Synthesis and biochemical evaluation of guanidino-alkyl-ribitol derivatives as nucleoside hydrolase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.03.027

Nucleoside hydrolase (NH) is a key enzyme in the purine salvage pathway. The purine specificity of the IAG-NH from Trypanosoma vivax is at least in part due to cation-π-stacking interactions. Guanidinium ions can be involved in cation-π-stacking interactions, therefore a series of guanidino-alkyl-ribitol derivatives were synthesized in order to examine the binding affinity of these compounds towards the target enzyme. The compounds show moderate to good inhibiting activity towards the IAG-NH from T. vivax.

Full text of DOI:10.1016/j.ejmech.2007.03.027

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 315e326
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biochemical evaluation of guanidino-alkyl-ribitol
derivatives as nucleoside hydrolase inhibitors
A. Goeminne a, M. McNaughton a, G. Bal a, G. Surpateanu a, P. Van Der Veken a,
b
S. De Prol , W. Versees , J. Steyaert , A. Haemers , K. Augustyns
b
b
a
a,
*
´
a Department of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
b Department of Ultrastructure, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
Received 18 December 2006; received in revised form 16 March 2007; accepted 19 March 2007
Available online 10 April 2007
Abstract
Nucleoside hydrolase (NH) is a key enzyme in the purine salvage pathway. The purine specificity of the IAG-NH from Trypanosoma vivax is
at least in part due to cationep-stacking interactions. Guanidinium ions can be involved in cationep-stacking interactions, therefore a series of
guanidino-alkyl-ribitol derivatives were synthesized in order to examine the binding affinity of these compounds towards the target enzyme. The
compounds show moderate to good inhibiting activity towards the IAG-NH from T. vivax.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Nucleoside hydrolase; Inhibitor; Cationep-stacking interactions; Guanidino-alkyl-ribitol
1. Introduction
salvage by purine auxotrophic parasites, since it is the step
preceding the phosphoribosyltransferase reaction responsible
for the synthesis of purine nucleotides [3].
Parasitic infections are a continuing health problem, espe-
cially in developing countries. Amongst these infections, try-
panosomiasis and leishmaniasis are responsible for more than
110 000 deaths a year. Drugs that are currently in use suffer
from disadvantages such as toxicity, upcoming resistance,
complex administration mode, high costs or are simply not ef-
fective in certain stages of the disease. Hence, there is an urgent
need for new drugs in the treatment of trypanosomiasis [1].
One approach for the development of new anti-trypanoso-
mal compounds is based on the knowledge that parasitic
protozoa lack the ability to synthesize purines de novo [2].
Protozoa use the purine salvage pathway to obtain purine ba-
ses from nucleosides of the host. A key enzyme in the purine
salvage pathway is nucleoside hydrolase (NH). This enzyme
hydrolytically cleaves the N-glycosidic bond of nucleosides
to give a nucleobase and ribose. It plays a central role in purine
During cleavage of a nucleoside by nucleoside hydrolase,
an oxocarbenium ion-like transition state is formed in which
the ribose-oxygen carries a partial positive charge in which
N7 of the purine is protonated [4]. At present, four types of
NH’s are known, each differing in substrate specificity [5].
The target enzyme of our research is the NH from Trypano-
soma vivax. This NH shows a high specificity for inosine,
adenosine and guanosine (IAG-NH). The purine specificity
is imposed by parallel aromatic stacking of the purine ring be-
tween Trp83 and Trp260 (Fig. 1) [5,6]. These stacking interac-
tions contribute in raising the pKa of N7 of the purine, allowing
direct protonation of the base by solvent molecules, hence
functioning as an alternative to general acid catalysis [7].
The active site and hence the catalytic mechanism of the
IAG-NH is different from that of the mammalian nucleoside
phosphorylase, the enzyme that cleaves nucleosides in mam-
malian cells. Therefore selective inhibition of the former
enzyme could be an effective way to kill the parasite without
causing toxicity towards the host [2].
* Corresponding author. Tel.: þ32 3 8202703; fax: þ32 3 8202739.
E-mail address: koen.augustyns@ua.ac.be (K. Augustyns).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.027

316
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
containing a three-carbon atom spacer (10) are slightly hin-
dered in the NH active pocket.
As these docking studies suggested that guanidino-alkyl-
ribitol derivatives could be able to fit into the active site of the
target enzyme, we synthesized guanidino-alkyl-ribitol deriva-
tives (3 and 8e10). Concurrently we synthesized compounds
lacking a basic group (1 and 7) and a quaternary ammonium
salt (2), to prove that the guanidino function contributes favour-
ably to the binding interactions of the compounds with the
enzyme.
A further objectivewas to apply the transition-state-analogue
hypothesis proposed by Schramm and Horenstein [9]. They pro-
posed that the partial positive charge that develops in the ribose
ring during hydrolysis can be mimicked by substituting the ri-
bose-oxygen for a protonated nitrogen and developed nM active
inhibitors, characterized by a deaza-purine substituted iminori-
bitol moiety. (Fig. 1b) [10]. The best docking pose obtained for
compound 20, containing a guanidino-ethyl arm, confirms the
cationep-stacking interaction with the Trp83 and Trp260 side
Fig. 1. Rational design of the inhibitors, based on the structure of the transition
state (top drawing). (a) Guanidinium ions can mimic the partial positive charge
in the purine ring. (b) Iminoribitol derivatives can mimic the partial positive
charge in the ribose ring.
2. Results and discussion
´
chains (Fig. 2). As revealed by Versees et al. [11] the iminoribi-
2.1. Design of the inhibitors
tol moiety is stabilized in the active pocket by an additional
interaction of the imino group with Asn186. The same stabiliza-
tion occurs when the guanidine-alkyl chain was introduced on
the imino group (26 and 27). The length of the alkyl chain deter-
mines the correct position of the guanidine group for stacking.
The spacer arm separating the iminoribitol and guanidine moi-
eties should minimally consist of three methylene units (27)
for a right assignment of both groups.
To enhance the aromatic stacking interactions, we also de-
veloped the aromatic guanidine and amidine compounds 32
and 33. The aromatic side chains occupy the same location
as the purine ring of ImmH [(1S )-1-(9-deazahypoxanthin-
9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] [11].
In our search for new inhibitors against NH we wanted to
mimic the partial positive charge on the purine ring by other
functional groups that would also be able to participate in cati-
onep-stacking interactions (Fig. 1a). Guanidinium ions are
candidates to get involved in this type of interactions [8] (Fig. 2).
Docking results confirm the cationep-stacking interaction
potential involving the guanidinium fragment. This moiety is
situated between the Trp83 and Trp260 side chains, both re-
sponsible for stacking with the purine ring. The docking study
points out that the length of the alkyl chain separating the ribi-
tol and the guanidine moieties could be relevant for correct
stacking. The best stacking interaction is observed with a linker
of two methylene units (8).
2.2. Synthesis
A closer vicinity of the guanidinium to the ribitol moiety (3)
increases the inhibitor’s tendency to adopt unfavourable con-
formations stabilized by intramolecular H-bonding. Inhibitors
Synthesis of compounds 1e3 (Scheme 1) started from
commercially available b-D-ribofuranose-1-acetate-2,3,5-
tribenzoate. Reaction with Me3SiCN selectively afforded the
b-cyanide 4 [12]. Stirring 4 in NH3 saturated MeOH resulted
in the amide 1. Reduction of nitrile 4 was performed with
NaBH4/TFA [13]. The resulting amine 5 underwent methylation
to yield, after deprotection, the quaternary ammonium salt 2. In-
troduction of the guanidino group was achieved by coupling
amine
5
with N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-
1-carboxamidine as the guanylating reagent [14]. Deprotection
of 6 gave the guanidino-methyl-ribitol 3.
Compounds 4e7 were synthesized from protected D-ribose
(Scheme 2) [15]. The protected ribose 11 underwent a Moffate
Wittig reaction with the appropriate phosphorylide reagent
[16]. After chromatographic separation of the a- and b-isomer
(1:3 ratio), the b-isomer was carried into the next step: reduc-
tion of the ester 12a resulted in the primary alcohol which was
transformed into the corresponding mesylate 13. Substitution
of the mesyl group of 13 by sodium azide and deprotection
afforded azide 7. Reduction of the protected azide 14, reaction
with the guanylating reagent and subsequent deprotection
Fig. 2. Aromatic stacking of the purine ring of 3-deaza-adenosine (pdb code
1HP0) (carbons in orange) and the guanidino-alkyl-iminoribitol 20 (carbons
in green) with Trp83 and Trp260 (carbons in grey) (For interpretation of the
references to colour in figure legends, the reader is refered to the web version
of this article).

