Novel hamamelitannin analogues for the treatment of biofilm related MRSA infections–A scaffold hopping approach

Article abstract of DOI:10.1016/j.ejmech.2016.10.056

Antimicrobial research is increasingly being focused on the problem of resistance and biofilm formation. Hamamelitannin (HAM) was recently identified as an antimicrobial potentiator for conventional antibiotics towards Staphylococcus aureus. This paper de

Full text of DOI:10.1016/j.ejmech.2016.10.056

European Journal of Medicinal Chemistry xxx (2016) 1e14
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel hamamelitannin analogues for the treatment of biofilm related
MRSA infectionseA scaffold hopping approach
Arno Vermote a, Gilles Brackman b, Martijn D.P. Risseeuw a, Tom Coenye b,
,
*
Serge Van Calenbergh a
a Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
b Laboratory for Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Antimicrobial research is increasingly being focused on the problem of resistance and biofilm formation.
Hamamelitannin (HAM) was recently identified as an antimicrobial potentiator for conventional anti-
biotics towards Staphylococcus aureus. This paper describes the synthesis and biological evaluation of
novel hamamelitannin analogues with alternative central scaffolds. Via a ligand-based approach, several
interesting compounds with improved synthetic accessibility were identified as potentiators for van-
comycin in the treatment of MRSA infections.
Received 17 September 2016
Received in revised form
20 October 2016
Accepted 24 October 2016
Available online xxx
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Hamamelitannin analogues
Scaffold hopping
MRSA
Antibiotic potentiators
Quorum sensing
1. Introduction
unavoidable with agents that target bacterial viability, prompted
the scientific community to explore alternative approaches, such as
The utility of conventional antibiotics has become compromised
by the emergence of antimicrobial resistance (AMR). In a recently
published report, it is estimated that by 2050 10 million lives a year
will be at risk due to the rise of drug-resistant infections [1].
Antibiotic resistance is not only a future threat, but poses a serious
global problem to patients and healthcare providers right now.
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible
for nosocomial as well as community-acquired infections and
represents a serious health threat [2]. Compounding the problem
even further is the fact that Staphylococcus aureus (S. aureus) is
notorious for its ability to form biofilms, surface-associated com-
munities of cells in a polymer-based matrix. Biofilm formation
provides a multi-level protection to microbes against antibiotics. It
alters growth rate and metabolism, raises the fraction of dormant
persister cells and hampers the penetration of antimicrobial agents
[3]. The global problem of AMR is further aggravated by a lack of
novel antibiotic development, which is fraught with scientific risk
and business challenges [4]. The fact that resistance development is
neutralizing virulence mechanisms. Therapeutic strategies to
reduce the expression of bacterial virulence factors would render
pathogens more susceptible to natural host defenses while main-
taining the normal microbiota. In S. aureus, biofilm formation and
virulence in general are regulated via quorum sensing (QS), an
intercellular communication system that controls gene expression.
Recently, the natural product hamamelitannin (HAM, 1, Fig. 1)
was identified as a quorum sensing inhibitor (QSI) in S. aureus [5].
We previously demonstrated that HAM and optimized derivative 2
interfere with the RAP-TRAP (RAP-targeting of the RNAIII-activating
peptide) signal transduction system, one of the regulators that is
believed to control the production of toxic shock syndrome toxin-1,
enterotoxins, proteases and d-hemolysins, which allow S. aureus to
survive, disseminate, and establish an infection [6]. HAM and the
metabolically stable lead compound 2 were also able to potentiate
the effect of several classes of antibiotics in vitro and to increase the
effect of cefalexin in a murine mastitis model of S. aureus infection.
[7] [8], Previous studies of our group have focused on the structural
optimization of the substituents at both the 5- and the 20-position
of the molecule [8e10]. Several HAM analogues were shown to be
active potentiators of vancomycin (VAN), a drug of last resort for
the treatment of MRSA infections. After having investigated the
* Corresponding author.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
http://dx.doi.org/10.1016/j.ejmech.2016.10.056
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: A. Vermote, et al., Novel hamamelitannin analogues for the treatment of biofilm related MRSA infectionseA
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2
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Fig. 1. Structure of hamamelitannin (HAM, 1) and optimized lead 2.
structure-activity relationship (SAR) of both ‘wings’ of HAM, we
explored alternatives for the central hamamelose-like scaffold in 2,
in the search for derivatives with improved synthetic accessibility
and/or potentiator activity.
hydroxyl group of 2 (II). Based on compounds c and d, a 2,5-
substituted 1,4-dioxane core (III) was put forward as possible bio-
isostere of the tetrahydrofuran ring present in 2. Also 1,3-
substituted morpholine (IV) and 2,4-substituted pyrrolidine (V)
emerged as alternative scaffolds.
2. Ligand-based virtual screening
3. Chemistry
HAM and optimized derivatives interfere with the RAP/TRAP QS
system in S. aureus. Prior to the discovery of these small-molecule
modulators of TRAP, the only TRAP inhibitors reported were RIP, a
heptapeptide (YSPXTNF, where X can be a Cys, a Trp or a modified
amino acid) originally isolated from culture supernatants of
S. xylosis, and synthetic variants [11e13]. Although the structure of
the membrane-associated signal transduction protein TRAP was
reported [14], in the absence of X-ray data of TRAPeinhibitor
complexes, structure-based drug design remains elusive. Hence, in
the present study, we followed a ligand-based approach to discover
TRAP inhibitors with alternative scaffolds. The open source plat-
form KNIME was used as interface and a publically available ZINC
[15] database of more than 9 million compounds was used for
virtual screening. A combined substructure search and 2D finger-
print similarity search protocol was used (Fig. 2). 2D fingerprints
are binary vectors in which the individual bits (1 or 0) encode the
presence (1) or absence (0) of certain fragments. The degree of
resemblance between the reference and a database molecule can
be calculated, which allows to arrange the database in descending
order of similarity with the target structure. Unfortunately, how-
ever, 2D fingerprints tend to lose information about the connec-
tivity of atoms. This means that although a database compound
may contain all of the fragments present in the reference structure,
the fragments are not necessarily connected in the ‘correct’ way.
Therefore, prior to the 2D fingerprint similarity searching, we
subjected the database to a substructure search using 2-chloro-N-
(2-methoxyethyl)benzamide as reference fragment. The latter
seems a privileged substructure according to our previous findings
[9]. With this more focused library of 2564 molecules, we started
the 2D fingerprint based similarity search using lead compound 2
as a probe. MACCS fingerprints were generated and for each
fingerprint a Tanimoto coefficient (Tc) was determined. A score of 1
means identity, 0 means no similarity at all. The obtained hit list
from this combined procedure was screened manually: the most
appealing structures and ideas extracted from this virtual screening
approach were used to guide us in the design of novel HAM ana-
logues with alternative core structure. Thus, in the present study,
we were not interested in the library compounds as such; we
wanted to use the hit list merely as a means and as a source of
inspiration for the design of HAM analogues in order to get insight
into the requirements for optimal potentiating activity. An impor-
tant factor that was taken into account was the synthetic feasibility
of the scaffold (and ultimately the final compound). An overview of
the selected scaffolds, together with a selection of database com-
pounds that inspired us, is given in Fig. 2. Inspired by compounds a
and b (Fig. 2), a first scaffold was obtained by transposing the 2-
benzamidomethyl moiety to position 1 of the tetrahydrofuran
core (I). A second central scaffold was achieved by deleting the 3-
Antimicrobial potentiator
2 consists of a 5-ortho-chlor-
obenzamidomethyl and a 2’-benzamidomethyl moiety, linked to a
central (3S,4R)-tetrahydrofuran-3,4-diol scaffold (Fig. 1). While
maintaining both aromatic moieties intact, we first investigated the
impact of transposing the 2-benzamidomethyl fragment to position
1. The synthesis of 9, a rather conservative scaffold hop (reflected in
a Tc of 0.909), starts from the known azidolactole 3 [16] (Scheme 1).
