditions: (i) MsCl, pyr, 0 °C rt, 1.5 h, from 30L 95%,
from 32L 84%;34 (ii) MeNH2 (33% in EtOH), 1,4-dioxane, rt, 16 h,
from 31L 95%, from 33L 96%; (iii) 10% Pd−C, H2, NaOAc, 1,4-
dioxane, rt, 3 h, then 2 M HCl, 19 h, from 34L, 94%, from 35L, 90%.
a
Scheme 4. Synthesis of Manno- and Gluco-Configured Proline Amides
a
Reagents and conditions: (i) Tf2O, pyr, DCM, −40 °C, 1 h, 99%;34 (ii) 10% Pd−C, H2, 1,4-dioxane, rt, 1 h;34 (iii) MeNH2 (33% in EtOH), rt, 18
h; then 10% Pd−C, H2, 1,4-dioxane−2 M HCl, rt, 18 h, 99% over three steps; (iv) MsCl, pyr, rt, 2 h, 99%;34 (v) MeNH2 (33% in EtOH), 16 h, rt,
92%; (vi) 10% Pd−C, H2, NaOAc, 1,4-dioxane, rt, 18 h, then Boc2O, rt, 24 h, 52%; (vii) 10% Pd−C, H2, 1,4-dioxane−2 M HCl, rt, 20 h 97%.
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(97%). The enantiomeric L-manno 41L and L-gluco 46L amides
were prepared in comparable yields via the enantiomeric route.
In the final two routes toward altro- and allo-configured
targets, the two lactones 47D and 49D were treated identically
(Scheme 5). Both lactones were activated as the mesylates 48D
accessed on a 1 g scale via the longest synthetic route from D-
glucuronolactone 15D (20 steps) in an overall yield of 16%.
2. Azetidine Acetamide 14. The 5-hydroxymethylazetidine
ester 5533 was activated as the mesylate 56 by esterification with
MsCl in pyridine (88%, Scheme 6). Treatment of the mesylate
a
Scheme 5. Synthesis of Altro- and Allo-Configured Proline
Amides
Scheme 6. Synthesis of Azetidine Acetamide
a
a
Reagents and conditions: (i) MsCl, pyr, rt, 1.5 h, 88%; (ii) NaN3,
DMF, 60 °C, 24 h, 78%; (iii) LiAlH4 (1 M in THF), THF, −78 °C,
3.5 h, then Ac2O, H2O, rt, 15 h, 79% over two steps; (iv) NaOMe,
MeOH, 60 °C, 25 h, 32%; (v) 10% Pd−C, H2, 1 M HCl−1,4-
dioxane−H2O, rt, 5 d, 99%.
56 with sodium azide proceeded smoothly to afford the azetidine
azide 57 (78%); this reaction occurred without neighboring
group participation and subsequent ring expansion.46,47
Reduction of both the ester and azide moieties was achieved
with lithium aluminum hydride, with peracetylation affording the
diacetate 58 (79% over two steps). Selective deprotection of the
O-acetyl group was achieved with sodium methoxide in methanol
and gave the protected acetamide 59 (32%). Final acidic
hydrogenation afforded the hydrochloride salt of the azetidine
acetamide 14 (99%).
Glycosidase Inhibition. The comparative inhibition of the
16 N-methyl proline amides against a panel of glycosidases
showed that all eight enantiomers with a (3R)-hydroxyl
configuration are low micromolar to nanomolar inhibitors of a
range of HexNAcases (β-N-acetylhexosaminidases) (Table 1).
The only amide with a (3S)-configuration that showed significant
inhibition is L-manno 41L (Jack bean HexNAcase: IC50 7.3 μM),
which is the enantiomer of the strongest inhibitor 41D (IC50
0.033 μM). For many glycosidases with potent iminosugar
a
Reagents and conditions: (i) MsCl, pyr, 0 °C−rt, 2 h, from 47D,
70%, from 49D, 84%;34 (ii) MeNH2 (33% in EtOH), 1,4-dioxane, rt,
17−18 h, from 48D, 69%, from 50D, 91%; (iii) 10% Pd−C, H2,
NaOAc, 1,4-dioxane, rt, 3−3.5 h, then 2 M HCl, 19−24 h, from 51D,
94%, from 52D, 91%.
(70%) and 50D (84%), respectively.34 Aminolysis with methyl-
amine gave the open-chain amides 51D (69%) and 52D (91%),
where in both cases no epimerization was observed under the
reaction conditions. Finally, acidic hydrogenation afforded the
target proline amides D-altro 53D (94%) and D-allo 54D (91%).
The enantiomeric targets L-altro 53L and L-allo 54L were
accessed by the same route in comparable yields.
The methodology developed to access the proline amide
targets is simple, robust, and scalable. The D-allo isomer 54D was
Figure 3. HexNAcase and proline amide SAR.
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Table 1. Concentration of Proline Amides Giving 50% Inhibition (IC50) of Various Glycosidases
a
b
Red: (2S). Blue: (3R). NI: no inhibition (less than 50% inhibition at 1000 μM). Value in parentheses: inhibition % at 1000 μM.
inhibition there is a 100- to 1000-fold difference for the same
enzymes between enantiomeric inhibitors.48,49
respectively). Their inhibition may be due to an equivalent
orientation between the C3, C4, and C5 hydroxyls of the
iminosugars and of the GalNAc substrate TS (Figure 4).
DGJNAc remains the only potent inhibitor of α-GalNAcase
reported,29 although DGJNAc is also a powerful inhibitor of
HexNAcase and a modest inhibitor of α-galactosidases.13
N-Methyl proline amide stereoisomers bearing a (2S,3R)-trans
configuration, such as D-talo 37D (IC50 0.15 μM), are
significantly more potent inhibitors than those with a (2R,3R)-
cis motif, such as the corresponding C2-epimer D-galacto 36D
(IC50 9.0 μM). Comparison of the strongest inhibitor D-manno
41D with its (3R)-epimer D-altro 53D (IC50 249 μM) shows a
10000-fold decrease in inhibition (Figure 3). If the stereo-
chemistry in D-manno 41D is inverted (2S,3R → 2R,3S) the
corresponding configuration is the weak inhibitor D-allo 54D
(IC50 219 μM). Change of configuration of C2 (2S→2R) yields
D-gluco 46D (IC50 0.46 μM) gives a better tolerated
epimerization (a 10-fold decrease). Alteration of the disposition
of other hydroxyls leads to similar decreases in activity: C4 leads
to D-talo 37D (5-fold decrease), C5 affords L-gulo 22L (40-fold
decrease), and changing both C4 and C5 gives L-allo 54L (10-
fold decrease).
None of the proline amides were found to inhibit β-
glucuronidases (E. coli and bovine liver), and only two were
found to show modest inhibition of α-GalNAcase (chicken
liver): D-galacto 36D and D-talo 37D (IC50 99 and 312 μM,
Figure 4. Comparison of GalNAc TS and the structures of the D-galacto
and D-talo proline amide inhibitors.
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Table 2. Comparison of Known AMDP-acetamides and Proline Amides
a
b
Red: (2S). Blue: (3R). Streptomyces cerevisiae. NI: no inhibition observed up to 1000 pM (Streptomyces plicatus). No biological data for R =
CH2NHAc L-ido,55 L-gulo,56 and L-gluco.57 No known synthesis of R = CH2NHAc D-allo, L-allo, L-altro, D-galacto, D-gulo, D-ido.
Figure 5. Pochonicine and D-talo proline amide comparison and prediction of pyrrolizidine inhibitors.
The structure−activity relationship (SAR) of the N-methyl
proline amides parallels that of the known ADMDP-acetamides
4D, 4L, 60D, 61D, 61L, 62D, and 63L12,50−55 designed by Wong
as pyrrolidine-based transition-state inhibitors (Table 2). The
ADMDP-acetamides possess inhibition similar to that of the
proline amides but are less specific in their glycosidase inhibition
profiles. Pochonicine 1 (IC50 0.0016 μM, Figure 5) is a
conformationally restricted pyrrolizidine analogue of D-talo
37D (IC50 0.15 μM).17 This constitutes a 100-fold improvement
over the proline amide equivalent. On this basis, it may be
predicted that (i) 4-epi-pochonicine 64, bearing a D-manno
configuration, should be an extremely potent inhibitor and (ii)
N-methylamide 65 of naturally occurring,56 but as yet not
synthesized, 7a-epi-alexaflorine 66 also bearing a D-manno
configuration, should also be an extremely potent inhibitor.
inhibitor (Jack bean HexNAcase: IC50 2.8 μM) (Table 3);33 the
ring-contraction leads to only a 2- to 7-fold drop in potency.