A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
317
Scheme 1. Reagents and conditions: (a) Ref. [12]: Me3SiCN, SnCl4, ACN, rt, 10 min; (b) NH3, MeOH, rt, 4 days; (c) Ref. [13]: NaBH4, TFA, rt, 18 h; (d) CH3I,
DMF, rt, 20 h; (e) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, CHCl3, rt, 24 h; (f) TFA:DCM 1:1, rt, 18 h.
resulted in the guanidino-ethyl-ribitol 8. Guanidino-methyl-
ethyl-ribitol 9 was synthesized from ketone 12b via a reductive
amination with ammonium formate as the amine source, fol-
lowed by reaction with the guanylating agent and subsequent
deprotection. Substitution of mesylate 13 with NaCN afforded
nitrile 17. Reduction, reaction with the guanylating reagent and
deprotection resulted in the guanidino-propyl-ribitol 10.
For the synthesis of compound 20 (Scheme 3), protected imi-
noribitol 21, synthesized from D-gulonolactone by the method
described by Fleet and Son [17], was oxidized to nitrone 22
and addition of lithiated acetonitrile selectively afforded
b-cyanide 23 [18]. Compound 23 was reduced, treated with
the guanylating reagent and deprotected to give the guanidino-
iminoribitol target compound 20.
Compounds 26 and 27 were synthesized starting from
2-bromoethylamine hydrobromide and 3-bromopropylamine
hydrobromide, respectively (Scheme 4). After Boc-protection
of the amines, the bromides were substituted by the protected
iminoribitol 21 [17]. The guanidino group was introduced as
before and deprotection afforded guanidine iminoribitols 26
and 27.
For the synthesis of compounds 32 and 33, the protected imi-
noribitol 21 [17] was reacted with the appropriate benzylbro-
mide (Scheme 5). The nitrobenzyl derivative 35a was reduced
with SnCl2 in ethanol and the resulting amine was treated
with the guanylating reagent. Deprotection yielded the target
compound 32. Nitrile derivative 35b was converted into an
amidine with lithium hexamethyldisilazide [19]. Deprotection
Scheme 2. Reagents and conditions: (a) Ref. [16]: Ph3P]CHCOOMe or Ph3P]CHCOCH3, ACN, reflux, 18 h; (b) LiAlH4, Et2O, 0 ꢀC, 15 min; (c) MsCl,
pyridine, DMAP, rt, 2 h; (d) NaN3, DMF, 100 ꢀC, 7 h; (e) TFA:H2O 1:1, rt, 18 h; (f) LiAlH4, Et2O, 0 þꢀC, 3 h; ꢁ(g) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-
ꢀ
˚
1-carboxamidine, CHCl3, rt, 24 h; (h) NaCN, NaI, DMF, 100 C, 7 h; (i) LiAlH4, Et2O, rt, 18 h; (j) NH4 HCOO , NaCNBH3, 3 A MS, MeOH, rt, 18 h.

318
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
Scheme 3. Reagents and conditions: (a) Ref. [18]: SeO2, H2O2, acetone, <4 ꢀC, 3e4 h; (b) Ref. [18]: n-BuLi, ACN, ꢁ78 ꢀC, 0.5 h; (c) Ref. [18]: Zn dust, AcOH,
rt, 6 h; (d) Ref. [18]: (Boc)2O, CHCl3, rt, 18 h; (e) LiAlH4, THF, rt, 3 h; (f) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, CHCl3, rt, 24 h;
(g) TFA:H2O 1:1, rt, 18 h.
resulted in the target compound 33. Deprotection of 21 [17]
yielded iminoribitol 34. An intermediate in the synthesis of imi-
noribitol 21 is the protected benzyl derivative 37 (Scheme 6),
which, after deprotection, led to benzyl iminoribitol 38.
analogue 8, supporting the transition-state-analogue hypothesis
described earlier. N-Guanidino-ethyl-iminoribitol 26 is less ac-
tive than its propyl equivalent 27, confirming the optimal chain
length conclusions of the modeling study. The inhibitory activ-
ity of benzylguanidine 32 and benzylamidine 33 appears higher
(7.0 and 6.7 mM) and shows that the aromatic ring improves the
stacking properties substantially. The guanidine and amidine
moieties are clearly important, increasing the activity 10-fold
compared to the non-substituted benzyl derivative 38. The loss
in activity of compound 38 compared to unsubstituted iminori-
bitol 34 could be due to an increase in conformational freedom
for compound 38. Substitution of the benzyl moiety of 38 with
a guanidine or amidinegroup possibly results in an additional in-
teraction with the enzyme, which allows more favourable orien-
tation of the aromatic moiety within the active site.
2.3. Biochemical results
The target compounds were tested as inhibitors against
IAG-NH isolated from T. vivax and the results are shown in
Table 1. The neutral ribitol derivatives 1 and 7, and the
quaternary ammonium 2 showed poor inhibiting activity,
with Ki values in the mM range. Compared to this, guanidino-
alkyl-ribitol derivatives 3 and 8e10 showed a 10-fold de-
crease in Ki values, compound 8 with a C2 linker being the
most active one confirming the results obtained by the dock-
ing studies. It can therefore be concluded that the guanidine
function plays a role in the binding of the inhibitor to the
enzyme, as the binding of D-ribose in the target enzyme shows
a Krib of 148 mM [7]. Guanidino-alkyl-iminoribitol 20 shows
a significant increase in potency compared to its ribitol
3. Conclusion
Our hypothesis e adding a guanidino group to a ribitol moi-
ety improves the binding of ribitol derivatives with nucleoside
Scheme 4. Reagents and conditions: (a) di-tert-butyl dicarbonate, Et3N, DCM, rt, 2 h to overnight; (b) 21 [17], K2CO3, DMF, 60 ꢀC, 24 h; (c) TFA:DCM 1:1, rt,
30 min; (d) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, Et3N, CHCl3, rt, 20 h to 3 days; (e) TFA:H2O 1:1, rt, 42e60 h.

A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
319
Scheme 5. Reagents and conditions: (a) 4-nitrobenzylbromide, DMF, K2CO3, 40 ꢀC, 3 h or a-bromo-para-tolunitrile, DCM, H2O, Na2CO3, reflux, 3 h; (b) SnCl2,
EtOH, N2, 70 ꢀC, 30 min; (c) (1) N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, CHCl3, reflux, 48 h; (2) TFA:H2O 1:1, rt, 18 h; (d) n-BuLi, HMDS,
Et2O, N2, 0 ꢀC to rt, 2 h; (e) aqueous 6 N HCl, MeOH, rt, 20 h.
hydrolase e was confirmed. Although the inhibiting activity of
these guanidino-alkyl-ribitol derivatives is rather poor, it was
observed that the guanidine function does contribute to the bind-
ing of the inhibitor with the enzyme, possibly by catione
p-stacking interactions. Using an iminoribitol scaffold im-
proved the activity drastically and low mM active compounds
(20, 27, 32 and 33) were obtained. These compounds are now
being used as leads for further inhibitor design.
on an ion trap mass spectrometer (Bruker DaltonicsÒ esquireÔ
3000plus). LCeMS spectra were recorded on an Agilent 1100
Series HPLC system equipped with a HILIC Silica column
(2.1 ꢂ 100 mm, 5 mm, Atlantis HILIC, Waters) coupled with
a Bruker DaltonicsÒ esquireÔ 3000plus mass spectrometer (sol-
vent A: H2O with 0.1% formic acid, solvent B: ACN with 0.1%
formic acid, gradient 2: 90% B to 40% B, 12 min, 0.2 mL/min).
4.2. Chemistry
4. Experimental protocols
4.2.1. (2R,3S,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydro-2-furancarboxamide (1)
4.1. General procedures
2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl cyanide (4) [12]
(0.90 g, 1.9 mmol) was dissolved in a solution of 7 N NH3
in methanol (20 mL) and stirred at rt for 4 days. The reaction
mixture was concentrated, the residue dissolved in water and
washed with CHCl3. The aqueous layer was concentrated
and the residue was purified by column chromatography
(CHCl3:MeOH, 4:1) to yield the title compound as a gum
(0.11 g, 34%); MS (ESI): m/z 178.0 (MHþ); LCeMS: rt
All starting materials were obtained from Acros Organics or
Aldrich. Tris-(2-aminoethyl)-amine polystyrene resin was ob-
tained from Novabiochem. NMR spectra were recorded on
a Bruker AvanceÒ DRX-400 spectrometer (400 MHz), coupling
constants are reported in Hz. Column chromatography was
performed on a Flashmaster II (Jones Chromatography) with
IsoluteÒ columns pre-packed with silica gel (30e90 mM) for
normal phase chromatography and C18 (30e90 mM) for reversed
phase chromatography. Melting points were determined on an
ElectrothermalÒ digital melting point apparatus and are uncor-
rected. Electrospray Ionisation (ESI) mass spectra were acquired
1
2.6 min, m/z 177.9 (MHþ); H NMR (D2O): d 3.70 (dd, 1H,
J ¼ 12.6, J0 ¼ 4.3), 3.84 (dd, 1H, J ¼ 12.6, J0 ¼ 2.8), 4.01
(m, 1H), 4.06 (dd, 1H, J ¼ 6.6, J0 ¼ 4.9), 4.23 (m, 1H), 4.30
(d, 1H, J ¼ 3.8); 13C NMR (D2O): d 60.4, 70.3, 74.5, 82.3,
83.2, 175.9.