Horner-Wadsworth-Emmons olefination of 3 with methyl (tri-
phenylphosphoranylidene)acetate and treatment of the resulting
a,b-unsaturated carbonyl intermediate with a methanolic sodium
methoxide solution afforded methyl ester 4 as an inseparable
mixture of cis and trans epimers [17]. Saponification yielded a
mixture of epimeric carboxylic acids 5, which were converted into
the separable Boc-protected amines by Curtius rearrangement.
TFA-mediated Boc-deprotection of
6 and subsequent EDC-
mediated acylation with benzoic acid gave benzamide 8. The
structure and stereochemistry of the latter was confirmed by a 2D
1H-1H NOESY experiment (Supporting information). The benza-
mide was subjected to Staudinger reduction and the resulting
amine was converted into an ortho-chlorobenzamide. Acidic hy-
drolysis of the acetonide gave 9, a regioisomer of 2 (Scheme 1).
Phthalimide 10 [8] served as an orthogonally protected inter-
mediate for the synthesis of analogue 21, the 3-deoxy analogue of 2
(Tc ¼ 0.982). Acidic deprotection of the acetonide, treatment with
ethanolic hydrazine and Boc-protection of the resulting amine
afforded 13 (Scheme 2). Selective acetylation of the secondary hy-
droxyl group of 13 gave acetate 14. To circumvent the use of toxic
MOM-Cl, methoxymethylation of the tertiary alcohol group was
performed by treatment of 14 with dimethoxymethane and phos-
phorus pentoxide [18]. Interestingly, this reaction also yielded a
heterocyclic spiro compound (16) in which the Boc-protected
amine and the tertiary alcohol are connected via a methylene
group to form an oxazolidine ring. Although 1H NMR and HRMS
confirmed the structure of 16, further structural proof was given by
13C NMR and a 2D 13C-1H HSQC experiment, in which a cross peak
was observed between the protons at the hemiaminal carbon and
their proximate 13C (Supporting information). Removal of the ac-
etate in 15 under standard conditions gave alcohol 17. The latter
was acylated with thiocarbonyldiimidazole to yield azidothio-
carbamate 18, which was the substrate for the simultaneous radical
deoxygenation and azide reduction with tributyltin hydride and a
catalytic amount of azoisobutyronitrile in refluxing toluene. Final
3-deoxy derivative 21 was obtained via standard transformations of
amine 19.
Inspired by the results from the substructure search and 2D
fingerprint-based screening, a 1,4-dioxane motif was explored as
central scaffold. Derivative 29 contains a similar atom connectivity
Please cite this article in press as: A. Vermote, et al., Novel hamamelitannin analogues for the treatment of biofilm related MRSA infectionseA
scaffold hopping approach, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.056

Fig. 2. Overview of scaffold hopping strategies.
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Scheme 1. Reagents and conditions: (a) [i] Ph3P¼CHCO2Me, MeCN, 85 ꢀC, [ii] NaOMe, MeOH, 46%; (b) NaOH, H2O/dioxane, q.; (c) DPPA, Et3N, tBuOH, 100 ꢀC, 46% for 6, 13% for 7; (d)
[i] TFA, 1,2-DCE, [ii] PhCOOH, EDC$HCl, DIPEA, HOBt, DMF, 80%; (e) [i] PMe3, THF, 3 h, [ii] H2O, 45 min, [iii] 2-chlorobenzoic acid, EDC$HCl, DIPEA, HOBt, DMF, 80%; (f) 35% TFA in H2O,
q.
Scheme 2. Reagents and conditions: (a) 35% TFA in H2O; (b) N2H2$H2O, toluene/MeOH, 60 ꢀC; (c) (Boc)2O, K2CO3, H2O/dioxane, 85% from 10; (d) Ac2O, pyr, 76%; (e) P2O5,
dimethoxymethane, CHCl3, 24 h, 27% for 15, 40% for 16; (f) NaOMe, MeOH, q.; (g) TCDI, toluene, 125 ꢀC, 65%; (h) Bu3SnH, AIBN, toluene, 85 ꢀC, 69%; (i) 2-chlorobenzoic acid,
EDC$HCl, DIPEA, HOBt, DMF, 54%; (j) [i] 35% TFA in H2O, [ii] BzCl, Et3N, DCM, 71%.
as 2 and shows a Tc of 0.754. The synthesis of 29 starts with the
reaction of benzyl alcohol with (R)-epichlorohydrin (Scheme 3).
The resulting (R)-1-(benzyloxy)-3-chloropropan-2-ol was treated
with (S)-glycidyl tosylate to give intermediate 23. Treatment of the
latter with aqueous NaOH solution lead to intramolecular ring
closure in a 6-exo-tet process to give monoprotected cis-dioxane-
dimethanol intermediate 24. Subsequent transformation of 24 into
heterobisbenzamide 29 occurred via known procedures (Scheme
3).
Next, we also synthesized two analogues with a morpholine
scaffold (Scheme 4). (R)-benzylglycidyl ether was treated with 2-
aminoethylsulfate in the presence of aqueous NaOH. Intra-
molecular ring closure via a 6-exo-tet process gave an intermediate
morpholine, the amino function of which was subsequently Boc-
protected to give 30. Hydrogenolysis of the benzyl protecting
group and conversion of the resulting primary alcohol into the
corresponding azide gave intermediate 31 with two orthogonally
protected amino groups. Installation of an ortho-chlorobenzamide
and a benzamide on either side of the molecule gave final com-
pounds 34 and 35 (Scheme 4), both with a Tc of 0.597.
Nitrile 39 was a key intermediate in the synthesis of the
pyrrolidine-containing derivatives (Scheme 5). It was synthesized
according to the method described by Zlatopolskiy et al. [19]
Borane-mediated reduction of the carboxylic acid in Boc-
protected hydroxyproline 36 yielded the corresponding primary
alcohol 37. Selective protection of the latter as a silyl ether gave 38.
