N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase

Article abstract of DOI:10.1016/j.bmc.2008.05.056

A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino-N-(8-quinolinyl)methyl-d-ribitol (UAMC-00115, K_i 10.8 nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K_i 4.1 nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K_i 4.4 nM) being the three most active compounds. Docking studies of the most active inhibitors revealed several important interactions with the enzyme. Among these interactions are aromatic stacking of the nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhibitors and amino acid residues in the active site. During the course of these docking studies we also identified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an interaction which has not previously been considered as being important.

Full text of DOI:10.1016/j.bmc.2008.05.056

Bioorganic & Medicinal Chemistry 16 (2008) 6752–6763
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine
specific nucleoside hydrolase
Annelies Goeminne a, Maya Berg a, Michael McNaughton a, Gunther Bal a, Georgiana Surpateanu a,
Pieter Van der Veken a, Stijn De Prol b, Wim Versées b, Jan Steyaert b, Achiel Haemers a, Koen Augustyns a,
*
a Department of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, Antwerp B-2610, Belgium
b Group of Structural Biology Brussels, Department of Molecular and Cellular Interactions, VIB, Vrije Universiteit Brussel, Pleinlaan 2, B-1050, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a
good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a
novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several
of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino-N-(8-quinoli-
Received 25 March 2008
Revised 21 May 2008
Accepted 28 May 2008
Available online 18 June 2008
nyl)methyl-D-ribitol (UAMC-00115, Ki 10.8 nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-
imino- -ribitol (Ki 4.1 nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino-
D
D
-ribitol (Ki
Keywords:
4.4 nM) being the three most active compounds. Docking studies of the most active inhibitors revealed
several important interactions with the enzyme. Among these interactions are aromatic stacking of the
nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhib-
itors and amino acid residues in the active site. During the course of these docking studies we also iden-
tified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an
interaction which has not previously been considered as being important.
Nucleoside hydrolase
Purine-specific
Inhibitor
Antiparasitic
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
quent release of the purine bases. Four types of NH are currently
known.3 Our chosen target enzyme is a purine specific NH (IAG-
Parasitic infections remain an important health issue in devel-
oping countries. Among the parasites, different species from the
Trypanosomatidae family are pathogenic to humans and cause
African trypanosomiasis (sleeping sickness, Trypanosoma brucei),
American trypanosomiasis (Chagas’ disease, T. cruzi) or leishmani-
asis (Leishmania spp.). Together these three infections are respon-
sible for over one hundred thousand deaths annually and severe
disabilities in surviving patients.1 Other parasites from the same
family can cause disease in animals and are therefore a threat to
livestock. Drugs that are currently used to treat these parasitic
infections are old and often toxic. Moreover, resistance is also a
growing problem.
In the search for new antiparasitic drugs, it has been proposed
that the purine metabolism of the parasite could provide a good
drug target.2 In contrast to mammals, parasites are unable to syn-
thesize purines de novo, and rely instead on the purine salvage
pathway to obtain purines which are essential for their survival.
Nucleoside hydrolase (NH) is an enzyme from the purine pathway
which hydrolyses nucleosides obtained from the host, with subse-
NH), preferring inosine, adenosine, and guanosine as substrates,
and is isolated from Trypanosoma vivax, a parasite that causes a
sleeping-sickness like disease in cattle.4 It should be noted that
NH-activity is not present in mammalian cells. The active site
and hence the catalytic mechanism of the IAG-NH is different from
that of the mammalian purine nucleoside phosphorylase (PNP), the
enzyme that cleaves nucleosides in mammalian cells. Substrate
specificity is also different between the two enzymes, with IAG-
NH cleaving inosine, adenosine, and guanosine, whereas the phys-
iological substrates for mammalian PNP are inosine, guanosine and
deoxyguanosine, with no significant substrate activity for adeno-
sine.5 Based on this information, selective inhibition of IAG-NH
seems possible, and would be an effective way to kill the parasite
without causing toxicity toward the host.2
2. Inhibitor design
The catalytic mechanism of NH proceeds via an oxocarbenium
ion-like transition state.6 Transition state analogs are generally
very good inhibitors, with the iminoribitol (1,4-dideoxy-1,4-imi-
no-D-ribitol) scaffold providing a good transition state mimic for
NH.7,8 The purine substrate specificity of IAG-NH from T. vivax
(TvNH) is imposed by parallel aromatic stacking interactions
* Corresponding author. Tel.: +32 38202703; fax: +32 38202739.
E-mail address: koen.augustyns@ua.ac.be (K. Augustyns).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.056

A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
6753
between the purine ring of the substrate and two Trp-residues
3. Results and discussion
3.1. Chemistry
(Trp83 and Trp260) in the active site.9 These stacking interactions
contribute to raising the pKa of N7 of the purine substrate, allowing
direct protonation by solvent molecules. Hence these stacking
interactions function as an alternative to general acid catalysis.10
In the natural substrates, as well as in the known inhibitors of
IAG-NH (Chart 1: 3-deazaadenosine,4 Immucillin-H11,12 and Immu-
cillin-A11,12) the purine is attached to the C1 position of the ribose
or iminoribitol moiety. From preliminary modeling studies we ob-
served that the purine analogs could also be placed on the imino-
ribitol-N, and the correct distance between the sugar and
substituent could be provided by a methylene group. Such struc-
tures would provide a novel class of NH-inhibitors, which are syn-
thetically easier to access than C1-substituted iminoribitols. That
the inhibitory activity is conserved or even improved with N-sub-
stitution of iminoribitol was confirmed by earlier results from our
group, where iminoribitol was substituted with an alkylguanidine
group or a phenyl, both on C1 (Chart 1, structure 113 and 314) or via
a methylene-linker on the iminoribitol-N (Chart 1, structure 213
and 413). Other N-substituted iminoribitols developed by our group
also showed promising results as inhibitors of TvNH.13 During the
time of our research, Evans et al. reported the synthesis of the N-
substituted analog of Immucillin-H as an inhibitor of PNP.15 Here
we describe a different synthetic approach toward this compound
and report for the first time its inhibitory activity against IAG-NH.
As substituents for the iminoribitol-N, a wide range of aromatic
moieties were chosen with preference for nitrogen-containing aro-
matics. Commercial available aromatic derivatives were preferred
in order to allow rapid generation of a library of inhibitors. This en-
abled us to confirm our hypothesis on substitution of the iminorib-
itol-N. Three main types of aromatic derivatives were used in the
synthesis of our target compounds: arylmethylhalides (usually –
bromide), arylcarboxaldehydes, and indole derivatives, which were
‘coupled’ to the iminoribitol moiety via three different methods:
(1) N-Alkylation; (2) Reductive amination; and (3) Mannich
reaction.
The iminoribitol scaffold was synthesized first and subse-
quently derivatized to obtain the target compounds. Two methods
were applied for the synthesis of the iminoribitol scaffold. The first
method was reported by Fleet et al. and starts from
tone. An important intermediate in this synthesis is 5-O-tert-butyl-
dimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene- -ribi-
tol (Scheme 2, compound 10), which can be treated with aqueous
D-gulonolac-
D
acid to obtain 1,4-dideoxy-1,4-imino-D-ribitol (Scheme 1, com-
pound 9).16,17 Due to the lengthy character and variable yields
experienced for several steps of this synthesis, an alternative meth-
od, based on a publication of Qiu et al., was investigated (Scheme
1).18 In the synthesis reported by Qui et al., trans-4-hydroxy-
L-pro-
line is used as the starting material. Compound 618 is obtained as
an intermediate, and Qiu et al. suggested to protect the 20-hydroxyl
group as a TBDPS ether.18 However, for our purposes, we wanted a
protecting group which could be easily cleaved under acidic condi-
tions. As the protecting group needs bulk to ensure stereoselective
dihydroxylation in the subsequent reaction, two possibilities were
investigated (Scheme 1, step a): protection as a triphenylmethyle-
ther (7a) or as a TBDMS ether (7b). Comparison of the NMR spectra
of iminoribitol 9 obtained after deprotection of both intermediate
8a and 8b with iminoribitol obtained from D-gulonolactone, con-
firmed that both protecting groups furnished the desired ribitol
stereochemistry after stereoselective dihydroxylation.
Both methods applied for the synthesis of the iminoribitol scaf-
fold are rather lengthy and time consuming, with comparable
overall yields in the range of 15–20%. For the majority of our
synthetic purposes, the TBDMS and isopropylidene protected imi-
noribitol (10) were required. The synthesis starting from D-gulon-
olactone appeared to be the most convenient method to obtain
this compound. However, unprotected iminoribitol (9) was synthe-
sized from trans-4-hydroxy-L-proline.
Three strategies were used to substitute the iminoribitol nitro-
gen. A first strategy was N-alkylation. For this, protected iminorib-
itol 10 was reacted with the appropriate arylmethylbromide,
followed by deprotection with aqueous trifluoracetic acid (Scheme
2). For the synthesis of compound 44, 7-methylquinoline (59) was
brominated in the benzylic position and the resulting aryl-
methylbromide 60 was used in a similar N-alkylation procedure
(Scheme 2). Protected iminoribitol 10 was also employed for the
second strategy, namely reductive aminations, in which it was re-
acted with the appropriate arylcarboxaldehyde in the presence of
polymer supported cyanoborohydride, followed by deprotection
with aqueous trifluoroacetic acid (Scheme 2).
Mannich reactions on similar azasugar scaffolds have been re-
ported using the unprotected azasugar and a purine derivative.19,20
We applied this Mannich reaction to the synthesis of the N-substi-
Chart 1. Inhibitors against purine specific NH (TvNH = IAG-NH from T. vivax;
TbbNH = IAG-NH from T. brucei brucei). Data taken from aJ. Mol. Biol. 2001, 307,
1363–1379, b J. Mol. Biol. 2006, 359, 331–346, cBiochemistry 1999, 38, 13147–13154,
dEur. J. Med. Chem. 2008, 43, 315–326, eunpublished results from our group.
Scheme 1. Reagents and conditions: (a) TrCl, pyridine, rt, 24 h, or TBDMSCl,
imidazol, Et3N, CH2Cl2, rt, 18 h; (b) K2OsO4.2H2O, 4-methylmorpholine N-oxide,
acetone, H2O, rt, 18 h; (c) TFA/H2O 1:1, rt, 18 h.