4.2.2. 2,5-Anhydro-1-deoxy-1-(trimethylammonio)-D-allitol
iodide (2)
A solution of 1-amino-2,5-anhydro-3,4,6-tri-O-benzoyl-
1-deoxy-D-allitol (5) [12,13] (0.13 g, 0.27 mmol) and MeI
(1.5 mL, 24 mmol) in dry DMF was stirred under a N2 atmo-
sphere for 24 h. Subsequently the reaction mixture was co-
evaporated with hexane, EtOAc was added to the residue
and the precipitate that formed was filtered off. The filtrate
was evaporated and the residue purified by column chromatog-
raphy (CHCl3:MeOH, 4:1) to yield 3,4,6-O-tribenzoyl-2,
5-anhydro-1-deoxy-1-(trimethylammonio)-D-allitol iodide as
Scheme 6. Reagents and conditions: (a) TFA:H2O 1:1, rt, 18 h.

320
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
Table 1
Inhibition of IAG-nucleoside hydrolase isolated from T. vivax by the target
compounds (Ki ¼ inhibition constant)
(quantitative yield). The TFA salt of 3,4,6-O-tribenzoyl-1-gua-
nidino-2,5-anhydro-1-deoxy-D-allitol was dissolved in a solu-
tion of 7 N NH3 in methanol (10 mL) and stirred at rt for 4
days. The reaction mixture was concentrated, the residue dis-
solved in water and washed with CHCl3. The aqueous layer
was concentrated and the residue was purified by reversed
phase column chromatography (H2O:ACN, 9:1) to yield the ti-
tle compound as a gum (80 mg, 98%); MS (ESI): m/z 206.0
Compds
Ki (mM)
1
4500 ꢃ 1307
2283 ꢃ 357
545 ꢃ 63
5640 ꢃ 1725
309 ꢃ 32
685 ꢃ 74
677 ꢃ 107
18 ꢃ 5
2
3
7
8
1
(MHþ); LCeMS: rt 10.3 min, m/z 205.9 (MHþ); H NMR
9
(CD3OD): d 3.32 (dd, 1H, J ¼ 14.5, J0 ¼ 6.7), 3.48 (dd, 1H,
J ¼ 14.5, J0 ¼ 2.9), 3.61 (dd, 1H, J ¼ 11.9, J0 ¼ 4.2), 3.70
(dd, 1H, J ¼ 11.9, J0 ¼ 3.4), 3.82e3.91 (m, 3H), 4.01 (t, 1H,
J ¼ 5.0); 13C NMR (CD3OD): d 44.9, 62.8, 72.6, 73.7, 82.9,
86.5, 159.6.
10
20
26
27
32
33
34
38
286 ꢃ 58
40 ꢃ 3
7.0 ꢃ 1.4
6.7 ꢃ 0.4
6.1 ꢃ 0.6
59 ꢃ 18
4.2.5. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-1-O-
(methylsulfonyl)-7-O-trityl-D-allo-heptitol (13)
a syrup (0.10 g, 70%). 3,4,6-O-Tribenzoyl-2,5-anhydro-1-
deoxy-1-(trimethylammonio)-D-allitol iodide (0.10 g, 1.19
mmol) was dissolved in a solution of 7 N NH3 in methanol
(5 mL). The reaction mixture was stirred at rt for 4 days, con-
centrated under reduced pressure, and the residue dissolved in
water and washed with CHCl3. The aqueous layer was concen-
trated and the residue was recrystallized from hot EtOH to
yield the title product as a white crystalline powder (22 mg,
58%); mp 156e158 ꢀC; MS (ESI): m/z 206.3 (Mþ); LCe
To a suspension of LiAlH4 (0.17 g, 4.6 mmol) in dry Et2O
(10 mL) at 0 ꢀC, a solution of 12a [16] (0.74 g, 1.5 mmol) in
dry Et2O (7.5 mL) was added dropwise. The reaction mixture
was stirred at rt for 18 h, cooled to 0 ꢀC and the excess of
LiAlH4 was destroyed by the dropwise addition of water
(2 mL). After the reaction mixture was washed with water
and brine, the Et2O layer was dried (Na2SO4) and concentrated
to yield the alcohol derivative as a sticky white gum (0.52 g,
74%) which was used without further purification. The alcohol
derivative (0.52 g, 1.1 mmol) was dissolved in pyridine
(2.5 mL), and mesylchloride (259 mL, 2.26 mmol) together
with a catalytic amount of DMAP was added while stirring.
The reaction mixture was stirred at rt for 2 h. The pyridine
was evaporated and the residue dissolved in CHCl3, washed
with water, dried (Na2SO4) and concentrated. Purification by
column chromatography (Hex:EtOAc, 1:1) yielded the title
compound as a syrup (0.45 g, 74%); MS (ESI): m/z 561.2
(MNaþ); 1H NMR (CDCl3): d 1.32 (s, 3H), 1.52 (s, 3H),
1.99 (m, 1H), 2.15 (m, 1H), 2.97 (s, 3H), 3.18 (dd, 1H,
J ¼ 10.1, J0 ¼ 4.8), 3.29 (dd, 1H, J ¼ 10.1, J0 ¼ 3.7), 4.01
(m, 1H), 4.12 (m, 1H), 4.34e4.42 (m, 3H), 5.59 (dd, 1H,
J ¼ 6.7, J0 ¼ 3.8), 7.21e7.32 (m, 9H), 7.42e7.45 (m, 6H).
1
MS: rt 11.6 min, m/z 205.9 (Mþ); H NMR (D2O): d 3.19
(s, 9H), 3.59 (m, 3H), 3.70 (m, 1H), 3.87 (m, 1H), 4.01 (dd,
2H, J ¼ 13.5, J0 ¼ 3.4), 4.21 (m, 1H); 13C NMR (D2O):
d 56.7, 64.3, 71.4, 73.0, 75.9, 78.2, 88.4.
4.2.3. N,N0-Bis(tert-butoxycarbonyl)-1-guanidino-2,5-
anhydro-3,4,6-tri-O-benzoyl-1-deoxy-D-allitol (6)
1-Amino-2,5-anhydro-3,4,6-tri-O-benzoyl-1-deoxy-D-alli-
tol (5) [12,13] (2.21 g, 4.65 mmol) was dissolved in CHCl3
and N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxami-
dine (1.49 g, 4.80 mmol) was added. After stirring at rt for
24 h, the solvent was removed under reduced pressure and
the residue was redissolved in DCM and tris-(2-aminoethyl)-
amine polystyrene resin was added to scavenge the excess of
N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine.
After 3 h of stirring at rt, the resin was filtered off and the sol-
vent was evaporated. The residue was purified by reversed
phase column chromatography (H2O:ACN, 1:1) to obtain 6
4.2.6. 3,6-Anhydro-1-azido-1,2-dideoxy-4,5-
O-isopropylidene-7-O-trityl-D-allo-heptitol (14)
To 13 (0.44 g, 0.82 mmol) in dry DMF (8 mL) was added
NaN3 (0.27 g, 4.1 mmol). The reaction mixture was stirred
at 100 ꢀC for 7 h, cooled to rt and diluted with EtOAc. The or-
ganic solution was washed with brine and the aqueous layer
was extracted twice with EtOAc. The combined organic
phases were dried (Na2SO4) and concentrated. Purification
by column chromatography (Hex:EtOAc, 4:1) yielded the de-
sired product 14 as a sticky yellow gum (0.33 g, 83%); MS
1
as a syrup (0.73 g, 22%); MS (ESI): m/z 718.4 (MHþ); H
NMR (CDCl3): d 1.40 (s, 9H), 1.42 (s, 9H), 3.85 (m, 2H),
4.51 (m, 1H), 4.55e4.61 (m, 2H), 4.67 (dd, 1H, J ¼ 13.5,
J0 ¼ 5.0), 5.48 (dd, 1H, J ¼ 7.2, J0 ¼ 5.7), 5.73 (dd, 1H,
J ¼ 5.6, J0 ¼ 3.7), 7.35 (m, 4H), 7.41 (t, 2H, J ¼ 7.5), 7.55
(m, 3H), 7.90, 7.97, 8.09 (3dd, 3 ꢂ 2H, J ¼ 8.4, J0 ¼ 1.4).