The secondary hydroxyl group was mesylated and subsequent
displacement by cyanide anion provided nitrile 39. Reduction of
this nitrile with NaBH4/CoCl2 gave the corresponding primary
amine, which was converted into a benzamide via EDC-mediated
acylation with the appropriate benzoic acid (40 and 41). TBAF-
mediated liberation of the primary alcohol and transformation of
the latter into the corresponding azide gave 42 and 43. Reduction of
this azide
functionality with trimethylphosphine
and
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
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Scheme 3. Reagents and conditions: (a) [i] BF3$OEt2, 1,2-DCE, 16 h, [ii] reflux, 2 h, 48%; (b) BF3$OEt2, 1,2-DCE, 16 h, 50%; (c) [i] 1.0 M NaOH, 2.5 h, [ii] 90 ꢀC, 4 h, [iii] rt, 16 h, [iv] 90 ꢀC,
2 h, 66%; (d) [i] MsCl, Et3N, DCM, [ii] NaN3, DMF, 60 ꢀC, 91%; (e) [i] PMe3, THF, 3 h, [ii] H2O, 45 min, [iii] PhCOOH, EDC$HCl, DIPEA, HOBt, DMF, 90%; (f) H2, Pd/C, MeOH, q.; (g) [i] MsCl,
Et3N, DCM, [ii] NaN3, DMF, 60 ꢀC, 69%; (h) [i] PMe3, THF, 3 h, [ii] H2O, 45 min, [iii] 2-chlorobenzoic acid, EDC$HCl, DIPEA, HOBt, DMF, 74%.
Scheme 4. Reagents and conditions: (a) [i] NaOH, H2O, MeOH, 50 ꢀC, 1 h, [ii] NaOH, toluene, 65 ꢀC, 16 h; (b) (Boc)2O, K2CO3, acetone/H2O, 2 h, 43%; (c) [i] H2, Pd/C, MeOH, [ii] MsCl,
Et3N, DCM, [iii] NaN3, DMF, 60 ꢀC, 81%; (d) [i] TFA, DCM, [ii] RCOOH, EDC$HCl, DIPEA, HOBt, DMF, 82% for 32, 79% for 33; (e) [i] PMe3, THF, 3 h, [ii] H2O, 45 min, [iii] RCOOH, EDC$HCl,
DIPEA, HOBt, DMF, 62% for 34, 91% for 35.
carbodiimide-mediated acylation of the resulting amine with the
appropriate benzoic acid, furnished derivatives 46 and 47 after final
deprotection (Tc ¼ 0.661).
alone, VAN resulted only in a minor reduction of the number of
S. aureus sessile cells (30 14% compared to an untreated control,
Table 1). In contrast, combined treatment of VAN with 2 resulted in
significantly more killing of bacterial biofilm cells, both under
pretreatment and under combined treatment regimens (Table 1).
Initially, all of the compounds with alternative scaffolds were tested
4. Results and discussion
in a concentration of 100 mM. For the most active derivatives, the
For all final compounds, minimum inhibitory concentrations
(MIC) against S. aureus Mu50 were determined to rule out a direct
effect on growth. In all cases, MIC values were higher than 500 mM,
being the highest concentration tested (data not shown). Subse-
quently, the HAM derivatives were tested for their in vitro effect on
S. aureus biofilm susceptibility to VAN, both under pretreatment
and combination treatment regimens. To evaluate the effect of
pretreatment, S. aureus Mu50 was allowed to form a biofilm in the
presence of the HAM analogues, after which the biofilm was treated
effect on biofilm susceptibility towards VAN was tested in lower
concentrations, which allowed us to determine an EC50 value. The
latter is defined as the concentration of the analogue needed to
double the activity of VAN, as measured by the number of surviving
cells.
When comparing the potentiating activity of lead compound 2
with its isomer 9, it can be concluded that this scaffold rear-
rangement is well-tolerated (Table 1). Bisbenzamide 9 shows very
good potentiating activity in the combination treatment setup
with VAN (20
mg/mL). In the combination treatment setup, the
(EC50 ¼ 3.708
mM, compared to 7.976 mM for 2). This is encouraging,
as clinicians in daily practice are often confronted with already
bacteria were allowed to form a mature biofilm after which a HAM
analogue and VAN were administered simultaneously. When used
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6
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Scheme 5. Reagents and conditions: (a) BH3$THF, 96%; (b) TBDPSCl, imidazole, DMF, 63%; (c) [i] MsCl, Et3N, DCM, [ii] nBu4NþCNꢁ, MeCN, 65 ꢀC, 19%; (d) [i] NaBH4, CoCl2, MeOH, [ii]
RCOOH, EDC$HCl, DIPEA, HOBt, DMF, 68% for 40, 60% for 41; (e) [i] TBAF, THF, [ii] MsCl, Et3N, DCM, [iii] NaN3, DMF, 60 ꢀC, 55% for 42, 55% for 43; (f) [i] PMe3, THF, 3 h, [ii] H2O,
45 min, [iii] RCOOH, EDC$HCl, DIPEA, HOBt, DMF, 38% for 44, 66% for 45; (g) TFA, DCM, q. for 46, q. for 47.
Table 1
Microbiological evaluation of HAM analogues with different scaffold.a: Percentage reduction in Colony Forming Units (CFU's) per biofilm when biofilms are treated with VAN
alone (20
m
g/mL) or in combination with HAM or a HAM-analogue (100
m
M) compared to the untreated (negative) control.* significantly different from treatment with
HAM þ VAN (p < 0.05).
Compound
R1 ¼ oCl Ph
R2 ¼ Ph
Reduction in CFU's compared to control (%)a
EC50 (mM)
Pretreatment
30 14*
Combination treatment
30 14*
Pretreatment
Combination treatment
VAN alone
e
e
e
e
HAM, 1
2
57 13
88 2*
57 22
92 4*
145.5
0.3890
165.1
7.976
9
93 6*
95 2*
65 14
2.598
n.d.
3.708
n.d.
21
21 11*
29
95 3*
91 1*
5.568
13.12
34
35
46
ꢁ6 42*
ꢁ7 31*
51
5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
41 37
54
8
50
7
47
62 24
47 11
n.d.
n.d.
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
7
infected and biofilm-related wound infections. Moreover, the
improved synthetic accessibility (compared to a longer route to-
wards 2) makes compound 9 even more interesting. Deletion of the
3-hydroxyl function in 2 completely abolishes activity, which sug-
gests that the 3-OH group exhibits signature interactions with the
target. In the combination treatment regimen, dioxane derivative
29 caused little change in activity compared to 2 and 9. This com-
pound contains to a large extend the same connectivity of atoms
relative to 2 and 9. With 29 being almost equipotent in the com-
bination treatment setup, this scaffold hop proves that it is possible
to replace the central hamamelose-like central scaffold. Morpho-
line derivatives 34 and 35 were completely devoid of activity.
Finally, pyrrolidines 46 and 47 lacked activity as well. The absence
of both hydroxyl groups may account for the poor potentiating
properties, as well as the fact that the protonated molecules might
interact with the negatively charged extracellular DNA in the bio-
film matrix, as previously demonstrated for positively-charged
nanoparticles [20].
6.1.1. General procedure 1: EDC-mediated amide formation
To a solution of compound 19 or the crude amine obtained from
6 or 39, in DMF (25 mL/mmol) were added the appropriate organic
acid (1.5 equiv per amine), EDC.HCl (2 equiv per amine), diisopro-
pylethylamine (4 equiv per amine) and a catalytic amount of 1-
hydroxybenzotriazole. The reaction mixture was stirred overnight
at rt. The mixture was concentrated and partioned between water
and EtOAc. The organic layer was dried over sodium sulphate,
filtered and concentrated in vacuo. The products were purified by
column chromatography with appropriate eluents.