The enantiomeric amide 12D, lacks the (2S,3R)-motif and
possesses little activity (IC50 797 μM),33 analogous to the
contracted proline amides 22D (IC50 119 μM) and 54D (IC50
219 μM). The inhibitory profile of the azetidine acetamide 14
showed only modest inhibition of HexNAcases (IC50 358 μM).
Table 3. Concentration of Azetidine Acetamide Giving 50%
Inhibition (IC50) of Various Glycosidases
a
Red: (2S). Blue:(3R). NI: no inhibition (less than 50% inhibition at
b
1000 μM). Values in parentheses: inhibition % at 1000 μM.
CONCLUSION
■
The 16 stereoisomeric N-methyl proline amides prepared from
the enantiomers of glucuronolactone constitute a remarkable
new family of specific HexNAcase inhibitors; that 9 of the 16
amides are low to submicromolar inhibitors is noteworthy
(Figure 7). The SAR of the N-methyl proline amides correlates
with HexNAcase data for the isosteric Wong ADMDP-acetamide
series. Two amides demonstrate moderate activity against α-
GalNAcase. In addition, a 4-membered azetidine-acetamide was
also synthesized and shown to be an inhibitor of HexNAcases; it
Figure 6. Azetidine SAR comparison.
The N-methyl azetidine amides33 12D and 12L correlate with
the SAR established for the proline amides and ADMDP-
acetamides (Figure 6). The azetidine amide 12L may be
considered as a ring-contracted analogue of the L-gulo 22L and
L-allo 54L amides while maintaining the crucial (2S,3R)-
configuration (Figure 3). Azetidine amide 12L is a comparable
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Figure 7. Comparison of the N-methyl amides of piperidine, pyrrolidine, and azetidine carboxylic acids.
signals where used as internal references; however, where D2O was used
as the solvent acetonitrile was used as internal reference. All chemical
shifts (δ) are quoted in ppm and coupling constants (J) in Hz and were
assigned used 2D COSY and HSQC spectra. Low-resolution mass
spectra (m/z) were recorded on ESI ToF spectrometer. High-resolution
mass spectra (HRMS m/z) were carried out on a ToF spectrometer
(resolution = 10000 fwhm).
is likely that other azetidine and pipecolic acid derivatives will
also provide a new range of hexosaminidase inhibitors. The
inhibitory profile of the proline amides makes them important
scaffolds in the exploitation of their therapeutic potential toward
a range of pathologies. The D-manno N-methylproline amide
41D is among the most potent inhibitors of HexNAcases yet
described and is easy to make in gram-quantities; further study of
its derivatives is in progress to evaluate its future as a
chemotherapeutic agent. The N-methyl amide 65 of 7a-epi-
alexaflorine 66 would provide a conformationally locked
equivalent of 41D and may prove to be an even more potent
inhibitor.
Synthesis of Gulo- And Ido-Configured Proline Amides
(Scheme 2). Methyl 2,5-Dideoxy-2,5-imino-L-gulonamide 22L.
From triflate 19L: Palladium (10% on carbon, 12 mg) was added to a
solution of the triflate 19L34 (64 mg, 0.21 mmol) in 1,4-dioxane (0.6
mL). The reaction vessel was evacuated and flushed with hydrogen and
stirred for 1.5 h, after which TLC analysis (2:1, cyclohexane−ethyl
acetate) indicated the complete consumption of starting material (Rf
0.59) and the formation of a major product (Rf 0.19). The reaction
mixture was filtered through glass microfiber (GF/B). Methylamine
(33% in ethanol, 0.01 mL, 0.23 mmol) was added to the filtrate and the
reaction mixture stirred at rt for 16 h, after which TLC analysis (ethyl
acetate) indicated the complete consumption of the intermediate
bicyclic lactone 20L (Rf 0.29) and the formation of a single product (Rf
0.02). The reaction mixture was concentrated in vacuo to afford the
crude dibenzyl amide 21L, which was reacted on without further
purification. Partial data for 21L. δC (100 MHz, CD3OD): 26.2 (N-Me),
62.0 (C-5), 66.6 (C-6), 70.5 (C-4), 74.3 (C-2), 75.2 (C-3), 128.7, 128.8,
128.9, 128.9, 129.3, 129.4, 129.5, 129.5 (ArCH), 139.3, 139.5 (ArCC),
174.6 (C-1). m/z (ESI +ve): 371 (M + H+, 80), 393 (M + Na+, 85), 741
(2M + H+, 100), 763 (2M + Na+, 96). HRMS m/z (ESI +ve): found
371.1962 [M + H+], C21H27N2O4 requires 371.1965. Palladium (10% on
carbon, 4.5 mg) was added to a solution of the crude dibenzyl amide 21L
in a 5:1 1,4-dioxane−2 M aqueous hydrochloric acid mixture (2.5 mL).
The flask was evacuated and flushed with hydrogen and stirred for 16 h,
after which the reaction mixture was filtered through glass microfiber
(GF/B) and concentrated in vacuo. The crude residue was dissolved in 2
M aqueous hydrochloric acid and loaded onto a short column of Dowex
(50W-X8, H+) and the resin washed with water. The L-gulo amide 22L
was liberated with 2 M aqueous ammonia, and the ammoniacal fractions
were concentrated in vacuo to afford the L-gulo amide 22L (15.5 mg,
66% over three steps) as a yellow oil. From Boc-protected 27L:
Palladium (10% on carbon, 24 mg) was added to a solution of the
dibenzyl ether 27L (61 mg, 0.13 mmol) in a 1:1 1,4-dioxane−2 M
aqueous hydrochloric acid mixture (2.5 mL). The reaction vessel was
evacuated and flushed with hydrogen and stirred for 16 h. The reaction
mixture was filtered and the filtrate stirred at 50 °C for 2 h and
concentrated in vacuo. The crude residue was loaded onto a short
column of Dowex (50W-X8, H+) and the resin washed with water. The
L-gulo amide 22L was liberated with 2 M aqueous ammonia and the
ammoniacal fractions concentrated in vacuo to afford the L-gulo amide
EXPERIMENTAL SECTION
■
In Vitro Enzyme Inhibition: IC50 Determination. The enzymes β-
N-acetylhexosaminidases (from human placenta, bovine kidney, jack
bean, and Aspergillus oryzae), α-N-acetylgalactosaminidase (from
chicken liver), β-N-acetylgalactosaminidase (from Aspergillus oryzae),
β-glucuronidases (Escherichia coli and bovine liver), and p-nitrophenyl
glycosides were purchased from Sigma-Aldrich Co. The cell lysate of
human acute amyloid leukemia cell line HL-60 (RBRC) was cultured in
RPMI 1640 medium containing 10% fetal calf serum, 100 units·mL−1 of
penicillin, and 100 μg·mL−1 streptomycin (Invitrogen) at 37 °C under
5% CO2 and used as the source of β-N-acetylhexosaminidase and β-N-
acetylgalactosaminidase. The glycosidase activities were determined
using an appropriate p-nitrophenyl glycoside as substrate at the
optimum pH of each enzyme (the HL60 lysate assay was performed
at pH 4.5). The reaction mixture (1 mL) contained 2 mM of the
substrate and the appropriate amount of enzyme. The reaction was
stopped by adding 2 mL of 400 mM Na2CO3. The released p-
nitrophenol was measured spectrometrically at 400 nm.
General Experimental Methods. All commercial reagents were
used as supplied. Pyridine and DMF were purchased dry. All other
solvents were used as supplied, without prior purification. All reactions
were performed under an inert nitrogen or argon atmosphere and were
maintained by an inflated balloon. All solutions are saturated unless
otherwise stated. Thin-layer chromatography (TLC analysis) was
performed on aluminum sheets coated with 60 F254 silica. Sheets were
visualized using a dip of 0.2% w/v cerium(IV) sulfate and 5%
ammonium molybdate in 2 M sulfuric acid solution. Flash
chromatography was performed on Sorbsil C60 40/60 silica. Melting
points were recorded on a Kofler hot block and are uncorrected. Optical
rotations were recorded on a polarimeter with a path length of 1 dm.