1
4.2.4. 1-Guanidino-2,5-anhydro-1-deoxy-D-allitol (3)
(ESI): m/z 508.2 (MNaþ); H NMR (CDCl3): d 1.81e2.00
A solution of 6 (0.27 g, 0.38 mmol) in DCM:TFA (1:1,
2 mL) was stirred at rt overnight. The solvents were evapo-
rated to yield, without further purification, 3,4,6-O-triben-
zoyl-1-guanidino-2,5-anhydro-1-deoxy-D-allitol as a TFA salt
(m, 2H), 3.17 (dd, 1H, J ¼ 10.1, J0 ¼ 4.7), 3.27 (dd, 1H,
J ¼ 10.1, J0 ¼ 3.9), 3.44 (m, 1H), 3.96 (m, 1H), 4.12 (m,
1H), 4.38 (dd, 1H, J ¼ 6.6, J0 ¼ 5.2), 4.58 (dd, 1H, J ¼ 6.7,
J0 ¼ 3.7), 7.21e7.32 (m, 9H), 7.41e7.46 (m, 6H).

A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
321
4.2.7. 3,6-Anhydro-1-azido-1,2-dideoxy-D-allo-heptitol (7)
Azide 14a (0.46 g, 0.95 mmol) was dissolved in TFA:H2O
(1:1, 10 mL) and stirred at rt for 18 h. The solvent was evap-
orated and the residue was purified by column chromatogra-
phy (CHCl3:MeOH, 9:1) to furnish the title compound as
a gum (0.18 g, 91%); MS (ESI): m/z 226.0 (MNaþ); LCe
MS: rt 1.7 min, m/z 225.8 (MNaþ); 1H NMR (CD3OD):
d 1.78 (m, 1H), 1.91 (m, 1H), 3.46 (m, 2H), 3.57 (dd, 1H,
J ¼ 11.9, J0 ¼ 4.7), 3.67 (dd, 1H, J ¼ 11.9, J0 ¼ 3.5), 3.73
(m, 1H), 3.80 (m, 2H), 3.95 (m, 1H); 13C NMR (CD3OD):
d 33.8, 49.2, 63.4, 72.7, 76.3, 81.0, 85.9.
1H NMR (CDCl3): d 1.32 (s, 3H), 1.42 (s, 9H), 1.50 (s, 9H),
1.52 (s, 3H), 1.83 (m, 1H), 1.94 (m, 1H), 3.24 (d, 2H,
J ¼ 4.4), 3.49 (m, 1H), 3.68 (m, 1H), 3.93 (m, 1H), 4.14 (m,
1H), 4.36 (m, 1H), 4.56 (dd, 1H, J ¼ 6.6, J0 ¼ 3.5), 7.20e
7.31 (m, 9H), 7.40e7.46 (m, 6H).
4.2.11. N,N0-Bis(tert-butoxycarbonyl)-2-guanidino-4,
7-anhydro-1,2,3-trideoxy-5,6-O-(1-methylethylidene)-
8-O-trityl-D-allo-octitol (16b)
Starting from 15b (0.84 g, 1.8 mmol), the same procedure
was followed as described for 16a, to yield 16b as an insepa-
rable mixture of diastereoisomers (0.26 g, 20%); MS (ESI):
m/z 716.2 (MHþ), 738.2 (MNaþ).
4.2.8. 1-Amino-3,6-anhydro-1,2-dideoxy-4,5-
O-isopropylidene-7-O-trityl-D-allo-heptitol (15a)
A solution of 14a (0.31 g, 0.64 mmol) in dry Et2O (3 mL)
was added at 0 ꢀC to a suspension of LiAlH4 (0.10 g,
2.6 mmol) in dry Et2O (10 mL). The mixture was stirred for
3 h at 0 ꢀC under a N2 atmosphere, diluted with Et2O and sat-
urated aqueous Na2SO4 was gradually added at 0 ꢀC. After
being stirred at rt for 30 min, the mixture was extracted twice
with Et2O. The combined organic layers were washed with
brine, dried (Na2SO4) and concentrated to yield the title com-
pound as a syrup (0.27 g, 92%); MS (ESI): m/z 460.1 (MHþ),
482.2 (MNaþ); 1H NMR (CDCl3): d 1.32 (s, 3H), 1.52 (s, 3H),
1.80 (m, 2H), 2.17 (s, 2H), 2.89 (dt, 2H, J ¼ 6.7, J0 ¼ 1.8), 3.17
(dd, 1H, J ¼ 10.0, J0 ¼ 4.8), 3.26 (dd, 1H, J ¼ 10.0, J0 ¼ 4.0),
3.96 (m, 1H), 4.10 (m, 1H), 4.37 (dd, 1H, J ¼ 6.6, J0 ¼ 5.4),
4.56 (dd, 1H, J ¼ 6.8, J0 ¼ 3.8), 7.20e7.33 (m, 9H), 7.44e
7.47 (m, 6H).
4.2.12. 1-Guanidino-3,6-anhydro-1,2-dideoxy-D-
allo-heptitol$TFA (8)
Starting from 16a (0.36 g, 0.51 mmol), the same procedure
was followed as described for 7. Purification by reversed phase
column chromatography (H2O:ACN, 9:1) yielded the TFA salt
of the title compound as a gum (53 mg, 31%); MS (ESI): m/z
220.0 (MHþ); LCeMS: rt 10.3 min, m/z 219.9 (MHþ); H
1
NMR (CD3OD): d 1.75 (m, 1H), 1.96 (m, 1H), 3.34 (m,
2H), 3.58 (dd, 1H, J ¼ 11.8, J0 ¼ 5.2), 3.69 (d, 1H, J ¼ 3.5),
3.72 (d, 1H, J ¼ 5.4), 3.80 (m, 2H), 3.94 (t, 1H, J ¼ 5.1);
13C NMR (CD3OD): d 33.6, 39.9, 63.5, 72.7, 76.3, 81.8,
86.3, 158.8.
4.2.13. 2-Guanidino-4,7-anhydro-1,2,3-trideoxy-D-
allo-octitol$TFA (9)
Starting from 16b (0.23 g, 0.32 mmol), the same procedure
was followed as described for 7. Purification by reversed phase
column chromatography (H2O:ACN, 9:1) yielded the TFA salt
of the title compound as an inseparable mixture of diastereo-
isomers (38 mg, 44%); MS (ESI): m/z 234.1 (MHþ); LCe
MS: rt 10.2 min, m/z 233.9 (MHþ); 1H NMR (CD3OD):
d 1.27 (d, 3H), 1.66e1.88 (m, 2H), 3.58 (m, 1H), 3.70 (m,
2H), 3.75e3.85 (m, 3H), 3.95 (m, 1H); 13C NMR (CD3OD),
d isomer A: 20.8, 41.5, 47.2, 63.3, 72.5, 76.7, 81.0, 86.2,
157.7; isomer B: 21.4, 41.2, 46.8, 63.5, 72.6, 76.4, 80.2,
86.1, 158.2.
4.2.9. 2-Amino-4,7-anhydro-1,2,3-trideoxy-5,6-
O-isopropylidene-8-O-trityl-D-allo-octitol (15b)
A mixture of 12b [16] (0.85 g, 1.8 mmol), ammonium for-
mate (1.25 g, 19.9 mmol), sodium cyanoborohydride (0.62 g,
˚
9.9 mmol) and 3 A molecular sieves (1 g) in MeOH (20 mL)
was stirred at rt for 18 h. The solution was filtered through
Celite and evaporated. The residue was taken up in EtOAc,
washed with brine and dried (Na2SO4). Evaporation of the sol-
vent yielded the product as an inseparable mixture of diaste-
reoisomers which was used without further purification
(0.84 g, 98%); MS (ESI): m/z 474.2 (MHþ).
4.2.10. N,N0-Bis(tert-butoxycarbonyl)-1-guanidino-3,6-
anhydro-1,2-dideoxy-4,5-O-(1-methylethylidene)-7-O-trityl-
D-allo-heptitol (16a)
4.2.14. 4,7-Anhydro-2,3-dideoxy-5,6-O-isopropylidene-8-
O-trityl-D-allo-octononitrile (17)
Mesylate 13a (0.26 g, 0.48 mmol), sodium cyanide (0.19 g,
3.9 mmol) and sodium iodide (0.063 g, 0.42 mmol) in dry
DMF (5 mL) were stirred under a N2 atmosphere at 100 ꢀC
for 7 h. Water was added and extracted with EtOAc. The or-
ganic washings were combined, washed with brine, dried
(Na2SO4), and evaporated. Purification by column chromatog-
raphy (Hex:EtOAc, 3:1) yielded the product as a gum (0.14 g,
61%); MS (ESI): m/z 492.2 (MNaþ); 1H NMR (CDCl3): d 1.35
(s, 3H), 1.55 (s, 3H), 1.93 (m, 1H), 2.06 (m, 1H), 2.50 (m, 2H),
3.20 (dd, 1H, J ¼ 10.1, J0 ¼ 4.6), 3.30 (dd, 1H, J ¼ 10.1,
J0 ¼ 3.6), 3.96 (m, 1H), 4.18 (m, 1H), 4.40 (dd, 1H, J ¼ 6.5,
J0 ¼ 5.3), 4.63 (dd, 1H, J ¼ 6.6, J0 ¼ 3.5), 7.24e7.34 (m,
9H), 7.44e7.48 (m, 6H).