6.1.2. General procedure 2: Staudinger reduction and subsequent
EDC-mediated acylation of the resulting amine with the appropriate
benzoic acid
A
solution of compound 8, 25, 28, 32, 33, 42 or 43
(0.4e1.4 mmol) in THF (10 mL/mmol) was treated with Me3P (1 M
solution, 5 equiv) and the reaction mixture was stirred for 3 h.
Water (13 equiv) was added and the solution was stirred for
another hour, after which it was concentrated. The residue was co-
evaporated with toluene. The obtained crude amine was used
without further purification. To a solution of this crude amine in
DMF (25 mL/mmol) were added the appropriate organic acid (1.5
equiv), EDC.HCl (2 equiv), diisopropylethylamine (4 equiv) and a
catalytic amount of 1-hydroxybenzotriazole and the reaction
mixture was stirred overnight at rt. The reaction mixture was
concentrated and partioned between water and EtOAc. The organic
layer was dried over sodium sulphate, filtered and evaporated. The
products were then purified by column chromatography with
appropriate eluents.
5. Conclusion
Several practical pathways towards the synthesis of novel HAM
analogues with an alternative central scaffold were successfully
pioneered. The resulting compounds were tested for their ability to
potentiate the activity of VAN in S. aureus biofilms in vitro. The
readily accessible 2,5-anhydro- -allitol derivative 9 and dioxane
D
derivative 29 showed comparable activity to that of lead compound
2 in the combination treatment setup. These findings imply that
there is room for structural improvement in the central part of the
molecule and several analogues represent attractive starting points
for further lead optimization of the potentiating activity.
6.1.3. General procedure 3: TFA in water e mediated deprotection
A known amount of the isopropylidene protected compound 20
or the crude obtained from 8 was treated with a 35% aq. CF3COOH
solution (30 mL/mmol) overnight at room temperature. When TLC
indicated that the deprotection was complete, the reaction mixture
was concentrated and, if required, purified by column
chromatography.
6. Experimental
6.1. Chemistry
All reactions described were performed under an argon atmo-
sphere and at ambient temperature unless stated otherwise. All
reagents and solvents were purchased from Sigma-Aldrich (Die-
gem, Belgium), Acros Organics (Geel Belgium), TCI Europe (Zwijn-
drecht, Belgium) or Carbosynth Ltd (Compton Berkshire, United
Kingdom) and used as received. NMR solvents were purchased
from Eurisotop (Saint-Aubin, France). Reactions were monitored by
TLC analysis using TLC aluminium sheets (Macherey-Nagel, Alu-
gram Sil G/UV254) with detection by UV or by spraying with a so-
lution of (NH4)6Mo7O24$4H2O (25 g/L) and (NH4)4Ce(SO4)4$2H2O
(10 g/L) in H2SO4 (10%) followed by charring or an aqueous solution
of KMnO7 (20 g/L) and K2CO3 (10 g/L) or an ethanolic solution of
ninhydrin (2 g/L) and acetic acid (1% v/v) followed by charring.
Silica gel column chromatography was performed manually using
6.1.4. General procedure 4: Boc deprotection and subsequent EDC-
mediated acylation of the resulting amine with the appropriate
benzoic acid
A solution of compound 31 (248 mg, 1.02 mmol) in 20% tri-
fluoroacetic acid in CH2Cl2 (10 mL/mmol) was stirred for 3 h. When
finished, volatile organics were evaporated under reduced pressure
and the resulting crude amine was taken up in DMF (25 mL/mmol).
The appropriate organic acid (1.5 equiv per amine), EDC.HCl (2
equiv per amine), diisopropylethylamine (4 equiv per amine) and a
catalytic amount of 1-hydroxybenzotriazole were added. The re-
action mixture was stirred overnight at rt. The mixture was
concentrated and partioned between water and EtOAc. The organic
layer was dried over sodium sulphate, filtered and concentrated in
vacuo. The products were purified by column chromatography with
appropriate eluents.
Grace Davisil 60 Å silica gel (40e63 mm) or automated using a Grace
Reveleris X2 system and the corresponding flash cartridges. High
resolution spectra were recorded with a Waters LCT Premier XE
Mass spectrometer. 1H and 13C NMR spectra were recorded with a
Varian Mercury-300BB (300/75 MHz) spectrometer. Chemical
6.1.5. (3aR,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro
[3,4-d][1,3]dioxol-4-ol (3)
See reference [8] and [15]. Spectroscopy data for 3 are consistent
with those published previously [8,16].
shifts are given in ppm (d) relative to tetramethylsilane as an in-
ternal standard (1H NMR) or the NMR solvent (13C NMR). In 19F
NMR, signals have been referred to CDCl3 or DMSO-d6 lock reso-
nance frequency according to IUPAC referencing with CFCl3 set to
0 ppm. Coupling constants are given in Hz. Preparative HPLC pu-
rifications were carried out using a Laprep preparative HPLC system
6.1.6. Methyl 2-((3aS,6R,6aR)-6-(azidomethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)acetate (4)
A solution of lactol 3 (4.00 g, 18.6 mmol) in acetonitrile (60 mL)
was flushed with N2 gas. Methyl (triphenylphosphoranylidene)ac-
etate (6.84 g, 20.5 mmol) was added and the reaction mixture was
heated to reflux for 90 min. Then, the mixture was concentrated in
equipped with a Xbridge Prep C18 column (19 ꢂ 250 mm, 5
mm)
using a water/acetonitrile/formic acid gradient system.
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8
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
vacuo and the residue was taken up in MeOH (40 mL), after which a
methanolic sodium methoxide solution (5.4 M, 370 L) was added.
This mixture was stirred for another 90 min and then neutralized
with Amberlite (IR 120 H-form). The suspension was filtered and
the filtrate was concentrated. FCC of the residue (toluene/EtOAc
1:1) gave methyl esters 4 as a pale oil in 46% yield. Epimeric
6.1.9. Tert-butyl (((3aS,4R,6R,6aR)-6-(azidomethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)carbamate
(7)
m
Compound 7 was obtained according to the procedure described
for compound 6. 1H NMR (300 MHz, CDCl3)
d ppm 1.33 (s, 3 H) 1.45
(s, 9 H) 1.49 (s, 3 H) 3.23e3.48 (m, 4 H) 4.07e4.15 (m, 1 H) 4.17e4.25
(m, 1 H) 4.62 (dd, J ¼ 6.2, 1.5 Hz, 1 H) 4.74 (dd, J ¼ 6.2, 4.2 Hz, 1 H)
4.94 (br. s., 1 H). HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
ratio ¼ 94:6. Major epimer: 1H NMR (300 MHz, CDCl3)
d ppm 1.34
(s, 3 H) 1.54 (s, 3 H) 2.65 (dd, J ¼ 15.8, 7.0 Hz, 1 H) 2.74 (dd, J ¼ 15.8,
5.4 Hz, 1 H) 3.34 (dd, J ¼ 13.2, 4.7 Hz, 1 H) 3.54 (dd, J ¼ 13.2, 4.0 Hz,
1 H) 3.71 (s, 3 H) 4.05e4.12 (m, 1 H) 4.27e4.33 (m, 1 H) 4.52e4.63
C
14H25N4Oþ5 329.18195; Found 329.1831.
