Physiochemical Tuning of Potent Escherichia coli Anti-Adhesives by Microencapsulation and Methylene Homologation

Article abstract of DOI:10.1002/cmdc.201700061

Thiazolylaminomannosides (TazMan) are FimH antagonists with anti-adhesive potential against adherent-invasive Escherichia coli (AIEC) promoting gut inflammation in patients with Crohn's disease. The lead TazMan is highly potent in vitro, but shows limited in vivo efficiency, probably due to low pH stability and water solubility. We recently developed a second generation of stable TazMan, but the anti-adhesive effect was lower than the first. Herein we report a co-crystal structure of the lead TazMan in FimH, revealing that the anomeric NH group and the second thiazole moiety provide a positive hydrogen bonding interaction with a trapped water molecule, and π-stacking with Tyr48 of FimH, respectively. Consequently, we developed NeoTazMan homologated with a methylene group for low-pH and mannosidase stability with a conserved NH group and bearing various heterocyclic aglycones. Microencapsulation of the lead NeoTazMan in γ-cyclodextrin dramatically improved water solubility without disrupting the affinity for FimH or the anti-adhesive effect against AIEC isolated from patients with Crohn's disease.

Full text of DOI:10.1002/cmdc.201700061

Accepted Article
Title: Physiochemical tuning of potent E. coli antiadhesives by
microencapsulation and methylene homologation
Authors: Dimitri alvarez dorta, Thibaut Chalopin, Adeline Sivignon,
Jerome De Ruyck, Tanya dumych, Rostyslav Bilyy, david
deniaud, nicolas barnich, Julie Bouckaert, and Sébastien G.
Gouin
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To be cited as: ChemMedChem 10.1002/cmdc.201700061
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700061
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Physiochemical tuning of potent E. coli antiadhesives by
microencapsulation and methylene homologation.
Dimitri Alvarez Dorta,[a] Thibaut Chalopin,[a] Adeline Sivignon,[b] Jérôme de Ruyck,[c] Tanya I.
Dumych,[d] Rostyslav O. Bilyy,[d] David Deniaud,[a] Nicolas Barnich,[b] Julie Bouckaert,[c] and Sébastien
G. Gouin[a]*
[a]
Dr D.A. Dorta, Dr T. Chalopin, Prof. D. Deniaud, Dr S.G. Gouin
LUNAM Université, CEISAM, Chimie Et Interdisciplinarité, Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR des Sciences et des Techniques,
2, rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France
E-mail: sebastien.gouin@univ-nantes.fr
[b]
[c]
[d]
Dr A. Sivignon, Dr N. Barnich
Université Clermont Auvergne, Inserm U1071, M2iSH, USC-INRA 2018, F-63000 Clermont-Ferrand, France,
63000 Clermont-Ferrand, France
Dr J. Bouckaert, Dr J. de Ruyck
Université Lille, Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR8576 UGSF,
FRABio FR3688F-59000 Lille, France
Dr R.O. Bilyy, Dr T.I. Dumych
Danylo Halytsky Lviv National Medical University,
Pekarska Str. 69, 79010 Lviv,Ukraine
Supporting information for this article is given via a link at the end of the document
Abstract: Thiazolylaminomannosides (TazMan) are FimH
antagonists with anti-adhesive potential against adherent-invasive
Escherichia coli (AIEC) promoting gut inflammation in patients with
Crohn’s disease (CD). The lead TazMan is highly potent in vitro but
shows limited in vivo efficiency probably due to low pH stability and
water solubility. We recently developed a second generation of stable
TazMan but the anti-adhesive effect was decreased compared to the
first. Here, we report the co-crystal structure of the lead TazMan in
FimH, revealing that the anomeric NH and the second thiazole moiety
provide a positive H-bonding interaction with a trapped water
molecule, and π-stacking with Tyrosine 48 of FimH, respectively.
Consequently, we have developed NeoTazMan homologated with a
methylene group for low pH and mannosidase stability with a
conserved NH group and bearing various heterocyclic aglycons.
Microencapsulation of the lead NeoTazMan in a γ-cyclodextrin
dramatically improved the water solubility without disrupting the FimH
affinity or the anti-adhesive effect against AIEC isolated from patients
with CD.
treatments for urinary tract infections (UTIs) at the academic and
industrial level9. Potent in vitro10 and in vivo11–14 anti-adhesive
effects were observed, clearly suggesting the high potential of the
approach for the treatment of E.coli-induced UTIs.
Complementarily, multivalent glycoconjugates bearing multiples
copies of specific mannosides were also extensively investigated
by us15–17 and others18–21. This particular class of compounds was
shown to induce the formation of bacterial aggregates and to be
more effective in vitro and in vivo compared to their monovalent
analogues.16,18,22
Recently, we investigated the potential of FimH antagonists for
treating E. coli-induced inflammation in Crohn’s disease (CD).23–
25 CD is characterized by a dysfunction of the immune system in
response to an altered microbiota. A pathogenic group of bacteria
called adherent-invasive E. coli (AIEC) has been shown to induce
inflammatory cytokine expression after adhesion to
a
mannosylated receptor (CEACAM6), overexpressed in the ileum
of CD patients compared to healthy controls. The synthetic
mannosides were shown to decrease AIEC colonization in the
feces, gut and ileum of the CEABAC 10 mouse model mimicking
CD after oral administration at a dose of 10 mg/kg.24,25 Importantly,
this was correlated with a decrease in the inflammatory syndrome.
These results strongly suggested an alternative approach for CD
patients to the current treatment based on the administration of
immunosuppressive agents (such as anti TNF-α).
Introduction
The anti-adhesive strategy is an appealing alternative approach
to antibiotic treatments that consist in preventing or disrupting
bacterial adherence to the host cells.1–4 One of the most studied
target is the FimH adhesin, a mannose-binding lectin situated at
the tip of rod-like organelles (pil) expressed by most E. coli.5,6 The
concept was proposed more than 30 years ago, when aryl-
mannosides were shown to be FimH antagonists, with anti-
adhesive effects observed in cell-based assays.7 Alkyl-
mannosides were identified as a second class of potent inhibitors,
with heptylmannosides (HM) being the most potent of the series,
and showing an in vivo anti-adhesive effect when coadministred
with uropathogenic E. coli strains to mouse bladder.8 These
pioneering works drived the development of alternative
The
thiazolylaminomannosides
(TazMans)
family
(i.e.
compounds 1 and 2, Figure1), recently developed in the group,23
are highly potent E. coli anti-adhesives in eukaryotic cells,
preventing the adhesion of a broad range of E. coli strains isolated
from patients with UTIs, CD or osteoarticular infections.26
However, this first generation suffered from an anomerization of
the heteroarylamino groups at low pH from the active α to the
inactive β-form. This may be problematic in a potential oral
administration, in which the compound passes through the
stomach (pH = 2). We recently designed a second generation of
TazMan,27 in which the anomeric NH was replaced by O-CH2, S-
1
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CH2, CH2-CH2, CH2-S or O-CH2CH2 groups. These compounds Results and Discussion
proved to be stable in acidic media but in vitro tests showed a
decreased in anti-adhesive potency compared to 1, probably due
in part to the absence of a stabilizing hydrogen bonding between
anomeric NH and a water molecule trapped in the FimH binding
sites as seen in the 1-FimH co-crystal structure (Figure 1B). Such
a stabilizing interaction has recently been observed by Janetka
and co-workers with biphenyl-C-mannosides bearing anomeric
hydroxyalkyl groups.28 The compounds included in our recent
study also lacked the second thiazole and pyrazine ring of 2, the
most potent FimH antagonist of the serie, that showed
outstanding in vitro anti-adhesive effects23,26. In this work, we
developed a new series of TazMans based on a co-crystal
structure of 2-FimH (Figure 1C). The anomeric amino linkage was
replaced by a methylamino group to improve the chemical stability
at low pH and prevent hydrolysis by glycosidases. The pyrazine
Prior to starting our investigation, we wanted to gain more insight
into the generally high anti-adhesive potency of the TazMan
series and particularly of the best compound 2 which is around
100-fold more potent than the reference compound heptyl-
mannoside (HM), and 50 to 100-fold more potent than 1.26 Co-
crystals were obtained using the vapor diffusion method similar to
a
previously published protocol.8 The obtained complex
crystallized in tetragonal space group P43212, with two molecules
in the asymmetric unit. Data collection and refinement statistics
are presented as supplemental information (Table S1). The
orientation of the ligand is similar to that observed in compound 1.
Nevertheless, the additional thiazol moiety born by 2 interacts
more evidently through π-π stacking with phenol ring of Tyr48,
keeping it into it the half-open tyrosine gate conformation. The N-
glycosidic linker atom is hydrated like in the crystal structure of
FimH in complex with compound 1,23 and forms a weak hydrogen
bond (3.2 – 3.4 Å depending on the molecule in the asymmetric
unit).
pharmacophore of
2 was replaced by a small library of
heterocyclic aglycons to modulate both the FimH affinity and
water solubility of the compounds. The FimH affinity of the small
library was evaluated as well as their potency in inhibiting AIEC
adhesion to intestinal cells.
Figure 1. A) Chemical structure of previously described TazMans 1 and 2.23 2 is around 50-100 fold more potent as an E. coli anti-adhesive compared to 1 but the
aglycone group partially anomerizes at low pH from the α to the inactive β form. To prevent anomerization and improve chemical and enzymatic stability, we
developed a new TazMan family homologated with a methylene group and bearing diverse pharmacophores. B) The structure of 1 co-crystalized with FimH(PDB
entry 3zl2, 1.25 Å resolution)23 shows anomericNH bonding with a water molecule. C) A similar interaction is observed in the co-cristal structure of 2-FimH (PDB
entry 5MTS, 2.6 Å resolution).
The chemical synthesis of the homologated TazMans required
protected α-mannosides with the methylthiourea group in the
anomeric position to be obtained for the addition-cyclization
mannose hydroxyl groups were both synthesized (Scheme1),
as reactivity during the critical thiazole formation was
differently affected depending on the substrates.
step leading to
compounds 7 and 10 bearing benzyles and acetates on the
a
thiazole ring. Armed and disarmed
The synthesis of benzyl-protected 7 started from α-C-
mannoside 3 obtained in seven steps from mannose as we
2
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previously reported.27 The mesylate group of 3 was substituted
by an azido-group with sodium azide and tert-butylammonium
iodide and then the crude compound 4 obtained was further
reduced by a Staudinger procedure to form amine 529 in 60%
yield over two steps. 5 was directly converted into the benzoyl-
protected thiourea
6 with 86% yield using potassium
isothiocyanate and benzoyl chloride in acetone. After flash
chromatography purification, the benzoyl group was easily
deprotected with sodium hydroxide to form 7.
The synthesis of acetyl protected thiourea 10 started from
aminomethylmannoside 8 obtained in three steps and as a
pure alpha-form using a previously described procedure.30 The
amino group of 8 was first converted to an isothiocyanate in a
mixture of calcium carbonate and carbone disulfide and the
product was acetylated to form 9.29 Finally, treatment of
isothiocyanate 8 with HMDS in DMF yielded thiourea 10.
Scheme1. Synthesis of thiourea 7 and 10. Reagents and conditions: a) NaN3,
DMF, 71%; b) PPh3, THF/H2O, 84%, c) KNCS, BzCl, acetone, 86%; d)
NaOH, MeOH, 94%; e) CS2, CaCO3 then Ac2O, Pyr 38% two steps; f) HMDS,
DMF, 79%.
After addition of DMF-DMA, the corresponding benzyl or acetyl
protected C-mannosides were engaged in the critical addition-
cyclization step with diverse chloroketones to form protected
compounds 11-22. The benzyl and acetate groups of the
corresponding
cycloadducts
were
removed
with
trichloroborane or sodium methanolate, respectively, leading
to unprotected C-mannoside 23-34 with yields ranging from 64
to 96% (Figure 2).
Figure 2. Structure of cycloadducts 11-33. Reagents and conditions a) DMF-DMA, chlorocetone, Et3N, KI, THF; b) BCl3, DCM for Bn;
c) MeOH, MeONa for acetates; d) LiOH, MeOH/H2O.