Concentrations are quoted in g·100 mL−1. Infrared spectra were
recorded using thin films on either NaCl or Ge plates. Only the
characteristic peaks are quoted. Nuclear magnetic resonance (NMR)
spectra were recorded in the deuterated solvent stated where residual
22L (19 mg, 77%) as a pale-yellow oil: [α]25 −9.0 (c 0.32 in H2O)
D
[lit.57 [α]25D −4.3 (c 0.675 in H2O)]. νmax (thin film): 3325 (br s, NH,
3404
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OH), 1648 (s, CO), 1562 (s, CO). δH (400 MHz, D2O): 2.63 (3 H, s, N-
Me), 3.40 (1 H, a-q, H-5, J5,4 = J5,6 = J5,6′ 5.6), 3.50 (1 H, d, H-2, J2,3 2.5),
3.56 (1 H, dd, H-6, J6,5 6.8, J6,6′ 11.2), 3.68 (1 H, dd, H-6′, J6′,5 6.1, J6′,6
11.2), 3.98 (1 H, dd, H-4, J4,3 2.6, J4,5 4.8), 4.06 (1 H, a-t, H-3, J3,2 = J3,4
2.6). δC (100 MHz, D2O): 26.1 (N-Me), 61.1 (C-5), 61.4 (C-6), 66.4
(C-2), 76.6 (C-4), 80.8 (C-3), 175.6 (C-1). HRMS m/z (ESI +ve):
found 213.0850 [M + Na+], C7H14N2NaO4 requires 213.0846. m/z (ESI
+ve): 191 (M + H+, 100). For the enantiomer 22D; [α]25D +7.9 (c 0.30
in H2O).
(2M + Na+, 100). HRMS m/z (ESI +ve): found 493.2310 [M + Na+],
C26H34N2NaO6 requires 493.2309. For the enantiomer 28D (Rf 0.36):
[α]25D +15.8 (c 0.96 in CHCl3).
Methyl 2,5-Dideoxy-2,5-imino-L-idonamide 29L. Palladium (10%
on carbon, 20 mg) was added to a solution of the Boc-protected amide
28L (48 mg, 0.102 mmol) in a 1:1 1,4-dioxane−2 M aqueous
hydrochloric acid mixture (4 mL). The reaction vessel was evacuated
and flushed with hydrogen and stirred for 16 h. The reaction mixture was
filtered and the filtrate stirred at 45 °C for 2 h and concentrated in vacuo.
The crude residue was loaded onto a short column of Dowex (50W-X8,
H+) and the resin washed with water. The product was liberated with 2
M aqueous ammonia, and the ammoniacal fractions were combined and
concentrated in vacuo to afford the L-ido amide 29L as a pale yellow oil
(14.5 mg, 75%): [α]25D +44.3 (c 0.92 in CHCl3). νmax (thin film): 3450
(br s, NH, OH), 1646 (s, CO), 1560 (s, CO). δH (500 MHz, D2O): 2.68
(3 H, s, N-Me), 3.69 (1 H, m, H-5), 3.70 (1 H, dd, H-6, J6,5 6.8, J6,6′ 11.4),
3.79 (1 H, dd, H-6′, J6′,5 6.1, J6′,6 11.4), 4.18 (1 H, d, H-2, J2,3 4.5), 4.25 (1
H, dd, H-4, J4,3 1.0, J4,5 3.3), 4.32 (1 H, dd, H-3, J3,4 1.0, J3,2 4.5). δC (125
MHz, D2O): 26.2 (N-Me), 58.9 (C-6), 62.7 (C-5), 63.8 (C-2), 76.4 (C-
4), 77.2 (C-3), 170.1 (C-1). m/z (ESI +ve): 191 (M + H+, 100). HRMS
m/z (ESI +ve): found 213.0843 [M + Na+], C7H14N2NaO4 requires
213.0846. For the enantiomer 29D: [α]25D −46.7 (c 0.95 in H2O).
Synthesis of Galacto- and Talo-Configured Proline Amides
(Scheme 3). Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-metha-
nesulfonyl-L-talonamide 34L/Methyl 5-Azido-3,6-di-O-benzyl-5-
deoxy-2-O-methanesulfonyl-L-galactonamide 35L. From lactone
31L: Methylamine (33% in ethanol, 90 μL, 2.13 mmol) was added to
a solution of the lactone 31L34 (197 mg, 0.426 mmol) in 1,4-dioxane (10
mL). The reaction was stirred for 16 h, after which TLC analysis (3:7,
cyclohexane−ethyl acetate) indicated the almost complete consumption
of the starting material (Rf 0.96) and the formation of a major (Rf 0.35)
and a minor product (Rf 0.64). The reaction mixture was concentrated in
vacuo and the crude residue purified by flash chromatography (7:3 to
1:4, cyclohexane−ethyl acetate) to afford the L-talo amide 34L (Rf 0.35,
165 mg, 84%) as a clear, viscous oil and the minor L-galacto amide 35L
(Rf 0.64, 22 mg, 11%) as a clear oil which crystallized on standing. From
lactone 33L: Methylamine (33% in EtOH, 0.11 mL, 0.89 mmol) was
added to a solution of the lactone 33L (137 mg, 0.297 mmol) in 1,4-
dioxane (2.5 mL) under argon. The reaction mixture was stirred for 16 h,
after which TLC analysis (3:7, cyclohexane−ethyl acetate) indicated the
complete consumption of the starting material (Rf 0.96) and the
formation of a major (Rf 0.64) and a minor product (Rf 0.35). The
reaction was concentrated in vacuo and the crude residue was purified by
flash chromatography (7:3 to 11:9, cyclohexane−ethyl acetate) to afford
the L-talo amide 34L (Rf 0.35, 7 mg, 5%) as a clear oil and the L-galacto
amide 35L (Rf 0.64, 133 mg, 91%) as a white crystalline solid. Data for L-
talo amide 34L: [α]25D +66.6 (c 2.01 in CHCl3). νmax (thin film): 3418
(br s, OH), 2105 (s, N3), 1666 (s, CO), 1560 (s, CO), 1455 (s, SO2),
1179 (s, SO2). δH (400 MHz, CDCl3): 2.65 (1 H, d, 4-OH, JOH,4 8.8),
2.83 (3 H, d, N-Me, JH,NH 5.1), 3.05 (3 H, s, SO2-Me), 3.76 (1 H, dd, H-
6, J6,5 5.1, J6,6′ 9.9), 3.78 (1 H, dd, H-6′, J6′,5 6.6, J6′,6 9.9), 3.82 (1 H, ddd,
H-4, J4,5 1.3, J4,OH 8.8, J4,3 9.3), 3.85 (1 H, ddd, H-5, J5,4 1.3, J5,6 5.1, J5,6′
6.6), 4.24 (1 H, dd, H-3, J3,2 1.4, J3,4 9.3), 4.55 (1 H, d, OCH2Ph, Jgem
11.9), 4.57 (1 H, d, OCH2Ph, Jgem 10.9), 4.59 (1 H, d, OCH2Ph, Jgem
11.9), 4.77 (1 H, d, OCH2Ph, Jgem 10.9), 5.46 (1 H, d, H-2, J2,3 1.4), 6.46
(1 H, q, N-H, JNH,H 5.1), 7.30−7.40 (10 H, m, Ar-H). δC (100 MHz,
CDCl3): 26.4 (N-Me), 39.1 (SO2-Me), 60.7 (C-5), 70.3 (C-4), 71.3 (C-
6), 73.4, 73.8 (OCH2Ph), 79.0 (C-3), 79.4 (C-2), 127.9, 128.1, 128.6,
128.8 (ArCH), 136.5, 137.4 (ArCC), 167.4 (C-1). m/z (ESI +ve): 515
(M + Na+, 100). (ESI −ve): 491 ([M − H]−, 64), 527 (M + 35Cl−, 52),
529 (M + 37Cl−, 18), 983 ([2M − H]−, 100). HRMS m/z (ESI +ve):
found 515.1568 [M + Na+], C22H28N4NaO7S requires 515.1571. For the
Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-methanesulfonyl-L-
idonamide 25L/Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-meth-
anesulfonyl-L-gulonamide 26L. Methylamine (33% in ethanol, 0.11