(m, 2 H). 13C NMR (75 MHz, CDCl3)
d
ppm 25.6, 27.5, 38.1, 52.0, 52.4,
81.0, 82.2, 83.2, 84.3, 115.1, 170.9. Minor epimer: 1H NMR
(300 MHz, CDCl3) ppm 1.33 (s, 3 H) 1.49 (s, 3 H) 2.76e2.79 (m, 2 H)
6.1.10. N-(((3aS,4S,6R,6aR)-6-(azidomethyl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)benzamide
(8)
d
3.24e3.47 (m, 2 H) 3.71 (s, 3 H) 4.18e4.24 (m, 1 H) 4.41 (td, J ¼ 6.8,
Boc-protected amine 6 (100 mg, 0.305 mmol) was taken up in
dry 1,2-dichloroethane (2 mL) and molecular sieves (3 Å rods) were
added. The flask was purged with N2 gas and cooled on ice to 0 ꢀC.
Trifluoroacetic acid (0.179 mL, 2.34 mmol) was added and the re-
action mixture was stirred for 3 h. After filtration, the filtrate was
taken to dryness and the residue was adsorbed onto celite. FCC
(CH2Cl2/MeOH/NH4OH 100:0:0.1 / 90:10:0.1) gave the corre-
sponding primary amine, which was subjected to general proced-
4.0 Hz,1 H) 4.64 (dd, J ¼ 6.2,1.2 Hz,1 H) 4.80 (dd, J ¼ 6.2, 4.1 Hz,1 H).
13C NMR (75 MHz, CDCl3)
d ppm 25.1, 26.4, 34.4, 51.7, 51.9, 77.5, 81.4,
82.8, 83.4, 115.1, 170.9. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
C
11H18N3Oþ5 272.12410; Found 272.1240.
6.1.7. 2-((3aS,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro
[3,4-d][1,3]dioxol-4-yl)acetic acid (5)
To a solution of methyl esters 4 (0.950 g, 3.50 mmol) in dioxane
(10 mL), was added a 1 M NaOH solution (4.20 mL). After 150 min of
stirring, TLC analysis (toluene/EtOAc 4:1) showed complete con-
sumption of starting material and presence of a lower-running spot.
The pH of the reaction mixture was adjusted to approximately 2 by
addition of HCl (1 M solution). The reaction mixture was extracted
with EtOAc. The organic layers were pooled, dried over Na2SO4,
filtered and concentrated in vacuo. The product was obtained as a
transparent oil without further purification (q.). Epimeric
ure 1. Transparent oil, 80%. 1H NMR (300 MHz, CDCl3)
d ppm 1.32 (s,
3 H) 1.53 (s, 3 H) 3.44 (dd, J ¼ 13.0, 4.0 Hz, 1 H) 3.64e3.76 (m, 2 H)
3.85 (ddd, J ¼ 14.1, 6.6, 5.1 Hz, 1 H) 4.06e4.14 (m, 1 H) 4.16e4.23 (m,
1 H) 4.51e4.60 (m, 2 H) 6.69 (br. s., 1 H) 7.37e7.55 (m, 3 H)
7.75e7.87 (m, 2 H). 13C NMR (75 MHz, CDCl3)
d ppm 25.5, 27.5, 41.6,
52.4, 81.6, 82.5, 83.1, 83.3, 115.0, 127.1, 128.7, 131.7, 134.3, 167.8.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C16H21N4Oþ4 333.15573;
Found 333.1563.
ratio ¼ 76:24. Major epimer: 1H NMR (300 MHz, CDCl3)
d
ppm 1.35
6.1.11. N-(((2R,3S,4R,5S)-5-(benzamidomethyl)-3,4-
(s, 3 H) 1.55 (s, 3 H) 2.63e2.74 (dd, J ¼ 16.1, 7.3 Hz, 1 H) 2.74e2.82
(m, 1 H) 3.35 (dd, J ¼ 13.1, 4.5 Hz, 1 H) 3.56 (dd, J ¼ 13.1, 3.8 Hz, 1 H)
4.08e4.13 (m, 1 H) 4.26e4.34 (m, 1 H) 4.52e4.58 (dd, J ¼ 6.7, 4.4 Hz,
1 H) 4.58e4.63 (dd, J ¼ 6.7, 4.1 Hz, 1 H) 10.82 (br. s., 1 H). 13C NMR
dihydroxytetrahydrofuran-2-yl)methyl)-2-chlorobenzamide (9)
Compound 8 was subjected to general procedure 2. 1H NMR
(300 MHz, CDCl3) d ppm 1.32 (s, 3 H) 1.53 (s, 3 H) 3.52e3.86 (m, 4 H)
4.10e4.21 (m, 2 H) 4.49e4.60 (m, 2 H) 6.88 (br. s., 1 H) 7.00 (br. s.,
1 H) 7.21e7.41 (m, 5 H) 7.42e7.51 (m, 1 H) 7.55e7.65 (m, 1 H)
(75 MHz, CDCl3)
d ppm 25.5, 27.4, 38.1, 52.3, 80.6, 82.0, 83.1, 84.1,
115.2, 176.1. Minor epimer: 1H NMR (300 MHz, CDCl3)
d
ppm 1.34
7.75e7.81 (m, 2 H). 13C NMR (75 MHz, CDCl3)
d ppm 25.5, 27.4,
(s, 3 H) 1.50 (s, 3 H) 2.72e2.89 (m, 2 H) 3.32 (dd, J ¼ 12.9, 4.7 Hz,1 H)
3.44 (d, J ¼ 12.9, 6.2 Hz, 1 H) 4.18e4.24 (m, 1 H) 4.37e4.44 (m, 1 H)
4.66 (dd, J ¼ 6.2,1.2 Hz,1 H) 4.81 (dd, J ¼ 6.2, 4.1 Hz,1 H) 10.82 (br. s.,
42.16, 42.22, 82.79, 82.84, 83.6, 83.8, 114.7, 127.19, 127.23, 128.6,
130.1, 130.4, 130.7, 131.5, 131.6, 134.1, 135.1, 167.3, 167.9. HRMS (ESI-
TOF) m/z: [MþH]þ Calcd for C23H25ClN2Oþ5 445.15248; Found
445.1523. The isopropylidene protected derivative was subjected to
general procedure 3. White foam, 80% from 8. 1H NMR (300 MHz,
1 H). 13C NMR (75 MHz, CDCl3)
d ppm 25.0, 26.2, 34.4, 51.8, 77.3,
81.3, 82.7, 83.3,113.2,176.8. HRMS (ESI-TOF) m/z: [M ꢁ H]ꢁ Calcd for
C
10H14N3Oꢁ5 256.09389; Found 256.0939.