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A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
Table 1
Inhibition constants (Ki) against TvNH of group I target compounds
Compound
R=
Ki (lM)
4a
5.9 ꢀ 101 1.8 ꢀ 101
4.3 1.2
28
29
30
31
32
Scheme 2. Reagents and conditions: (a) Procedure A: arylmethylhalide, Na2CO3,
CH2Cl2, H2O, reflux, 3–20 h, Procedure B: arylmethylhalide, K2CO3 or KHCO3, DMF,
40 °C, 1.5–4 h; (b) TFA/H2O 1:1, rt, 18 h; (c) Procedure for the synthesis of 52:
4-pyridinecarboxaldehyde, NaCNBH3, MeOH, rt, 24 h; Procedure for the synthesis of
target compounds 53–58: arylcarboxaldehyde, CH2Cl2/AcOH 10/1, (polystyrylm-
ethyl)trimethylammonium cyanoborohydride, rt, 28 h; (d) N-bromosuccinimide,
dibenzoylperoxide, CCl4, reflux, 18 h.
7.5 2.2
2.6 ꢀ 101 0.3 ꢀ 101
2.3 ꢀ 101 0.1 ꢀ 101
3.8 1.7
33
34
3.4 0.2
1.0 0.1
35
36
1.2 0.1
1.2 0.1
Scheme 3. Reagents and conditions: (a) 9-deazahypoxanthine or Boc-protected 9-
deaza-adenine, aq CH2O, NaOAc, H2O, 95 °C, 17 h; (b) indole or 7-methoxy-indole,
aq CH2O, H2O/dioxane 1:1, 95 °C, 20 h; (c) TFA/H2O 1:1, rt, 18 h.
tuted analog of Immucillin-H, compound 61 (Scheme 3). The
hydrochloric acid salt of iminoribitol 9 was treated with aqueous
formaldehyde in the presence of 9-deaza-hypoxanthine. The latter
compound was synthesized from isoxazole following a method
published by Furneaux et al.21 This synthesis of compound 61 is
an alternative to the reductive amination approach reported by
Evans et al.15 The same approach (Mannich reaction) was used
for the synthesis of the novel N-substituted analog of Immucillin-
A, compound 62. The Mannich reaction procedure as described
for compounds 61and 62 was also applied to the synthesis of in-
dole derivatives 65 and 66. However, we were unable to obtain
the desired compounds. Similar reaction conditions were more
successful when applied to the TBDMS and isopropylidene pro-
tected iminoribitol derivative 10 (Scheme 3).
37
52
53
54
0.063 0.007
2.0 ꢀ 101 0.7 ꢀ 101
1.2 ꢀ 102 0.1 ꢀ 102
3.2 ꢀ 101 0.3 ꢀ 101
4.3 ꢀ 101 0.6 ꢀ 101
3.2. Biochemical results
55
All target compounds were tested as inhibitors of the IAG-NH
isolated from T. vivax (TvNH). The target compounds can be divided
into three groups, based on their general structure, with each
group showing interesting structure–activity relationships. A first
group of compounds includes iminoribitol derivatives substituted
with a single aromatic ring and non-fused biaryl compounds. The
biochemical results of group I compounds are shown in Table 1.
Group II includes naphthalene and (iso)quinoline derivatives
(Table 2). Group III includes iminoribitols with indole, benzimid-
azole, benzotriazole, and 9-deaza-hypoxanthine substituents
(Table 3). For group I compounds, a first observation is that com-
pounds with only one aromatic ring as substituent, are less active
inhibitors compared to the biaryl compounds. Comparing the
a
Eur. J. Med. Chem. 2008, 43, 315–326).
biochemical results for the non-fused biaryl compounds of group
I with the fused aromatics of groups II and III, it appears that the
latter are in general more active. It is known that stacking interac-
tions are more favorable with aromatics containing multiple rings
than with monocyclics.22
The series of naphthalene and (iso)quinoline derivatives in
group II were developed to investigate the role of a nitrogen in
the nucleobase mimic. From the biochemical results in Table 2 it

A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
6755
Table 2
appears that there is a significant difference in activity between
these compounds, with the 8-methylquinolinyl compound 42
(UAMC-00115) being the strongest inhibitor developed in this
group, showing a Ki of 10.8 nM. The results from group II com-
pounds suggest that there is a correlation between the inhibitory
activity of the (iso)quinoline derivatives and the position of the
(iso)quinoline-nitrogen in these compounds. This hypothesis was
further investigated in the molecular modeling study discussed
below.
In group III (Table 3), Immucillin-H analog 61 and Immucillin-A
analog 62 are by far the strongest inhibitors, with Ki’s of, respec-
tively, 4.4 and 4.1 nM. Comparing the Ki of compound 61 with
the Ki of 6.2 nM for both ‘lead-compounds’, Immucillin-H and
Immucillin-A, confirms once again our hypothesis that placing
the nucleobase on the iminoribitol-N via a methylene linker results
in very good inhibitors of nucleoside hydrolase. It appears that the
other compounds of group III do not have enough favorable fea-
tures to bind tightly in the active site of the target enzyme.
Inhibition constants (Ki) against TvNH of group II target compounds
Compound
R=
Ki (lM)
40
41
2.0 0.6
0.18 0.05
42
43
0.0108 0.0012
0.75 0.09
3.3. Molecular modeling study
Docking studies were performed in an attempt to understand
the difference in biochemical activity observed with the naphtha-
lene and (iso)quinoline derivatives and to further investigate the
structure–activity-relationship. In particular, the possible role of
the nitrogen in the (iso)quinoline nucleobase mimic was investi-
gated. The compounds in this study included 39, 40, 41, 42, 43,
44
56
0.72 0.13
2.0 ꢀ 101 0.2 ꢀ 101
44, 56, 57, 61, and 62, ranging in activity from 4.1 nM to 29 lM.
They were docked in the active site of the target enzyme, which
was obtained from the pdb structure of TvNH co-crystallized with
Immucillin-H (pdb code 2FF2).11 The structure of this complex is
illustrated in Figure 1.
Immucillin-H interacts with different active site residues of the
enzyme through the hydroxyl groups and the ring-N of its imino-
ribitol moiety. The 9-deazahypoxanthine ring of Immucillin-H is
orientated almost parallel between two Trp-residues (Trp83 and
57
58
2.9 ꢀ 101 0.3 ꢀ 101
1.0 0.1
Table 3
Inhibition constants (Ki) against TvNH of group III target compounds
Compound
R=
Ki (lM)
38
39
2.1 0.3
0.72 0.07
61
0.0044 0.0005
62
65
66
0.0041 0.0007
1.2 ꢀ 101 0.1 ꢀ 101
2.3 0.3
Figure 1. The active site of Tv NH co-crystallized with Immucillin-H (pdb code
2FF2).11 Interaction distances are given in Å. The active site residues are depicted in
grey (carbons), blue (nitrogens), and red (oxygens). The carbons of the inhibitor are
depicted in green, the color codes for nitrogens and oxygens are the same as for the
active site residues. Water molecules are depicted as red spheres, the Ca2+ ion is
depicted as a magenta sphere. The image was made with PyMOL (PyMOL Molecular
Graphics System, DeLano Scientific, San Carlos, CA).

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A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
Figure 2. Inhibitor 61 in the active site of the target enzyme. Interaction distances
are given in Å. Color codes are the same as for Figure 1. The images were made with
PyMOL (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA).
Figure 4. Binding of inhibitor 42 in the active site of the target enzyme. The
nucleobase mimic is orientated parallel between Trp83 and Trp260, enabling
aromatic stacking interactions. The active site residues are depicted in grey
(carbons), blue (nitrogens), and red (oxygens). The carbons of the inhibitor are
depicted in green, the color codes for nitrogens and oxygens are the same as for the
active site residues. Water molecules are depicted as red spheres, the Ca2+ ion is
depicted as a magenta sphere. The colors of the enzyme surface are according to the
elements. The image was made with PyMOL (PyMOL Molecular Graphics System,
DeLano Scientific, San Carlos, CA).
Trp260) of the active site, allowing for aromatic stacking interac-
tions. The 9-deazahypoxanthine ring is also involved in several
hydrogen bonds: a first hydrogen bond is formed between N7
and a water molecule in the active site, a second hydrogen bond
is formed between the carbonyl-O6 and amino acid residue
Arg252, and a third hydrogen bond is formed between N3 and ami-
no acid residue Asp40. The pKa of Asp40 was calculated to be
approximately 7.2–7.8, making it mostly protonated at our exper-
imental conditions (pH 7.0) and thus allowing a hydrogen bond
with N3.23
An automatic docking protocol and minimization was used to
position our inhibitors into the active site. The interactions for
our inhibitors with the target enzyme were compared to the inter-
actions for Immucillin-H. Where possible, the interactions de-
scribed for Immucillin-H were also observed with our inhibitors.
In Figure 2, docking of inhibitor 61 is illustrated. This compound
is the N-substituted analog of Immucillin-H. Comparison of Figures
1 and 2 clearly demonstrates that the interactions between the
inhibitor and the enzyme are very similar.
Our most active inhibitors among the docked compounds are
62, 61, 42, 41, 44, 39, and 43 with inhibition constants of 4.1,
4.4, 10.8, 180, 720, 720, and 750 nM, respectively. All these com-
pounds appear to have a common feature as observed in this
modeling study: the preferred conformation obtained by the
docking experiments allows an interaction between a nucleo-
base-N and the Asp40 residue, an interaction that was also de-
scribed with Immucillin-H.11 This observation leads us to put
forward the hypothesis that Asp40 plays an important role in
binding of the inhibitor to the enzyme. Previous results from
our group also support this hypothesis.24 The presence of this
N-Asp40 interaction was recently confirmed by a crystallography
study of TvNH in complex with inhibitor 42.25 Docking of inhib-
itor 42 is illustrated in Figure 3, clearly showing the N-Asp40
interaction.
A further observation made during the docking study was that
the orientation of our inhibitors, as obtained from the docking
experiments, always favored aromatic stacking interactions. As
illustrated in Figure 4, the nucleobase mimic of the inhibitor is ori-
entated parallel between the aromatic side chains of Trp83 and
Trp260, allowing stacking interactions to occur.
4. Conclusions
We have synthesized a small library of novel N-arylmethyl
substituted iminoribitols, which were screened for inhibitory
activity against IAG-NH from T. vivax. These tests identified three
very potent inhibitors, 62 with Ki = 4.1 nM, 61 with Ki = 4.4 nM,
Figure 3. Inhibitor 42 in the active site of the target enzyme. Interaction distances
are given in Å. Color codes are the same as for Figure 1. The images were made with
PyMOL (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA).

A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
6757
and 42 with Ki = 10.8 nM. These compounds are among the most
active inhibitors of IAG-NH reported. This confirms our hypothesis
that N-arylmethyl substitution of iminoribitol is allowable for
inhibitors of this target enzyme. Docking experiments on a series
of the most active inhibitors revealed an interaction between a
nitrogen in the nucleobase mimic and the Asp40 residue in the ac-
tive site of the target enzyme. We propose here that this interac-
tion plays an important role in the binding of an inhibitor in the
enzyme. Inhibitor 42 was shown to be the one of the most active
inhibitors developed in this series (Ki 10.8 nM) and this compound
provides us with a good lead structure for further discovery of
more potent NH-inhibitors.