3
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adhesion of the pathogenic AIEC LF82 strain (isolated from
patients with CD) to T84 intestinal epithelial cells. T84 express
the mannosylated CEACAM6 protein, a GPI-anchored protein
abnormally expressed at the ileal mucosa of CD, and allowing
FimH-mediated AIEC attachment to the cells. The residual
percentage of bacterial adhesion obtained in the presence of a
10 µM concentration of the compounds, compared to the non-
treated wells is presented in Figure 3. HM was included in the
assay as an internal reference. HM has previously been shown
to display a low nanomolar affinity for FimH (5 nM by SPR) and
a strong in vitro anti-adhesive effect against AIEC LF82, but it
failed to reduce AIEC levels in vivo using a transgenic mouse
model mimicking CD.25
The presence of the conjugated aglycons was shown to
significantly lower the water solubility of the compounds. In
order to obtain fully water-soluble C-mannosides, we designed
a subset of compounds bearing hydrophilic moieties at position
2 of the second thiazole starting from alkynes 16 or 17
(Scheme 2). The new pharmacophores should not disrupt
FimH binding as they point out of the protein-carbohydrate
binding domain. 16 or 17 were reacted in a CuAAC protocol
with highly hydrophilic groups which were an azido-
functionalized triethyleneglycol, a C-mannoside or a beta-
cyclodextrin.
Scheme2. Synthesis of hydrophilic analogues 38-40. Reagents and
conditions: a) sodium ascorbate, CuSO4, dioxane/H2O; b) BCl3, DCM for Bn;
c) MeOH, MeONa for acetates.
Figure 3. Inhibitory effect of the compounds on the ability of the LF82 strain
to adhere to intestinal epithelial cells T84. Cells were infected at a multiplicity
of infection of 10 bacteria per cell, for a 3-hour period. Compounds were
In addition, we synthesized the highly polar compound 43
bearing a permanent positive charge on the nitrogen atom of
DABCO (Scheme 3). 43 was obtained in three steps starting
from the hetero-functionalized tetraethyleneglycol 41 and
alkyne 17. After insertion of the linker by CuAAc, protective
acetates were removed from the sugar and the mesylate was
substituted with DABCO.
incubated with AIEC bacteria for 1h before infection of cells at
a
concentration of 10µM. Results are expressed in percentage of bacteria
associated with the cells (n=6 experiments, means ± SEM; *: p < 0.05; **: p
< 0.01; ***: p < 0,001). LF82 infection in the absence of treatment was
normalized to 100%.
Although a larger number of molecules would be required in
the library to investigate structure-activity relationships
extensively, interesting information can be extracted from the
results. Most of the compounds were shown to prevent
significantly the bacterial adhesion to the cells, with seven out
of the fifteen compounds tested being more effective than the
HM reference compound. The only exception was compound
23, lacking additional substituents after the methylcarbonyl
group. To observe a significant reduction in the bacterial
attachment the concentration of 23 had to be increased to 50
µM, which was 5 times higher than the concentration used to
assay the whole set of molecules (data not shown). Thus, the
addition of a second heterocycle (substituted thiazole, pyrrole
or isoxazole) after the carbonyl moiety was highly beneficial for
improving the anti-adhesive effect in all cases studied here.
This can be rationalized by the crystal structure of 2-FimH
showing a stacking interaction between Tyr 48 and the second
thiazole ring. Although Tyr48 can adopt different conformation,
the X-ray structure of 1-FimH shows a parallel but staggered
orientation of the thiazole ring of 1 relative to the phenyl group
of Tyr 48. Modifications beyond the second heterocycle also
impact the anti-adhesive effect but to a lesser extent as the
inhibition values ranged from 16% to 62% at a fixed
concentration of 10 µM for all compounds (Figure 3). As seen
in the 2-FimH co-crystal, the additional pyrazine group is
Scheme 3. Synthesis of hydrophilic compound 43. Reagents and
conditions: a) sodium ascorbate, CuSO4, dioxane/H2O, 98%; b) NaOMe,
MeOH; c) AcOEt, DABCO, MeOH, 90% two steps.
The whole set of compounds was evaluated side-by-side in a
cell-based assay to measure their potency in preventing the
4
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pointing out of the binding domain were possible steric clashes
and positive interactions with the protein are more limited.
Substitution of this group by hydrophilic substituents enabled
us to design the water-soluble compounds 38-40 and 43 with
conserved anti-adhesive potencies. It should be noted that
compound 39 can potentially bind two FIMH due to the
presence of two mannose moieties.
Compound 24, the homologated analogue of 2, was
significantly more potent, with a residual adhesion level of only
16%. This value also exceeded the anti-adhesive effects that
we observed with the same pre-incubation protocol and at the
same concentration with our previously published second
generation of enzymatically stable TazMans (values ranging
from 44 to 78%),27 and heptyl-mannoside derivatives (37-
95%).24
Addition of the pyrzinylthiazolyl moiety also significantly altered
the water solubility of compounds and stock solutions of 2 and
24 had to be prepared in DMSO prior to running the in vitro
assays. Although we showed that DMSO did not impact the
result in cell-based assays, this vector is not optimal for in vivo
evaluation. To overcome this disadvantage, we planned to
encapsulate the compounds by host molecules possessing a
hydrophobic cavity. Cyclodextrins are particularly suited for
microencapsulation and are extensively used in formulation to
improve the therapeutic index of hydrophobic molecules. 2 and
24 were mixed with α-, β- and γ-cyclodextrins (CD) possessing
hydrophobic cavities of 4.5, 7 and 8Å, respectively. After
addition of ten volumes of water to the compounds-CD
mixtures dissolved in DMSO, the samples were lyophilized. 2
and 24 were shown to form 1-1 stoechiometric inclusion
complexes in γCD but not in α or β. The solubility of the
compounds was dramatically improved allowing around 10
mg.mL-1 of the two complexes 2@γCD and 24@γCD to be
dissolved in water.
The low residual adhesion value of 17% for 24 at 10 µM is
promising considering that compounds with higher levels of in
vitro anti-adhesive effect (ranging from 40-75%) showed AIEC
decolonization and reduced inflammatory syndromes in a CD
mouse model after oral administration (10 mg/kg).25
We previously identified compounds 1 and 2 as very potent
FimH antagonists in vitro.23 However these two compounds
were not suited for in vivo application due to their
anomerization to the inactive β-form at the low pH encountered
in the stomach and their potential instability towards
mannosidases hydrolysis. The homologation by a methylene
group to form analogs 23 and 24, respectively now fully
prevents these phenomena. To quantify better the potential
loss of affinity provided by such homologation, the binding
affinity for FimH of 1, 2, 23 and 24 was compared side by side
in an enzyme-linked lectinosorbent assay (ELLSA). In this
assay, the highly mannosylated RNAseB protein was coated
on the surface of immunological wells and FimH was added in
the presence or absence of inhibitors. The surface-bound
FimH was detected spectrophotometrically with anti-FimH and
The affinity of the two complexes was then evaluated in our
ELLSA and the IC50 values were 109 and 135 nM for 2@γCD
and 24@γCD, respectively. Thus, the complexes display
similar level of affinity for their targets compared to the free
molecules, meaning that 2 and 24 can easily “escape” from the
CD ring to interact with FimH. γCD was also included in the
assay, but no inhibition was observed at the higher dose tested
(1 mM).
2@γCD and 24@γCD (dissolved in water) were then tested
using the preincubation protocol to measure their AIEC anti-
adhesive efficiency. The inclusion complexes were tested at
five different concentrations of 100, 10, 1, 0.1 and 0.01 µM in
order to provide an estimation of the IC50, defined as the
concentration of compounds required to decrease the AIEC
adhesion level by 50%. Compound 2 (dissolved in DMSO) was
also included in the assay as a reference to account for a
potential loss of affinity with 2@γCD. The results presented in
Figure 5 clearly show that no loss of anti-adhesive effect was
observed with 2@γCD compared to 2, consistent with the
results from the ELLSA assay. This was confirmed by the
calculation of the IC50 which were virtually identical and equal
to 0.7 µM for 2 and 2@γCD.
Thus, microencapsulation does not impair FimH binding and is
an interesting strategy to enhance the water-solubility of anti-
adhesive compounds without altering their anti-adhesive effect.
The determination of the IC50 for 24@γCD was more
approximate because the residual adhesion at concentrations
of 0.1 and 0.01 exceeded 100%, a phenomenon that we
previously observed with heptavalent HM covalently linked to
a cyclodextrin core.25 Curve fitting gave an IC50 of 2 µM for
24@γCD which is around three time higher than for 2@γCD.
Thus, the results obtained in the cell-based assay are
consistent with the loss of FimH affinity by compound 24
compared to 2. This decreased efficiency is limited considering
that 2 is very potent in preventing AIEC attachment to intestinal
cells at around 10000-fold and 100-fold lower concentrations
than mannose and the potent FimH antagonist HM,
respectively.23
secondary-labeled
antibodies.
Dose-response
curves
obtained from testing compounds at eight different
concentrations enabled the determination of the minimal
inhibitory concentration to achieve 50% inhibition (IC50). All
four compounds showed an IC50 below 500 nM, and down to
70 nM for the best compound 2. The homologated 23 and 24
were 2.4-fold (494 vs. 205 nM) and 2.8-fold (194 vs. 70 nM)
less potent than 1 and 2, respectively (Figure 4). The
decreased FimH affinity is therefore significant after
homologation but remains acceptable considering the very
high in vitro potency of
1 and 2. Interestingly, the
pyrazinylthiazolyl moiety improved FimH binding to a similar
level of 2.5 and 2.9 fold when switching from 1 to 2 and 23 to
24, respectively.
Figure 4. Binding affinity of TazMans 1, 2, 23, and 24 for FimH determined
by ELLSA. Average of three measurements. IC50 values expressed in
nanomolar.
5
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Ionisation (ESI) on a Micromass-Waters Q-TOF Ultima Global or with
a Bruker Autoflex III SmartBeam spectrometer (MALDI). Low-resolution
mass spectra (MS) were recorded with a Thermo electron DSQ
spectrometer. All reagents were purchased from Acros Organics or
Aldrich and were used without further purification. Column
chromatography was conducted on silica gel Kieselgel SI60 (40-63 μm)
from Merck. Reactions requiring anhydrous conditions were performed
under argon. Dichloromethane was distilled from calcium hydride under
nitrogen prior to use. Microwave experiments were conducted in sealed
vials in commercial microwave reactors especially designed for
synthetic chemistry. (MultiSYNTH, Milestone). The instrument features
a special shaking system that ensures high homogeneity of the reaction
mixtures. Optical rotations were measured on a 343 PERKIN ELMER
at 20°C in a 1cm cell in the stated solvent; [α]D values are given in 10-1
deg.cm2 g-1 (concentration c given as g/100 mL).
GP1 = First general procedure for the addition-cyclization step The
thiourea 7 or 10 (1eq) was dissolved in acetonitrile or THF (20
mL/mmol), DMF-DMA (1.3 eq) was added and the mixture was warmed
at 60°C for 40 min. After completion, as indicated by TLC, -
halogenoketone (1.2 eq) was added with a catalytic amount of
potassium iodide (0.05 eq). After 15 min of stirring at room temperature,
triethylamine (2 eq) was added and the mixture was heated at 60°C
until reaction completion, as indicated by TLC. The mixture was
washed with brine, extracted by AcOEt, dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel.
Figure 5. Inhibitory effect, measured at five different concentrations of 2,
2@γCD and 24@γCD on the ability of the LF82 strain to adhere to
intestinal epithelial cells.
Conclusions
The co-crystal structure of the potent TazMan 2 in the FimH
binding site and the ligands and protein amino-acid (Tyr48 and
Tyr137) conformations were compared with the previously
published structure of 1-FimH. An anomeric NH bonding
interaction with a water molecule was conserved in both
structures. The Tyr 48 orientation has significantly shifted to
form a stabilizing stacking interaction with the second thiazole
of 2, probably explaining its higher FimH affinity compared to
1. Based on these structures, and to provide chemically and
enzymatically stable FimH antagonists for potential in vivo
applications, we designed fifteen homologated C-mannosides
with an NH group, and functionalized thiazole, pyrrole or
isoxazole as a second heterocyclic moiety for a π-stacking
interaction withTyr 48. The most potent compound of the serie
was 24, the analogue of 2 homologated by a methylene group.
This new compound should be fully stable towards enzymatic
and acidic hydrolysis and showed a limited affinity loss for
FimH compared to 2. 24 has the higher AIEC anti-adhesive
effect measured so far in vitro for a stable TazMan. The
compound was formulated as a water soluble γCD complex
that escape from the hydrophobic cavity to interact with FimH.
24@γCD is a promising formulation in an E. coli anti-adhesive
therapy considering that heptylmannoside derivatives with
much lower in vitro potency have proved effective in reducing
bacterial levels and inflammatory syndromes in a transgenic
mouse model of Crohn’s disease.