CDCl3)
7.29e7.58 (m, 7 H) 7.77e7.88 (m, 2 H) 8.37e8.52 (m, 2 H). 13C NMR
(75 MHz, CDCl3) ppm 41.3, 41.8, 72.1, 72.3, 81.4, 81.6, 127.0, 127.2,
d ppm 3.21e3.55 (m, 4 H) 3.73e3.90 (m, 4 H) 4.86 (br. s., 2 H)
6.1.8. Tert-butyl (((3aS,4S,6R,6aR)-6-(azidomethyl)-2,2-
d
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)carbamate
(6)
128.2, 128.9, 129.5, 129.9, 130.7, 131.1, 134.4, 137.0, 166.5, 166.6.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C20H22ClN2Oþ5 405.12118;
Found 405.1213.
A flask containing carboxylic acids 5 (1.20 g, 4.66 mmol) and
molecular sieves in tert-butanol (90 mL) was purged with N2 gas.
Tiethylamine (0.715 mL, 5.13 mmol) and diphenylphosphoryl azide
(1.10 mL, 5.13 mmol) were added and the reaction mixture was
heated to reflux for 20 h. TLC analysis (hexane/EtOAc 85:15)
showed complete consumption of starting material. The reaction
mixture was filtered and the filtrate was concentrated in vacuo. The
residue was taken up in EtOAc and washed with brine. The organic
layer was dried over Na2SO4, filtered and concentrated in vacuo.
This residue was adsorbed onto celite and purified via FCC (hexane/
EtOAc 10:0 / 8:2). Both compound 6 (46% yield) and its trans
epimer 7 (13% yield) were obtained as transparent oils. 1H NMR
6.1.12. 2-(((3aS,6R,6aR)-6-(azidomethyl)-2,2-dimethyldihydrofuro
[3,4-d][1,3]dioxol-3a(4H)-yl)methyl)isoindoline-1,3-dione (10)
See reference [8]. Spectroscopy data for 10 are consistent with
those published previously [8].
6.1.13. 2-(((3S,4R,5R)-5-(azidomethyl)-3,4-
dihydroxytetrahydrofuran-3-yl)methyl)isoindoline-1,3-dione (11)
Compound 10 (920 mg, 2.57 mmol) was dissolved in a 35% TFA
in H2O solution (30 mL) and put at sonication overnight. MS anal-
ysis showed complete consumption of starting material. Reaction
mixture was concentrated in vacuo and the remaining nacre-
looking solid (i.e. the title compound) was used without further
purification in the next step. Yield was determined over several
(300 MHz, CDCl3)
3.29e3.49 (m, 3 H) 3.60 (dd, J ¼ 13.0, 3.7 Hz,1 H) 3.98e4.09 (m, 2 H)
4.46e4.57 (m, 2 H) 4.91 (br. s., 1 H). 13C NMR (75 MHz, CDCl3)
ppm
d ppm 1.33 (s, 3 H) 1.45 (s, 9 H) 1.53 (s, 3 H)
d
25.5, 27.5, 28.4, 42.5, 52.4, 79.6, 81.8, 82.4, 83.1, 83.5, 114.9, 156.1.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C14H25N4Oþ5 329.18195;
Found 329.1829.
steps. 1H NMR (300 MHz, CDCl3)
d
ppm 2.82 (d, J ¼ 7.0 Hz, 1 H) 3.38
(dd, J ¼ 13.2, 4.7 Hz, 1 H) 3.58e3.67 (dd, J ¼ 13.2, 2.9 Hz, 1 H)
3.77e4.09 (m, 7 H) 7.75e7.82 (m, 2 H) 7.86e7.93 (m, 2 H). 13C NMR
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
9
(75 MHz, CDCl3)
d
ppm 43.5, 52.1, 74.6, 75.5, 78.6, 81.9, 124.0, 131.7,
J ¼ 6.1, 3.7 Hz, 1 H) 4.76 (app. s, 2 H) 4.95 (d, J ¼ 6.4 Hz, 1 H) 5.38 (t,
134.8, 169.6. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C14H15N4Oþ5
J ¼ 5.1 Hz, 1 H). 13C NMR (75 MHz, CDCl3)
d ppm 20.9, 28.4, 43.2,
319.10370; Found 319.1024.
52.3, 55.9, 72.2, 74.9, 79.7, 81.0, 83.3, 92.5, 156.3, 170.6. HRMS (ESI-
TOF) m/z: [MþH]þ Calcd for C15H27N4Oþ7 375.18743; Found
375.1887.
6.1.14. 2R,3R,4S)-4-(aminomethyl)-2-(azidomethyl)
tetrahydrofuran-3,4-diol (12)
To a solution of compound 11 (obtained in the previous step) in
toluene/MeOH 1:1 (26 mL) was added hydrazine monohydrate
(0.312 mL, 6.43 mmol). The reaction mixture was heated to 60 ꢀC
for 4 h. Flocculation was seen and when the reaction was complete,
the suspension was filtered and the filtrate concentrated in vacuo.
Crude amine 12 (pale oil) was used in the next step without further
purification. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C6H13N4Oþ3
189.09822; Found 189.0983.
6.1.18. Tert-butyl (5S,8R,9R)-9-acetoxy-8-(azidomethyl)-1,7-dioxa-
3-azaspiro[4.4]nonane-3-carboxylate (16)
Compound 16 was obtained as a side product in the synthesis of
compound 15. 1H NMR (300 MHz, CDCl3)
d ppm 1.48 (s, 9 H) 2.12 (s,
3 H) 3.38 (dd, J ¼ 13.3, 4.5 Hz, 1 H) 3.52 (d, J ¼ 10.8 Hz, 1 H) 3.60 (dd,
J ¼ 13.5, 3.2 Hz, 1 H) 3.77 (d, J ¼ 10.8 Hz, 1 H) 3.89 (d, J ¼ 9.7 Hz, 1 H)
3.96 (d, J ¼ 9.7 Hz, 1 H) 4.12 (ddd, J ¼ 6.3, 4.5, 3.5 Hz, 1 H) 4.91 (br. s.,
2 H) 5.03 (d, J ¼ 6.4 Hz, 1 H). 13C NMR (75 MHz, CDCl3)
d ppm 20.8,
28.4, 50.5, 52.0, 73.9, 75.9, 79.3, 80.9, 81.0, 86.0, 152.7, 170.3. HRMS
(ESI-TOF) m/z: [MþH]þ Calcd for C14H23N4Oþ6 343.16121; Found
343.1611.