5.2.2. (2S)-N-tert-Butoxycarbonyl-2-tert-butyldimethylsiloxym-
ethyl-3-pyrroline (7b)
A solution of imidazole (0.17 g, 2.5 mmol) and TBDMSCl (0.38 g,
2.5 mmol) in CH2Cl2 (15 mL) was added dropwise to a stirred solu-
tion of compound 618 and Et3N (0.35 mL) in CH2Cl2 (5 mL). The
reaction mixture was stirred at room temperature for 18 h, then
it was concentrated under reduced pressure. The residue was dis-
solved in EtOAc, this was washed with water and brine. The organ-
ic layer was dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by column chromatography
(hexane to hexane/EtOAc 4:1) to yield the product as a syrup
(0.32 g, 61%). MS (ESI) m/z 314.5 [M+H]+.
5.2.3. N-tert-Butoxycarbonyl-1,4-dideoxy-1,4-imino-5-O-
triphenylmethyl-D-ribitol (8a)
5. Experimental
Compound 7a (11 g, 25 mmol) and 4-methylmorpholine N-
oxide (10 g, 74 mmol) were dissolved in acetone (250 mL) and
H2O (60 mL) and this was cooled to 0 °C. K2OsO4.2H2O (50%,
0.12 g, 0.16 mmol) was added and the resulting mixture was al-
lowed to warm to rt and was then stirred at rt for 18 h. NaHSO3
(5 g) was added and after 30 min of stirring, the mixture was ex-
tracted three times with EtOAc. The combined organic layers were
washed with brine, dried (Na2SO4), and the solvent was removed
under reduced pressure. The crude product was purified by column
chromatography (hexane/EtOAc 1:1) to yield the title compound as
a syrup (7.2 g, 62%). MS (ESI) m/z 475.0 [M+Na]+.
5.1. General procedures
All starting materials and dry solvents were obtained from
Acros Organics or Aldrich. The arylmethylhalides used for
N-alkylation were obtained from Acros Organics or Maybridge. 7-
Methylquinoline was obtained from TCI. (Polystyrylmethyl)trim-
ethylamonium cyanoborohydride and tris-(2-aminoethyl)-amine
polystyrene resin were obtained from Novabiochem. Column
chromatography was performed on a Flashmaster II (Jones Chro-
matography) with IsoluteÒ columns pre-packed with silica gel
(30–90 lM) for normal phase and C18 (30–90 lM) for reversed
phase chromatography. Melting points were determined on an
ElectrothermalÒ digital melting point apparatus and are uncor-
rected. NMR-spectra were recorded on a Bruker AvanceÒ DRX-
400 spectrometer (400 MHz), coupling constants are reported in
Hz. Electrospray Ionization (ESI) mass spectra were acquired on
an ion trap mass spectrometer (Bruker DaltonicsÒ esquireTM
3000plus). LC-MS spectra were recorded on an Agilent 1100 Series
5.2.4. N-tert-Butoxycarbonyl-1,4-dideoxy-1,4-imino-5-O-tert-
butyldimethylsilyl-D-ribitol (8b)
Compound 7b (0.32 g, 1.0 mmol) and 4-methylmorpholine N-
oxide (0.41 g, 3.1 mmol) were dissolved in acetone (15 mL) and
H2O (3 mL) and this was cooled to 0 °C. OsO4 (0.006 g, 0.02 mmol)
was added and the resulting mixture was allowed to warm to rt
and was then stirred at rt for 18 h. NaHSO3 (0.25 g) was added
and after 30 min of stirring, the mixture was extracted three times
with EtOAc. The combined organic layers were washed with brine,
dried (Na2SO4), and the solvent was removed under reduced pres-
sure. The crude product was purified by column chromatography
(hexane to hexane/EtOAc 1:4) to yield the title compound as a syr-
up (0.14 g, 39%). MS (ESI) m/z 348.5 [M+H]+.
HPLC system equiped with a C18 column (2.1 ꢀ 50 mm, 5
Supelco, Sigma–Aldrich) for system I, and a HILIC Silica column
m, Atlantis HILIC, Waters) for system II, both
lm,
(2.1 ꢀ 100 mm, 5
l
coupled with a Bruker DaltonicsÒ esquireTM 3000plus mass spec-
trometer (solvent A: H2O with 0.1% formic acid, solvent B: ACN
with 0.1% formic acid; gradient I-A: 5% B to 100% B, 20 min,
0.2 mL/min; gradient I-B: 0% B to 100% B, 20 min, 0.2 mL/min; gra-
dient II-A: 90% B to 50% B, 20 min, 0.2 mL/min; gradient II-B: 90% B
to 40% B, 12 min, 0.2 mL/min). HPLC was performed on a Gilson
5.2.5. 1,4-Dideoxy-1,4-imino-
D
-ribitolꢁHCl (9ꢁHCl)
Method A. Compound 8a (7.2 g, 15 mmol) was dissolved in
TFA/H2O (1:1, 15 mL) and this was stirred at rt for 18 h. The reac-
tion mixture was concentrated under reduced pressure, and the
residue was dissolved in aq 2 N HCl. This was extracted twice
with EtOAc. The combined organic phases were dried (Na2SO4),
and the solvent was removed by evaporation under reduced pres-
sure to yield the iminoribitol compound as a crystalline solid
(2.5 g, 97%). MS, LC-MS, and NMR analysis showed that the com-
instrument equiped with a C18 column (4.6 ꢀ 25 cm, 5
sphereTM ODS) for system I, and
HILIC Silica column
(2.1 ꢀ 100 mm, 5 m, Atlantis HILIC, Waters) for system II (system
lm, Ultra-
a
l
I: solvent A: H2O with 0.1% trifluoroacetic acid, solvent B: ACN with
1% trifluoroacetic acid; gradient I-A: 10% B to 100% B, 36 min, 1 mL/
min; gradient I-B: 5% B to 50% B, 36 min, 1 mL/min; system II: sol-
vent A: H2O with 0.1% formic acid, solvent B: ACN with 0.1% formic
acid; gradient II: 90% B to 50% B, 20 min, 0.4 mL/min).
pound was identical to iminoribitol 9 obtained from D-gulonolac-
tone (Method C). Method B. Compound 8b (0.14 g, 0.4 mmol) was
dissolved in TFA/H2O (1:1, 2 mL) and this was stirred at rt for
30 h. The reaction mixture was concentrated under reduced pres-
sure and the residue was dissolved in aq 2 N HCl. This was ex-
tracted twice with EtOAc. The combined organic phases were
dried (Na2SO4) and the solvent was removed by evaporation un-
der reduced pressure to yield the iminoribitol compound as a
crystalline solid. MS, LC-MS, and NMR analysis showed that the
5.2. Chemistry
5.2.1. (2S)-N-tert-Butoxycarbonyl-2-triphenylmethoxymethyl-
3-pyrroline (7a)
Compound 618 (5.4 g, 27 mmol) was dissolved in pyridine
(22 mL) and triphenylmethylchloride (9.0 g, 32 mmol) was added.
The reaction mixture was stirred at rt for 24 h. The solvent was re-
moved under reduced pressure to give a syrup which was dis-
solved in EtOAc. This was washed with H2O and the aqueous
phase was extracted with EtOAc. The combined organic layers
were dried (Na2SO4) and concentrated under reduced pressure.
The residue was purified by column chromatography (hexane to
hexane/EtOAc 4:1) to yield the product as a syrup (11 g, 92%).
MS (ESI) m/z 463.6 [M+Na]+.
compound was identical to iminoribitol 9 obtained from
olactone (Method C). Method C. 5-O-tert-Butyldimethylsilyl-1,4-
dideoxy-1,4-imino-2,3-O-isopropylidene-
-ribitol 1016 (0.22 g,
D-gulon-
D
0.76 mmol) was dissolved in MeOH (1 mL), aq 6 N HCl (3 mL)
was added and the solution was stirred at rt for 20 h. The reaction
mixture was separated between H2O and CHCl3, the aqueous
layer was concentrated under vacuum and the residue was

6758
A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
recrystallized from hot EtOH yielding the title compound as a
crystalline solid (0.13 g, 96%), mp 125–128 °C. MS (ESI) m/z
134.1 [M+H]+; LC-MS (II-B) Rt 10.2 min, m/z 134.1 [M+H]+; 1H
5.2.9. 1,4-Dideoxy-1,4-imino-N-(4-cyanobenzyl)-
D-ribitol (30)
a-Bromo-p-tolunitrile was used in procedure A to obtain, after a
second purification by column chromatography, the title com-
pound as a yellow sticky gum (0.0061 g, 28%). MS (ESI) m/z 271.1
[M+Na]+; LC-MS (I-B) Rt 3.1 min, m/z 249.1 [M+H]+; HPLC (I-A,
k = 254 nm) Rt 4.1 min (100%); 1H NMR (CD3OD) d 2.37–2.43 (m,
1 H), 2.71–2.76 (m, 1H), 3.01–3.07 (m, 1H), 3.54–3.65 (m, 3H),
3.88 (dd, 1H, J = 9.7, J0 = 4.8), 3.99–4.05 (m, 1H), 4.13 (dd, 1H,
J = 13.8, J0 = 4.0), 7.52–7.56 (m, 2H), 7.64–7.68 (m, 2H); 13C NMR
(CD3OD) d 59.2, 60.4, 63.0, 71.2, 72.5, 74.3, 111.8, 119.8, 130.8,
133.2, 146.8.
NMR (D2O)
d 3.34 (d, 1H, J = 13.0), 3.47 (dd, 1H, J = 12.9,
J0 = 3.9), 3.57–3.63 (m, 1H), 3.80 (dd, 1H, J = 12.6, J0 = 6.0), 3.94
(dd, 1H, J = 12.6, J0 = 3.3), 4.18 (dd, 1H, J = 8.5, J0 = 4.2), 4.33–4.37
(m, 1H); 13C NMR (D2O) d 49.7, 58.1, 61.9, 69.5, 71.2.