GP2 = General procedure for the benzyl deprotection step The
protected carbohydrate (1eq) was dissolved in DCM (2mL/mmol) under
inert atmosphere and the solution was stirred at -10°C. BCl3 1M in DCM
(3eq per function) was added dropwise and the mixture was stirred at
rt for 20h. Methanol was added slowly and the mixture was
concentrated under vacuum. This operation was repeated 4 times.
Then the resulting mixture was purified by flash chromatography on
silica gel.
GP3 = General procedure for the acetyl deprotection step The
protected carbohydrate (1 equiv.) was dissolved in dry MeOH (30 mL)
and sodium methoxide (1 M solution in MeOH, 10% mol per AcO) was
added. The mixture was stirred for 4 h, neutralized with Amberlite IR120
(H), filtered and the solvents evaporated to dryness. The substrate was
dissolved in water and subjected to lyophilization.
2,3,4,6-tetra-O-benzyl-1-azidomethyl--D-mannopyranose
(4).
Compound 327 (900 mg, 1.42 mmol) was dissolved in DMF (15 mL),
NaN3 (462 mg, 6 eq) and TBAI (1eq, 523 mg) were added. The mixture
was heated to 110°C for 24h then extracted by Et2O. The organic layer
was washed with brine 5 times, dried over MgSO4 and concentrated
under vacuum. The residue was purified on silica gel (PE/AcOEt 9:1)
[ ]20
to afford 587mg (71% yield) of 4 as a colourless oil. α = +28 (c = 0.5,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ = 7.18-7.36 (20H, m, HBn), 4.41-
3
3
3
4.57 (8H, m, CH2Bn), 4.10 (1H, ddd, J5-4=2.6 Hz, J5-6’=6.0 Hz, J5-
6=6.4 Hz, H-5), 4.06 (1H, ddd, J=4.5 Hz, 3J=5.5 Hz, 3J=8.5 Hz, H-1),
3
3.82 (1H, dd, 3J6-5=6.6 Hz, 2J6-6’=10.1 Hz, H-6), 3.76-3.81 (2H, m, H-3,
H-4), 3.76 (1H, dd, J=2.8 Hz, 3J=8.5Hz, H-2),3.71 (1H, dd, 3J5-6’= 6.0
3
Hz, 2J6-6’=10.1Hz, H-6’), 3.40-3.48 (2H, m, H7); 13C NMR (100 MHz,
CDCl3): δ = 138.4 (CBnIV), 138.1 (CBnIV), 138.0 (CBnIV), 137.9 (CBnIV),
127.7-128.6 (CBn), 74.6 (C-5), 74.2 (C-4), 73.8 (C-3), 73.7 (C-2), 73.3
(CH2Bn), 72.6 (CH2Bn), 72.3 (CH2Bn), 71.6 (CH2Bn), 69.9 (C-1), 68.2
(C-6), 51.7 (C-7); HRMS (ES+) m/z calcd for C35H38N3O5: 580.2811,
found 580.2787.
Experimental Section
General experimental details. NMR spectra were recorded at room
temperature with a Bruker Avance 300 Ultra Shield or eBruker Avance
III 400 spectrometer and chemical shifts are reported in parts per million
relative to tetramethylsilane or a residual solvent peak peak (CHCl3: 1H:
δ=7.26, 13C: δ=77.2; DMSO-d6: 1H: δ=2.54, 13C: δ=40.4). Peak
multiplicity is reported as: singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Peak multiplicity and chemical shifts are
reported for α compounds in case of anomeric mixtures in equilibrium.
High resolution mass spectra HRMS where obtained by Electrospray
2,3,4,6-tetra-O-benzyl-1-aminomethyl--D-mannopyranose (5). 4
(587 mg, 1.01 mmol) was dissolved in THF (15mL) and a few drops of
water were added. Triphenylphosphine (345 mg, 1.3 eq) was added
and the mixture was heated to reflux for 2h. The mixture was
concentrated in vacuo, rinsed by PE, concentrated and dissolved in
6
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10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
Et2O. The triphenylphosphine oxide precipitated and was filtered off.
The mixture was dried over MgSO4, filtered,and concentrated in
vacuum. The residue was purified on silica gel (CHCl3/MeOH 1:0 to
[ ]20
+20 (c = 0.5, CHCl3); NMR previously described in litt;29 HRMS (MALDI)
m/z calcd for C35H40NO5: 554.2901, found 554.2873.
(PE/AcOEt 3:6) and a second purification (CHCl3/AcOEt 7:3), 152 mg
[ ]20
(83% yield) of 11 was obtained as a slightly yellow oil. α = +14 (c =
D
0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 7.76 (1H, s, H-9), 7.16-
97:3) to afford the 469 mg (84% yield) of 5 as a colourless oil. α
=
7.37 (20H, m, H-Bn), 6.05 (1H, bt, NH), 4.36-4.55 (8H, m, H-CH2Bn),
4.03-4.10 (2H, m, H-1, H-5) , 3.79-3.86 (2H, m, H-4, H-6), 3.67-3.72
D
2
3
(2H, m, H-2, H-3), 3.61 (1H, dd, J6-6’ =10.3Hz, J6’-5 = 5.6 Hz, H-6’),
3.43-3.61 (2H, m, H-7), 2.43 (3H, s, H-12); 13C NMR (100 MHz, CDCl3):
δ = 189.2 (C-11), 175.1 (C-8), 148.1 (C-9), 138.2 (C-BnIV), 137.9 (2C-
BnIV), 137.6 (C-BnIV), 129.4 (C-10), 127.8-128.8 (C-Bn), 74.9 (C-5),
74.4 (C-2), 74.3 (C-3), 74.3 (C-4), 73.4 (C-CH2Bn), 72.9 (C-CH2Bn),
72.4 (C-CH2Bn), 71.6 (C-CH2Bn), 68.2 (C-1), 67.99 (C-6), 46.5 (C-7),
26.1 (C-12); HRMS (MALDI) m/z calcd for C40H43N2O6S: 679.2836,
found 679.2836.
N-benzoyl-N’-(2,3,4,6-tetra-O-benzyl--D-mannopyranosyl)
methylthiourea (6). Potassium isothiocyanate (3 eq, 411 mg), was
dissolved in acetone (10 mL). Benzoyl chloride (2 eq, 400 mL) was
added and the white suspension was stirred for 20 min, then 5 (780 mg,
1.41 mmol) diluted in DCM (5 mL) was added to the mixture. After 10
min, the reaction was complete. The mixture was washed by brine,
extracted by DCM, dried over MgSO4 and concentrated under vacuum.
The resulting oil was purified by flash chromatography on silica gel
(PE/AcOEt 8:2 to 7:3) to afford 870 mg (86% yield) of 6 as a colourless
oil. Broad NMR signals were obtained due to the presence of rotamers;
HRMS (MALDI) m/z calcd for C43H44N2O6SNa: 739.2812, found
739.2783.
5-(4-methyl-2-(pyrazin-2-yl)thiazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-
O-benzyl--D-mannopyranosyl)methylaminothiazole
Prepared following GP 1, starting from 7 (300 mg, 0.49mmol) and 2-
bromo-1-(4-methyl-2-(pyrazine-2-yl)thiazol-5-yl)ethanone. After
(12).
purification over silica gel (PE/AcOEt 4:6) 249 mg (60% yield) of 12 was
[ ]20
obtained as a yellow oil. α = +2 (c = 0.5, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ = 9.46 (1H, d,3J19-18 =1.5Hz, H-19), 8.64 (1H, d,3J17-18 =2.5Hz,
N-(2,3,4,6-tetra-O-benzyl--D-mannopyranosyl)methylthiourea (7).
6 (870 mg, 1.21 mmol) was diluted in MeOH (6mL), sodium hydroxide
pellets were added. After 10 min, the reaction was complete. The
mixture was filtered, then neutralised by 2M HCl, extracted by DCM,
washed by brine, dried over MgSO4 and concentrated under vacuum.
The resulting oil was purified by flash chromatography on silica gel
(PE/AcOEt 1:1) to afford 698 mg (94% yield) of 7 as colourless oil.
Severals conformers were observed by NMR and broad signals were
obtained; 1H NMR (400 MHz, CD3OD) δ = 7.21-7.53 (20H, m, 4 x BnO),
4.42-4.55 (8H, m, 4 x BnO), 4.07 (1H, m, H-1), 3.86-3.93 (3H, m, H5,
H-6, H-7), 3.72-3.79 (3H, m, H-3, H-4, H-7’), 3.58-3.63 (2H,m, H-2, H-
6’); 13C NMR (127 MHz, CD3OD): δ = 45.8 (CH2, C-7), 68.1 (CH2, C-
6, several rotamers), 69.7-79.2 (5CH, 4CH2, several rotamers), 128.3-
133.7 (20CH, 4 x BnO, several rotamers), 139.4-139.5 (4C, 4 x BnO),
183.7 (C, thiourea); HRMS (MALDI) m/z calcd for C36H41N2O5S:
613.2731, found 613.2706.
H-17), 8.58 (1H, dd, 3J17-18 =2.5Hz, , 3J19-18 =1.5Hz, H-18), 7.96 (1H, s,
H-9), 7.17-7.37 (20H, m, H-Bn), 6.24 (1H, bt, NH), 4.36-4.56 (8H, m, H-
CH2Bn), 4.05-4.13 (2H, m, H-1, H-5) , 3.81-3.87 (2H, m,H-4, H-6), 3.68-
3
3.73 (2H, m, H-2, H-3), 3.61 (1H, dd, 2J6-6’ =10.2Hz, J6’-5 = 5.6 Hz, H-
6’),3.47-3.63 (2H, m, H-7), 2.75 (3H, s, H-14); 13C NMR (100 MHz,
CDCl3): δ = 177.3 (C-11), 175.4 (C-8), 166.2 (C-15), 159.4 (C-12),
149.9 (C-9), 146.5 (C-16), 145.9 (C-17), 144.2 (C18), 142.0 (C-19),
138.1 (C-BnIV) ,137.9 (C-BnIV), 137.6 (C-BnIV), 130.3 (C-13), 130.1 (C-
10), 127.8-128.8 (C-Bn), 75.0 (C-5), 74.3 (C-4), 73.6 (C-3), 73.4 (C-2),
73.4 (C-CH2Bn), 73.0 (C-CH2Bn), 72.3 (C-CH2Bn), 71.6 (C-CH2Bn),
68.0 (C-1), 68.0 (C-6), 46.7 (C-7), 20.0 (C-14); HRMS (MALDI) m/z
calcd for C47H46N5O6S2: 840.2884, found 840.2857.
5-(2-benzoylamino-4-methylthiazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-
O-benzyl--D-mannopyranosyl)methylaminothiazole
(13).
Prepared following GP 1, starting from 7 (50 mg, 0.082 mmol) and N-
(5-(2-chloroacetyl)-4-methylthiazol-2-yl)benzamide (36 mg, 0.123
mmol) as starting materials, the derivative 13 (54 mg, 0.061 mmol,
75%) was obtained after purification by silica gel column
Compound (9). To a solution of amine 8 (1.88 g, 9.741 mmol) in 6:4
H2O/acetone (50 mL) was added CaCO3 (2.92 g, 29.223 mmol) and
CSCl2. After 4 h, the mixture was filtered under Celite pad and
concentrated. The isothiocyanate crude was solved in Py (20 mL) and
Ac2O (20 mL) and DMAP (20 mg) were added. After 8 h, the reaction
mixture was concentrated and the obtained crude was purified by silica
gel column chromatography (PE/EtOAc 7/3 as eluent) to give the
protected isothiocyanate 10 (1.53 g, 3.796 mmol, 39%), whose
spectroscopic dates are in concordance with the bibliographic ones.29
chromatography (petroleum ether/EtOAc, 1:1 as eluents) as
a
[ ]20
yellowish oil. α = +48 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3) δ
D
= 2.47 (3H, s, methylthiazol), 3.52 (1H, dd, J7a,7b = 12.9 Hz, J7a,1 = 6.6
Hz, H-7a), 3.62-3.68 (2H, m, H-7b, H-6a), 3.71-3.76 (2H, m, H-2, H-3),
3.82-3.89 (2H, m, H-4, H-6b), 4.09-4.17 (2H, m, H-1, H-5), 4.41-4.57
(8H, m, 4 x BnO), 6.94 (1H, bs, NH), 7.21-7.38 (20H, m, 4 x BnO), 7.54
(2H, bt, J = 7.5 Hz, benzamide), 7.63 (1H, bt, J = 7.5 Hz, benzamide),
7.94 (1H, s, H9), 7.98 (1H, bd, J = 7.5 Hz, benzamide); 13C NMR (127
MHz, CHCl3): δ = 17.5 (CH3, methylthiazol), 46.8 (CH2, C-7), 67.9 (CH2,
C-6), 68.3 (CH, C-1), 71.3, 72.1, 72.6, 73.2 (4CH2, 4 x BnO), 73.4 (CH,
C-4), 73.6, 74.1 (CH, C-2, C-3), 74.6 (CH, C-5), 121 (C), 127.6-128.5
(21CH, 4 x BnO, benzamide), 129.0 (CH, benzamide), 129.9 (C), 131.7
(C), 133.2 (CH, benzamide), 137.4 (C, BnO), 137.7 (2C, 2 x BnO),
137.9 (C, BnO), 148.4 (CH, C-9), 154.2 (C), 159.6 (C), 165.4 (C), 175.1
(C), 175.2 (C, C-10); HRMS (ESI) m/z calcd for C50H48N4O7S2 [M +
H]+ 881.3027, found 881.3043.