6.1.15. Tert-butyl (((3S,4R,5R)-5-(azidomethyl)-3,4-
dihydroxytetrahydrofuran-3-yl)methyl)carbamate (13)
Diol-amine 12 (obtained in the previous step) was dissolved in a
dioxane/water 3:1 mixture (28 mL in total). K2CO3 (1.07 g) was
added, followed by di-tert-butyl dicarbonate (0.589 g) and the re-
action mixture was stirred vigorously for 4 h. The dioxane was
evaporated and the remaining water layer was extracted with
CH2Cl2 (4 ꢂ 40 mL). The organic layers were pooled, dried over
Na2SO4, filtered and concentrated in vacuo. FCC afforded title
compound 13 as a pale yellow oil (85% over 3 steps from 10). 1H
6.1.19. Tert-butyl (((3S,4R,5R)-5-(azidomethyl)-4-hydroxy-3-
(methoxymethoxy)tetrahydrofuran-3-yl)methyl)carbamate (17)
By addition of a methanolic solution of NaOMe (30% wt), the pH
of a solution of compound 16 (86.0 mg, 0.230 mmol) in MeOH
(5 mL) was adjusted to 9e10. The reaction mixture was stirred
overnight. TLC analysis (toluene/EtOAc 6:4) showed presence of 1
lower-running spot. Amberlite (IR 120 H-form) was added until pH
was neutral. The suspension was filtered and the filtrate concen-
trated in vacuo. The pure title compound was obtained as a colorless
NMR (300 MHz, CDCl3)
d ppm 1.45 (s, 9 H) 3.19e3.41 (m, 3 H)
3.55e3.94 (m, 6 H) 4.42 (br. s., 1 H) 5.39 (br. s., 1 H). 13C NMR
(75 MHz, CDCl3)
d
ppm 28.3, 46.0, 52.0, 74.0, 75.1, 78.6, 80.8, 82.1,
oil in quantitative yield. 1H NMR (300 MHz, CDCl3)
d ppm 1.45 (s,
158.2. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C14H15N4Oþ5
9 H) 3.02 (br. s., 1 H) 3.33e3.56 (m, 6 H) 3.58e3.66 (m, 1 H)
3.84e3.91 (m, 3 H) 4.06 (d, J ¼ 10.5 Hz, 1 H) 4.82e4.91 (m, 2 H) 5.13
319.10370; Found 319.1024. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
C
11H21N4Oþ5 289.15065; Found 289.1512.
(br. s., 1 H). 13C NMR (75 MHz, CDCl3)
d ppm 28.4, 43.2, 52.3, 56.1,
71.9, 75.9, 80.0, 82.3, 83.3, 92.2,156.4. HRMS (ESI-TOF) m/z: [MþH]þ
6.1.16. (2R,3R,4S)-2-(azidomethyl)-4-(((tert-butoxycarbonyl)
amino)methyl)-4-hydroxytetrahydrofuran-3-yl acetate (14)
Calcd for C13H25N4Oþ6 333.17686; Found 333.1779.
To an ice-cold solution of diol 13 (340 mg, 1.18 mmol) in pyridine
(12 mL) was added acetic anhydride. The reaction mixture was
stirred for 16 h. Volatile organics were removed under reduced
pressure and the residue was taken up in EtOAc and washed with
HCl (0.1 N solution), NaHCO3 (sat. aq. soln.) and brine. The organic
layer was dried over Na2SO4, filtered and concentrated in vacuo. FCC
(hexane/EtOAc 10:0 / 4:6) afforded pure acetate 14 as a colorless
6.1.20. O-((2R,3R,4S)-2-(azidomethyl)-4-(((tert-butoxycarbonyl)
amino)methyl)-4-(methoxymethoxy)tetrahydrofuran-3-yl) 1H-
imidazole-1-carbothioate (18)
To a solution of alcohol 17 (76.0 mg, 0.229 mmol) in toluene
(2.5 mL) was added thiocarbonyldiimidazole (61.3 mg,
0.344 mmol). The solution was heated to reflux overnight. Reaction
mixture was filtered and the filtrate was concentrated in vacuo. The
residue obtained was purified by silica-gel chromatography
(toluene/EtOAc 10:0 / 5:5) to give thiocarbamate 18 as a pale
oil in 78% yield. 1H NMR (300 MHz, CDCl3)
d ppm 1.45 (s, 9 H) 2.14
(s, 3 H) 3.27e3.47 (m, 3 H) 3.62 (dd, J ¼ 13.2, 3.2 Hz, 1 H) 3.69 (d,
J ¼ 9.7 Hz, 1 H) 3.91 (d, J ¼ 9.7 Hz, 1 H) 3.99e4.10 (m, 2 H) 4.83 (d,
yellow oil (65%). 1H NMR (300 MHz, CDCl3)
d ppm 1.45 (s, 9 H)
J ¼ 6.2 Hz, 1 H) 5.33 (br. s., 1 H). 13C NMR (75 MHz, CDCl3)
d
ppm
3.34e3.48 (m, 4 H) 3.56e3.77 (m, 3 H) 3.94e4.03 (m, 2 H)
4.18e4.25 (m, 1 H) 4.71 (d, J ¼ 7.6 Hz, 1 H) 4.74 (d, J ¼ 7.6 Hz, 1 H)
5.25 (t, J ¼ 5.9 Hz, 1 H) 5.82 (d, J ¼ 5.0 Hz, 1 H) 7.03e7.10 (t,
J ¼ 0.9 Hz, 1 H) 7.65 (t, J ¼ 1.3 Hz, 1 H) 8.36 (app. s, 1 H). HRMS (ESI-
TOF) m/z: [MþH]þ Calcd for C17H27N6O6Sþ 443.17073; Found
443.1707.
20.8, 28.3, 46.1, 52.2, 74.6, 74.7, 79.4, 80.4, 81.3, 157.8, 170.5. HRMS
(ESI-TOF) m/z: [MþH]þ Calcd for C13H23N4Oþ6 331.16121; Found
331.1604.
6.1.17. (2R,3R,4S)-2-(azidomethyl)-4-(((tert-butoxycarbonyl)
amino)methyl)-4-(methoxymethoxy)tetrahydrofuran-3-yl acetate
(15)
A solution of compound 14 (0.280 mg, 0.848 mmol) and mo-
lecular sieves in CHCl3 (8.5 mL) was purged with N2 gas. P2O5
(1.55 g; 10.9 mmol) was added as well as dimethoxymethane
(3.86 mL, 43.6 mmol) and the reaction mixture was flushed again.
After 24 h of stirring, the suspension was filtered with CHCl3 and
the filtrate was washed with Na2CO3 (sat. aq. soln.). The organic
layer was dried over Na2SO4, filtered and concentrated in vacuo.
6.1.21. Tert-butyl (((3S,5S)-5-(aminomethyl)-3-(methoxymethoxy)
tetrahydrofuran-3-yl)methyl)carbamate (19)
Thiocarbamate 18 (66.0 mg, 0.149 mmol) was dissolved in
toluene (5 mL) and purged with N2 gas. Azoisobutyronitrile (cata-
lytic quantity, 5 mg) was added and the solution was heated to
85 ꢀC. Tributyltinhydride (0.201 mL, 0.745 mmol) was added and
the reaction mixture was stirred for 3 h at 85 ꢀC, after which volatile
organics were removed under reduced pressure. FCC afforded 19 as
Flash silica-gel chromatography (toluene/EtOAc 10:0
/
6:4)
a colorless oil in 69% yield. 1H NMR (300 MHz, CDCl3)
d ppm 1.45 (s,
afforded title compound 15 as a colorless oil in 27% yield, along
with some left over starting material 14 (colorless oil, 8%) and spiro
9 H) 1.53e1.69 (m, 3 H) 2.14 (dd, J ¼ 13.3, 6.6 Hz, 1 H) 2.73 (dd,
J ¼ 13.2, 6.4 Hz, 1 H) 2.85 (dd, J ¼ 13.2, 3.8 Hz, 1 H) 3.34e3.47 (m,
5 H) 3.78 (d, J ¼ 10.0 Hz, 1 H) 3.86 (d, J ¼ 9.7 Hz, 1 H) 4.08e4.19 (m,
1 H) 4.67e4.78 (m, 2 H) 5.15 (br. s., 1 H). 13C NMR (75 MHz, CDCl3)
compound 16 (colorless oil, 40%). 1H NMR (300 MHz, CDCl3)
d ppm
1.45 (s, 9 H) 2.13 (s, 3 H) 3.32e3.48 (m, 6 H) 3.58 (dd, J ¼ 13.2, 3.8 Hz,
1 H) 3.92 (d, J ¼ 10.3 Hz, 1 H) 3.98 (d, J ¼ 10.3 Hz, 1 H) 4.08 (app. td,
d
ppm 28.5, 38.0, 45.6, 46.1, 55.8, 74.6, 80.7, 92.3, 110.1, 156.5. HRMS
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10
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
(ESI-TOF) m/z: [MþH]þ Calcd for C13H27N2Oþ5 291.19145; Found
colorless liquid in 50% yield. 1H NMR (300 MHz, CDCl3)
d ppm 2.44
291.1919.