5.2.6. General procedures for N-alkylation
Procedure A. The protected iminoribitol 1016 (1 equiv) was dis-
solved in CH2Cl2 (10 mL per mmol iminoribitol) and a solution of
Na2CO3 (2 equiv) in H2O (4 mL per mmol Na2CO3) was added. The
arylmethylhalide (1 equiv) was added, and the reaction mixture
was stirred under reflux for 3 h. The organic layer was separated,
and the aqueous layer was extracted twice with CH2Cl2. The com-
bined organic extracts were dried (Na2SO4), and the solvent was
evaporated under reduced pressure. The crude product was dis-
solved in TFA/H2O (1:1, 10 mL per mmol product), and this was
stirred at rt overnight. H2O was added, and this was extracted
twice with CH2Cl2. The aqueous layer was concentrated under re-
duced pressure and the residue was purified by column chroma-
tography (CHCl3/MeOH 4:1) to yield the final product. Procedure
B. The protected iminoribitol 1016 (1 equiv) was dissolved in
DMF (2 mL per 0.3 mmol iminoribitol). K2CO3 (2 equiv.) and the
arylmethylhalide (1.1 equiv) were added and this was stirred at
40 °C for 3–4 h. EtOAc and H2O were added and the mixture
was separated. The organic layer was washed twice with H2O
and the combined aqueous layers were extracted with EtOAc.
The combined organic layers were dried (Na2SO4) and concen-
trated under reduced pressure. The crude product was dissolved
in TFA/H2O (1:1, 10 mL per mmol product) and this was stirred
at rt overnight. H2O was added and this was extracted twice with
CH2Cl2. The aqueous layer was concentrated under reduced pres-
sure and redissolved in a mixture of H2O and methanol (usually
1:1, depending on the solubility of the product) and stirred with
Amberlyst A26(OH) ion exchange resin until the pH was neutral.
The resin was removed by filtration and rinsed extensively with
H2O and methanol. The solvents were evaporated under reduced
pressure and the residue was purified by column chromatography
(CH2Cl2/MeOH 4:1 or 3:1) to yield the final product.
5.2.10. 1,4-Dideoxy-1,4-imino-N-(3-nitrobenzyl)-D-ribitol (31)
3-Nitrobenzyl bromide was used in procedure A. The crude
product was dissolved in a mixture of H2O and methanol (1:1)
and stirred with Amberlyst A26(OH) ion exchange resin until the
pH was neutral. The resin was removed by filtration and rinsed
extensively with H2O and methanol. The solvents were evaporated
under reduced pressure, and the residue was purified by column
chromatography (CH2Cl2/methanol 4:1) to obtain the title com-
pound as a brown sticky gum (0.0082 g, 11%). MS (ESI) m/z 291.1
[M+Na]+; LC-MS (I-B) Rt 3.5 min, m/z 269.0 [M+H]+; HPLC (I-A,
k = 254 nm) Rt 7.3 min (100%); 1H NMR (CD3OD) d 2.43 (dd, 1H,
J = 9.5, J0 = 6.8), 2.75 (dd, 1H, J = 9.1, J0 = 4.6), 3.06 (dd, 1H, J = 9.5,
J0 = 5.8), 3.57–3.68 (m, 3H), 3.92 (t, 1H, J = J0 = 5.0), 4.04 (dd, 1H,
J = 12.2, J0 = 5.7), 4.18 (d, 1H, J = 13.6), 7.56 (t, 1H, J = J0 = 7.9), 7.77
(d, 1H, J = 7.6), 8.12 (dd, 1H, J = 8.1, J0 = 1.6), 8.25 (s, 1H); 13C
NMR (CD3OD) d 59.1, 59.9, 63.1, 71.1, 72.4, 74.3, 123.0, 124.5,
130.4, 136.2, 143.2, 149.8.
5.2.11. 1,4-Dideoxy-1,4-imino-N-(3-cyanobenzyl)-
D-ribitol (32)
a-Bromo-m-tolunitrile was used in procedure A. The crude
product was dissolved in a mixture of H2O and methanol (1:1)
and stirred with Amberlyst A26(OH) ion exchange resin until the
pH was neutral. The resin was removed by filtration and rinsed
extensively with H2O and methanol. The solvents were evaporated
under reduced pressure and the residue was purified by column
chromatography (CH2Cl2/methanol 4:1) to obtain the title com-
pound as a yellow sticky gum (0.0046 g, 6%). MS (ESI) m/z 249.1
[M+H]+; LC-MS (II-B) Rt 8.9 min, m/z 249.0 [M+H]+; HPLC (I-B,
k = 254 nm) Rt 11.2 min (100%), (I-B, k = 214 nm) Rt 10.0 min
1
(88%); H NMR (CD3OD) d 2.40 (dd, 1H, J = 9.5, J0 = 6.8), 2.73 (dd,
5.2.7. 1,4-Dideoxy-1,4-imino-N-(4-iodobenzyl)-
(28ꢁTFA)
D
-ribitolꢁTFA
1H, J = 8.9, J0 = 4.5), 3.03 (dd, 1H, J = 9.5, J0 = 5.8), 3.56–3.65 (m,
3H), 3.91 (t, 1H, J = J0 = 5.0), 4.03 (dd, 1H, J = 12.2, J0 = 5.8), 4.07 (d,
1H, J = 13.6), 7.49 (t, 1H, J = J0 = 7.7), 7.60 (d, 1H, J = 7.7), 7.66 (d,
1H, J = 7.8), 8.75 (s, 1H); 13C NMR (CD3OD) d 59.0, 59.9, 62.9,
71.1, 72.4, 74.2, 113.3, 119.8, 130.4, 131.9, 133.5, 134.7, 142.6.
4-Iodobenzyl bromide was used in procedure A to obtain the ti-
tle compound as a brown amorphous solid (0.099 g, 45%). MS (ESI)
m/z 350.0 [M+H]+ LC-MS (I-A) Rt 9.5 min, m/z 350.1 [M+H]+; HPLC
(I-A, k = 254 nm) Rt 11.7 min (100%); 1H NMR (CD3OD) d 3.33 (d,
1 H, J = 15.5), 3.53 (dd, 1H, J = 12.6, J0 = 4.1), 3.59–3.62 (m, 2H),
3.74–3.80 (m, 1H), 4.13 (dd, 1H, J = 6.7, J0 = 4.2), 4.27 (d, 1H,
J = 3.4), 4.41 (d, 1H, J = 12.9), 4.58 (d, 1H, J = 12.8), 7.32 (d, 2H,
J = 8.1), 7.84 (d, 2H, J = 8.1); 13C NMR (CD3OD) d 58.6, 58.7, 61.0,
70.5, 72.4, 72.9, 97.1, 118.1 (q, CF3COOH), 131.1, 133.9, 139.6,
163.0 (q, CF3COOH).
5.2.12. 1,4-Dideoxy-1,4-imino-N-[3-(1H-pyrrol-1-yl)benzyl]-
D-ribitol (33)
1-[3-(Bromomethyl)phenyl]-1H-pyrrole was used in procedure
B to obtain the title compound as a yellow sticky gum (0.0051 g,
6%). MS (ESI) m/z 289.0 [M+H]+; LC-MS (I-B) Rt 8.7 min, m/z
289.0 [M+H]+; HPLC (I-B, k = 254 nm) Rt 19.6 min (100%), (I-B,
k = 214 nm) Rt 21.3 min (95%); 1H NMR (CD3OD) d 2.60 (m, 1H),
2.86 (d, 1H, J = 4.0), 3.16 (dd, 1H, J = 9.9, J0 = 5.6), 3.61 (d, 2H,
J = 4.3), 3.71 (d, 1H, J = 13.3), 3.95 (t, 1H, J = J0 = 5.1), 4.07 (dd, 1H,
J = 11.3, J0 = 5.6), 4.17 (d, 1H, J = 13.0), 6.27 (s, 2H), 7.19 (s, 2H),
7.24 (d, 1H, J = 4.5), 7.38–7.41 (m, 2H), 7.51 (s, 1H); 13C NMR
(CD3OD) d 59.2, 60.9, 62.3, 71.0, 72.5, 74.2, 111.4, 120.1, 120.2,
121.9, 127.4, 129.9, 130.8, 132.4, 140.9, 142.4.
5.2.8. 1,4-Dideoxy-1,4-imino-N-(4-nitrobenzyl)-
(29ꢁTFA)
D
-ribitolꢁTFA
4-Nitrobenzyl bromide was used in procedure A (reaction time
20 h) to obtain the title compound as a brownish syrup (0.039 mg,
94%). MS (ESI) m/z 269.1 [M+H]+; LC-MS (II-B) Rt 8.9 min, m/z 268.9
[M+H]+; HPLC (I-A, k = 254 nm) Rt 6.9 min (100%); 1H NMR
(CD3OD) d 3.19–3.22 (m, 1H), 3.47–3.51 (m, 2H), 3.63–3.78 (m,
2H), 4.07–4.12 (m, 1H), 4.22–4.26 (m, 1H), 4.43 (d, 1H, J = 11.0),
4.67 (d, 1H, J = 13.0), 7.78 (d, 2H, J = 8.6), 8.30 (d, 2H, J = 8.7); 13C
NMR (CD3OD) d 59.1, 59.6, 61.1, 70.7, 72.8, 73.2, 125.1, 133.0,
140.2, 150.1, 163.0 (br signal, CF3COOH).
5.2.13. 1,4-Dideoxy-1,4-imino-N-[4-(1H-pyrrol-1-yl)benzyl]-
D-ribitol (34)
1-[4-(Bromomethyl)phenyl]-1H-pyrrole was used in procedure
B to obtain the title compound as an amorphous solid (0.0044 g,

A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
6759
6%). MS (ESI) m/z 289.1 [M+H]+; LC-MS (I-B) Rt 7.5 min, m/z 289.0
[M+H]+; HPLC (I-A, k = 254 nm) Rt 12.6 min (100%); 1H NMR
(CD3OD) d 2.47 (dd, 1 H, J = 9.5, J0 = 7.1), 2.73–2.75 (m, 1H), 3.09
(dd, 1H; J = 9.6, J0 = 5.9), 3.55–3.61 (m, 3H), 3.92 (t, 1H,
J = J0 = 5.0), 4.01–4.08 (m, 2H), 6.27 (t, 2H, J = J0 = 2.1), 7.16 (t, 2H,
J = J0 = 2.1), 7.42 (s, 4H); 13C NMR (CD3OD) d 59.2, 60.3, 63.0, 71.1,
72.6, 74.5, 111.4, 120.2, 121.0, 131.6, 137,3, 141.4.
amorphous solid (0.066 g, 85%). MS (ESI) m/z 279.0 [M+H]+; LC-
MS (II-B) Rt 13.3 min, m/z 278.9 [M+H]+; HPLC (I-B, k = 254 nm)
Rt 10.0 min (100%), (I-B, k = 214 nm) Rt 9.6 min (100%); 1H NMR
(CD3OD) d 2.48 (dd, 1H, J = 9.5, J0 = 7.0), 2.78 (dd, 1H, J = 8.9,
J0 = 4.5), 3.05 (dd, 1H, J = 9.5, J0 = 5.8), 3.57–3.64 (m, 2H), 3.69 (d,
1H, J = 13.1), 3.95 (t, 1H, J = J0 = 4.9), 4.05 (dd, 1H, J = 12.2,
J0 = 5.9), 4.19 (d, 1H, J = 13.1), 4.29 (s, 3H), 7.58 (d, 1H, J = 8.6),
7.66 (d, 1H, J = 8.6), 7.89 (s, 1H); 13C NMR (CD3OD) d 34.7, 59.0,
60.5, 62.9, 71.1, 72.4, 74.3, 110.9, 119.3, 130.5, 134.4, 137.3,
146.7.