N-(2,3,4,6-tetra-O-acetyl--D-mannopyranosyl)methylthiourea
(10). To a solution of 9 (50 mg, 0.124 mmol) in DMF (0.6 mL), at 0° C
and under N2, was added HMDS (258 µL, 1.240 mmol). After 8 h, the
mixture was concentrated and the crude was purified by silica gel
column chromatography (EtOAc as eluent) to give the thiourea 10 (41
[ ]20
mg, 0.098 mmol, 79%) as a colorless oil. α = +12 (c = 0.9, CHCl3);
D
1H NMR (400 MHz, CDCl3) δ = 1.94 (3H, s, AcO), 1.97 (3H, s, AcO),
1.99 (6H, s, 2 x AcO), 3.74 (2H, m, H-7), 3.89-4.15 (2H, m, H-1, H-5,
H-6a), 4.36 (1H, m, H-6b), 4.97-5.11 (2H, m, H-4, H-2), 5.15 (1H, dd,
J3,4 = 7.5 Hz, J3,2 = 3.3 Hz, H-3), 6.42 (2H, bs, NH2), 7.37 (1H, bs,
NH); 13C NMR (127 MHz, CDCl3): δ = 20.4, 20.47, 20.49, 20.55 (4CH3,
4 x AcO), 43.5 (CH2, C-7), 61.4 (CH2, C-6, several rotamers), 66.8 (CH,
C-4, several rotamers), 67.6 (CH, C-3, several rotamers), 68.1 (CH, C-
3, several rotamers), 68.4 (CH, C-1), 70.9-72.1 (2CH, C-1, C-5, several
rotamers), 169.4 (2C, 2 x AcO), 169.9 (C, AcO), 170.7 (C, AcO), 183.7
(C, thiourea); HRMS (ESI) m/z calcd for C16H25N2O9S [M + H]+
421.1273, found 421.1275.
5-(2-bromo-4-methylthiazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-O-
benzyl--D-mannopyranosyl)methylaminothiazole (14). Prepared
following GP 1, starting from 7 (50 mg, 0.082 mmol) and 1-(2-bromo-4-
methylthiazol-5-yl)-2-chloroethanone (31 mg, 0.123 mmol) as starting
materials, the derivative 14 (48 mg, 0.057 mmol, 70%) was obtained
after purification by silica gel column chromatography (petroleum
[ ]20
ether/EtOAc, 70:30 as eluents) as a yellowish oil. α = +18 (c = 1.3,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.64 (3H, s, methylthiazol),
3.49 (1H, dd, J7a,7b = 12.7 Hz, J7a,1 = 6.2 Hz, H-7a), 3.57-3.65 (2H, m,
H-7b, H-6a), 3.67-3.71 (2H, m, H-2, H-3), 3.81-3.87 (2H, m, H-4, H-
6b), 4.04-4.13 (2H, m, H-1, H-5), 4.34-4.56 (8H, m, 4 x BnO), 6.26 (1H,
5-acetyl-2-(2,3,4,6-tetra-O-benzyl--D-mannopyranosyl)methyl
aminothiazole (11). Prepared following GP 1, starting from 7 (166 mg,
0.271mmol) and chloroacetone. After purification over silica gel
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
bs, NH), 7.17-7.36 (20H, m, 4 x BnO), 7.82 (1H, s, H9); 13C NMR (127
MHz, CDCl3): δ = 17.7 (CH3, methylthiazol), 46.8 (CH2, C-7), 67.7 (CH2,
C-6), 68.0 (CH, C-1), 71.3, 72.1, 72.6, 73.2 (4CH2, 4 x BnO), 73.0 (CH,
C-4), 73.4, 74.0 (CH, C-2, C-3), 74.7 (CH, C-5), 127.6-128.5 (20CH, 4
x BnO), 128.9 (C), 131.2 (C), 137.3 (C, BnO), 137.6 (2C, 2 x BnO),
137.8 (C, BnO), 148.7 (CH, C-9), 157.4 (C), 175.3 (C), 175.5 (C);
HRMS (ESI) m/z calcd for C43H43BrN3O6S2 [M + H]+ 840.1774, found
840.1771.
(1H, m, thiazol); 13C NMR (127 MHz, CDCl3): δ = 18.4 (CH3,
methylthiazol), 20.69, 20.75, 20.77, 20.81 (4CH3, 4 x AcO), 34.7 (CH2,
C-7), 45.4 (CH2, propargylamine), 60.7 (CH2, C-6), 67.0, 67.4, 67.9,
68.9 (4CH), 72.9 (CH, propargylamine), 73.4 (CH, C-1), 78.0 (C,
propargylamine), 116.6 (C), 130.5 (C), 146.0 (CH, thiazol), 158.4(C),
169.26, 169.49, 169.64, 169.75 (4C, 4 x AcO), 171.0, 173.9, 176.0
(3C); HRMS (ESI) m/z calcd for C26H31N4O10S2 [M + H]+ 623.1484,
found 623.1476.
5-(2-bromo-4-methylthiazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-O-
acetyl--D-mannopyranosyl)methylaminothiazole (15). Prepared
following GP 1, using the thiourea 10 (30 mg, 0.0714 mmol) and 1-(2-
bromo-4-methylthiazol-5-yl)-2-chloroethanone (25 mg, 0.0928 mmol)
as starting materials, 15 (39 mg, 0.0615 mmol, 86%) was obtained after
purification by silica gel column chromatography (petroleum
5-(3-ethoxycarbonylisoxazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-O-
benzyl--D-mannopyranosyl)methylaminothiazole (18). Prepared
following GP 1, starting from 7 (50 mg, 0.082 mmol) and ethyl 5-(2-
bromoacetyl)isoxazole-3-carboxylate as starting material, the
derivative 18 (47 mg, 0.058 mmol, 71%) was obtained after purification
by silica gel column chromatography (petroleum ether/EtOAc, 70:30 →
[ ]20
[ ]20
ether/EtOAc, 30:70 as eluents) as a yellowish oil. α = +63 (c = 0.8,
50:50 as eluents) as a yellowish oil. α = +63 (c = 0.8, CHCl3); 1H
D
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.07 (3H, s, AcO), 2.09 (3H, s,
NMR (400 MHz, CDCl3) δ = 1.36 (3H, t, J = 7.13 Hz, COOEt), 3.45 (1H,
AcO), 2.10 (3H, s, AcO), 2.12 (3H, s, AcO), 2.62 (3H, methylthiazol),
3.60 (2H, m, H-7), 3.97 (1H, dd, J6a,6b = 12.1 Hz, J6a,5 = 3.5 Hz, H-6a),
4.05 (1H, m, H-5), 4.27 (1H, m, H-1), 4.77 (1H, dd, J6b,6a = 12.1 Hz, J6a,5
= 8.3 Hz, H-6b), 4.98 (1H, dd, J4,3 = 5.5 Hz, J4,5 = 3.6 Hz, H-4), 5.11 (1H,
dd, J7a,7b = 13.4 Hz, J7a,1 = 6.3 Hz, H-7a), 3.51-3.59 (2H, m, H-7b, H-
6a), 3.61-3.65 (2H, m, H-2, H-3), 3.75-3.81 (2H, m, H-4, H-6b), 3.99-
4.09 (2H, m, H-1, H-5), 4.27-4.49 (10H, m, COOEt, 4 x BnO), 7.00 (1H,
bs, NH), 7.11-7.29 (21H, m, isoxazole, 4 x BnO), 8.41 (1H, s, H-9); 13
C
dd, J2,1 = 7.9 Hz, J2,3 = 3.3 Hz, H-2), 5.32 (1H, dd, J3,4 = 5.3 Hz, J3,2
=
NMR (100 MHz, CDCl3): δ = 14.0 (CH3, COOEt), 46.9 (CH2, C-7), 62.4
(CH2, COOEt), 67.6 (CH2, C-6), 67.8 (CH, C-1), 71.2, 72.0, 72.6, 72.9
(4 x CH2, 4 x BnO), 73.2 (CH, C-4), 73.3, 73.9 (CH, C-2, C-3), 74.7
(CH, C-5), 108.2 (CH, isoxazole), 126.5 (C), 127.6-128.5 (20CH, 4 x
BnO), 137.3, 137.63, 137.65, 137.9 (4C, 4 x BnO), 152.6 (CH, C-9),
159.1, 168.4, 169.1 (3C), 176.2 (C, COOEt); HRMS (ESI) m/z calcd for
C45H45N3O9SNa [M + Na]+ 826.2770, found 826.2774.
3.3 Hz, H-3), 6.61 (1H, bs, NH), 7.80 (1H, s, thiazol); 13C NMR (127
MHz, CDCl3): δ = 18.4 (CH3, methylthiazol), 20.69, 20.75, 20.77, 20.81
(4CH3, 4 x AcO), 34.7 (CH2, C-7), 60.7 (CH2, C-6), 67.0, 67.4, 67.9,
68.9 (4CH), 73.4 (CH, C-1), 116.6 (C), 130.5 (C), 146.0 (CH, thiazol),
158.4(C), 169.26, 169.49, 169.64, 169.75 (4C, 4 x AcO), 171.0, 173.9,
176.0 (3C); HRMS (ESI) m/z calcd for C23H27BrN3O10S2 [M + H]+
648.0321, found 648.0329.
5-(3-phenylisoxazol-5-ylcarbonyl)-2-(2,3,4,6-tetra-O-acetyl--D-
mannopyranosyl)methylaminothiazole (19). Prepared following GP
1, using the thiourea 10 (30 mg, 0.0714 mmol) and 2-bromo-1-(3-
phenylisoxazol-5-yl)ethanone (25 mg, 0.0928 mmol) as starting
materials, the derivative 19 (35 mg, 0.0568 mmol, 80%) was obtained
after purification by silica gel column chromatography (petroleum
5-(2-(prop-2-yn-1-yl)amino-4-methylthiazol-5-ylcarbonyl)-2-
(2,3,4,6-tetra-O-benzyl--D-mannopyranosyl)methylaminothiazole
(16). According to the general procedure GP 1, using the thiourea 7
(130 mg, 0.212 mmol) and 2-chloro-1-(2-(prop-2-yn-1-ylamino)thiazol-
5-yl)ethanone chloroacetone alkyne (63 mg, 0.276 mmol) as starting
materials, the derivative 16 (125 mg, 0.153 mmol, 72%) was obtained
after purification by silica gel column chromatography (EtOAc as
[ ]20
ether/EtOAc, 30:70 as eluents) as a yellowish oil. α = +58 (c = 0.7,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.08 (6H, s, 2 x AcO), 2.1 (6H,
[ ]20
eluents) as a yellowish oil. α = +35 (c = 1.4, CHCl3); 1H NMR (400
s, 2 x AcO), 3.63 (2H, m, H-7), 3.99 (1H, dd, J6a,6b = 12.3 Hz, J6a,5 = 3.8
Hz, H-6a), 4.11 (1H, ddd, J5,6 = 8.4 Hz, J5,6 = 3.5 Hz, J5,4 = 3.5 Hz, H-
5), 4.31 (1H, m, H-1), 4.79 (1H, dd, J6b,6a = 12.0 Hz, J6a,5 = 8.5 Hz, H-
6b), 5.00 (1H, dd, J4,3 = 5.3 Hz, J4,5 = 3.5 Hz, H-4), 5.13 (1H, dd, J2,1
= 8.0 Hz, J2,3 = 3.2 Hz, H-2), 5.34 (1H, dd, J3,4 = 5.3 Hz, J3,2 = 3.3 Hz,
H-3), 7.30 (1H, s, phenylisoxazol), 7.48 (3H, m, phenylisoxazol), 7.85
(2H, m, phenylisoxazol), 8.54 (1H, s, thiazol); 13C NMR (127 MHz,
CDCl3): δ = 20.69, 20.72, 20.79, 20.80 (4CH3, 4 x AcO), 45.7 (CH2, C-
7), 60.5 (CH2, C-6), 66.9 (CH, C-2), 67.2 (CH, C-3), 67.9 (CH, C-4),
68.4 (CH, C-1), 73.8 (CH, C-5), 106.3 (CH, phenylisoxazol), 126.8-
129.1 (5 x CH, phenylisoxazol), 127.9 (C, phenylisoxazol), 130.5 (C,
phenylisoxazol), 151.9 (C, phenylisoxazol), 162.6 (C), 162.7 (CH,
thiazol), 167.4 (C), 169.2-170.27 (4C, 4 x AcO); HRMS (ESI) m/z calcd
for C28H30N3O11S [M + H]+ 616.1595, found 616.1596.