Amine 15a (0.25 g, 0.54 mmol) was dissolved in CHCl3
and N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxami-
dine (0.18 g, 0.54 mmol) was added. The reaction mixture
was stirred at rt for 24 h. The solvent was removed under re-
duced pressure and the residue was purified by column chro-
matography (Hex:EtOAc, 1:1). The product obtained was
dissolved in DCM and tris-(2-aminoethyl)-amine polystyrene
resin was added to scavenge the remaining excess of N,N0-
bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine. After
3 h of stirring at rt, the resin was filtered off and the solvent
was evaporated to yield the title compound 16a as a gum
(0.36 g, 94%); MS (ESI): m/z 702.3 (MHþ), 724.4 (MNaþ);

322
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
4.2.15. 1-Amino-4,7-anhydro-1,2,3-trideoxy-5,6-
O-isopropylidene-8-O-trityl-D-allo-octitol (18)
J0 ¼ 2.9), 3.77 (m, 1H), 3.84e4.13 (m, 2H), 4.44 (d, 1H,
J ¼ 5.4), 4.69 (dd, 1H, J ¼ 5.6, J0 ¼ 1.1).
To nitrile 17 (0.14 g, 0.30 mmol) in dry THF (3 mL) was
added LiAlH4 (0.13 g, 3.4 mmol). The reaction mixture was
stirred under a N2 atmosphere at rt for 18 h. Water was added
and extracted with EtOAc. The organic washings were com-
bined, washed with brine, dried (Na2SO4), and evaporated.
Purification by column chromatography (Hex:EtOAc, 1:1)
yielded the product as a gum (0.063 g, 45%); MS (ESI): m/z
474.1 (MHþ); 1H NMR (CDCl3): d 1.34 (s, 3H), 1.54 (s,
3H), 1.66 (m, 2H), 1.86 (m, 2H), 3.20 (m, 1H), 3.27 (m,
1H), 3.76 (m, 2H), 3.92 (m, 1H), 4.13 (m, 1H), 4.35 (m,
1H), 4.57 (m, 1H), 7.20e7.32 (m, 9H), 7.46e7.49 (m, 6H).
4.2.18. (1S )-1,4-Dideoxy-1-(2-guanidinoethyl)-1,4-imino-
D-ribitol$TFA (20)
A solution of 25 (19 mg, 0.028 mmol) in TFA:H2O (1:1,
1 mL) was stirred at rt for 36 h. The solvent was evaporated
and the residue was dissolved in MeOH (0.5 mL) and 5 drops
of EtOAc were added. Subsequent cooling resulted in the for-
mation of a precipitate, which was filtered and redissolved in
MeOH:H2O (1:1). Evaporation of the MeOH:H2O mixture
yielded the TFA salt of the title compound 20 as a gum
(3.6 mg, 59%); MS (ESI): m/z 219.1 (MHþ); LCeMS: rt
1
13.5 min, m/z 218.9 (MHþ); H NMR (CD3OD): d 2.08 (dd,
2H, J ¼ 14.4, J0 ¼ 7.2), 3.41 (m, 2H), 3.50 (m, 1H), 3.61
(dd, 1H, J ¼ 8.1, J0 ¼ 3.9), 3.81 (m, 2H), 4.03 (dd, 1H,
J ¼ 7.4, J0 ¼ 4.9), 4.12 (m, 1H); 13C NMR (CD3OD): d 30.8,
39.4, 59.8, 60.9, 67.4, 72.5, 75.8, 158.8.
4.2.16. 1-Guanidino-4,7-anhydro-1,2,3-trideoxy-D-
allo-octitol$TFA (10)
Starting from 18 (0.063 g, 0.13 mmol), the same procedure
was followed as described for 16a, to yield the fully protected
intermediate 19 (0.040 g, 42%); MS (ESI): m/z 716.4 (MHþ).
A solution of 19 (0.040 g, 0.056 mmol) in TFA:H2O (1:1,
2 mL) was stirred at rt for 18 h. The solvent was evaporated
and the residue was dissolved in EtOH. Filtration and subse-
quent concentration of the filtrate furnished the TFA salt of
the title compound as a gum (17 mg, 59%); MS (ESI): m/z
4.2.19. tert-Butyl 2-bromoethylcarbamate (28a)
Di-tert-butyl dicarbonate (2.9 g, 13 mmol) was added to
stirred solution of 2-bromoethylamine hydrobromide
a
(3.0 g, 15 mmol) in DCM (80 mL). Et3N (3.0 g, 29 mmol)
was added dropwise to the solution. This was stirred at rt
for 1.5 h, then aqueous 2 N HCl was added and the layers
were separated. The organic layer was washed with aqueous
2 N HCl and brine, dried (Na2SO4) and the solvent was evap-
orated under reduced pressure. The residue was purified by
flash chromatography (Hex:EtOAc, 3:2) to yield the title com-
pound as an oil (2.1 g, 72%); MS (ESI): m/z 246.0 (MHþ) and
248.0 (MNaþ).
234.1 (MHþ); LCeMS: rt 11.0 min, m/z 233.9 (MHþ); H
1
NMR (CD3OD): d 1.58 (m, 1H), 1.70e1.79 (m, 3H), 3.22
(t, 2H, J ¼ 6.7), 3.57 (dd, 1H, J ¼ 11.8, J0 ¼ 5.2), 3.70e3.74
(m, 3H), 3.80 (dd, 1H, J ¼ 8.4, J0 ¼ 4.8), 3.91 (m, 1H); 13C
NMR (CD3OD): d 26.5, 31.3, 42.2, 63.5, 72.7, 76.3, 83.6,
86.0, 158.7.
4.2.17. N,N,N-Tris(tert-butoxycarbonyl)-1-guanidino-7-O-
tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-
4.2.20. N-[2-Amino-N-tert-butoxycarbonyl-ethyl]-5-O-tert-
butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-D-allo-heptitol (25)
isopropylidene-D-ribitol (29a)
A solution of N-tert-butoxycarbonyl-7-O-tert-butyldime-
thylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-D-allo-
heptononitrile 23 [18] (0.19 g, 0.45 mmol) in dry THF (5 mL)
was cooled to 0 ꢀC, LiAlH4 (0.080 g, 2.1 mmol) was added,
and the reaction mixture was stirred at rt for 3 h. The reaction
was quenched with water, diluted with EtOAc, washed with
water and brine, dried (Na2SO4) and concentrated to yield the
intermediate 24 (0.12 g, 62%) which was used without further
purification. A solution of 24 (0.12 g, 0.28 mmol) was dissolved
in CHCl3 and N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine (0.13 g, 0.42 mmol) was added. This was stirred
at rt for 24 h. The solvent was removed under reduced pressure
and the residue was redissolved in DCM and tris-(2-amino-
ethyl)-amine polystyrene resin was added to scavenge the
excess of N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-car-
boxamidine. After 3 h of stirring at rt, the resin was filtered off
and the solvent was evaporated. Further purification by column
chromatography (Hex:EtOAc, 3:1) yielded the title compound
25 (19 mg, 10%); MS (ESI): m/z 673.6 (MHþ), 695.5
To a solution of the protected iminoribitol 21 [17] (0.50 g,
1.7 mmol) in DMF (15 mL), K2CO3 (0.48 g, 3.5 mmol) and
tert-butyl 2-bromoethylcarbamate (0.56 g, 2.5 mmol) were
added. After stirring at 60 ꢀC for 24 h, EtOAc and H2O
were added. The organic layer was separated and washed
twice with H2O and brine. The combined aqueous layers
were extracted with EtOAc. The combined organic layers
were dried (Na2SO4) and evaporated under reduced pressure.
The residue was purified by flash chromatography to yield
the title compound as a syrup (Hex:EtOAc, 4:1) (0.59 g,
78%); MS (ESI): m/z 431.4 (MHþ).