5.2.14. 1,4-Dideoxy-1,4-imino-N-[4-(1H-pyrazol-1-yl)benzyl]-D-
ribitol (35)
1-[4-(Bromomethyl)phenyl]-1H-pyrazole was used in procedure
B to obtain the title compound as a green gum (0.069 g, 85%). MS
(ESI) m/z 290.0 [M+H]+; LC-MS (I-B) Rt 9.0 min, m/z 290.0 [M+H]+;
HPLC (I-B, k = 254 nm) Rt 15.4 min (100%), (I-B, k = 214 nm) Rt
5.2.19. 1,4-Dideoxy-1,4-imino-N-(2-naphtyl)methyl-
D-
ribitolꢁTFA (40ꢁTFA)
2-(Bromomethyl)naphtalene was used in procedure A to obtain
the title compound as a light brown gum (0.077 g, 80%). MS (ESI)
m/z 274.1 [M+H]+; LC-MS (I-B) Rt 10.1 min, m/z 274.1 [M+H]+;
1
14.6 min (100%); H NMR (CD3OD) d 2.48 (dd, 1H, J = 9.5, J0 = 7.0),
2.75 (dd, 1H, J = 8.9, J0 = 4.5), 3.09 (dd, 1H, J = 9.5, J0 = 5.9), 3.56–
3.65 (m, 3H), 3.95 (t, 1H, J = J0 = 5.0), 4.03–4.09 (m, 2H), 6.51 (t, 1H,
J = J0 = 2.1), 7.46 (d, 2H, J = 8.4), 7.67 (d, 2H, J = 8.4), 7.70 (d, 1H,
J = 1.7), 8.17 (d, 1H, J = 2.4); 13C NMR (CD3OD) d 59.1, 60.0, 62.8,
71.0, 72.4, 74.4, 108.6, 120.3, 129.0, 131.3, 138.8, 140.4, 142.0.
HPLC (I-A, k = 254 nm) Rt 12.3 min (100%); 1H NMR (CD3OD)
d
2.78 (br s, 1H), 3.04 (br s, 1H), 3.21 (dd, 1H, J = 10.3, J0 = 5.4),
3.56–3.66 (m, 2H), 3.93–4.01 (m, 2H), 4.10 (dd, 1H, J = 10.8,
J0 = 5.4), 4.35 (d, 1H, J = 12.8), 7.45–7.51 (m, 2H), 7.53 (dd, 1H,
J = 8.5, J0 = 1.5), 7.83–7.87 (m, 4H); 13C NMR (CD3OD) d 58.9, 60.0,
62.3, 70.7, 72.4, 73.4, 118.0 (q, CF3COOH), 128.8, 129.1, 129.8,
131.1, 132.3, 134.6, 134.9, 163.1 (br, CF3COOH).
5.2.15. 1,4-Dideoxy-1,4-imino-N-[4-(1H-1,2,4-triazol-1-yl)
benzyl]-D-ribitol (36)
1-[4-(Bromomethyl)phenyl]-1H-1,2,4-triazole was used in pro-
cedure B to obtain the title compound as a white amorphous solid
(0.072 g, 89%). MS (ESI) m/z 291.0 [M+H]+; LC-MS (II-B) Rt 10.6 min,
m/z 290.9 [M+H]+; HPLC (I-B, k = 254 nm) Rt 11.8 min (100%), (I-B,
k = 214 nm) Rt 11.0 min (100%); 1H NMR (CD3OD) d 2.46 (dd, 1 H,
J = 9.5, J0 = 7.0), 2.75 (dd, 1H, J = 9.0, J0 = 4.5), 3.08 (dd, 1H, J = 9.5,
J0 = 5.9), 3.58–3.66 (m, 3H), 3.94 (t, 1H, J = J0 = 5.0), 4.05 (dd, 1H,
J = 12.3, J0 = 5.9), 4.10 (d, 1H, J = 13.3), 7.53 (d, 2H, J = 8.3), 7.75 (d,
2H, J = 8.4), 8.15 (s, 1H), 9.04 (s, 1H); 13C NMR (CD3OD) d 59.1,
60.0, 62.9, 71.1, 72.4, 74.3, 120.9, 131.4, 137.3, 140.9, 142.9, 152.8.
5.2.20. 1,4-Dideoxy-1,4-imino-N-(2-quinolinyl)methyl-
ribitolꢁTFA (41ꢁTFA)
D-
2-(Chloromethyl)quinoline was used in procedure A (reaction
time 16 h, addition of 2 equiv NaI) to obtain the title compound
as a gum (0.0020 g, 3%). MS (ESI) m/z 275.2 [M+H]+; LC-MS (I-B)
Rt 10.2 min, m/z 275.2 [M+H]+; HPLC (I-A, k = 214 nm) Rt 9.3 min
(100%); 1H NMR (CD3OD) d 2.60–2.64 (m, 1H), 2.89 (s, 1H), 3.17–
3.20 (m, 1H), 3.58–3.73 (m, 2H), 3.93–4.00 (m, 2H), 4.06–4.09
(m, 1H), 4.36 (d, 1H, J = 14), 7.59 (t, 1H, J = J0 = 7.5), 7.64 (d, 1H,
J = 8.5), 7.76 (t, 1H, J = J0 = 7.1), 7.92 (d, 1H, J = 7.9), 8.02 (d, 1H,
J = 8.6), 8.31 (d, 1H, J = 8.6); 13C NMR (CD3OD) d 59.5, 61.7, 62.4,
71.3, 72.4, 74.1, 122.6, 127.6, 128.6, 128.9, 129.7, 131.0, 132.4,
138.5, 148.2.
5.2.16. 1,4-Dideoxy-1,4-imino-N-(5-methyl-2-phenyl-2H-1,2,
3-triazol-4-yl)methyl-
D-ribitol (37)
4-(Bromomethyl)-5-methyl-2-phenyl-2H-1,2,3-triazole
was
used in procedure B to obtain the title compound as a white amor-
phous solid (0.055 g, 65%). MS (ESI) m/z 305.1 [M+H]+; LC-MS (I-B)
Rt 9.6 min, m/z 305.1 [M+H]+; HPLC (I-B, k = 254 nm) Rt 20.3 min
(100%), (I-B, k = 214 nm) Rt 19.6 min (100%); 1H NMR (CD3OD) d
2.38 (s, 3H), 2.61 (dd, 1H, J = 9.6, J0 = 7.0), 2.80 (dd, 1H, J = 9.2,
J0 = 4.6), 3.15 (dd, 1H, J = 9.5, J0 = 5.9), 3.60–3.70 (m, 2H), 3.77 (d,
1H, J = 13.9), 3.92 (t, 1H, J = J0 = 5.0), 4.05 (dd, 1H, J = 12.4, J0 = 5.8),
4.10 (d, 1H, J = 13.9), 7.32 (t, 1H, J = J0 = 7.4), 7.47 (m, 2H), 7.97 (d,
2H, J = 7.7); 13C NMR (CD3OD) d 10.1, 49.4, 59.1, 62.9, 71.0, 71.9,
74.3, 119.3, 128.1, 130.3, 141.0, 145.9, 146.0.
5.2.21. 1,4-Dideoxy-1,4-imino-N-(8-quinolinyl)methyl-
ribitolꢁTFA (42ꢁTFA)
D-
8-(Bromomethyl)quinoline was used in procedure B to obtain
the title compound as a white amorphous solid (0.062 g, 72%).
MS (ESI) m/z 275.0 [M+H]+ and m/z 297.0 [M+Na]+; LC-MS (I-B)
Rt 4.5 min, m/z 275.0 [M+H]+; HPLC (I-B, k = 254 nm) Rt 14.8 min
(100%); 1H NMR (CD3OD) d 2.70 (br s, 1H), 3.02–3.10 (m, 2H),
3.71 (dd, 1H, J = 11.7, J0 = 4.7), 3.89–4.03 (m, 4H), 4.96 (d, 1H,
J = 12.7), 7.51–7.58 (m, 2H), 7.73 (d, 1H, J = 6.9), 7.89 (d, 1H,
J = 8.2), 8.34 (d, 1H, J = 8.3), 8.84–8.86 (m, 1H); 13C NMR (CD3OD)
d 57.3, 59.4, 62.2, 71.0, 71.5, 73.7, 118.0 (q, CF3COOH), 122.6,
127.5, 129.5, 130.2, 131.9, 138.6, 147.5, 150.4, 150.6, 163.3 (d,
CF3COOH).
5.2.17. 1,4-Dideoxy-1,4-imino-N-(1-methyl-1H-benzimidazol-
2-yl)methyl-D-ribitol (38)
2-(Bromomethyl)-1-methyl-1H-benzimidazole was used in
procedure A to obtain the title compound as a yellow amorphous
solid (0.013 g, 15%). MS (ESI) m/z 278.2 [M+H]+ and m/z 300.2
[M+Na]+; LC-MS (I-B) Rt 7.8 min, m/z 278.1 [M+H]+; HPLC (I-A,
k = 254 nm) Rt 6.5 min (100%); 1H NMR (CD3OD) d 2.67 (dd, 1 H,
J = 9.5, J0 = 6.6), 2.87 (dd, 1H, J = 8.8, J0 = 4.4), 3.08 (dd, 1H, J = 9.4,
J0 = 5.7), 3.52–3.64 (m, 2H), 3.88 (s, 3H), 3.91 (t, 1H, J = J0 = 4.9),
3.96 (d, 1H, J = 14.2), 4.04 (dd, 1H, J = 11.2, J0 = 5.5), 4.34 (d, 1H,
J = 14.3), 7.21–7.31 (m, 2H), 7.47 (d, 1H, J = 8.0), 7.59 (d, 1H,
J = 7.9); 13C NMR (CD3OD) d 30.4, 52.4, 59.4, 63.2, 71.5, 72.3,
74.5, 110.8, 119.4, 123.3, 124.0, 137.2, 142.4, 153.8.