D
MHz, CDCl3) δ = 2.35 (1H, t, J = 2.5 Hz, propargyamine), 2.57 (3H, s,
methylthiazol), 3.49 (1H, dd, J7a,7b = 13.4 Hz, J7a,1 = 6.6 Hz, H-7a), 3.57-
3.63 (2H, m, H-7b, H-6a), 3.67-3.72 (2H, m, H-2, H-3), 3.81-3.87 (2H,
m, H-4, H-6b), 4.05-4.11 (2H, m, H-1, H-5), 4.14 (2H, d, J = 2.5 Hz,
propargyamine), 4.37-4.55 (8H, m, 4 x BnO), 6.36 (1H, bs, NH), 7.18-
7.36 (20H, m, 4 x BnO), 7.87 (1H, s, thiazol); 13C NMR (127 MHz,
CDCl3): δ = 18.3 (CH3, methylthiazol), 34.8 (CH2, propargyamine), 46.5
(CH2, C-7), 67.8 (CH2, C-6), 68.9 (CH, C-1), 71.4, 72.2, 72.7, 73.2
(4CH2,
4 x BnO), 73.2, 73.4, 74.1, 74.7 (4CH), 77.2 (CH,
propargyamine), 77.9 (C, propargyamine), 117.2 (C), 127.7-128.5
(20CH, 4 x BnO), 130.1 (C), 132.0 (C), 137.4, 137.70, 137.73, 137.9
(4C, 4 x BnO), 146.3 (CH, thiazol), 169.0 (C), 174.1 (C), 176.0 (C);
HRMS (ESI) m/z calcd for C46H47N4O6S2 [M + H]+ 815.2941, found
815.2932.
5-(2,4-difluorobenzoyl)-2-(2,3,4,6-tetra-O-benzyl--D-
5-(2-(prop-2-yn-1-yl)amino-4-methylthiazol-5-ylcarbonyl)-2-
(2,3,4,6-tetra-O-acetyl--D-mannopyranosyl)methylaminothiazole
(17). Prepared following GP1, using the thiourea 10 (200 mg, 0.476
mmol) and 2-chloro-1-(2-(prop-2-yn-1-ylamino)thiazol-5-yl)ethanone
chlorocetone alkyne (142 mg, 0.619 mmol) as starting materials, 17
(249 mg, 0.399 mmol, 84%) was obtained after purification by silica gel
column chromatography (EtOAc as eluents) as a yellowish amorphous
mannopyranosyl)methylaminothiazole (20). Prepared following
GP1, using the thiourea 7 (50 mg, 0.082 mmol) and 2’-chloro-2,4-
difluoroacetophenone (20 mg, 0.106 mmol) as starting materials, the
derivative 20 (39 mg, 0.050 mmol, 61%) was obtained after purification
by silica gel column chromatography (petroleum ether/EtOAc, 80:20 as
[ ]20
eluents) as a yellowish oil. α = +13 (c = 0.7, CHCl3); 1H NMR (400
D
MHz, CDCl3) δ = 3.45 (1H, dd, J7a,7b = 12.8 Hz, J7a,1 = 6.1 Hz, H-7a),
[ ]20
solid. α = +61 (c = 1.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.06
3.54-3.60 (2H, m, H-7b, H-6a), 3.64-3.68 (2H, m, H-2, H-3), 3.75-3.81
(2H, m, H-4, H-6b), 3.99-4.07 (2H, m, H-1, H-5), 4.30-4.49 (8H, m, 4 x
BnO), 6.44-6.56 (2H, m, difluoroacetophenone), 7.13-7.33 (21H, m, 4
x BnO, difluoroacetophenone), 7.53 (1H, s, H-9); 13C NMR (127 MHz,
CDCl3): δ = 46.5 (CH2, C-7), 67.7 (CH2, C-6), 68.1 (CH, C-1), 71.3,
72.1, 72.6, 73.2 (4 x CH2, 4 x BnO), 73.2 (CH, C-4), 73.4, 74.0 (CH, C-
D
(3H, s, AcO), 2.03 (3H, s, AcO), 2.099 (3H, s, AcO), 2.104 (3H, s, AcO),
2.34 (1H, t, J = 2.5 Hz, propargylamine), 2.56 (3H, s, thiazol), 3.57 (2H,
m, H-7), 4.02 (1H, dd, J6a,6b = 12.0 Hz, J6a,5 = 3.7 Hz, H-6a), 4.07-4.14
(3H, m, propargylamine, H-5), 4.25 (1H, m, H-1), 4.69 (1H, dd, J6b,6a
12.0 Hz, J6b,5 = 8.2 Hz, H-6a), 4.99 (1H, dd, J2,3 = 5.5 Hz, J2,1 = 3.8 Hz,
H-2), 5.12 (1H, dd, J4,5 = 7.6 Hz, J4,3 = 3.4 Hz, H-4), 5.32 (1H, dd, J3,2
5.5 Hz, J3,4 = 3.3 Hz, H-3), 6.92 (1H, bs, NH), 7.23 (1H, bs, NH), 7.85
=
=
2, C-3), 74.7 (CH, C-5), 102.9 (CH, d, JC,F
difluoroacetophenone), 104.7 (CH, d, JC,F
=
24.7 Hz,
22.2 Hz,
=
8
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10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
difluoroacetophenone), 124.0 (C,
d, JC,F
=
2.4
Hz,
5-(4-methyl-2-(pyrazin-2-yl)thiazol-5-ylcarbonyl)-2-(-D-
difluoroacetophenone), 127.6-128.5 (20CH, 4 x BnO), 128.8 (C), 132.2
(CH, d, JC,F = 10.8 Hz, difluoroacetophenone), 137.4, 137.69, 137.72,
137.9 (4C, 4 x BnO), 150.2 (CH, C-9), 152.7 (C, d, JC,F = 10.3 Hz,
mannopyranosyl)methylaminothiazole (24). Prepared following
GP2 starting from 12 (239 mg, 0.284 mmol). After purification over silica
gel (DCM/MeOH 8:2) and lyophilisation, 130 mg (95% yield) of 24 was
[ ]20
difluoroacetophenone), 164.7 (C, d, JC,F
=
247.9 Hz,
obtained as a yellow powder. α = +29 (c = 0.5, DMSO); 1H NMR
D
difluoroacetophenone), 175.1 (C), 187.5 (C, C-10); HRMS (ESI) m/z
calcd for C45H42F2N2O6SNa [M + Na]+ 776,2732, found 776,2729.
(400 MHz, CDCl3) δ = 9.36 (1H, d, 3J19-18 =1.4Hz, H-19), 8.94 (1H, bs,
NH) 8.82 (1H, d, 3J17-18 =2.5Hz, H-17), 8.77 (1H, dd, 3J17-18 =2.5Hz, 3J19-
2
18 =1.4Hz, H-18), 8.01 (1H, s, H-9), 4.83 (1H, d, J=5.0 Hz, OH), 4.79
(1H, d, 2J=4.3 Hz, OH), 4.70 (1H, d, 2J =5.4 Hz, OH), 4.41 (1H, dd,
2J=5.1 Hz, 2J =6.5 Hz, OH), 3.86 (1H, m, H-1), 3.57-3.71 (4H, m, H-3,
H-4, H-6, H-7), 3.42-3.57 (4H, m, H-2, H-5 H-6’, H-7’), 2.62 (3H, s, H-
14); 13C NMR (100 MHz, DMSO): δ = 183.0 (C-11), 176.5 (C-8), 166.0
(C-15), 157.6 (C-12), 151.6 (C-9), 147.1 (C-17), 145.7 (C-16), 145.2
(C-18), 141.2 (C-19), 130.8 (C-13), 127.9 (C-10), 78.2 (C-2), 72.2 (C-
1), 71.3 (C-3), 68.9 (C-5), 67.81 (C-4), 60.9 (C-6, C-7), 17.9 (C-14);
HRMS (ESI) m/z calcd for C19H21N5O6S2Na: 502.0831, found
502.0841.
5-((4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-ylcarbonyl)-2-
(2,3,4,6-tetra-O-benzyl--D-mannopyranosyl)methylaminothiazole
(21). Prepared following GP 1, starting from 7 (50 mg, 0.082 mmol) and
2-chloro-1-[1-(4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl]ethanone
(33 mg, 0.123 mmol) as starting materials, the derivative 21 (38 mg,
0.045 mmol, 55%) was obtained after purification by silica gel column
chromatography (petroleum ether/EtOAc, 80:20 as eluents) as a
[ ]20
yellowish oil. α = +31 (c = 1.3, CDCl3); 1H NMR (400 MHz, CDCl3) δ
D
= 1.95 (3H, s, methylpyrrol), 2.22 (3H, s, methylpyrrol), 3.46 (1H, dd,
J7a,7b = 13.1 Hz, J7a,1 = 6.7 Hz, H-7a), 3.52-3.61 (2H, m, H-7b, H-6a),
3.66-3.69 (2H, m, H-2, H-3), 3.75-3.81 (2H, m, H-4, H-6b), 3.99-4.09
(2H, m, H-1, H-5), 4.35-4.49 (8H, m, 4 x BnO), 6.32 (1H, bs,
methylpyrrol), 7.12-7.31 (24H, m, 4 x BnO, fluorophenyl), 7.81 (1H, s,
H-9); 13C NMR (127 MHz, CDCl3): δ = 12.6 (2 x CH3, methylpyrrol),
46.2 (CH2, C-7), 67.9 (CH2, C-6), 68.5 (CH, C-1), 71.5, 72.3, 72.6, 73.2
(4 x CH2, 4 x BnO), 73.5 (CH, C-4), 73.8, 74.2 (CH, C-2, C-3), 74.6
(CH, C-5), 101.8 (CH, methylpyrrol), 116.3 (2 x CH, d, JC,F = 23.0 Hz,
fluorophenyl), 119.2 (C, fluorophenyl), 127.6-128.4 (20CH, 4 x BnO),
128.8 (C), 129.8 (2 x CH, d, JC,F = 8.9 Hz, fluorophenyl), 130.8 (C),
133.5 (C, d, JC,F = 3.3 Hz, fluorophenyl), 135.8 (C), 137.5, 137.79,
137.81, 138.0 (4C, 4 x BnO), 146.3 (CH, C-9), 162.4 (C, d, JC,F = 10.3
Hz, fluorophenyl), 173.6 (C), 181.8 (C, C-10); HRMS (ESI) m/z calcd
for C51H51FN3O6S [M + H]+ 852.3506, found 852.3483.
5-(2-benzoylamino-4-methylthiazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (25). Prepared following GP
2, using the derivative 13 (37 mg, 0.042 mmol) as starting materials,
the derivative 25 (20 mg, 0.038 mmol, 91%) was obtained after
purification by silica gel column chromatography (DCM/MeOH, 80:20
[ ]20
as eluents) as an amorphous white solid. α = +32 (c = 0.6, MeOH);
D
1H NMR (400 MHz, DMSO) δ = 2.52 (3H, s, methylthiazol), 3.34-3.70
(H, m), 3.87 (1H, m, H-1), 7.55 (2H, bt, J = 7.5 Hz, benzamide), 7.66
(1H, bt, J = 7.5 Hz, benzamide), 7.93 (1H, s, thiazol), 8.11 (1H, bd, J =
7.5 Hz, benzamide), 8.94 (1H, bs, NH), 13.0 (1H, bs, NH); 13C NMR
(127 MHz, DMSO): δ = 17.6 (CH3, methylthiazol), 49.9 (CH2, C-7), 60.4
(CH2, C-6), 67.5 (CH), 68.5 (CH), 70.8 (CH), 71.9 (CH, C-1), 77.7 (CH),
127.9 (C), 128.3, 128.7 (CH, benzamide), 131.6 (C), 133.0 (CH,
benzamide), 137.3 (C), 149.1 (CH, thiazol), 153.1 (C), 159.1 (C), 162.4
(C), 176.7 (C); HRMS (ESI) m/z calcd for C22H25N4O7S2 [M + H]+
521.1166, found 521.1159.