4.2.21. N-[2-N,N-Bis(tert-butoxycarbonyl)-guanidinopropyl]-
5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-D-ribitol (31a)
Compound 29a (0.59 g, 1.4 mmol) was stirred at rt in
TFA:DCM (1:1, 4 mL). After 30 min, the mixture was evapo-
rated and the residue dissolved in CHCl3 (15 mL) and Et3N
(1.2 mL, 4.1 mmol) was added. After 15 min of stirring,
N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine
(0.51 g, 1.6 mmol) was added. After 3 days of stirring at rt,
tris-(2-aminoethyl)-amine polystyrene resin was added to
1
(MNaþ); H NMR (CDCl3): d 0.06 (d, 6H, J ¼ 5.6), 0.89 (s,
9H), 1.26 (s, 3H), 1.32 (s, 3H), 1.45e1.50 (m, 27H), 1.65e
2.05 (m, 2H), 3.40e3.65 (m, 2H), 3.71 (dd, 1H, J ¼ 10.3,

A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
323
scavenge the excess of N,N0-bis(tert-butoxycarbonyl)-1H-pyr-
azole-1-carboxamidine, and this was stirred for 2 h at rt. The
resin was removed by filtration and the filtrate was evaporated
under reduced pressure. The residue was purified by column
chromatography (Hex:EtOAc, 4:1) to furnish the title com-
pound as a syrup (0.17 g, 22%); MS (ESI): m/z 573.5 (MHþ).
excess of N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-car-
boxamidine, this was stirred at rt for 2 h. The resin was removed
by filtration and the filtrate was evaporated under reduced pres-
sure. The residue was purified by column chromatography
(Hex:EtOAc, 4:1) to yield the title compound as a syrup
(0.12 g, 30%); MS (ESI): m/z 587.50 (MHþ).
4.2.22. 1,4-Dideoxy-(2-guanidinoethyl)-1,4-
4.2.26. 1,4-Dideoxy-(3-guanidinopropyl)-
imino-D-ribitol$TFA (26)
1,4-imino-D-ribitol$TFA (27)
Compound 31a (0.13 g, 0.23 mmol) was stirred in
TFA:H2O (1:1, 3 mL) for 60 h at rt. The solvent was removed
under reduced pressure to yield the TFA salt of the title com-
pound (54 mg, 70%); MS (ESI): m/z 219.13 (MHþ); LCeMS:
rt 12.6 min, m/z 218.7 (MHþ); 1H NMR (CD3OD): d 3.31 (m,
4H), 3.45 (m, 1H), 3.57 (m, 1H), 3.76 (m, 1H), 3.83 (dd, 1H,
J ¼ 12.3, J0 ¼ 6.9), 3.97 (dd, 1H, J ¼ 3.3, J0 ¼ 12.3), 4.06 (m,
1H), 4.26 (m, 1H); 13C NMR (CD3OD): d 38.6, 57.9, 59.7,
59.8, 70.9, 73.3, 73.5, 159.1.
Compound 31b (0.12 g, 0.2 mmol) was stirred in TFA:H2O
(1:1, 3 mL) for 42 h at rt. The solvent was removed under re-
duced pressure to obtain the TFA salt of the title compound
(44 mg, 63%); MS (ESI): m/z 233.1 (MHþ); LCeMS: rt
1
11.5 min, m/z 233.1 (MHþ); H NMR (CD3OD): d 2.00 (m,
2H), 3.24 (m, 4H), 3.47 (m, 2H), 3.69 (m, 1H), 3.79 (dd,
1H, J ¼ 12.4, J0 ¼ 5.7), 3.92 (dd, 1H, J ¼ 12.4, J0 ¼ 3.1),
4.05 (m, 1H), 4.22 (m, 1H); 13C NMR (CD3OD): d 26.0,
39.6, 56.7, 59.0, 59.2, 70.6, 72.9, 73.2, 158.9.
4.2.23. tert-Butyl 3-bromopropylcarbamate (28b)
4.2.27. 5-O-tert-Butyldimethylsilyl-1,4-dideoxy-1,4-imino-
2,3-O-isopropylidene-N-(4-nitrobenzyl)-D-ribitol (35a)
Di-tert-butyl dicarbonate (1.8 g, 8.2 mmol) was added to
a stirred solution of 3-bromopropylamine hydrobromide
(2.0 g, 9.1 mmol) in DCM (60 mL). Et3N (3.8 mL, 27 mmol)
was added dropwise to the solution. After stirring overnight
at rt, aqueous 2 N HCl was added and the layers were sepa-
rated. The organic layer was washed with aqueous 2 N HCl
and brine, dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by flash chromatography
(Hex:EtOAc, 7:3) to yield the title compound as an oil
(0.86 g, 44%); MS (ESI): m/z 260.0 (MHþ) and 262.0 (MNaþ).
Protected iminoribitol 21 [17] (0.63 g, 2.2 mmol) was dis-
solved in DMF (5 mL) and K2CO3 (0.61 g, 4.4 mmol) was
added. 4-Nitrobenzylbromide (0.52 mg, 2.4 mmol) was added
and the mixture was stirred at 40 ꢀC for 3 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 concentrated under vacuum. The residue
was redissolved in CHCl3 and stirred overnight at rt with
tris-(2-aminoethyl)-amine polystyrene resin to scavenge the
excess of 4-nitrobenzylbromide. The resin was removed by fil-
tration and the solvent was evaporated to yield the product as
a yellowish syrup (0.72 g, 78%); MS (ESI): m/z 423.4
4.2.24. N-[2-Amino-N-tert-butoxycarbonyl-propyl]-5-O-
tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-D-ribitol (29b)
1
To a solution of the protected iminoribitol 21 [17] (0.50 g,
1.7 mmol) in DMF (15 mL), K2CO3 (0.48 g, 3.5 mmol) and
tert-butyl 3-bromopropylcarbamate (0.45 g, 1.9 mmol) were
added. After stirring at 60 ꢀC for 24 h, EtOAc and H2O
were added. The organic layer was separated and washed
with H2O and brine. The combined aqueous layers were
washed with EtOAc. The combined organic layers were dried
(Na2SO4) and evaporated under reduced pressure. The residue
was purified by flash chromatography (Hex:EtOAc, 4:1) to
yield the title compound as a syrup (0.30 g, 38%); MS
(ESI): m/z 445.4 (MHþ).
(MHþ); H NMR (CDCl3): d 0.06 (d, 6H, J ¼ 6.0 Hz), 0.89
(s, 9H), 1.34 (s, 3H), 1.43 (s, 3H), 2.74 (d, 1H, J ¼ 10.4),
3.06 (s, 1H), 3.10 (dd, 1H, J ¼ 10.3, J0 ¼ 5.4), 3.65 (dd, 1H,
J ¼ 10.7, J0 ¼ 4.2), 3.77 (dd, 1H, J ¼ 10.7, J0 ¼ 3.8), 3.89 (d,
1H, J ¼ 14.7), 4.10 (d, 1H, J ¼ 12.7), 4.56 (d, 1H, J ¼ 6.3),
4.67 (m, 1H), 7.52 (d, 2H, J ¼ 8.4), 8.16 (d, 2H, J ¼ 8.5); 13C
NMR (CDCl3): d ꢁ5.7, ꢁ5.3, 18.3, 25.1, 26.0, 27.3, 56.3,
59.4, 63.3, 69.3, 79.8, 83.4, 112.1, 123.7, 129.0, 147.3, 147.8.
4.2.28. 5-O-tert-Butyldimethylsilyl-1,4-dideoxy-1,4-imino-
2,3-O-isopropylidene-N-(4-aminobenzyl)-D-ribitol (36)
Compound 35a (0.72 g, 1.7 mmol) was dissolved in EtOH
(10 mL) and SnCl2 (1.6 g, 8.5 mmol) was added. This was
stirred at 70 ꢀC for 30 min under N2 atmosphere. The reaction
mixture was allowed to cool to rt and poured into ice. The pH
was adjusted to pH 7e8 with sat. aq. NaHCO3 before being ex-
tracted with EtOAc. The organic phase was washed with brine,
dried (Na2SO4) and the solvent was removed under reduced
pressure. Purification by column chromatography (Hex:EtOAc
1:1) yielded the title compound as a gum (0.12 g, 18%); MS
4.2.25. N-[2-N,N-Bis(tert-butoxycarbonyl)-guanidinopropyl]-
5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-
isopropylidene-D-ribitol (31b)
Compound 29b (0.30 g, 0.67 mmol) was stirred at rt in
TFA:DCM (1:1, 3 mL). After 45 min, the mixture was evapo-
rated under reduced pressure and the residue was dissolved in
CHCl3 (7 mL) and Et3N (0.3 mL, 2.0 mmol). This was stirred
at rt for 15 min and N,N0-bis(tert-butoxycarbonyl)-1H-pyra-
zole-1-carboxamidine (0.25 g, 0.81 mmol) was added. The
reaction mixture was stirred at rt for 20 h, then tris-(2-amino-
ethyl)-amine polystyrene resin was added to scavenge the
1
(ESI): m/z 393.4 (MHþ); H NMR (CDCl3): d 0.07 (d, 6H,
J ¼ 5.3), 0.91 (s, 9H), 1.33 (s, 3H), 1.55 (s, 3H), 2.68 (dd,
1H, J ¼ 10.3, J0 ¼ 3.0), 2.97 (d, 1H, J ¼ 2.1), 3.10 (dd, 1H,

324
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
J ¼ 10.3, J0 ¼ 5.6), 3.59 (d, 1H, J ¼ 13.0), 3.65 (dd, 1H,
J ¼ 10.6, J0 ¼ 4.3), 3.76 (dd, 1H, J ¼ 10.6, J0 ¼ 4.5), 3.90 (d,
1H, J ¼ 13.0), 4.54 (dd, 1H, J ¼ 6.5, J0 ¼ 2.2), 4.64 (m, 1H),
6.62 (d, 2H, J ¼ 8.4), 7.11 (d, 2H, J ¼ 8.3).