mL, 0.86 mmol) was added to a solution of the mesylate 24L34 (132 mg,
0.29 mmol) in 1,4-dioxane (6 mL) at rt. The reaction mixture was stirred
for 16 h, after which TLC analysis (3:7, cyclohexane−ethyl acetate)
indicated the complete consumption of the starting material (Rf 0.72)
and the formation of two major, cospotting products (Rf 0.60). The
reaction mixture was concentrated in vacuo and coevaporated with
dichloromethane (3 × 10 mL) and the residue purified by flash
chromatography (9:1 to 1:1 cyclohexane−ethyl acetate) to afford an
inseparable 1.3:1 (A:B) epimeric mixture of the L-gulo amide 26L and
the L-ido amide 25L (140 mg, 99%) as a yellow oil. νmax (thin film): 3405
(br s, OH), 2100 (s, N3), 1668 (s, CO), 1558 (s, CO), 1358 (s, SO2),
1176 (s, SO2). δH (400 MHz, CD3OD): 2.75 (3 H, s, N-MeB), 2.76 (3 H,
s, N-MeA), 3.14 (3 H, s, SO2-MeB), 3.14 (3 H, s, SO2-MeA), 3.52−3.56
(2 H, m, H-6A, H-6B), 3.61−3.67 (2 H, m, H-6′A, H-6′B), 3.75 (1 H, ddd,
H-5A or H-5B, J 3.7, J 5.6, J 6.9), 3.77−3.80 (1 H, m, H-5A or H-5B), 3.82
(1 H, dd, H-4B, J4,5 4.5, J4,3 5.4), 3.90 (1 H, dd, H-4A, J4,3 4.3, J4,5 5.6), 3.96
(1 H, dd, H-3A, J3,4 4.3, J3,2 5.1), 4.05 (1 H, dd, H-3B, J3,2 4.3, J3,4 5.4), 4.49
(1 H, d, OCH2Ph, Jgem 11.9), 4.50 (1 H, d, OCH2Ph, Jgem 12.1), 4.53 (1
H, d, OCH2Ph, Jgem 11.3), 4.54 (1 H, d, OCH2Ph, Jgem 11.9), 4.56 (1 H,
d, OCH2Ph, Jgem 12.1), 4.62 (1 H, d, OCH2Ph, Jgem 10.9), 4.64 (1 H, d,
OCH2Ph, Jgem 11.3), 4.70 (1 H, d, OCH2Ph, Jgem 10.9), 5.13 (1 H, d, H-
2B, J2,3 4.3), 5.18 (1 H, d, H-2A, J2,3 5.1), 7.26−7.35 (20 H, m, Ar-HA, Ar-
HB). δC (100 MHz, CD3OD): 26.5, 26.5 (N-MeA, N-MeB), 38.4, 38.6
(SO2-MeA, SO2-MeB), 63.1, 64.4 (C-5A, C-5B), 71.0, 71.1 (C-6A, C-6B),
71.6 (C-4B), 71.8 (C-4A), 74.2, 74.3, 75.2, 76.7 (OCH2PhA, OCH2PhB),
79.0 (C-2A), 79.8 (C-2B), 80.6, 80.6 (C-3A, C-3B), 128.8, 128.9, 129.0,
129.4, 129.4, 129.5 (ArCHA, ArCHB), 138.9, 139.2, 139.3 (ArCCA,
ArCCB), 169.6 (C-1A, C-1B). m/z (ESI +ve): 515 (M + Na+, 18), 1007
(2M + Na+, 100). (ESI −ve): 491 ([M − H]−, 65), 983 ([2M − H]−,
100). HRMS m/z (ESI +ve): found 515.1567 [M + Na+],
C22H28N4NaO7S requires 515.1571. For the enantiomeric mixture
25D/26D: −.
Methyl 2-N-(tert-Butoxycarbonyl)-3,6-di-O-benzyl-2,5-dideoxy-
2,5-imino-L-gulonamide 27L/Methyl 2-N-(tert-Butoxycarbonyl)-3,6-
di-O-benzyl-2,5-dideoxy-2,5-imino-L-idonamide 28L. Palladium
(10% on carbon, 54 mg) and sodium acetate (45 mg, 0.46 mmol)
were added to a solution of the inseparable L-gulo and L-ido amides 25L/
26L (1:1.3 mixture, 134 mg, 0.27 mmol) in 1,4-dioxane (5 mL). The
reaction vessel was evacuated and flushed with hydrogen and stirred for
3 h, after which TLC analysis (2:1, toluene−acetone) indicated the
complete consumption of the starting materials (Rf 0.54) and the
presence of a major product (Rf 0.07). The reaction was filtered through
glass microfiber (GF/A), and di-tert-butyl dicarbonate (45 mg, 0.46
mmol) was added portionwise to the filtrate. This reaction mixture was
stirred for 16 h, after which TLC analysis (2:1, toluene−acetone)
indicated the complete consumption of the intermediate product (Rf
0.07) and the formation of two products (Rf 0.54, 0.36). The reaction
mixture was concentrated in vacuo and the crude residue purified by
flash chromatography (1:0 to 3:2, toluene−acetone) to afford the
cyclized Boc-protected amides, L-gulo 27L (Rf 0.54, 54 mg, 42%) and L-
ido 28L (Rf 0.36, 56 mg, 44%) as clear oils. Partial data for L-gulo amide
27L (Rf 0.54): [α]25D −10.0 (c 0.58 in CHCl3). νmax (thin film): 3321 (br
s, OH), 1666 (s, CO), 1552 (s, CO). m/z (ESI +ve): 493 (M + Na+, 30),
963 (2M + Na+, 100). HRMS m/z (ESI +ve): found 493.2306 [M +
Na+], C26H34N2NaO6 requires 493.2309. For the enantiomer 27D (Rf
0.54): [α]25D +2.9 (c 1.0 in CHCl3). Partial data for L-ido amide 28L (Rf
0.36): [α]25D −21.7 (c 1.17 in CHCl3). νmax (thin film): 3326 (br s, OH),
1657 (s, CO), 1550 (s, CO). m/z (ESI +ve): 493 (M + Na+, 25), 963
enantiomer 34D: [α]25 −71.1 (c 2.05 in CHCl3). Data for L-galacto
D
amide 35L: mp 114−116 °C. [α]25D +33.9 (c 1.08 in CHCl3). νmax (thin
film): 3407 (br s, OH), 2105 (s, N3), 1668 (s, CO), 1555 (s, CO), 1455
(s, SO2), 1178 (s, SO2). δH (400 MHz, CDCl3): 2.86 (3 H, d, N-Me,
JH,NH 4.5), 3.11 (3 H, s, SO2-Me), 3.15 (1 H, d, 4-OH, JOH,4 8.2), 3.70 (1
H, ddd, H-4, J4,5 1.3, J4,OH 8.2, J4,3 9.3), 3.75 (1 H, ddd, H-5, J5,4 1.3, J5,6
4.3, J5,6′ 6.6), 3.84 (1 H, dd, H-6, J6,5 4.3, J6,6′ 10.4), 3.87 (1 H, dd, H-6′,
J6′,5 6.6, J6′,6 10.4), 4.30 (1 H, dd, H-3, J3,2 1.7, J3,4 9.3), 4.52 (1 H, d,
3405
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OCH2Ph, Jgem 11.1), 4.58 (3 H, a-d, 3 × OCH2Ph, J 11.4), 5.32 (1 H, d,
H-2, J2,3 1.7), 6.63 (1 H, q, N-H, JNH,H 4.5), 7.24−7.37 (10 H, m, Ar-H).
δC (100 MHz, CDCl3): 26.6 (N-Me), 38.3 (SO2-Me), 59.9 (C-5), 70.4
(C-4), 71.5 (C-6), 73.8, 75.2 (OCH2Ph), 78.7 (C-3), 79.2 (C-2), 127.9,
128.1, 128.2, 128.4, 128.7, 128.7 (ArCH), 137.1, 137.3 (ArCC), 168.4
(C-1). m/z (ESI +ve): 515 (M + Na+, 100). HRMS m/z (ESI +ve):
found 515.1575 [M + Na+], C22H28N4NaO7S requires 515.1571. For the
enantiomer 35D: mp 114−116 °C. [α]25D −36.1 (c 1.10 in CHCl3).
Methyl 2,5-Dideoxy-2,5-imino-L-galactonamide 36L. Palladium
(10% on carbon, 75 mg) was added to a solution of the L-talo amide 34L
(165 mg, 0.34 mmol) and sodium acetate (72 mg, 0.88 mmol) in 1,4-
dioxane (8 mL). The flask was evacuated and flushed with hydrogen and
stirred for 6 h, after which TLC analysis (99:1, ethyl acetate−
triethylamine) indicated the complete consumption of the starting
material (Rf 0.72) and the formation of a major product (Rf 0.08).