5-(benzo[d]thiazol-2-ylcarbonyl)-2-(2,3,4,6-tetra-O-benzyl--D-
mannopyranosyl)methylaminothiazole (22). Prepared following GP
1, starting from 7 (75 mg, 0.122 mmol) and 1-(benzo[d]thiazol-2-yl)-2-
bromoethanone (34 mg, 0.134 mmol) as starting materials, the
derivative 22 (61 mg, 0.076 mmol, 63%) was obtained after purification
by silica gel column chromatography (petroleum ether/EtOAc, 80:20 as
5-(2-chloro-4-methylthiazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (26). Prepared following GP
3, using the derivative 14 (30 mg, 0.036 mmol) as starting materials,
the derivative 26 (14 mg, 0.032 mmol, 89%) was obtained after
purification by silica gel column chromatography (DCM/MeOH, 85:15
[ ]20
eluents) as a yellowish oil. α = +18 (c = 0.9, CDCl3); 1H NMR (400
D
MHz, CDCl3) δ = 3.60 (1H, dd, J7a,7b = 13.1 Hz, J7a,1 = 6.6 Hz, H-
[ ]20
7a), 3.66-3.72 (2H, m, H-7b, H-6a), 3.76-3.79 (2H, m, H-2, H-3), 3.86-
3.94 (2H, m, H-4, H-6b), 4.11-4.21 (2H, m, H-1, H-5), 4.41-4.56 (8H, m,
4 x BnO), 7.22-7.39 (24H, m, 4 x BnO), 7.54 (2H, m, benzothiazol),
7.99 (1H, m, benzothiazol), 8.22 (2H, m, benzothiazol), 9.04 (1H, s,
H9); 13C NMR (127 MHz, CHCl3): δ = 46.8 (CH2, C-7), 67.7 (CH2, C-
6), 68.1 (CH, C-1), 71.3, 72.1, 72.6, 73.2 (4CH2, 4 x BnO), 73.2 (CH,
C-4), 73.3, 74.0 (CH, C-2, C-3), 74.6 (CH, C-5), 122.1, 125.2, 126.7,
as eluent) as an amorphous white solid. α = +21 (c = 1.1, MeOH);
D
1H NMR (400 MHz, MeOD) δ = 2.53 (3H, s, methylthiazol), 3.62-3.78
(6H, m), 3.82 (1H, dd, J2,3 = 4.9 Hz, J2,1 = 3.2 Hz, H-2), 3.89 (1H, dd,
J6b,6a = 11.9 Hz, J6b,5 = 6.8 Hz, H-6b), 4.07 (1H, m, H-1), 7.89 (1H,
s, thiazol); 13C NMR (127 MHz, MeOD): δ = 17.7 (CH3, methylthiazol),
45.4 (CH2, C-7), 62.2 (CH2, C-6), 69.4 (CH, C-2), 69.8 (CH), 72.4 (CH),
74.6 (CH, C-1), 78.2 (CH), 129.2 (C), 131.3 (C), 151.6 (CH, thiazol),
151.3(C),154.1 (C), 156.7 (C), 177.1 (C); HRMS (ESI) m/z calcd for
C15H19ClN3O6S2 [M + H]+ 436.0396, found 436.0398.
127.0 (4CH, benzothiazol), 127.6-128.4 (20CH, 1C,
4 x BnO,
benzothiazol), 136.6 (C), 137.4 (C, BnO), 137.7 (2 x C, 2 x BnO), 137.9
(C, BnO), 153.6 (CH, C-9), 153.7 (C), 167.2 (C), 174.8 (C), 176.5 (C,
C-10); HRMS (ESI) m/z calcd for C46H44N3O6S2 [M + H]+ 798.2686,
found 798.2672.
5-(2-bromo-4-methylthiazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (27). Prepared following
GP3, using the derivative 15 (25 mg, 0.039 mmol) as starting material,
the derivative 27 was obtained after lyophilization (17 mg, 0.036 mmol,
5-acetyl-2-(-D-mannopyranosyl)methylaminothiazole
(23).
[ ]20
Prepared following GP 2, starting from 11 (152mg, 0.224mmol). After
purification over silica gel (DCM/MeOH 9:1) and lyophilisation,
[ ]20
94%) as an amorphous white solid. α = +42 (c = 1.1, CD3OD); 1H
D
NMR (400 MHz, CD3OD) δ = 2.56 (3H, s, methylthiazol), 3.64-3.83 (6H,
52.15mg (73% yield) of 23 was obtained as a light white solid. α
=
m), 3.81 (1H, dd, J2,3 = 5.2 Hz, J2,1 = 3.2 Hz, H-2), 3.90 (1H, dd,
J6a,6b = 11.8 Hz, J6a,5 = 6.9 Hz, H-6a), 4.06 (1H, dt, J = 8.0 Hz, J =
4.8 Hz, H-1), 7.89 (1H, s, thiazol); 13C NMR (127 MHz, MeOD): δ = 17.6
(CH3, methylthiazol), 45.6 (CH2, C-7), 62.2 (CH2, C-6), 69.3 (CH, C-
2), 69.9 (CH), 72.4 (CH), 74.4 (CH, C-1), 78.4 (CH), 129.2 (C), 132.9
(C), 139.2 (C), 150.9 (CH, thiazol), 158.1 (C), 176.5 (C), 177.0 (C);
HRMS (ESI) m/z calcd for C15H19BrN3O6S2 [M + H]+ 479.9893, found
479.9880.
D
+44 (c = 0.5, H2O); 1H NMR (400 MHz, D2O) δ = 8.15 (1H, s, H-9), 4.27
(1H, m, H-1), 4.06 (1H, t, 3J=3.2 Hz, H-2), 3.84-3.93 (4H, m, H-3, H-6,
H7’), 3.79 (1H, t, 3 =8.3 Hz, H-4), 3.67-3.74 (2H, m, H-5, H-7’) 4.03-4.10
(2H, m, H-1, H-5) , 3.79-3.86 (2H, m,H-4, H-6), 3.67-3.72 (2H, m, H-2,
2
3
H-3), 3.61 (1H, dd, J6-6’ =10.3Hz, J6’-5= 5.6 Hz, H-6’), 3.43-3.61 (2H,
m, H-7), 2.43 (3H, s, H-12); 13C NMR (100 MHz, D2O): δ = 193.5 (C-
11), 174.1 (C-8), 144.9 (C-9), 126.8 (C-10), 75.3 (C-5), 74.9 (C-1), 70.7
(C-3), 68.7 (C-2), 67.4 (C-4), 60.8 (C-6), 43.9 (C-7), 25.2 (C-12); HRMS
(MALDI) m/z calcd for C12H19N2O6S: 319.0958, found 319.0949.
5-(2-(prop-2-yn-1-yl)amino-4-methylthiazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (28). Prepared following
GP3, using 17 (29 mg, 0.046 mmol) as starting materials, 28 (20 mg,
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
[ ]20
0.044 mmol, 96%) was obtained after lyophilization. α = +37 (c = 1.3,
materials, the derivative 32 (16 mg, 0.0385 mmol, 75%) was obtained
D
CD3OD); 1H NMR (400 MHz, CD3OD) δ = 2.49 (3H, s, methylthiazol),
2.69 (1H, t, J = 2.5 Hz, propargylamine), 3.59-3.79 (8H, m), 3.83 (1H,
dd, J2,3 = 4.7 Hz, J2,1 = 3.4 Hz, H-2), 3.88 (1H, dd, J6b,6a = 11.8 Hz, J6b,5
= 7.1 Hz, H-6b), 4.06 (1H, dt, J = 7.8 Hz, 4.9, H-1), 4.16 (1H, d, J = 2.5
Hz, propargylamine), 7.85 (1H, s, thiazol); 13C NMR (127 MHz,
CD3OD): δ = 18.6 (CH3, methylthiazol), 34.5 (CH2, propargylamine),
45.3 (CH2, C-7), 62.3 (CH2, C-6), 69.5(CH), 69.9 (CH), 72.5 (CH, C-1),
73.2 (CH), 74.7 (CH), 78.2 (CH, propargylamine), 79.9 (C,
propargylamine), 117.2 (C), 130.3 (C), 147.9 (CH, thiazol), 160.1 (C),
171.3 (C), 175.5 (C), 178.1 (C); HRMS (ESI) m/z calcd for
C18H22N4O6S2 [M + H]+ 455.1063, found 455.1054.
after purification by silica gel column chromatography (AcOEt/MeOH,
[ ]20
70:30 as eluent) as an amorphous white solid. α = +42 (c = 1.4,
D
CD3OD); 1H NMR (400 MHz, CD3OD) δ = 3.62-3.77 (6H, m), 3.81 (1H,
dd, J = 4.9 Hz, J = 3.1 Hz), 3.89 (1H, dd, J = 11.9 Hz, J = 7.1 Hz, H-
6b), 4.06 (1H, m, H-1), 6.55-7.65 (3H, m, difluoroacetophenone), 7.54
(1H, s, thiazol); 13C NMR (127 MHz, CD3OD): δ = 45.4 (CH2, C-7), 62.3
(CH2, C-6), 69.5 (CH), 69.9 (CH), 72.5 (CH), 74.6 (CH, C-1), 78.3 (CH),
104.2 (CH, d, JC,F = 25.0 Hz, difluoroacetophenone), 105.9 (CH, d,
JC,F = 22.5 Hz, difluoroacetophenone), 128.6 (C), 133.3 (C, d, JC,F =
11.1 Hz, difluoroacetophenone), 132.2 (CH, d, JC,F = 10.8 Hz,
difluoroacetophenone), 152.1 (CH, thiazol), 153.3 (C, d, JC,F = 10.3
Hz, difluoroacetophenone), 166.2 (C, d, JC,F
= 246.1 Hz,
difluoroacetophenone), 175.1 (C), 189.8 (C); HRMS (ESI) m/z calcd for
C17H19F2N2O6S [M + H]+ 417.0938, found 417.0926.
5-(3-ethoxycarbonylisoxazol-5-ylcarbonyl)-2-(-D-
5-((4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (33). Prepared following
GP2, using the derivative 21 (31 mg, 0.0364 mmol) as starting
materials, the derivative 33 (17 mg, 0.0345 mmol, 95%) was obtained
after purification by silica gel column chromatography (DCM/MeOH,
mannopyranosyl)methylaminothiazole (29). Prepared following GP
3, starting from 18 (40 mg, 0.049 mmol) as starting materials, the
derivative 29 (14 mg, 0.032 mmol, 64%) was obtained after purification
by silica gel column chromatography (DCM/MeOH, 80:20 as eluents)
[ ]20
as an amorphous white solid. α = +13 (c = 0.8, H2O); 1H NMR (400
D
[ ]20
80:20 as eluent) as an amorphous white solid. α = +19 (c = 0.3,
D
MHz, D2O) δ = 1.47 (3H, t, J = 7.2 Hz, COOEt), 3.68 (1H, dd, J7a,7b
MeOH); 1H NMR (400 MHz, CD3OD) δ = 2.01 (3H, s, methylpyrrol),
= 14.2 Hz, J7a,1 = 4.0 Hz, H-7a), 3.73 (1H, m, H-5), 3.81 (1H, t, J = 8.6
Hz, H-4), 3.83 (1H, dd, J6a,6b = 14.6 Hz, J6a,5 = 4.7 Hz, H-6a), 3.88-
3.93 (3H, m, H-3, H-6b, H-7b), 4.08 (1H, t, J = 3.0 Hz, H-2), 4.26 (1H,
ddd, J1,7b = 9.8 Hz, J1,7a = 4.1 Hz, J1,2 = 2.9 Hz, H-1), 4.53 (2H, q, J =
7.2 Hz, COOEt), 7.43 (1H, s, isoxazole), 8.32 (1H, s, thiazol); 13C NMR
(127 MHz, D2O): δ = 13.3 (CH3, COOEt), 43.3 (CH2, C-7), 60.9 (CH2,
C-6), 63.8 (CH2, COOEt), 67.3 (CH, C-4), 69.0 (CH, C-2), 70.8 (CH, C-
3), 75.1 (CH, C-5), 75.5 (CH, C-1), 108.5 (CH, isoxazole), 125.1 (C),
153.6 (CH, thiazol), 159.8, 159.9, 167.1, 169.9 (4C); HRMS (ESI) m/z
calcd for C17H21N3O9SNa [M + Na]+ 466.0895, found 466.0895.