J0 ¼ 5.4), 3.64 (dd, 1H, J ¼ 10.7, J0 ¼ 4.3), 3.76 (dd, 1H,
J ¼ 10.7, J0 ¼ 4.0), 3.84 (d, 1H, J ¼ 14.5), 4.05 (d, 1H,
J ¼ 14.5), 4.56 (dd, 1H, J ¼ 6.4, J0 ¼ 1.6), 4.66 (m, 1H),
7.46 (d, 2H, J ¼ 8.1), 7.59 (d, 2H, J ¼ 8.1).
4.2.29. 1,4-Dideoxy-1,4-imino-N-
(4-guanidinobenzyl)-D-ribitol$TFA (32)
4.2.31. 1,4-Dideoxy-1,4-imino-N-
(4-amidinobenzyl)-D-ribitol$TFA (33)
Compound 36 (0.12 g, 0.31 mmol) was dissolved in CHCl3
and N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxami-
dine (0.095 g, 0.31 mmol) was added. The solution was stirred
for 48 h under reflux. The solvent was removed under reduced
pressure and the residue was redissolved in DCM and tris-
(2-aminoethyl)-amine polystyrene resin was added to scavenge
the excess of N,N0-bis(tert-butoxycarbonyl)-1H-pyrazole-
1-carboxamidine. After 18 h of stirring at rt, the resin was
removed by filtration and the solvent was evaporated under
vacuum. Further purification by column chromatography
(Hex:EtOAc, 7:1) yielded the protected intermediate as a syrup
(22 mg, 11%); MS (ESI): m/z 635.7 (MHþ), 657.6 (MNaþ);
1H NMR (CDCl3): d 0.07 (d, 6H, J ¼ 6.0), 0.91 (s, 9H),
1.34 (s, 3H), 1.54 (s, 3H), 2.71 (dd, 1H, J ¼ 10.4, J0 ¼ 2.4),
3.00 (s, 1H), 3.10 (dd, 1H, J ¼ 10.3, J0 ¼ 5.6), 3.63e3.69
(m, 2H), 3.78 (dd, 1H, J ¼ 10.6, J0 ¼ 4.0), 3.98 (d, 1H,
J ¼ 13.5), 4.55 (d, 1H, J ¼ 4.8), 4.65 (m, 1H), 7.29 (d, 2H,
J ¼ 8.3), 7.53 (d, 2H, J ¼ 8.1). The protected intermediate
was dissolved in TFA:H2O (1:1, 1 mL) and this was stirred
at rt overnight. H2O was added and this was extracted twice
with DCM. The H2O layer was concentrated to obtain the
TFA salt of the title compound without further purification
(12 mg, 89%); MS (ESI): m/z 281.2 (MHþ); LCeMS: rt
13.8 min, m/z 280.9 (MHþ); 1H NMR (D2O): d 2.50 (dd,
1H, J ¼ 10.0, J0 ¼ 7.7), 2.73 (d, 1H, J ¼ 5.0), 3.01 (dd, 1H,
J ¼ 10.0, J0 ¼ 6.0), 3.54 (d, 2H, J ¼ 5.4), 3.90 (t, 1H,
J ¼ J0 ¼ 5.0), 3.96 (d, 1H, J ¼ 12.6), 4.01 (m, 1H), 7.26 (d,
2H, J ¼ 8.4), 7.41 (d, 2H, J ¼ 8.4); 13C NMR (D2O): d 61.1,
61.8, 69.1, 69.9, 70.9, 72.7, 125.2, 126.5, 130.8, 132.2,
133.7, 136.6, 156.4.
Compound 35b (90 mg, 0.22 mmol) was dissolved in dry
Et2O (2 mL) and cooled to 0 ꢀC under N2 atmosphere. To
this was added a solution of HMDS (0.10 mL, 0.44 mmol)
and n-BuLi (0.20 mL, 0.48 mmol) in 1 mL of dry Et2O. The
resulting mixture was allowed to stir at rt for 2 h. Subse-
quently, 2 mL of H2O was added and stirred at rt for
15 min. The reaction mixture was diluted with water and the
Et2O layer was separated from the H2O and evaporated. The
residue was dissolved in TFA:H2O (1:1, 2 mL) and stirred at
rt for 18 h. The mixture was extracted with DCM and the
H2O layer was evaporated. The residue was purified by re-
versed phase column chromatography on silica C18 (gradient:
H2O to H2O:MeOH 1:1) to yield the TFA salt of the title com-
pound as a gum (79 mg, 95%); MS (ESI): m/z 266.0 (MHþ);
LCeMS: rt 13.7 min, m/z 265.9 (MHþ); 1H NMR (D2O):
d 3.19 (d, 1H, J ¼ 12.3), 3.47e3.51 (m, 2H), 3.60 (dd, 1H,
J ¼ 12.7, J0 ¼ 3.8), 3.71 (dd, 1H, J ¼ 12.7, J0 ¼ 4.8), 4.12
(dd, 1H, J ¼ 6.9, J0 ¼ 4.7), 4.28 (m, 1H), 4.34 (d, 1H,
J ¼ 12.8), 4.54 (d, 1H, J ¼ 12.9), 7.67 (d, 2H, J ¼ 8.2), 7.81
(d, 2H, J ¼ 8.2); 13C NMR (D2O): d 57.4, 58.0, 60.0, 68.8,
70.6, 71.4, 128.6, 129.2, 131.5, 137.1, 166.4.
4.2.32. 1,4-Dideoxy-1,4-imino-D-ribitol$HCl (34)
Protected iminoribitol 21 [17] (0.22 g, 0.76 mmol) was dis-
solved in MeOH (1 mL), aqueous 6 N HCl (3 mL) was added
and the solution was stirred at rt for 20 h. The reaction mixture
was separated between H2O and CHCl3, the aqueous layer was
concentrated under vacuum and the residue was recrystallized
from hot EtOH yielding the HCl salt of the title compound as
a crystalline solid (0.13 g, 96%); mp 125e128 ꢀC; MS (ESI):
m/z 134.1 (MHþ); LCeMS: rt 10.2 min, m/z 134.1 (MHþ); 1H
NMR (D2O): d 3.34 (d, 1H, J ¼ 13.0), 3.47 (d, 1H, J ¼ 12.9,
J0 ¼ 3.9), 3.61 (m, 1H), 3.80 (dd, 1H, J ¼ 12.6, J0 ¼ 6.0),
3.94 (dd, 1H, J ¼ 12.6, J0 ¼ 3.3), 4.18 (dd, 1H, J ¼ 8.5,
J0 ¼ 4.2), 4.36 (m, 1H); 13C NMR (D2O): d 49.7, 58.1, 61.9,
69.5, 71.2.
4.2.30. 5-O-tert-Butyldimethylsilyl-1,4-dideoxy-1,4-imino-
2,3-O-isopropylidene-N-(4-cyanobenzyl)-methyl-D-ribitol
(35b)
Protected iminoribitol 21 [17] (0.22 g, 0.77 mmol) was dis-
solved in DCM (2 mL) and a solution of Na2CO3 (0.162 g,
1.53 mmol) in H2O (2 mL) was added. a-Bromo-para-toluni-
trile (0.15 mg, 0.77 mmol) was added and the mixture was
stirred under reflux for 3 h. The organic layer was separated
and the aqueous layer was extracted twice with DCM. The
combined extracts were dried (Na2SO4) and concentrated un-
der vacuum. The residue was redissolved in CHCl3 and stirred
overnight with tris-(2-aminoethyl)-amine polystyrene resin to
scavenge the remaining excess of a-bromo-para-tolunitrile.
The resin was filtered off and the solvent was evaporated.