Aqueous hydrochloric acid (2 M, 1.4 mL) was added, the reaction vessel
evacuated and flushed with hydrogen, and the reaction stirred for 16 h,
after which the reaction mixture was filtered through glass microfiber
(GF/A) and concentrated in vacuo. The crude residue was loaded onto
a short column of Dowex (50W-X8, H+) and the resin washed with
water. The product was liberated with 2 M aqueous ammonia, and the
ammoniacal fractions were combined and concentrated in vacuo to
The reaction was filtered through glass microfiber (GF/B) and
concentrated in vacuo. The crude residue was dissolved in 2 M aqueous
HCl and loaded onto a short column of Dowex (50W-X8, H+), the
column washed with water, and the product liberated with 2 M aqueous
ammonia to afford the proline amide 41D (58 mg, 99%) as a yellow oil:
[α]25D −3.8 (c 1.01 in MeOH). νmax (thin film): 3331 (br s, OH, NH),
1651 (s, CO), 1547 (s, CO). δH (400 MHz, D2O): 2.65 (3 H, s, N-Me),
3.00 (1 H, ddd, H-5, J5,6′ 4.2, J5,6 6.2, J5,4 7.2), 3.41 (1 H, d, H-2, J2,3 6.5),
3.50 (1 H, dd, H-6, J6,5 6.2, J6,6′ 11.6), 3.61 (1 H, dd, H-6′, J6′,5 4.2, J6′,6
11.6), 3.72 (1 H, dd, H-4, J4,3 6.5, J4,5 7.2), 3.94 (1 H, t, H-3, J3,2 = J3,4 6.5).
δC (100 MHz, D2O): 26.2 (N-Me), 62.3 (C-6), 63.0 (C-5), 64.2 (C-2),
78.1 (C-4), 80.5 (C-3), 175.1 (C-1). m/z (ESI +ve): 191 (M + H+, 100),
213 (M + Na+, 68), 403 (2M + Na+, 32). (ESI −ve): 189 ([M − H]−,
95), 225 (M + Cl−, 100), 379 ([2M − H]−, 52). HRMS m/z (ESI +ve):
found 191.1026 [M + H+], C7H15N2O4 requires 191.1026. For the
enantiomer 41L: [α]25D +1.9 (c 1.01 in MeOH).
Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-methanesulfonyl-D-
mannonamide 44D. Methylamine solution (33% in absolute ethanol,
0.24 mL, 1.90 mmol) was added to a solution of the mesylate 43D34
(0.18 g, 0.38 mmol) in 1,4-dioxane (3 mL) at rt. The reaction was stirred
for 16 h, after which TLC analysis (1:1, ethyl acetate−cyclohexane)
indicated the complete consumption of the starting material (Rf 0.83)
and the formation of a major product (Rf 0.29). The reaction was
concentrated in vacuo, and the remaining residue was purified by flash
chromatography (1:4, ethyl acetate−cyclohexane) to afford the amide
44D (170 mg, 92%) as a colorless oil: [α]20D −52.8 (c 0.71 in CHCl3).
νmax (thin film): 3394 (br s, OH, NH), 2101 (s, N3), 1668 (s, CO). δH
(400 MHz, CDCl3): 2.85 (3 H, d, N-Me, JH,NH 4.8), 3.06 (3 H, s, SO2-
Me), 3.51 (1 H, br s 4-OH), 3.58 (1 H, ddd, H-5, J5,6′ 3.4, J5,6 6.4, J5,4 9.6),
3.71 (1 H, m, H-4), 3.72 (1 H, dd, H-6, J6,5 6.4, J6,6′ 10.0), 3.89 (1 H, dd,
H-6′, J6′,5 3.4, J6′,6 10.0), 4.25 (1 H, dd, H-3, J3,4 1.4, J3,2 4.0), 4.57 (2 H, s,
OCH2Ph), 4.63 (1 H, d, OCH2Ph, Jgem 10.8), 4.75 (1 H, d, OCH2Ph,
Jgem 10.8), 5.25 (1 H, d, H-2, J2,3 4.0), 6.55 (1 H, q, N-H, JNH,H 4.8),
7.28−7.40 (10 H, m, Ar-H). δC (100 MHz, CDCl3): 26.6 (N-Me), 39.1
(SO2-Me), 61.9 (C-5), 70.3 (C-6), 70.4 (C-4), 73.7, 74.3 (OCH2Ph),
77.1 (C-3), 79.7 (C-2), 127.8, 128.0, 128.6, 128.7, 128.7, 128.7, 128.8
(ArCH), 136.8, 137.7 (ArCC), 167.1 (C-1). m/z (ESI +ve): 493 (M +
H+, 9), 515 (M + Na+, 30), 1007 (2M + Na+, 100). HRMS m/z (ESI
+ve): found 515.1567 [M + Na+], C22H28N4NaO7S requires 515.1571.
For the enantiomer 44L: [α]20D +59.2 (c 0.74 in CHCl3).
afford the L-galacto amide 36L (60 mg, 94%) as a yellow oil: [α]25
D
−41.1 (c 0.24 in H2O). νmax (thin film): 3332 (br s, OH, NH), 1645 (s,
CO), 1554 (s, CO). δH (400 MHz, D2O): 2.66 (3 H, a-s, N-Me), 3.34 (1
H, a-q, H-5, J5,4 = J5,6 = J5,6′ 5.8), 3.53 (1 H, dd, H-6, J6,5 6.3, J6,6′ 11.5),
3.61 (1 H, dd, H-6′, J6′,5 4.8, J6′,6 11.5), 3.81 (1 H, d, H-2, J2,3 6.1), 4.23 (1
H, dd, H-4, J4,3 5.5, J4,5 6.2), 4.27 (1 H, a-t, H-3, J3,2 = J3,4 5.8). δC (100
MHz, D2O): 25.9 (N-Me), 59.9 (C-5), 61.6 (C-6), 62.5 (C-2), 72.5 (C-
4), 72.8 (C-3), 174.6 (C-1). m/z (ESI +ve): 191 (M + H+, 100), 213 (M
+ Na+, 68). HRMS m/z (ESI +ve): found 191.1025 [M + H+],
C7H15N2O4 requires 191.1026. For the enantiomer 36D: mp 85−87 °C
(from 2-propanol). [α]25D +46.6 (c 0.26 in H2O).
Methyl 2,5-Dideoxy-2,5-imino-L-talonamide 37L. Palladium (10%
on carbon, 10 mg) was added to a solution of the mesylate 35L34 (22
mg, 0.04 mmol) and sodium acetate (16 mg, 0.20 mmol) in 1,4-dioxane
(2 mL). The flask was evacuated, flushed with hydrogen, and stirred for
6 h, after which TLC analysis (99:1, ethyl acetate−triethylamine)
indicated the complete consumption of the starting material (Rf 0.82)
and the formation of a major product (Rf 0.18). Aqueous hydrochloric
acid (2 M, 0.4 mL) was added, the reaction vessel evacuated and flushed
with hydrogen, and the reaction stirred for 16 h. The reaction mixture
was filtered through glass microfiber (GF/A) and concentrated in vacuo.
The crude residue loaded onto a short column of Dowex (50W-X8, H+)
and the resin washed with water. The product was liberated with 2 M
aqueous ammonia, and the ammoniacal fractions were combined and
concentrated in vacuo to afford the L-talo amide 37L (5.5 mg, 90%) as a
yellow oil: [α]25D −5.0 (c 0.28 in H2O). νmax (thin film): 3335 (br s, OH,
NH), 1648 (s, CO), 1558 (s, CO). δH (400 MHz, D2O): 2.66 (3 H, s, N-
Me), 3.30 (1 H, a-td, H-5, J5,4 4.1, J5,6 = J5,6′ 6.6), 3.44 (1 H, d. H-2, J2,3
8.2), 3.52 (1 H, dd, H-6, J6,5 6.6, J6,6′ 11.0), 3.67 (1 H, dd, H-6′, J6′,5 6.6,
J6′,6 11.0), 4.05 (1 H, dd, H-3, J3,4 4.1, J3,2 8.2), 4.08 (1 H, t, H-4, J4,3 = J4,5
4.1). δC (100 MHz, D2O): 26.3 (N-Me), 60.9 (C-5), 61.0 (C-6), 63.7
(C-2), 72.7 (C-4), 76.9 (C-3), 175.5 (C-1). m/z (ESI +ve): 191 (M +
H+, 100), 213 (M + Na+, 55). HRMS m/z (ESI +ve): found 191.1028
[M + H+], C7H15N2O4 requires 191.1026. For the enantiomer 37D:
[α]25D +6.9 (c 0.30 in H2O).
Methyl 2,5-Dideoxy-2,5-imino-D-gluconamide 46D. Palladium
(10% on carbon, 56 mg) and sodium acetate (47 mg, 0.57 mmol)
were added to a solution of the amide 44D (140 mg, 0.28 mmol) in 1,4-
dioxane (10 mL). The flask was evacuated and flushed three times with
argon and three times with hydrogen. The reaction was stirred at rt for
18 h, after which TLC analysis (1:1, ethyl acetate−cyclohexane)
indicated the complete consumption of starting material (Rf 0.29) and
the formation of a baseline product. The reaction mixture was filtered
through glass microfiber (GF/B) and was concentrated in vacuo to
afford a yellow oil. The crude residue was dissolved in 1,4-dioxane (10
mL), and di-tert-butyl dicarbonate (0.13 mL, 0.57 mmol) was added.