2.20 (3H, s, methylpyrrol), 3.61-3.79 (7H, m), 3.85 (1H, dd, J2,3 = 4.8
Hz, J2,1 = 3.3 Hz, H-2), 3.89 (1H, dd, J6b,6a = 11.9 Hz, J6b,5 = 6.9 Hz, H-
6b), 4.09 (1H, ddd, J = 10.2 Hz, J = 5.1 Hz, J = 5.1 Hz, H-1), 6.39 (1H,
bs, pyrrol), 7.29-7.32 (4H, m, fluorophenyl), 7.82 (1H, s, H-9); 13C NMR
(127 MHz, CD3OD):
δ = 12.7 (CH3, methylpyrrol), 12.9 (CH3,
methylpyrrol), 45.2 (CH2, C-7), 62.4 (CH2, C-6), 69.6, 69.8, 72.5 (3 x
CH), 74.9 (CH, C-1), 78.1 (CH), 109.0 (CH, methylpyrrol), 117.5 (2 x
CH, d, JC,F = 23.2 Hz, fluorophenyl), 120.3 (C, fluorophenyl), 130.6
(C), 131.3 (2 x CH, d, JC,F = 8.9 Hz, fluorophenyl), 134.9 (C, d, JC,F =
3.9 Hz, fluorophenyl), 137.1 (C), 148.3 (CH, thiazol), 163.9 (C, d, JC,F
= 246.9 Hz, fluorophenyl), 175.1 (C), 184.1 (C); HRMS (ESI) m/z calcd
for C23H27FN3O6S [M + H]+ 492.1597, found 492.1599.
5-(3-carboxyisoxazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (30). To a solution of 29 (33
mg, 0.0745 mmol) in a mixture 3:1 of MeOH/H2O (2 mL) was added
LiOH (3.6 mg, 0.149 mmol). The mixture was stirred at 50° C for 8 h,
neutralized with Amberlite IR120 (H), filtered and the solvents
evaporated to dryness. The substrate was dissolved in water and
subjected to lyophilization to give 30 (28 mg, 0.0675 mmol, 91%) as an
5-(benzo[d]thiazol-2-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (34). Prepared following GP
2, starting from 22 (30 mg, 0.0376 mmol) as starting materials, the
derivative 34 (15 mg, 0.0343 mmol, 91%) was obtained after
purification by silica gel column chromatography (AcOEt/MeOH, 70:30
[ ]20
amorphous white solid. α = +19 (c = 0.6, H2O); 1H NMR (400 MHz,
D
[ ]20
as eluent) as an amorphous white solid. α = +51 (c = 0.2, MeOH);
D
MeOD) δ = 3.67 (1H, dd, J6a,6b = 11.6 Hz, J6a,5 = 2.5 Hz, H-6a),
3.71-3.87 (6H, m), 4.03(1H, dd, J6b,6a = 11.6 Hz, J6b,5 = 7.8 Hz, H-
6b), 4.08 (1H, m, H-1), 7.52 (1H, s, isoxazole), 8.49 (1H, s, thiazol); 13
NMR (127 MHz, MeOD): δ = 47.2 (CH2, C-7), 61.4 (CH2, C-6), 68.2
(CH), 70.4 (CH), 71.9 (CH), 72.1 (CH), 79.7 (CH), 110.2 (CH,
isoxazole), 126.2 (C), 142.65 (CH, thiazol), 142.67, 159.0, 161.4,
168.0, 171.1 (5C); HRMS (ESI) m/z calcd for C15H18N3O9S [M + H]+
416.0754, found 416.0758.
1H NMR (400 MHz, DMSO-d6) δ = 3.47-3.70 (8H, m), 3.91 (1H,ddd, J
= 8.7 Hz, J = 4.9 Hz, J = 4.9 Hz, H-1), 4.50 (1H, bs, OH), 4.76 (1H, bs,
OH), 4.89 (2H, bs, OH), 7.64 (2H, m, benzothiazol), 8.25 (2H, bd, J =
7.9 Hz, benzothiazol), 8.89 (1H, s, thiazol), 9.34 (1H, bs, NH); 13C NMR
(127 MHz, DMSO-d6): δ = 49.8 (CH2, C-7), 60.4 (CH2, C-6), 67.4, 68.5,
70.8 (3CH), 71.8 (CH, C-1), 77.8 (CH, C-5), 122.7 (C), 122.9, 124.9,
127.4, 127.6 (4CH, benzothiazol), 135.8, 153.2 (2C, benzothiazol),
154.2 (CH, thiazol), 167.4 (C), 173.6 (C), 174.1 (C); HRMS (ESI) m/z
calcd for C18H20N3O6S2 [M + H]+ 438.0788, found 438.0790.
C
5-(3-phenylisoxazol-5-ylcarbonyl)-2-(-D-
mannopyranosyl)methylaminothiazole (31). Prepared following GP
3, using 19 (20 mg, 0.0325 mmol) as starting materials, the derivative
[ ]20
The following compounds were not named due to the complexity of the
structures.
31 (14 mg, 0.0313 mmol, 96%) was obtained after lyophilization. α
=
D
+12 (c = 0.5, MeOH); 1H NMR (400 MHz, DMSO-d6) δ = 3.45-3.57 (4H,
m), 3.59-3.76 (4H, m), 3.89 (1H, m, H-1), 7.56 (3H, m, phenylisoxazol),
7.89 (1H, s, phenylisoxazol), 8.01 (2H, m, phenylisoxazol), 8.43 (1H, s,
thiazol); 13C NMR (127 MHz, , DMSO-d6): δ = 49.5 (CH2, C-7), 60.3
(CH2, C-6), 67.2 (CH), 68.3 (CH), 68.4 (CH), 70.6 (CH), 71.6 (CH, C-
1), 77.7 (CH), 106.1 (CH, phenylisoxazol), 125.1 (C, phenylisoxazol),
126.8 (3CH, phenylisoxazol), 129.1 (2CH, phenylisoxazol), 130.6 (C,
phenylisoxazol), 152.3 (CH, thiazol), 162.4 (2 x C), 166.2 (C), 169.4
(C); HRMS (ESI) m/z calcd for C20H22N3O7S [M + H]+ 448.1173, found
448.1173.
Compound (35). To a solution of mannosyl alkyne 16 (20 mg, 0.0245
mmol) and azidotetraethylenglycol (11 mg, 0.0491 mmol) in a mixture
3:1 of DMF-H2O (0.8 mL) were added CuSO4 (1 mg, 0.005 mmol) and
sodium ascorbate (2 mg, 0.010 mmol) and the mixture was warmed to
70°C. After 8 h, the mixture was concentrated and the crude was
purified by silica gel column chromatography (AcOEt /MeOH: 95/5 →
90/10 as eluents) to give 35 (21 mg, 0.0203 mmol, 83%) as a colorless
[ ]20
oil. α = +51 (c = 0.7, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.56
D
(3H, s, methylthiazol), 3.45-3.85 (24H, m), 4.03-4.15 (2H, m, H-1, H-5),
4.30 (1H, m), 4.38-4.54 (8H, m, 4 x BnO), 4.64 (2H, s, propargylamine),
6.57 (1H, bs, NH), 7.18-7.37 (20H, m, 4 x BnO), 7.87 (1H, s, thiazol),
7.86 (1H, s, triazol); 13C NMR (127 MHz, CDCl3): δ = 18.4 (CH3,
methylthiazol), 40.7 (CH2, NH-CH2-triazol), 46.3 (CH2, C-7), 50.3 (CH2,
5-(2,4-difluorobenzoyl)-2-(-D-
mannopyranosyl)methylaminothiazole (32). Prepared following
GP2, using the derivative 20 (40 mg, 0.0515 mmol) as starting
10
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10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
CH2-triazol), 61.3 (CH2, TEG), 67.9 (CH2, C-6), 68.5 (CH, C-1), 69.2
(CH2, TEG), 70.16-70.39 (4CH2, 4 x BnO), 71.4, 72.4 (2CH2, TEG),
72.5 (2CH2, TEG), 73.2 (CH2, TEG), 73.4 (CH, C-4), 73.8, 74.1 (2CH,
C-2, C-3), 74.6 (CH, C-5), 116.0 (C), 123.6 (CH, triazol), 127.6-128.4
(20CH, 4 x BnO), 130.9 (C), 137.5 (C, BnO), 137.7 (2C, 2 x BnO), 137.9
(C, BnO), 143.4 (C), 146.2 (CH, C-9), 158.7 (C), 169.2 (C), 176.1 (C);
HRMS (ESI) m/z calcd for C54H64N7O10S2 [M + H]+ 1034.4161, found
1034.4151.
Compound (39). Prepared following GP2, using the derivative 36 (150
mg, 0.107 mmol) as starting materials, the derivative 39 (64 mg, 0.095
mmol, 89%) was obtained after purification by silica gel column
chromatography (AcOEt/MeOH, 70:30 as eluent) as an amorphous
[ ]20
white solid. α = +13 (c = 0.7, MeOH); 1H NMR (400 MHz, D2O) δ =
D
2.45 (3H, s, methylthiazol), 3.63-3.86 (12H, m), 3.99 (2H, q, J = 3.2 Hz),
4.19 (1H, m, H-1), 4.32 (1H, m, H-1’), 4.67 (1H, dd, J6a,6b = 15.0 Hz,
J6a,5 = 3.6 Hz, H-6a), 4.74 (2H, s, CH2-triazol), 4.89 (1H, dd, J6b,6a =
15.0 Hz, J6b,5 = 3.6 Hz, H-6b), 7.95 (1H, s, triazol), 8.16 (1H, s,
thiazol); 13C NMR (127 MHz, D2O): δ = 15.1 (CH3, methylthiazol), 40.9
(CH2, CH2-triazol), 44.3 (CH2, C-7’), 48.3 (CH2, C-7), 60.6, 60.7 (2CH2,
C-6, C-6’), 67.3, 67.4, 68.5, 68.6, 70.63, 70.66, 74.7, 75.2, 75.5, 76.0
(10CH), 115.1 (C), 125.4 (CH, triazol), 126.6, 141.5, 143.7 (3C), 149.4
(CH, thiazol), 169.3, 173.6, 176.3 (3C); HRMS (ESI) m/z calcd for
C25H36N7O11S2 [M + H]+ 674.1914, found 674.1921.
Compound (36). To a solution of mannosyl alkyne 16 (140 mg, 0.170
mmol) and 2,3,4,6-tetra-O-benzyl-1-azidomethyl-α-D-mannopyranose
(129 mg, 0.223 mmol) in a mixture 3:1 of 1,4-dioxane-H2O (3.7 mL)
were added CuSO4 (5.5 mg, 0.034 mmol) and sodium ascorbate (13
mg, 0.068 mmol) and the mixture was warmed up at 70°C. After 8 h,
the mixture was concentrated and the crude was purified by silica gel
column chromatography (DCM/AcOEt: 50/50 → 30/70 as eluents) to
[ ]20
give 36 (204 mg, 0.146 mmol, 86%) as a colorless oil. α = +10 (c =
Compound (40). According to the general procedure GP2, using 37
(30 mg, 0.0114 mmol) as starting materials, the derivative 40 (18 mg,
D
0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.58 (3H, s, methylthiazol),
[ ]20
3.48-3.86 (12H, m), 4.04-4.26 (4H, m, H-1, H-1’, H-5, H-5’), 4.37-4.55
(19H, m, 8 x BnO, CH2-triazol, H-7’a), 4.69 (1H, dd, J7a,7b = 14.4 Hz,
J7a,1 = 2.5 Hz, H-7’b), 6.83 (1H, bs, NH), 7.03 (1H, bs, NH), 7.18-7.37
(40H, m, 8 x BnO), 7.86 (1H, s, thiazol), 7.88 (1H, s, triazol); 13C NMR
(127 MHz, CDCl3): δ = 18.6 (CH3, methylthiazol), 40.3 (CH2, CH2-
triazol), 46.4 (CH2, C-7), 51.0 (CH2, C-7’), 67.7, 67.9 (2CH2, C-6, C-6’),
68.6 (2CH, C-1, C-1’), 71.2, 71.3, 71.9, 72.3, 72.5, 72.7, 73.0, 73.18
(8CH2, 8 x BnO), 72.6, 73.13, 73.4, 73.9, 74.1, 74.2, 74.5, 74.7 (8CH),
116.2 (C), 123.8 (CH, triazol), 127.5-128.4 (40CH, 8 x BnO), 128.9 (C),
137.4-137.9 (8C, 8 x BnO), 143.1 (C), 148.1 (CH, thiazol), 159.0, 169.3,
174.2, 176.0 (4C); HRMS (ESI) m/z calcd for C81H84N7O11S2 [M + H]+
1394.5684, found 1394.5665.