5.2.22. 1,4-Dideoxy-1,4-imino-N-(1-isoquinolinyl)methyl-D-
ribitol (43)
1-(Bromomethyl)isoquinoline was used in procedure A to ob-
tain the title compound as a yellow amorphous solid (0.018 g,
31%). MS (ESI) m/z 297.2 [M+Na]+; LC-MS (I-B) Rt 8.8 min, m/z
275.1 [M+H]+; HPLC (I-A, k = 254 nm) Rt 8.2 min (100%); 1H NMR
(CD3OD) d 2.66 (dd, 1H, J = 9.7, J0 = 6.5), 2.93 (dd, 1H, J = 8.9,
J0 = 4.4), 3.04 (dd, 1H, J = 9.6, J0 = 5.7), 3.58–3.70 (m, 2H), 3.93 (t,
1H, J = J0 = 5.1), 4.01 (dd, 1H, J = 10.7, J0 = 5.8), 4.26 (d, 1H,
J = 13.8), 4.72 (d, 1H, J = 13.8), 7.65–7.70 (m, 1H), 7.73–7.79 (m,
2H), 7.93 (d, 1H, J = 8.1), 8.36 (d, 1H, J = 5.8), 8.51 (d, 1H, J = 8.5);
13C NMR (CD3OD) d 59.2, 59.5, 63.0, 71.4, 72.7, 74.3, 122.2, 126.9,
128.3, 128.6, 128.8, 131.9, 138.0, 141.4, 159.7.
5.2.18. 1,4-Dideoxy-1,4-imino-N-(1-methyl-1H-1,2,3-benzo-
triazol-5-yl)methyl-D-ribitol (39)
5-(Bromomethyl)-1-methyl-1H-1,2,3-benzotriazole was used
in procedure B to obtain the title compound as a pale yellow

6760
A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
5.2.23. N-(7-Quinolinyl)-5-O-tert-butyldimethylsilyl-1,4-
dideoxy-1,4-imino-2,3-O-isopropylidene- -ribitol (27)
iminoribitol). The aldehyde (1.2 equiv) and (polystyrylmeth-
yl)trimethylammonium cyanoborohydride (2 equiv) were added.
This was stirred at rt for 24–48 h. The reaction mixture was filtered
to remove the polymer and the solvent was evaporated under re-
duced pressure. The residue was dissolved in TFA/H2O (1:1, 2 mL
per 0.3 mmol protected iminoribitol), and this was stirred at rt
for 18 h. The TFA and H2O were evaporated under reduced pres-
sure, and the residue was dissolved in methanol and H2O. Amber-
lyst A26(OH) ion exchange resin was added and the mixture was
stirred at rt until the pH was neutral or slightly basic. Then the
reaction mixture was filtered to remove the resin, which was
rinsed extensively with methanol and H2O. The solvents were
evaporated under reduced pressure to yield the crude product.
D
7-Methylquinoline 59 (0.10 g, 0.70 mmol), N-bromosuccinimide
(0.13 g, 0.73 mmol) and a catalytic amount of dibenzoylperoxide
were dissolved in carbontetrachloride (10 mL). The reaction mixture
was stirred under reflux for 18h. Protected iminoribitol compound
1016 (0.14 g, 0.49 mmol) was dissolved in dimethylformamide
(3 mL) and together with KHCO3 (0.10 g, 0.97 mmol) added to the
cooled (40 °C) reaction mixture containing 7-bromomethylquino-
line 60. This was stirred at 40 °C for 1.5 h. The reaction mixture
was filtered and the filtrate was concentrated under reduced pres-
sure. The residue was dissolved in EtOAc and washed with H2O,
dried (Na2SO4), and concentrated under reduced pressure. The resi-
due was purified by column chromatography (hexane/EtOAc 85:15)
to yield the protected intermediate product (0.15 g, 49%). MS (ESI)
m/z 429.3 [M+H]+; 1H NMR (CDCl3) d 0.04 (s, 3H), 0.07 (s, 3H), 0.88
(s, 9H), 1.33 (s, 3H), 1.58 (s, 3H), 2.80 (dd, J = 10.4, J0 = 2.5, 1H), 3.10
(dd, J = 5.8, J0 = 3.9, 1H), 3.18 (dd, J = 10.4,J = 5.5, 1H), 3.67 (dd,
J = 10.7, J0 = 4.2, 1H), 3.84 (dd,J = 10.7, J0 = 4.1, 1H), 3.98 (d, J = 13.7,
1H), 4.24 (d, J = 13.7, 1H), 4.58 (dd, J = 6.5, J0 = 2.0, 1H), 4.66–4.69
(m, 1H), 7.34–7.37 (m, 1H), 7.45 (d, J = 8.6, 1H), 7.65 (d, J = 8.4, 1H),
7.76 (d, J = 8.4, 1H), 8.03 (s, 1H), 8.11–8.17 (m, 1H).
5.2.27. 1,4-Dideoxy-1,4-imino-N-(2-aminopyridin-3-yl)methyl-
D-ribitol (53)
The general reductive amination procedure was followed with
2-amino-3-pyridinecarboxaldehyde (2 equiv) as the aldehyde.
The crude product obtained after deprotection was stirred at rt
overnight with tris-(2-aminoethyl)-amine polystyrene resin in
CH2Cl2/MeOH (3:1) to scavenge the excess of aldehyde. Column
purification (CH2Cl2/MeOH 3:1) yielded the final compound as a
white amorphous solid (0.019 g, 27%). MS (ESI) m/z 240.0 [M+H]+
and m/z 262.0 [M+Na]+; LC-MS (II-A) Rt 5.5 min, m/z 240.1
[M+H]+; HPLC (II, k = 214 nm) Rt 7.3 min (100%); 1H NMR (D2O) d
2.54 (t, 1H, J = 8.8), 2.84 (m, 1H), 3.09–3.14 (m, 1H), 3.58 (d, 1H,
J = 13.2), 3.65–3.78 (m, 2H), 4.01–4.05 (m, 2H), 4.14–4.18 (m,
1H), 6.83–6.87 (m, 1H), 7.63 (d, 1H, J = 7.0), 7.97 (d, 1H, J = 4.1);
13C NMR (D2O) d 56.8, 56.9, 61.3, 69.7, 70.2, 72.7, 114.6, 119.0,
139.9, 145.1, 157.3.
5.2.24. 1,4-Dideoxy-1,4-imino-N-(7-quinolinyl)methyl-D-
ribitolꢁTFA (44ꢁTFA)
Compound 27 (0.15 g, 0.34 mmol) was dissolved in TFA/water
(1:1, 2 ml). This was stirred at room temperature overnight. The
solvents were evaporated under reduced pressure. After purifica-
tion by column chromatography (DCM/MeOH 85:15) the TFA-salt
of the title compound was obtained as a white amorphous solid
(0.065 g, 49%). MS (ESI) m/z 275.1 [M+H]+; LC-MS (II-B) Rt
11.1 min, m/z 275.1 [M+H]+; HPLC (II, k = 214 nm) Rt 6.6 min
(100%); 1H NMR (MeOD) d 3.18 (d, J = 9.3, 1H), 3.40–3.47 (m, 2H),
3.59 (dd, J = 12,5, J0 = 3.5, 1H), 3.70 (dd, J = 12.0, J0 = 5.1, 1H), 4.07
(t, J = J0 = 6.8, 1H), 4.18–4.21 (m, 1H), 4.44 (d, J = 12.9, 1H), 4.69
(d, J = 13.0, 1H), 7.51–7.55 (m, 1H), 7.70 (d, J = 8.4, 1H), 7.98 (d,
J = 8.4, 1H), 8.11 (s, 1H), 8.34 (d, J = 8.3, 1H), 8.86 (br s, 1H); 13C
NMR (MeOD) d 59.0, 59.7, 62.0, 70.7, 72.7, 73.3, 116.1, 118.5 (q,
CF3COOH), 123.5, 129.5, 130.3, 131.4, 135.4, 138.2, 148.5, 152.2,
164.5 (q, CF3COOH).
5.2.28. 1,4-Dideoxy-1,4-imino-N-(6-methoxypyridin-3-yl)
methyl-D-ribitol (54)
The general reductive amination procedure was followed with
6-methoxy-3-pyridinecarboxaldehyde as the aldehyde. After puri-
fication by column chromatography (CH2Cl2/MeOH 3:1) the title
compound was obtained as a light yellow gum (0.053 g, 83%). MS
(ESI) m/z 255.2 [M+H]+ and 277.2 [M+Na]+; LC-MS (II-A) Rt
5.1 min, m/z 255.1 [M+H]+; HPLC (I-B, k = 254 nm) Rt 7.5 min
(100%), and (I-B, k = 214 nm) Rt 7.4 min (97%); 1H NMR (CD3OD)
d 2.62 (s, 1H), 2.88 (s, 1H), 3.11 (dd, 1H, J = 10.0, J0 = 5.4), 3.60 (d,
2H, J = 4.5), 3.67–3.71 (m, 1H), 3.95 (t, 1H, J = J0 = 5.2), 4.04–4.14
(m, 2H), 6.74–6.78 (m, 1H), 7.68–7.72 (m, 1H), 8.07–8.09 (m,
1H); 13C NMR (CD3OD) d 54.3, 57.7, 58.9, 62.1, 71.0, 72.4, 74.2,
111.7, 130.0, 132.5, 142.0, 148.7, 165.6.
5.2.25. 1,4-Dideoxy-1,4-imino-N-(4-pyridinyl)methyl-D-ribitol
(52)
The protected iminoribitol 1016 (0.084 g, 0.29 mmol) and 4-pyr-
idinecarboxaldehyde (0.03 mL, 0.31 mmol) were dissolved in
MeOH (2 mL). NaCNBH3 (0.13 g, 2.1 mmol) was added, and the
reaction mixture was stirred at rt for 24 h. The solvent was re-
moved under reduced pressure, and the residue was redissolved
in EtOAc. This was filtered through a pad of Silica. The solvent
was removed by evaporation under reduced pressure and the res-
idue was dissolved in TFA/H2O (1:1, 4 mL). This was stirred at rt
overnight. The reaction mixture was concentrated under reduced
pressure and the residue was purified by column chromatography
(CHCl3/MeOH 4:1) to yield the title compound as a gum (0.033 g,
48%). MS (ESI) 225.1 [M+H]+; LC-MS (II-B) Rt 11.6 min, m/z 225.1
[M+H]+; HPLC (II-B, k = 254 nm) Rt 8.7 min (88%); 1H NMR (CD3OD)
d 3.04–3.08 (m, 1H), 3.37–3.42 (m, 1H), 3.47 (dd, 1H, J = 11.6,
J0 = 4.7), 3.68–3.79 (m, 2H), 4.09 (dd, 1H, J = 6.8, J0 = 4.6), 4.21–
4.28 (m, 2H), 4.56 (d, 1H, J = 13.7), 7.61 (d, 2H, J = 5.4), 8.58–8.64
(br s, 2H); 13C NMR (CD3OD) d 59.2, 60.3, 60.6, 70.7, 73.0, 73.3,
118.2 (q, CF3COOH), 126.7, 145.1, 150.3, 163.2 (q, CF3COOH).