After 24 h, TLC analysis (2:1, toluene−acetone) indicated the
consumption of starting material and the formation of a major product
(Rf 0.42). The solvents were removed in vacuo, and the remaining
residue was purified by flash chromatography (9:1, toluene−acetone) to
yield the N-Boc-protected intermediate 45D as a yellow oil (69 mg,
52%). This intermediate was dissolved in a 1:1 1,4-dioxane−2 M HCl
mixture (2.8 mL), and palladium (10% on carbon, 27 mg) was added.
The flask was evacuated and flushed three times with argon and three
times with hydrogen. The reaction was stirred at rt for 20 h, after which
TLC analysis (2:1, toluene−acetone) indicated the complete
consumption of starting material (Rf 0.42) and the formation of a
baseline product. The reaction mixture was filtered through glass
microfiber (GF/B) and concentrated in vacuo, and the residue was
loaded onto a short column of Dowex (50W-X8, H+) and washed with
water. The product was liberated with 2 M aqueous ammonia, and the
ammoniacal fractions were combined and concentrated in vacuo to
Synthesis of Manno- and Gluco-Configured Proline Amides
(Scheme 4). Methyl 2,5-Dideoxy-2,5-imino-D-mannonamide 41D.
Palladium (10% on carbon, 32 mg) was added to a solution of triflate
39D34 (150 mg, 0.3 mmol) in 1,4-dioxane (2 mL). The reaction was
purged with argon and then hydrogen and allowed to stir for 1 h, after
which TLC analysis (2:1, cyclohexane−ethyl acetate) indicated the
complete consumption of the starting material (Rf 0.80) and the
formation of a major product (Rf 0.20). The reaction was filtered
through glass microfiber (GF/B) and methylamine (33% in EtOH, 0.07
mL, 0.58 mmol) was added. The reaction was stirred at rt for 18 h after
which TLC analysis indicated only baseline material. Palladium (10% on
carbon, 64 mg) and 2 M aqueous HCl (0.6 mL) were added, and the
reaction was purged with argon and then hydrogen and stirred for 18 h.
afford the proline amide 46D (27.0 mg, 97%) as a colorless oil: [α]25
D
+49.7 (c 0.86 in H2O) [lit.57 [α]23D +65.06 (c 0.8 in H2O)]. νmax (thin
film): 3304 (br s, OH, NH), 1636 (s, CO), 1544 (s, CO). δH (400 MHz,
3406
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D2O): 2.65 (3 H, s, N-Me), 3.04 (1 H, m, H-5), 3.52 (1H, dd, H-6, J6,5
6.4, J6,6′ 11.6), 3.59 (1 H, dd, H-6′, J6′,5 4.8, J6′,6 11.6), 3.80 (1 H, dd, H-4,
J4,5 4.0, J4,3 3.2), 3.86 (1 H, d, H-2, J2,3 5.8), 4.13 (1 H, dd, H-3, J3,4 3.2, J3,2
5.8). δC (100 MHz, D2O): 25.9 (N-Me), 62.8 (C-6), 63.7 (C-2), 64.7
(C-5), 77.9 (C-3), 78.2 (C-4), 173.9 (C-1). m/z (ESI +ve): 191 (M +
H+, 56), 213 (M + Na+, 100). HRMS m/z (ESI +ve): found 213.0849
[M + Na+], C7H14N2NaO4 requires 213.0846. For the enantiomer 46L:
[α]20D −54.0 (c 0.44 in H2O).
(thin film): 3402 (br s, OH), 2101 (s, N3), 1666 (s, CO), 1544 (s, CO),
1360 (s, SO2) 1176 (s, SO2). δH (400 MHz, CD3CN): 2.76 (3 H, d, N-
Me, JH,NH 4.8), 3.16 (3 H, s, SO2-Me), 3.63 (1 H, dd, H-6, J6,5 7.7, J6,6′
10.4), 3.69 (1 H, dd, H-6′, J6′,5 3.8, J6′,6 10.4), 3.71 (1 H, d, 4-OH, JOH,4
6.6), 3.80 (1 H, ddd, H-4, J4,5 3.0, J4,OH 6.6, J4,3 9.3), 3.87 (1 H, a-dt, H-5,
J5,4 = J5,6′ 3.5, J5,6 7.7), 4.05 (1 H, dd, H-3, J3,2 1.8, J3,4 9.3), 4.43 (1 H, d,
OCH2Ph, Jgem 11.1), 4.46 (1 H, d, OCH2Ph, Jgem 11.1), 4.48 (1 H, d,
OCH2Ph, Jgem 12.0), 4.51 (1 H, d, OCH2Ph, Jgem 12.0), 5.12 (1 H, d, H-
2, J2,3 1.8), 7.05 (1 H, br q, N-H, JNH,H 4.8), 7.25−7.38 (10 H, m, Ar-H).
δC (100 MHz, CD3CN): 26.5 (N-Me), 38.7 (SO2-Me), 64.5 (C-5), 70.3
(C-6), 71.0 (C-4), 73.8, 74.8 (OCH2Ph), 79.1 (C-2), 79.9 (C-3), 128.7,
128.8, 129.3, 129.4 (ArCH), 138.4, 139.1 (ArCC), 168.9 (C-1). m/z
(ESI +ve): 493 (M + H+, 16), 515 (M + Na+, 100). (ESI −ve): 491 ([M
− H]−, 100), 427 (M + 35Cl−, 65), 429 (M + 37Cl−, 25), 983 ([2M −
H]−, 45). HRMS m/z (ESI +ve): found 515.1570 [M + Na+],
C22H28N4NaO7S requires 515.1571. For the enantiomer 52L: mp 85−
87 °C. [α]25D +2.4 (c 1.60 in CHCl3).
Methyl 2,5-Dideoxy-2,5-imino-D-allonamide 54D. Palladium (10%
on carbon, 650 mg) and sodium acetate (1.08 g, 13.21 mmol) were
added to a solution of the amide 52D (3.25 g, 6.61 mmol) in 1,4-dioxane
(65 mL) at rt. The flask was evacuated and flushed with hydrogen and
stirred for 3.5 h, after which TLC analysis (1:1, toluene−ethyl acetate)
indicated the complete consumption of starting material (Rf 0.70) and
the formation of a major product (Rf 0.28). Aqueous hydrochloric acid
(2 M, 13 mL) was added to the reaction mixture and the flask evacuated
and flushed with hydrogen. The reaction mixture was stirred for 24 h.
The reaction was filtered glass microfiber (GF/B) and the filtrate
concentrated in vacuo. The crude residue was loaded onto a short
column of Dowex (50W-X8, H+) and the resin washed with water. The
product was liberated with 2 M aqueous ammonia and the ammoniacal
fractions were combined and concentrated in vacuo to afford the D-allo
amide 54D (1.15 g, 91%) as a yellow oil: [α]25D +14.6 (c 0.27 in H2O).
νmax (thin film): 3272 (br s, OH, NH), 1643 (s, CO), 1543 (s, CO). δH
(400 MHz, D2O): 2.66 (3 H, s, N-Me), 3.31 (1 H, a-td, H-5, J5,6′ 4.0, J5,4
= J5,6 6.1), 3.58 (1 H, dd, H-6, J6,5 5.6, J6,6′ 12.1), 3.69 (1 H, dd, H-6′, J6′,5
4.0, J6′,6 12.1), 3.74 (1 H, d, H-2, J2,3 4.3), 3.84 (1 H, dd, H-4, J4,3 4.7, J4,5
6.7), 4.07 (1 H, a-t, H-3, J3,2 = J3,4 4.5). δC (100 MHz, D2O): 26.3 (N-
Me), 61.1 (C-6), 63.2 (C-5), 64.8 (C-2), 71.9 (C-4), 75.0 (C-3), 172.8
(C-1). m/z (ESI +ve): 191 (M + H+, 100). HRMS m/z (ESI +ve): found
213.0842 [M + Na+], C7H14N2NaO4 requires 213.0846. For the
enantiomer 54L. [α]25D −17.3 (c 0.33 in H2O).