0.0111 mmol, 98%) was obtained after lyophilization. α = +71 (c =
D
0.3, MeOH); 1H NMR (400 MHz, CDCl3) δ = 2.55 (3H, s, methylthiazol),
3.51-5.38 (59H, m), 5.64 (1H, d, J = 4.0 Hz, H-1 CD), 6.42 (1H, bs, NH),
7.12 (1H, bs, NH), 7.69 (1H, s, triazol), 7.84 (1H, s, thiazol); HRMS
(ESI) m/z calcd for C60H91N7O40S2Na [M + Na]+ 1636.4635, found
1636.4590.
Compound (42). To a solution of mannosyl alkyne 17 (40 mg, 0.0642
mmol) and azidotetraethyleneglycolmethanesulfonate 41 (23 mg,
0.0770 mmol) in a mixture 3:1 of dioxane-H2O (2.6 mL) were added
CuSO4 (2 mg, 0.013 mmol) and sodium ascorbate (5 mg, 0.026 mmol)
and the mixture was warmed to 70°C. After 3 h, the mixture was
concentrated and the crude was purified by silica gel column
chromatography (AcOEt → AcOEt /MeOH: 90/10 as eluent) to give the
Compound (37). To a solution of mannosyl alkyne 17 (20 mg, 0.032
mmol) and azido-β-cyclodextrine (58 mg, 0.029 mmol) in a mixture 3:1
of DMF-H2O (1.3 mL) were added CuSO4 (1 mg, 0.0064 mmol) and
sodium ascorbate (2.5 mg, 0.0128 mmol) and the mixture was warmed
to 70°C. After 8 h, the mixture was concentrated and the crude was
purified by silica gel column chromatography (AcOEt → AcOEt /MeOH:
80/20 as eluents) to give 37 (34 mg, 0.0129 mmol, 41%) as a colorless
[ ]20
triazol 42 (58 mg, 0.0630 mmol, 98%) as a colorless oil. α = +51 (c
D
= 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.05 (3H, s, AcO), 2.07
(3H, s, AcO), 2.09 (3H, s, AcO), 2.10 (3H, s, AcO), 2.56 (3H, s,
methylthiazol), 3.04 (3H, s, MsO), 3.55-3.66 (12H, m, H-7a, H-7b, TEG),
3.74 (1H, m), 3.86 (2H, t, J = 4.9 Hz), 4.01-4.12 (2H, m, H-5, H-6a),
4.30 (1H, m, H-1), 4.35 (2H, m), 4.53 (2H, t, J = 4.9 Hz), 4.62 (1H, m,
H-6b), 4.70 (2H, s, CH2-triazol), 5.04 (1H, dd, J4,3 = 6.0 Hz, J4,5 = 4.5
Hz, H-4), 5.17 (1H, dd, J2,1 = 6.7 Hz, J2,3 = 3.2 Hz, H-2), 5.31 (1H, dd,
J3,4 = 6.1 Hz, J3,2 = 3.3 Hz, H-3), 7.42 (1H, bs, NH), 7.81 (1H, s,
thiazol), 7.89 (1H, s, triazol); 13C NMR (127 MHz, CDCl3): δ = 18.6
(CH3, methylthiazol), 20.69 (CH3, AcO), 20.76 (2CH3, 2 x AcO), 20.81
(CH3, AcO), 37.6 (CH3, MsO), 39.9 (CH2, CH2-triazol), 45.6 (CH2, C-7),
50.4 (CH2), 60.9 (CH2, C-6), 67.3 (CH, C-4), 67.6 (CH, C-3), 67.7 (CH,
C-2), 68.9, 69.2, 69.3 (3CH2), 69.5 (CH, C-1), 70.39, 70.42, 70.5, 70.6
(4CH2), 73.0 (CH, C-5), 115.7 (C), 123.4 (CH, triazol), 130.8 (C), 131.4
(C), 143.5 (C, triazol), 145.6 (CH, thiazol), 159.2 (C), 169.3, 169.6,
169.8, 170.9 (4C, 4 x AcO), 174.2 (C), 175.9 (C); HRMS (ESI) m/z calcd
for C35H50BrN7O16S3 [M + H]+ 920.2471, found 920.2460.
[ ]20
oil. α = +81 (c = 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 1.99
D
(3H, s, AcO), 2.01 (3H, s, AcO), 2.02 (3H, s, AcO), 2.03 (3H, s, AcO),
2.04 (3H, s, AcO), 2.05 (3H, s, AcO), 2.07-2.11 (48H, 16 x AcO), 2.13
(3H, s, AcO), 2.14 (3H, s, AcO), 2.55 (3H, s, methylthiazol), 3.51-5.38
(59H, m), 5.64 (1H, d, J = 4.0 Hz, H-1 CD), 6.42 (1H, bs, NH), 7.12 (1H,
bs, NH), 7.69 (1H, s, triazol), 7.84 (1H, s, thiazol); 13C NMR (127 MHz,
CDCl3): δ = 18.5 (CH3, methylthiazol), 20.7-20.8 (24CH3, 24 x AcO),
40.6 (CH2), 45.1 (CH2, C-7), 49.9 (CH2, C-6 CD), 60.7 (CH2, C-6), 62.2-
62.8 (6CH2, C-6 CD), 67.1-78.1 (33CH), 96.36 (CH, C-1 CD), 96.42
(CH, C-1 CD), 96.66 (2CH, 2 x C-1 CD), 96.8 (CH, C-1 CD), 96.9 (CH,
C-1 CD), 97.1 (CH, C-1 CD), 116.2 (C), 124.9 (CH, triazol), 130.8 (C),
131.4 (C), 143.3 (C, triazol), 145.7 (CH, thiazol), 162.5 (C), 169.3-171.1
(24C, 24 x AcO), 173.5 (C), 176.0 (C); HRMS (ESI) m/z calcd for
C108H141N7O64S2 [M + 2H]2+ 1311.8710, found 1311.8712.
Compound (43). According to the general procedure GP3, using the
methanesulfonate 42 (39 mg, 0.0424 mmol) as starting material, the
deprotected derivative was obtained. The crude was dissolved in a
mixture 3:1 of AcOEt-MeOH (3 mL) and DABCO (47 mg, 0.424 mmol)
was added. After 4 h, the mixture was concentrated and washed with
Et2O (2 mL x 4). The derivative 43 was obtained after lyophilization (33
Compound (38). Prepared following GP 2, using the derivative 35 (20
mg, 0.0194 mmol) as starting materials, 38 (12 mg, 0.0178 mmol, 92%)
was obtained after purification by silica gel column chromatography
[ ]20
(AcOEt/MeOH, 70:30 as eluent) as an amorphous white solid. α
=
D
[ ]20
+39 (c = 0.6, MeOH); 1H NMR (400 MHz, CD3OD) δ = 2.53 (3H, s,
mg, 0.038 mmol, 90%) as a colorless oil. α = +55 (c = 0.8, MeOH);
D
methylthiazol), 3.51-4.00 (22H), 4.07 (1H, m, H-1), 4.61 (2H, m, H-7),
1H NMR (400 MHz, D2O) δ = 2.31 (3H, s, methylthiazol), 2.77 (3H, s,
4.74 (2H, s, CH2-triazol), 7.96 (1H, s, thiazol), 8.14 (1H, s, triazol); 13
C
MsO), 3.13 (6H, bt, J = 7.4 Hz, DABCO), 3.41 (6H, bt, J = 7.4 Hz,
DABCO), 3.44-3.45 (16H), 3.89 (2H, t, J = 4.9 Hz), 3.95 (1H, t, J = 2.9
Hz, H-2), 4.12 (1H, m, H-1), 4.51 (2H, s, CH2-triazol), 4.57 (2H, bt, J =
4.7 Hz), 7.60 (1H, s, thiazol), 8.03 (1H, s, triazol); 13C NMR (127 MHz,
D2O): δ = 18.1 (CH3, methylthiazol), 38.4 (CH3, MsO), 42.8 (CH2, C-7),
44.1 (3CH2, DABCO), 50.5 (CH2), 52.9 (3CH2, DABCO), 60.8 (CH2, C-
6), 63.2, 63.6 (2CH2), 67.2, 68.7, 68.9 (3CH), 69.4-70.7 (6CH2), 75.0
(CH), 75.4 (CH, C-1), 114.9 (C), 124.7 (CH, triazol), 128.6 (C), 143.5
(C, triazol), 146.4 (CH, thiazol), 160.9, 170.9, 173.8, 176.1 (4C); HRMS
NMR (127 MHz, CD3OD): δ = 18.8 (CH3, methylthiazol), 40.7 (CH2,
CH2-triazol), 45.3 (CH2, C-7), 51.5 (CH2), 62.2, 62.3 (2CH2), 69.5,
69.8 (2CH), 70.4 (CH2, C-6), 71.38, 71.41, 71.49, 71.54 (4CH2), 72.5
(CH), 73.6 (CH2), 74.6 (CH, C-1)), 78.2 (CH), 116.9 (C), 125.4 (CH,
triazol), 130.5 (C), 145.3 (C, triazol), 147.7 (CH, thiazol), 160.5, 171.5,
175.4, 177.9 (4C); HRMS (ESI) m/z calcd for C26H39N7O10S2Na [M +
Na]+ 696.2089, found 696.2092.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700061
ChemMedChem
FULL PAPER
(ESI) m/z calcd for C32H50N9O9S2 [M – MsO]+ 768.3155, found
768.3167.
Keywords: Crohn’s disease • FimH antagonist • C-
Mannosides • Anti-adhesive therapy • Microencapsulation
Co-crystallization. Co-crystals were obtained by the vapor diffusion
method and 1.0 M lithium sulfate, 0.1 M Tris-HCl at pH 8.5 and 0.01 M
nickel (II) chloride as precipitant similar to a previously published
protocol.8 FimH was concentrated to 17.3 mg.ml-1 and 1 mM of
compound 2 was added prior to a 1:1 mix with the precipitant into 1 μl
hanging drops.
References:
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2
N. P. Pera and R. J. Pieters, MedChemComm, 2014, 5, 1027–1035.
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Lindhorst, M. Marradi, P. Messner, A. Molinaro, P. V. Murphy, C.
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Enzyme-Linked Lectinosorbent Assay (ELLSA). Immunosorbent
microplates (Nunc, Maxisorp) were coated with 100 μL of RNase B (5
mg/mL) in 100 mM carbonate/bicarbonate buffer, pH 9.6. Plates were
incubated at 4°C overnight and then washed (300 μL/well) three times
with 10 mM phosphate-buffered saline (PBS) containing 0.15% Tween-
20 (PBST). All wells were blocked with 200μL 3% bovine serum
albumin (BSA) in PBST and incubated for 2 h at 37°C. They were then
washed three times with PBST. Mannosides were dissolved in PBST
at the necessary concentrations and added to the microwells. FimH
was diluted in PBST to 0.07μM, added to each plate well and incubated
for 1h at room temperature. Wells were washed three times with PBST
and incubated with 100 μL of rabbit-antiFimH antibodies IgG (aFimH)
diluted 1:5000 in PBST for 1 h at room temperature. Then wells were
washed three times with PBST, and incubated with 100 μL of goat anti-
rabbit HRP-labeled secondary antibody (Enzo Life Sciences (2ndAb-
HRP)) diluted 1:10000 in PBST for 1 h at room temperature. The wells
were washed three times with PBST and 100 μL of 3,3′,5,5′-
tetramethylbenzidine (TMB) was added to each well and incubated in
darkness for 5-15 min. The reaction was stopped with 100 μL/well of
1N sulfuric acid. Plate absorbance was analyzed at 450 nm using a
microplate reader BioTekELx800.
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Bouckaert, PLOS ONE, 2008, 3, e2040.
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L. K. Mydock-McGrane, Z. T. Cusumano and J. W. Janetka, Expert
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Acknowledgements
This work was carried out with financial support from Enterome,
the Conseil Régional des Pays de La Loire (ERABAC project),
the Centre National de la Recherche Scientifique (CNRS), the
Ministère de l’Enseignement Supérieur et de la Recherche in
France and the Ministry of Healthcare in Ukraine (project
0116U000759). We acknowledge SOLEIL for provision of
synchrotron radiation facilities and we would like to thank
William Shepard for assistance in using beam line Proxima2A.
12
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13
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ChemMedChem
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Entry for the Table of Contents
We developed a new generation of potent anti-adhesives of
pathogenic E. coli strains promoting the gut inflammation in
Crohn’s disease. The neo-thiazolylmannosides were
homologated by a methyl group and included in a γ-
cyclodextrin to form a water soluble NeoTazMan@γCD
complex with retained anti-adhesive effect against E.coli.
14
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