5.2.29. 1,4-Dideoxy-1,4-imino-N-(4-methyl-2-phenylpy-
rimidin-5-yl)methyl-D-ribitol (55)
The general reductive amination procedure was followed with
4-methyl-2-phenyl-5-pyridimidinecarboxaldehyde as the alde-
hyde. After purification by column chromatography (CH2Cl2/MeOH
12:1) the title compound was obtained as a white amorphous solid
(0.034 g, 35%). MS (ESI) m/z 316.3 [M+H]+ and m/z 338.3 [M+Na]+;
LC-MS (II-A) Rt 4.9 min, m/z 316.3 [M+H]+; HPLC (I-B, k = 254 nm)
Rt 18.2 min (100%), and (I-B, k = 214 nm) 18.1 min (100%); 1H
NMR (CD3OD) d 2.47 (dd, 1H, J = 6.9, J0 = 2.3), 2.63 (s, 3H), 2.77
(dd, 1H, J = 9.1, J0 = 1.5), 3.03 (dd, 1H, J = 5.7, J0 = 3.5), 3.57–3.67
(m, 3H), 3.91 (t, 1H, J = J0 = 4.9), 4.03 (dd, 1H, J = 11.3, J0 = 5.6),
4.15 (d, 1H, J = 13.6), 7.44–7.48 (m, 3H), 8.33–8.36 (m, 2H), 8.62
(s, 1H); 13C NMR (CD3OD) d 22.2, 55.7, 59.3, 63.6, 71.4, 73.0, 74.6,
129.3, 129.6, 130.1, 131.7, 139.0, 158.3, 164.4, 168.7.
5.2.26. General procedure used for the reductive aminations
(except for compound 52)
5.2.30. 1,4-Dideoxy-1,4-imino-N-(3-quinolinyl)methyl-D-ribitol
(56)
The general reductive amination procedure was followed with
3-quinoline-carboxaldehyde as the aldehyde. After purification
The protected iminoribitol 1016(1 equiv) was dissolved in a
mixture of CH2Cl2/acetic acid (10:1, 2 mL per 0.3 mmol protected

A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
6761
by column chromatography (CH2Cl2/MeOH 3:1) the title com-
pound was obtained as a yellow sticky gum (0.023 g, 30%). MS
(ESI) m/z 275.2 [M+H]+; LC-MS (II-A) Rt 5.3 min, m/z 275.2
[M+H]+; HPLC (II-B, k = 214 nm) Rt 5.9 min (100%); 1H NMR
(CD3OD) d 2.50 (dd, 1H, J = 9.4, J0 = 6.8), 2.80 (dd, 1H, J = 8.9,
J0 = 4.4), 3.06 (dd, 1H, J = 9.5, J0 = 5.8), 3.59–3.64 (m, 2H), 3.76 (d,
1H, J = 13.5), 3.93 (t, 1H, J = J0 = 5.0), 4.04 (dd, 1H, J = 11.9, J0 = 6.0),
4.27 (d, 1H, J = 13.5), 7.56–7.61 (m, 1H), 7.70–7.75 (m, 1H), 7.91
(d, 1H, J = 8.1), 8.00 (d, 1H, J = 8.5), 8.27 (s, 1H), 8.85 (s, 1H); 13C
NMR (CD3OD) d 58.2, 59.2, 63.2, 71.3, 72.6, 74.4, 128.3, 129.0,
129.2, 129.7, 130.9, 133.9, 137.8, 148.1, 153.0.
5.2.34. N-(9-Deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-
D
-ribitolꢁCH3COOH (62)
6-Chloro-9-deazapurine26 (0.27 g, 1.7 mmol) was dissolved in a
mixture of aqueous ammonia (25% solution, 25 mL) and 1,4-diox-
ane (25 mL). The reaction was sealed and left at 160 °C for 20 h.
After cooling to rt, the reaction mixture was evaporated under re-
duced pressure and the residue was purified by column chroma-
tography (CH2Cl2/MeOH/Et3N 70:30:1) to obtain 9-deaza-adenine
as a light brown amorphous solid (0.19 g, 80%). MS (ESI) m/z
135.3 [M+H]+ and m/z 156.9 [M+Na]+; LC-MS (II-B) Rt 9.3 min, m/
z 135.0 [M+H]+; 1H NMR (DMSO) d 6.32 (s, 1H), 6.62 (br s, 2H),
7.48 (s, 1H), 8.07 (s, 1H), 10.87 (br s, 1H). 9-Deaza-adenine
(0.090 g, 0.67 mmol) was dissolved in 1,4-dioxane (5 mL). Et3N
(0.039 mL, 0.67 mmol), a catalytic amount of DMAP and Boc2O
(0.15 g, 0.67 mmol) were added, and the reaction mixture was stir-
red at rt for 24 h. TLC showed that not all starting material was dis-
appeared and another amount of Boc2O (0.10 g, 0.45 mmol) was
added. The reaction mixture was stirred for another 24 h, then con-
centrated under reduced pressure and the residue was dissolved in
EtOAc and washed with water. The organic phase was dried
(Na2SO4) and evaporated under reduced pressure. The crude prod-
uct was subjected to column chromatography (CH2Cl2/MeOH/Et3N
80:20:1) to obtain Boc-protected 9-deaza-adenine (0.11 g). This
was added to a solution of iminoribitol hydrochloride 9ꢁHCl
(0.073 g, 0.43 mmol), NaOAc (0.050 g, 0.56 mmol) and 37% aq
formaldehyde (0.049 mL, 0.56 mmol) in H2O (2 mL). The reaction
mixture was stirred at 95 °C for 3 h. After cooling down to rt, Silica
gel was added, and this was evaporated to dryness under reduced
pressure. Purification by column chromatography (CH2Cl2/MeOH/
NH4OH 5:4:2, two consecutive columns) yielded the product
(0.020 g, 14%). MS (ESI) m/z 280.1 [M+H]+; LC-MS (II-B) Rt
14.3 min, m/z 280.1 [M+H]+; HPLC (II-B, k = 214 nm) Rt 12.8 min
(98%) and (II-B, k = 254 nm) Rt 12.8 min (84%); 1H NMR (MeOD) d
3.09 (dd, 1H, J = 11.6, J0 = 4.2), 3.28–3.32 (m, 1H), 3.37 (dd, 1H,
J = 11.6, J0 = 4.8), 3.67–3.76 (m, 2H), 3.96–3.99 (m, 1H), 4.10 (dd,
1H, J = 9.4, J0 = 4.7), 4.29 (d, 1H, J = 13.7), 4.47 (d, 1H, J = 13.7),
7.63 (s, 1H), 8.16 (s, 1H); 13C NMR (MeOD) d 50.3, 58.6, 59.7,
70.3, 71.0, 73.2, 107.3, 115.5, 132.0, 146.5, 151.4, 152.5.
5.2.31. 1,4-Dideoxy-1,4-imino-N-(4-quinolinyl)methyl-D-ribitol
(57)
The general reductive amination procedure was followed
with 4-quinoline-carboxaldehyde as the aldehyde. After purifica-
tion by column chromatography (CH2Cl2/MeOH 4:1) the title
compound was obtained as an white amorphous solid (0.043 g,
57%). MS (ESI) m/z 275.1 [M+H]+ and m/z 297.1 [M+Na]+; LC-
MS (II-A) Rt 5.3 min, m/z 275.1 [M+H]+; HPLC (II-B, k = 214
nm) Rt 6.0 min (100%); 1H NMR (D2O) d 2.62 (dd, 1H, J = 9.6,
J0 = 7.6), 2.98 (dd, 1H, J = 4.7, J0 = 4.5), 3.01 (dd, 1H, J = 9.8,
J0 = 5.9), 3.60–3.67 (m, 2H), 3.90–3.93 (m, 2H), 4.02 (dd, 1H,
J = 12.3, J0 = 6.0), 4.47 (d, 1H, J = 13.4), 7.48 (d, 1H, J = 3.8) 7.60–
7.65 (m, 1H), 7.75–7.79 (m, 1H), 7.98 (d, 1H, J = 8.3), 8.16 (d,
1H, J = 8.2), 8.71 (d, 1H, J = 4.2); 13C NMR (CD3OD) d 58.0, 59.7,
63.7, 71.5, 73.1, 74.5, 122.6, 125.7, 128.0, 128.9, 129.7, 130.9,
148.1, 148.8, 151.1.
5.2.32. 1,4-Dideoxy-1,4-imino-N-(8-hydroxyquinolin-2-yl)
methyl-D-ribitol (58)
The general reductive amination procedure was followed with
8-hydroxyquinoline-2-carboxaldehyde as the aldehyde. After puri-
fication by column chromatography (CH2Cl2/MeOH 3:1) the title
compound was obtained as a brown amorphous solid (0.036 g,
44%). MS (ESI) m/z 291.2 [M+H]+ and m/z 313.1 [M+Na]+; LC-MS
(II-A) Rt 5.1 min, m/z 291.2 [M+H]+; HPLC (I-B, k = 254 nm) Rt
16.1 min (100%), and (I-B, k = 214 nm) Rt 15.5 min (100%); 1H
NMR (CD3OD) d 2.85 (br s, 1H), 3.18 (br s, 1H), 3.45 (br s, 1H),
3.70–3.80 (m, 2H), 4.00–4.08 (m, 1H), 4.13–4.26 (m, 2H), 4.58 (d,
1H, J = 14.8), 7.10 (d, 1H, J = 5.9), 7.31–7.44 (m, 2H), 7.51 (d, 1H,
J = 8.5), 8.24 (d, 1H, J = 8.3); 13C NMR (CD3OD) d 59.9, 61.5, 62.2,
70.9, 72.1, 73.7, 112.3, 118.7, 122.2, 128.5, 129.5, 138.2, 139.0,
154.1, 156.2.