The residue was purified further by thin layer chromatography
to yield the product as a yellowish syrup (90 mg, 30%); MS
4.2.33. N-Benzyl-1,4-dideoxy-1,4-imino-D-ribitol$TFA (38)
Protected benzyl derivative37 [17] (0.089 g, 0.32 mmol) was
dissolved in TFA:H2O (1:1, 2 mL) and the solution was stirred at
rt for 18 h. The reaction mixture was concentrated under re-
duced pressure and the residue was purified by column chroma-
tography (CHCl3:MeOH 4:1) to obtain the TFA salt of the title
compound as a gum (0.074 g, 97%); MS (ESI): m/z 224.1
(MHþ); LCeMS: rt 9.4 min, m/z 223.9 (MHþ); 1H NMR
(MeOD): d 3.32e3.36 (m, 1H), 3.50e3.54 (m, 2H), 3.58e
3.62 (m, 1H), 3.75 (dd, 1H, J ¼ 12.0, J0 ¼ 4.9), 4.14 (dd, 1H,
J ¼ 7.7, J0 ¼ 4.2), 4.25e4.28 (m, 1H), 4.45 (d, 1H, J ¼ 12.7),
4.61 (d, 1H, J ¼ 12.7), 7.45e7.48 (m, 3H), 7.53e7.56 (m,
1
(ESI): m/z 403.4 (MHþ); H NMR (CDCl3): d 0.06 (d, 6H,
J ¼ 5.4), 0.90 (s, 9H), 1.34 (s, 3H), 1.56 (s, 3H), 2.73 (dd,
1H, J ¼ 10.4, J0 ¼ 2.2), 3.04 (s, 1H), 3.09 (dd, 1H, J ¼ 10.4,

A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
325
2H); 13C NMR (MeOD): d 58.7, 58.8, 62.8, 70.5, 72.3, 72.9,
130.3, 131.2, 131.5, 131.9.
the Ca2þ ion and the water molecules placed within a distance
˚
of 6 A of the ligand. Hydrogen atoms were added and the
energy was minimized with MMFF94ꢂ force field (standard
parameters) keeping all heavy atoms fixed.
4.3. Biochemistry
The modeling experiments were performed using several
NH crystal structures available through PDB. Special attention
was paid to PDB structures 1HP0 [20] and 2FF2 [11]. The for-
mer is derived from T. vivax co-crystallized with the nucleo-
side analogue 3-deaza-adenosine. The latter corresponds to
a structure of NH co-crystallized with (1S )-1-(9-deazahypox-
anthin-9-yl)-1,4-imino-D-ribitol (Immucillin H). 2FF2 was
chosen for all docking studies reported here since it represents
a ‘‘closed’’ structure of the enzyme with all loops entirely or-
dered, possessing a small and narrow active pocket leading to
more reliable docking scores.
The ribitol moiety of 3-deaza-adenosine from 1HP0 was
used to construct our ribitol models. The contacts involving
the ribitol group were conserved as described [20] and the gua-
nidino-alkyl chains of guanidino-alkyl-ribitols 3 and 8e10,
differing in alkyl chain length, were constructed in the active
site and bonded to the ribitol moiety after systematic confor-
mational search. The iminoribitol target compounds were con-
structed using the iminoribitol moiety of ImmH from 2FF2.
The contacts involving the iminoribitol group were conserved
as described [11].
4.3.1. Protein expression and purification
Expression and purification of the wild type T. vivax IAG-
NH was performed as described previously [20]. Escherichia
coli cells (WK6) containing 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 NieNTA affinity
chromatographic step (Qiagen) and gel filtration on a Super-
dex-200 column (Amersham Bioscience). The concentration
of pure protein (expressed per monomer) was determined spec-
trophotometrically using a 3280 of 47752 Mꢁ1 cmꢁ1. Typically,
80 mg of purified protein was obtained from a 1 L fermenta-
tion. SDSepolyacrylamide gel electrophoresis was used to
confirm enzyme purity.
4.3.2. Enzyme inhibition studies
Inhibitor dissociation constants were determined by mea-
suring the initial rate of hydrolysis of a fixed concentration
of p-nitrophenyl-b-D-ribofuranoside at variable (at least five)
inhibitor concentrations, in a 50 mM phosphate buffer of pH
7.0 at 35 ꢀC. Hydrolysis of p-nitrophenyl-b-D-ribofuranoside
was followed by release of the p-nitrophenolate anion which
has strong absorbency at 400 nm with an extinction coefficient
of 12 mMꢁ1 cmꢁ1 under the assay conditions. Ki was deter-
mined by fitting of initial rates to the equation describing com-
petitive inhibition using the Dixon linearization (1/vi vs I ):
The automatic docking protocol with default parameters
was used to position the ligands in the active site of the en-
zyme. Subsequently, the top solution was chosen according
to the S (Score) function and was minimized as flexible ligand
in the rigid enzyme with MMFX94ꢂ force field. The docking
protocol was validated by ‘re-docking’ of ImmH in 2FF2.
KM
KM þ S
1=vi ¼
ꢂ I þ
Acknowledgments
Ki ꢂ kcat ꢂ E ꢂ S
kcat ꢂ E ꢂ S
This work was funded by a research project from the Insti-
tute for the Promotion of Innovation through Science and Tech-
nology in Flanders (IWT e Vlaanderen). A.G. has a Ph.D.
grant from the same institute. W.V. and P.V.D.V. have a postdoc-
toral grant from the Fund for Scientific Research e Flanders
(FWO e Vlaanderen).
where vi is the initial reaction rate, kcat is the catalytic turnover
number of p-nitrophenyl-b-D-ribofuranoside, KM is the Mi-
chaelis constant for p-nitrophenyl-b-D-ribofuranoside, Ki is
the dissociation constant of enzymeeinhibitor complex, I is
the inhibitor concentration, E is the enzyme concentration
and S is the substrate concentration.
The kcat and KM values of the substrate were determined by
direct non-linear fitting on the MichaeliseMenten equation us-
ing Microsoft Origin version 7.0.
References
[1] (a) World Health Report; 2002;
(b) www.who.int/tdr; 2005.
4.4. Docking of inhibitors in IAG-nucleoside hydrolase
of T. vivax
[2] M.H. El Kouni, Pharmacol. Ther. 99 (2003) 283e309.
[3] D.W. Parkin, B.A. Horenstein, D.R. Abdulah, B. Estupinan,
V.L. Schramm, J. Biol. Chem. 266 (1991) 20658e20665.
~´
The molecular modeling studies were performed using
MOE 2006.08 software (Chemical Computing Group). All
software necessary to build the compounds, perform minimi-
zations, alignments and superpositions is available in the
MOE package and was used with standard settings, unless oth-
erwise mentioned. Crystal structures of IAG-NH from T. vivax
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
~´
[4] B.A. Horenstein, D.W. Parkin, B. Estupinan, V.L. Schramm, Biochemistry
30 (1991) 10788e10795.
´
[5] W. Versees, J. Steyaert, Curr. Opin. Struct. Biol. 13 (2003) 731e738.
´
[6] W. Versees, K. Decanniere, E. Van Holsbeke, N. Devroede, J. Steyaert,
J. Biol. Chem. 277 (2002) 15938e15946.
´
[7] W. Versees, S. Loverix, A. Vandemeulebroucke, P. Geerlings, J. Steyaert,
J. Mol. Biol. 338 (2004) 1e6.
[8] E.A. Meyer, R.K. Castellano, F. Diederich, Angew. Chem. Int. Ed. 42
(2003) 1210e1250.
[9] B.A. Horenstein, V.L. Schramm, Biochemistry 32 (1993) 7089e7097.

326
A. Goeminne et al. / European Journal of Medicinal Chemistry 43 (2008) 315e326
[10] R.W. Miles, P.C. Tyler, G.B. Evans, R.H. Furneaux, D.W. Parkin,
V.L. Schramm, Biochemistry 38 (1999) 13147e13154.
[16] H. Ohrui, G.H. Jones, J.G. Moffat, M.L. Maddox, A.T. Christensen,
S.K. Byram, J. Am. Chem. Soc. 97 (1975) 4602e4613.
´
[11] W. Versees, J. Barlow, J. Steyaert, J. Mol. Biol. 359 (2006) 331e346.
´
[12] F.G. de las Heras, P. Fernandez-Resa, J. Chem. Soc., Perkin Trans 1
(1982) 903e907.
[17] G.W.J. Fleet, J.C. Son, Tetrahedron 44 (1988) 2637e2647.
[18] G.B. Evans, R.H. Furneaux, H. Hausler, J.S. Larsen, P.C. Tyler, J. Org.
Chem. 69 (2004) 2217e2220.
[13] D.R. Sauer, S.W. Schneller, Synthesis (1991) 747e750.
[14] B. Drake, M. Patek, M. Lebl, Synthesis (1994) 579e582.
[15] (a) H.R. Moon, W.J. Choi, H.O. Kim, L.S. Jeong, Tetrahedron: Asymmetry
13 (2002) 1189e1193;
[19] A. Thurkauf, A. Hutchinson, J. Peterson, L. Cornfield, R. Meade,
K. Huston, K. Harris, P.C. Ross, K. Gerber, T.V. Ramabhadran,
J. Med. Chem. 38 (1995) 2251e2255.
´
´
[20] W. Versees, K. Decanniere, R. Pelle, J. Depoorter, E. Brosens,
D.W. Parkin, J. Steyaert, J. Mol. Biol. 307 (2001) 1363e1379.
(b) S.B. Mandal, B. Achari, Synth. Commun. 23 (1993) 1239e1244.