Synthesis of the Azetidine-acetamide (Scheme 6). Methyl 2-
N,3-O-Dibenzyl-2,4-dideoxy-2,4-imino-5-O-methanesulfonyl-D-ri-
bonate 56. Methanesulfonyl chloride (0.08 mL, 2.45 mmol) was added
to a solution of the methyl ester 5533 (520 mg, 1.52 mmol) in pyridine
(5.0 mL) at rt under argon. The reaction mixture was stirred for 90 min,
after which TLC analysis (1:1, cyclohexane−ethyl acetate) indicated the
complete consumption of the starting material (Rf 0.46) and the
formation of a major product (Rf 0.64). The reaction mixture was diluted
with ethyl acetate (10 mL), and the mixture was washed with 2 M
aqueous hydrochloric acid (2 × 10 mL). The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo to afford the mesylate 56
(562 mg, 88%) as a yellow oil, which was reacted on without further
purification: [α]20D +39.0 (c 0.41 in CH3Cl). νmax (thin film): 1740 (s,
CO). δH (400 MHz, CDCl3): 2.93 (3 H, s, SO2-Me), 3.32 (1 H, a-q, H-4,
J4,3 = J4,5 = J4,5′ 5.0), 3.62 (1 H, d, H-2, J2,3 4.0), 3.62 (3 H, s, CO2-Me),
3.77 (1 H, d, NCH2Ph, Jgem 12.8), 3.84 (1 H, dd, H-5, J5,4 4.4, J5,5′ 11.2),
3.88 (1 H, d, NCH2Ph, Jgem 12.8), 4.00 (1 H, dd, H-5′, J5′,4 4.4, J5′,5 11.2),
4.10 (1 H, a-t, H-3, J3,2 = J3,4 5.2), 4.49 (1 H, d, OCH2Ph, Jgem 11.6), 4.60
(1 H, d, OCH2Ph, Jgem 11.6), 7.27−7.37 (10 H, m, Ar-H). δC (100 MHz,
CDCl3): 37.6 (SO2-Me), 52.5 (CO2-Me), 60.0 (NCH2Ph), 67.5 (C-2),
68.3 (C-5), 68.7 (C-4), 72.4 (OCH2Ph), 72.9 (C-3), 128.1, 128.4, 128.7,
128.7, 128.9, 130.1 (ArCH), 136,9 (ArCC), 169.7 (C-1). m/z (ESI +ve):
420 (M + H+, 100). HRMS m/z (ESI +ve): found 442.1299 [M + Na+],
C21H25NNaO6S requires 442.1295.
Synthesis of Altro- and Allo-Configured Proline Amides
(Scheme 5). Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-metha-
nesulfonyl-D-allonamide 51D. Methylamine (33% in ethanol, 0.11 mL,
0.85 mmol) was added to a solution of mesylate 48D34 (75 mg, 0.17
mmol) in 1,4-dioxane (3.8 mL). The reaction was stirred at rt for 17 h
under argon, after which TLC analysis (3:7, cyclohexane−ethyl acetate)
indicated the complete consumption of starting material (Rf 0.91) and
the formation of a major product (Rf 0.44). The reaction mixture was
concentrated in vacuo and purification by flash chromatography (4:1 to
1:1, cyclohexane−ethyl acetate) to afford the amide 51D (55 mg, 69%)
as a colorless oil: [α]25D +18.4 (c 0.89 in CHCl3). νmax (thin film): 3419
(br s, NH, OH), 2101 (s, N3), 1665 (s, CO). δH (400 MHz, CDCl3):
2.84 (3 H, d, N-Me, JH,NH 4.8), 3.04 (3 H, s, SO2-Me), 3.53 (1 H, dd, H-
6, J6,5 6.8, J6,6′ 11.4), 3.63−3.68 (2 H, m, H-5, H-6′), 3.95 (1 H, dd, H-4,
J4,5 2.5, J4,3 8.4), 4.20 (1 H, dd, H-3, J3,2 1.6, J3,4 8.4), 4.42 (1 H, d,
OCH2Ph, Jgem 11.1), 4.46 (1 H, d, OCH2Ph, Jgem 11.7), 4.49 (1 H, d,
OCH2Ph, Jgem 11.7), 4.67 (1 H, d, OCH2Ph, Jgem 11.1), 5.44 (1 H, d, H-
2, J2,3 1.6), 6.50 (1 H, d, N-H, JNH,H 4.8), 7.26−7.38 (10 H, m, Ar-H). δC
(100 MHz, CDCl3): 26.4 (N-Me), 39.1 (SO2-Me), 61.7 (C-5), 69.8 (C-
6), 71.2 (C-4), 72.3, 73.9 (OCH2Ph), 78.8 (C-3), 78.9 (C-2), 128.1,
128.2, 128.6, 128.7, 128.8, 128.8, 128.9 (ArCH), 136.5, 137.1 (ArCC),
167.6 (C-1). m/z (ESI +ve): 493 (M + H+, 51), 515 (M + Na+, 100).
(ESI −ve): 491 ([M − H]−, 34), 527 (M + 35Cl−, 100), 529 (M + 37Cl−,
60). HRMS m/z (ESI +ve): found 515.1567 [M + Na+],
C22H28N4NaO7S requires 515.1571. For the enantiomer 51L: [α]25
D
−17.7 (c 0.98 in CHCl3).
Methyl 2,5-Dideoxy-2,5-imino-D-altronamide 53D. Palladium
(10% on carbon, 11 mg) and sodium acetate (20 mg, 0.24 mmol)
were added to a solution of amide 51D (55 mg, 0.12 mmol) in 1,4-
dioxane (2.2 mL). The flask was evacuated and flushed three times with
argon and three times with hydrogen. The reaction was stirred at rt for 3
h under hydrogen, after which TLC analysis (ethyl acetate) indicated the
complete consumption of starting material (Rf 0.71) and the formation
of a single product (Rf 0.06). Aqueous hydrochloric acid (2 M, 0.44 mL)
was added to the reaction mixture and the flask evacuated and flushed
three times with argon and three times with hydrogen. The reaction was
stirred for a further 19 h. The reaction was filtered through glass
microfiber (GF/B) and the filtrate concentrated in vacuo. The crude
residue was dissolved in water and loaded onto a column of Dowex
(50W-X8, H+). The column was washed with water and the amide 53D
liberated with 2 M aqueous ammonia. The ammoniacal fractions were
concentrated in vacuo to afford the amide 53D (20 mg, 94%) as a yellow
solid: mp 154−156 °C. [α]25D −29.8 (c 0.83 in H2O). νmax (thin film):
3297 (br s, NH, OH), 1637 (s, CO). δH (400 MHz, D2O): 2.67 (3 H, s,
N-Me), 3.11 (1 H, ddd, H-5, J5,6′ 3.8, J5,6 5.4, J5,4 9.1), 3.54 (1 H, dd, H-6,
J6,5 5.4, J6,6′ 11.9), 3.72 (1 H, dd, H-6′, J6′,5 3.8, J6′,6 11.9), 3.84−3.88 (2 H,
m, H-2, H-4), 4.25 (1 H, a-t, H-3, J3,2 = J3,4 4.3). δC (100 MHz, D2O):
26.0 (N-Me), 61.8 (C-6), 62.0 (C-5), 62.8 (C-2 or C-4), 73.3 (C-3),
73.8 (C-2 or C-4), 173.5 (C-1). m/z (ESI +ve): 191 (M + Na+, 90), 213
(M + Na+, 100). HRMS m/z (ESI +ve): found 213.0849 [M + Na+],
C7H14N2NaO4 requires 213.0846. For the enantiomer 53L: mp 156−
158 °C. [α]25D +39.9 (c 0.88 in H2O).