5.2.35. 1,4-Dideoxy-1,4-imino-N-(3-indolyl)methyl-
ribitolꢁTFA (65ꢁTFA)
D-
The protected iminoribitol 1016 (0.20 g, 0.70 mmol) was dis-
solved in a mixture of H2O and 1,4-dioxane (1:1, 4 mL), and 37%
aq formaldehyde (0.14 mL, 1.4 mmol) was added. This was stirred
at rt for 1 h, then indole (0.090 mg, 0.77 mmol) was added. The re-
action mixture was stirred at 95 °C for 20 h. The reaction mixture
was concentrated under reduced pressure and the residue dis-
solved in EtOAc. This was washed with H2O, dried (Na2SO4), and
concentrated under reduced pressure. The residue was purified
by column chromatography (hexane to EtOAc) to yield the pro-
tected intermediate product. MS (ESI) m/z 417.4 [M+H]+ and m/z
439.4 [M+Na]+; 1H NMR (CDCl3) d 0.07 (2s, 6H), 0.91 (s, 9H), 1.32
(s, 3H), 1.50 (s, 3H), 2.83 (dd, 1H, J = 10.9, J0 = 2.8), 3.18–3.22 (m,
2H), 3.66 (dd, 1H, J = 10.6, J0 = 5.5), 3.75 (dd, 1H, J = 10.6, J0 = 4.3),
4.49 (dd, 1H, J = 6.4, J0 = 2.4), 4.64–4.68 (m, 1H), 5.12 (d, 1H,
J = 12.5), 5.21 (d, 1H, J = 12.5), 6.56–6.58 (m, 1H), 7.19–7.26 (m,
2H), 7.61 (d, 1H, J = 7.8), 7.67 (d, 1H, J = 8.0); The protected inter-
mediate was dissolved in TFA/H2O (1:1, 2 mL) and this was stirred
at rt overnight. The reaction mixture was concentrated and the re-
sidue was purified by column chromatography (CH2Cl2 to CH2Cl2/
methanol 4:1) to yield the TFA-salt of the title compound as an
amorphous solid (0.039 g, 15% calculated over both steps). MS
(ESI) m/z 263.2 [M+H]+ and m/z 285.1 [M+Na]+; LC-MS (II-B) Rt
9.6 min, m/z 262.9 [M+H]+; HPLC (II, k = 214 nm) Rt 4.6 min
5.2.33. N-(9-Deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-
imino-
D
HCl)-ribitolꢁHCl (61ꢁHCl)
Iminoribitol hydrochloride 9ꢁHCl (0.15 g, 0.89 mmol) and NaO-
Ac (0.073 g, 0.89 mmol) were dissolved in H2O (2 mL) and 37% aq
formaldehyde (0.070 mL, 0.89 mmol) and 9-deazahypoxanthine21
(0.096 g, 0.71 mmol) were added. The reaction mixture was stir-
red at 95 °C for 17 h. After cooling down to rt, Silica gel was
added and this was evaporated to dryness under reduced pres-
sure. Purification by column chromatography (CH2Cl2/MeOH/
NH4OH 8:2:1) yielded the product. This was dissolved in metha-
nol and converted into the hydrochloride salt by repeated evapo-
ration of concentrated HCl. The title compound was obtained as
an amorphous solid (0.033 g, 12%). MS (ESI) m/z 281.0 [M+H]+
and m/z 303.0 [M+Na]+; LC-MS (II-B) Rt 11.1 min, m/z 280.9
[M+H]+; HPLC (I-B, k = 254 nm) Rt 7.0 min (98%), and (II,
k = 214 nm) Rt 8.0 min (100%); 1H NMR (MeOD) d 3.43 (dd, 1H,
J = 12.6, J0 = 3.1), 3.64–3.69 (m, 2H), 3.80–3.89 (m, 2H), 4.11–
4.16 (m, 1H), 4.27 (dd, 1H, J = 7.7, J0 = 4.3), 4.67 (d, 1H, J = 14.0),
4.79 (d, 1H, J = 14.3), 7.85 (s, 1H), 8.72 (s, 1H); 13C NMR (MeOD)
d 51.2, 58.5, 58.7, 70.3, 71.7, 72.9, 104.8, 120.0, 133.2, 137.3,
145.9, 153.7.
1
(100%); H NMR (D2O) d 3.38 (dd, 1H, J = 12.9, J0 = 2.7), 3.51 (dd,
1H, J = 13.0, J0 = 4.4), 3.62–3.73 (m, 3H), 4.12–4.15 (m, 1H), 4.29

6762
A. Goeminne et al. / Bioorg. Med. Chem. 16 (2008) 6752–6763
(d, 1H, J = 4.4), 4.55 (d, 1H, J = 13.6), 4.60 (d, 1H, J = 13.6), 7.18–7.28
(m, 2H), 7.51–7.55 (m, 2H), 7.70 (d, 1H, J = 7.8); 13C NMR (D2O) d
52.3, 56.3, 57.2, 68.7, 69.2, 70.9, 103.1, 112.2, 116.2 (q, CF3COOH),
117.9, 120.4, 122.6, 126.5, 128.6, 136.0, 163.0 (q, CF3COOH).
Ki) + S), where vi is the initial reaction rate, kcat is the catalytic turn-
over number of p-nitrophenyl-b- -ribofuranoside, KM is the
Michaelis constant for p-nitrophenyl-b- -ribofuranoside, Ki is the
dissociation constant of enzyme-inhibitor complex, I is the inhibi-
tor concentration and S is the substrate concentration.
D
D
5.2.36. 1,4-Dideoxy-1,4-imino-N-(7-methoxy-indol-3-
yl)methyl-D-ribitol (66)
5.4. Molecular modeling
The protected iminoribitol 1016 (0.20 g, 0.70 mmol) was dis-
solved in a mixture of H2O and 1,4-dioxane (1:1, 4 mL) and 37%
aq formaldehyde (0.14 mL, 1.4 mmol) was added. This was stirred
at rt for 1 h, then 7-methoxyindole (0.10 mL, 0.77 mmol) was
added. The reaction mixture was stirred at 95 °C for 20 h. The reac-
tion mixture was concentrated under reduced pressure and the
residue dissolved in EtOAc. This was washed with H2O, dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by column chromatography (hexane to EtOAc) to
yield the protected intermediate product. MS (ESI) m/z 447.3
[M+H]+ and m/z 469.3 [M+Na]+; 1H NMR (CDCl3) d 0.01 (2s, 6H),
0.91 (s, 9H), 1.29 (s, 3H), 1.55 (s, 3H), 2.72–2.77 (m, 1H), 3.02 (br
s,1H), 3.14–3.18 (m, 1H), 3.67–3.72 (m, 1H), 3.78–3.87 (m, 2H),
4.03 (s, 3H), 4.19 (d, 1H, J = 13.2), 4.53–4.55 (m, 1H), 4.61–4.65
(m, 1H), 6.71 (d, 1H, J = 7.8), 7.01–7.07 (m, 1H), 7.25–7.27 (m,
1H), 7.36 (d, 1H, J = 7.7); The protected intermediate was dissolved
in TFA/H2O (1:1, 2 mL) and this was stirred at rt overnight. The
reaction mixture was concentrated and the residue was redis-
solved in H2O and this was stirred with Amberlyst A26(OH) ion ex-
change resin until the pH of the solution was neutral. The resin was
removed by filtration and rinsed extensively with H2O and metha-
nol. The solvents were evaporated under reduced pressure and the
residue was purified by column chromatography (CH2Cl2/MeOH/
Et3N 30:10:1) to yield the title compound as a brown gum
(0.026 g, 13% calculated over both steps). MS (ESI) m/z 292.9
[M+H]+ and m/z 314.8 [M+Na]+; LC-MS (II-B) Rt 9.6 min, m/z
292.9 [M+H]+; HPLC (II, k = 214 nm) Rt 4.6 min (100%); 1H NMR
(D2O) d 2.61 (br s, 1H), 2.79 (s, 1H), 3.04 (d, 1H, J = 5.6), 3.21 (s,
2H), 3.51 (s, 2H), 3.83 (s, 3H), 3.92–3.95 (m, 1H), 4.14 (d, 1H,
J = 12.8), 6.54 (d, 1H, J = 7.2), 6.85–6.89 (m, 1H), 7.12–7.18 (m,
2H); 13C NMR (D2O) d 51.2, 55.8, 59.0, 62.3, 70.8, 71.9, 74.3,
102.7, 111.6, 112.5, 120.8, 125.7, 128.3, 130.5, 147.9.
The molecular modeling studies were performed using MOE
2006.08 software (Chemical Computing Group). All software nec-
essary to build the compounds, perform minimizations, align-
ments, and superpositions is available in the MOE package and
was used with standard settings, unless otherwise mentioned.
Crystal structures of the target enzyme were downloaded from
the Protein Databank (PDB), and the ligands were checked and
corrected when necessary. Residues not belonging to the enzyme
or ligand were removed, except the Ca2+ ion and the water mol-
ecules placed within a distance of 6 Å of the ligand. Hydrogen
atoms were added, and the energy was minimized with
MMFF94x force field (standard parameters) keeping all heavy
atoms fixed.
The modeling experiments were performed using several NH
crystal structures available through PDB. Special attention was
paid to PDB file 2FF2, the structure of NH co-crystallized with
(1S)-1-(9-deazahypoxanthin-9-yl)-1,4-imino-D-ribitol (Immucil-
lin-H).11 2FF2 was chosen for all docking studies reported here
since it represents a ‘closed’ structure of the enzyme with all loops
entirely ordered, possessing a small and narrow active pocket lead-
ing to more reliable docking scores. The iminoribitol target com-
pounds were constructed using the iminoribitol moiety of
Immucillin-H from 2FF2.The contacts involving the iminoribitol
group were conserved as described.11
The automatic docking protocol with default parameters was
used to position the ligands in the active site of the enzyme. Sub-
sequently, the top solution was chosen according to the S (Score)
function and was minimized as flexible ligand in the rigid enzyme
with MMFX94x force field. The docking protocol was validated by
‘re-docking’ of Immucillin-H in 2FF2.
Acknowledgments
5.3. Biochemistry
This work was funded by a research project from the IWT-Vla-
anderen and by a research project from the Research Foundation
Flanders (FWO-Vlaanderen). A.G. had a Ph.D. Grant from the Insti-
tute for the promotion of Innovation through Science and Technol-
ogy in Flanders. M.B. has a Ph.D. Grant from the Research
Foundation Flanders (FWO-Vlaanderen). W.V. and P.VdV. have a
postdoctoral grant from the Research Foundation Flanders.
5.3.1. Protein expression and purification
Expression and purification of the wild type T. vivax IAG-NH
were performed as described previously.4 E. coli cells (WK6) con-
taining the IAG-NH ORF cloned in the pQE-30 expression vector
were used to express the protein. The presence of an N-terminal
His6-tag allowed for a two-step purification scheme, consisting of
a Ni–NTA affinity chromatography step (Qiagen) and gel filtration
on a Superdex-200 column (Amersham Bioscience). The concentra-
tion of pure protein (expressed per monomer) was determined
spectrophotometrically using a e280 of 47752 Mꢂ1cmꢂ1. Typically,
80 mg of purified protein was obtained from a 1-l fermentation.
SDS–polyacrylamide gel electrophoresis was used to confirm en-
zyme purity.
References and notes
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