Methyl 5-Azido-3,6-di-O-benzyl-5-deoxy-2-O-methanesulfonyl-D-
altronamide 52D. Methylamine (33% in ethanol, 2.75 mL, 22.3 mmol)
was added to a solution of the mesylate 50D34 (3.43 g, 7.43 mmol) in
1,4-dioxane (75 mL) at rt. The reaction was stirred for 18 h, after which
TLC analysis (3:1, toluene−acetone) indicated complete consumption
of the starting material (Rf 0.80) and the formation of a major product
(Rf 0.37). The reaction mixture was concentrated in vacuo and the
residue purified by flash chromatography (7:3 to 13:7, cyclohexane−
ethyl acetate) to afford the amide 52D (3.31 g, 91%) as a white
Methyl 5-Azido-2-N,3-O-dibenzyl-2,4,5-trideoxy-2,4-imino-D-ri-
bonate 57. Sodium azide (110 mg, 1.69 mmol) was added to a
solution of the mesylate 56 (500 mg, 1.30 mmol) in DMF (5.00 mL) at
rt. The reaction mixture was heated to 60 °C and stirred for 24 h, after
which TLC analysis (1:1, cyclohexane−ethyl acetate) indicated the
crystalline solid: mp 86−88 °C. [α]25 −1.50 (c 1.72 in CHCl3). νmax
D
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Article
complete consumption of the starting material (Rf 0.64) and the
formation of a major product (Rf 0.78). The reaction mixture was diluted
with ethyl acetate (10 mL) and washed with a 1:1 brine−water mixture
(2 × 10 mL). The organic layer was dried (MgSO4), filtered, and
concentrated in vacuo. The residue was purified by flash chromatog-
raphy (7:1 to 5:1, cyclohexane−ethyl acetate) to afford the azide 57
(372 mg, 78%) as a yellow oil: [α]20D +70.3 (c 0.92 in CH3Cl). νmax (thin
film): 2099 (s, N3), 1741 (s, CO). δH (400 MHz, CDCl3): 2.89 (1 H, dd,
H-5, J5,4 4.4, J5,5′ 12.8), 3.02 (1 H, d, H-5′, J5′4 4.8, J5′,5 12.8), 3.22 (1 H, a-
q, H-4, J4,3 = J4,5 = J4,5′ 4.8), 3.58 (1 H, d, H-2, J2,3 5.2), 3.66 (3 H, s, CO2-
Me), 3.74 (1 H, d, NCH2Ph, Jgem 12.6), 3.93 (1 H, d, NCH2Ph, Jgem
12.6), 4.10 (1 H, a-t, H-3, J3,2 = J3,4 5.2), 4.47 (1 H, d, OCH2Ph, Jgem
11.6), 4.61 (1 H, d, OCH2Ph, Jgem 11.6), 7.26−7.37 (10 H, m, Ar-H). δC
(100 MHz, CDCl3): 52.1 (CO2-Me), 52.4 (C-5), 61.3 (NCH2Ph), 68.5
(C-4), 69.7 (C-2), 71.9 (OCH2Ph), 74.3 (C-3), 127.8, 128.0, 128.1,
128.5, 128.6, 129.8 (ArCH), 137.4 (ArCC), 171.5 (C-1). m/z (ESI +ve):
367 (M + H+, 100). HRMS m/z (ESI +ve): found 389.1590 [M + Na+],
C20H22N4NaO3 requires 389.1584.
(0.05 mL) were added to a solution of dibenzyl 59 (62.0 mg, 0.18 mmol)
in 1:1 1,4-dioxane−water (2 mL). The reaction vessel was evacuated and
flushed with nitrogen, argon, and hydrogen gas sequentially. The
reaction mixture was stirred vigorously for 5 days at rt until mass
spectrometry indicated the absence of the starting material (m/z 355 [M
+ H+]). Completion of the reaction was confirmed by mass
spectrometry. The reaction mixture was filtered through glass microfiber
(GF/A) and concentrated in vacuo to afford the acetamide 14 (40.3 mg,
100%) as the hydrochloride salt and as a yellow gum: [α]20D +11.3 (c
0.42 in MeOH). νmax 3336 (br s, OH, NH), 1634 (s, CO). δH (400 MHz,
D2O): 1.91 (3 H, s, NHCOCH3), 3.49 (1 H, dd, H-5, J5,4 6.4, J5,5′ 15.0),
3.56 (1 H, dd, H-5′, J5′,4 4.4, J5′,5 15.0), 3.74 (1 H, dd, H-1, J1,2 3.2, J1,1′
13.2), 3.77 (1 H, dd, H-1′, J1′,2 3.2, J1,1′ 13.2), 4.17−4.23 (2 H, m, H-2, H-
4), 4.40 (1 H, t, H-3, J3,2 = J3,4 6.8). δC (100 MHz, D2O): 22.1
(NHCOCH3), 38.9 (C-5), 57.9 (C-1), 65.2, 65.4 (C-2, C-4), 67.1 (C-3),
176.1 (NHCOCH3). m/z (ESI +ve): 175 (M + H+, 100). HRMS m/z
(ESI +ve): found 175.1075 [M + H+], C7H15N2O3 requires 175.1077.
5-Acetamido-1-O-acetyl-2-N,3-O-dibenzyl-2,4,5-trideoxy-2,4-
imino-D-ribitol 58. Lithium aluminum hydride (1 M in THF, 2.84 mL,
2.84 mmol) was added dropwise to a solution of the azide 57 (237 mg,
0.71 mmol) in THF (5 mL) at −78 °C under argon. The reaction
mixture was stirred for 3.5 h, after which mass spectrometry indicated
the absence of a peak corresponding to the starting material (m/z 389
[M + Na+]) and the presence of a peak corresponding to the
intermediate amine (m/z 335 [M + Na+]). The reaction as quenched
with saturated aqueous ammonium chloride and the reaction mixture
concentrated in vacuo. A 1:1 acetic anhydride−water mixture (6 mL)
was added to the crude residue. The reaction mixture was stirred at rt for
15 h, after which the mixture was concentrated in vacuo and the crude
residue was purified by flash chromatography (1:1:0 to 0:100:1,
cyclohexane−ethyl acetate−methanol) to afford the diacetate 58 (207
ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of 1H and 13C NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: benayers@ntu.edu.sg
*E-mail: george.fleet@chem.ox.ac.uk.
*E-mail: kato@med.u-toyama.ac.jp.
■
Notes
The authors declare no competing financial interest.
mg, 79%) as a yellow oil: [α]20 −20.5 (c 0.39 in MeOH). νmax (thin
D
film): 1737 (s, CO), 1651 (s, CO). δH (400 MHz, CDCl3): 2.04 (6 H, a-
s, NHCOCH3, OCOCH3), 2.84 (1 H, br s, H-5), 3.09−3.33 (3 H, m, H-
2, H-4, H-5′), 3.68−3.74 (2 H, m, H-3, NCH2Ph), 3.86−3.91 (1 H, m,
NCH2Ph), 3.93 (1 H, dd, H-1, J1,2 5.6, J1,1′ 10.0), 3.98 (1 H, dd, H-1′, J1′,2
5.6, J1′,1 10.0), 4.44 (1 H, d, OCH2Ph, Jgem 12.0), 4.49 (1 H, d, OCH2Ph,
Jgem 12.0), 7.28−7.36 (10 H, m, Ar-H). δC (100 MHz, CDCl3): 21.1
(NHCOCH3, OCOCH3), 40.7 (C-5), 61.4 (NCH2Ph), 64.2 (C-1),
68.5, 69.5 (C-2, C-4), 72.0 (OCH2Ph), 73.0 (C-3), 128.0, 128.2, 128.2,
128.6, 128.8, 129.6 (ArCH), 137.4 (ArCC), 170.9, 171.0 (OCOCH3,
NHCOCH3). m/z (ESI +ve): 397 (M + H+, 100). HRMS m/z (ESI
+ve): found 397.2125 [M + H+], C23H29N2O4 requires 397.2122.
5-Acetamido-2-N,3-O-dibenzyl-2,4,5-trideoxy-2,4-imino-D-ribitol
59. Sodium methoxide (2.7 mg, 0.05 mmol) was added to a solution of
the diacetate 58 (191 mg, 0.48 mmol) in methanol (5 mL) at rt under
argon. The reaction mixture was heated to 60 °C for 25 h, after which
mass spectrometry indicated the consumption of a peak corresponding
to the starting material (m/z 397 [M + H+]) and the presence of a peak
corresponding to the monoacetylated product (m/z 335 [M + H+]).
The reaction mixture was concentrated in vacuo, and the residue
redissolved in a 1:2 1,4-dioxane−water mixture (5 mL) and loaded onto
a short column of Dowex (50W-X8, H+). The column was washed with
water and the product liberated with 2 M aqueous ammonia. The
ammoniacal fractions were combined and concentrated in vacuo to
afford the acetamido 59 (62 mg, 38%) as a light yellow oil: [α]20D −12.1
(c 0.77 in MeOH). νmax 3301 (br s, OH, NH), 1650 (s, CO). δH (400
MHz, CD3OD): 1.90 (3 H, s, NHCOCH3), 2.95 (1 H, dd, H-5, J5,4 5.2,
ACKNOWLEDGMENTS
This work was supported by the Newton Abraham Research
■
Fund (B.J.A.), Fundacion
́
Ramon
́
Areces (R.F.M.), Grant-in-Aid
for Scientific Research (C) (No. 23590127) (A.K.) from the
Japanese Society for the Promotion of Science (JSPS) and the
Leverhulme Trust (G.W.J.F.).
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