(s, 3 H) 2.94 (d, J ¼ 5.0 Hz, 1 H) 3.47e3.65 (m, 5 H) 3.67e3.76 (m,
2 H) 3.92e4.12 (m, 3 H) 4.53 (s, 2 H) 7.27e7.40 (m, 7 H) 7.74e7.83
6.1.22. Tert-butyl (((3S,5S)-5-((2-chlorobenzamido)methyl)-3-
(methoxymethoxy)tetrahydrofuran-3-yl)methyl)carbamate (20)
Compound 19 was subjected to general procedure 1. Colorless
(m, 2 H). 13C NMR (75 MHz, CDCl3)
d ppm 21.8, 44.0, 68.7, 69.8, 70.3,
71.3, 73.7, 79.9, 127.9, 128.09, 128.15, 128.7, 130.1, 132.8, 137.6, 145.2.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C20H26ClO6Sþ 429.11331;
Found 429.1139.
oil, 54%. 1H NMR (300 MHz, CDCl3)
d ppm 1.43 (s, 9 H) 1.71 (dd,
J ¼ 13.5, 9.7 Hz, 1 H) 2.21 (dd, J ¼ 13.5, 6.2 Hz, 1 H) 3.35e3.55 (m,
6 H) 3.73e3.89 (m, 3 H) 4.35 (dtd, J ¼ 9.5, 6.2, 6.2, 3.5 Hz, 1 H) 4.72
(d, J ¼ 7.6 Hz, 1 H) 4.75 (d, J ¼ 7.6 Hz, 1 H) 5.12 (br. s., 1 H) 6.59 (br. s.,
1 H) 7.28e7.43 (m, 3 H) 7.60e7.68 (m, 1 H). 13C NMR (75 MHz,
6.1.26. ((2R,5R)-5-((benzyloxy)methyl)-1,4-dioxan-2-yl)methanol
(24)
To a flask containing compound 23 (0.740 g, 1.73 mmol) was
added an aqueous NaOH solution (1 M, 6 mL, 6 mmol) and the
resulting biphasic mixture was stirred vigorously at room temper-
ature. After 150 min, the reaction mixture was heated to 90 ꢀC for
4 h and then cooled to room temperature again for reaction over-
night (16 h). Then, the mixture was heated to 90 ꢀC and stirred for
2 h. After that, the mixture was neutralized with HCl (1 M) and
extracted with CH2Cl2 (3 ꢂ 15 mL). The combined organic layers
were rewashed with a saturated aqueous solution of NaHCO3 and
brine, then dried over Na2SO4, filtered and concentrated in vacuo.
Silica-gel chromatography (CH2Cl2/MeOH 100:0 / 96:4) afforded
the title compound as a colorless oil in 66% yield. 1H NMR
CDCl3)
d ppm 28.5, 37.9, 43.0, 45.4, 55.9, 75.0, 77.8, 79.7, 87.4, 92.3,
127.2, 130.1, 130.3, 130.8, 131.4, 135.3, 156.3, 167.0. HRMS (ESI-TOF)
m/z: [MþH]þ Calcd for C20H30ClN2Oþ6 429.17869; Found 429.1797.
6.1.23. N-(((2S,4S)-4-(benzamidomethyl)-4-
hydroxytetrahydrofuran-2-yl)methyl)-2-chlorobenzamide (21)
Compound 20 (23.0 mg, 0.054 mmol) was subjected to general
procedure 3, after which the crude aminoalcohol obtained was
taken up in DCM (1 mL). Triethylamine (22.6
mL, 0.162 mmol) and
benzoyl chloride (6.62 L, 0.057 mmol) were added and the reac-
m
tion mixture was stirred overnight at room temperature. Then, the
mixture was washed with HCl (0.1 N solution), NaHCO3 (sat. aq.
soln.) and brine. The organic layer was dried over Na2SO4, filtered
and concentrated in vacuo. FCC (CH2Cl2/MeOH 10:0 / 9:1) affor-
ded 21 as a white foam in 71% yield. 1H NMR (300 MHz, CDCl3)
(300 MHz, CDCl3)
d ppm 2.36 (br. s., 1 H) 3.50e3.86 (m, 10 H)
4.51e4.59 (m, 2 H) 7.23e7.39 (m, 5 H). 13C NMR (75 MHz, CDCl3)
d
ppm 61.0, 63.9, 64.3, 68.2, 72.2, 73.5, 73.8, 127.77, 127.81, 128.5,
137.9. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C13H19O4þ 239.12779;
d
ppm 1.85 (dd, J ¼ 13.2, 9.1 Hz, 1 H) 2.09 (dd, J ¼ 13.0, 6.3 Hz, 1 H)
Found 239.1279.
3.22 (br. s., 1 H) 3.43e3.62 (m, 2 H) 3.68e3.81 (m, 3 H) 3.87 (d,
J ¼ 9.7 Hz, 1 H) 4.33e4.43 (m, 1 H) 6.67 (t, J ¼ 5.7 Hz, 1 H) 7.13 (t,
J ¼ 5.7 Hz, 1 H) 7.27e7.43 (m, 5 H) 7.46e7.53 (m, 1 H) 7.55e7.62 (m,
6.1.27. (2R,5R)-2-(azidomethyl)-5-((benzyloxy)methyl)-1,4-
dioxane (25)
1 H) 7.74e7.82 (m, 2 H). 13C NMR (75 MHz, CDCl3)
d ppm 40.9, 43.2,
48.0, 77.3, 77.7, 81.9, 127.29 (2 C), 128.7, 130.0, 130.4, 130.7, 131.6,
132.1, 133.7, 135.0, 167.3, 169.5. HRMS (ESI-TOF) m/z: [MþH]þ Calcd
for C20H22ClN2Oþ4 389.12626; Found 389.1264.
To a solution of compound 24 (530 mg, 2.22 mmol) and trie-
thylamine (0.618 mL, 4.44 mmol) in CH2Cl2 (12 mL) stirred at 0 ꢀC,
methanesulfonyl chloride (0.205 mL, 2.66 mmol) was added
dropwise. The reaction mixture was allowed to attain ambient
temperature. After 3 h, TLC analysis (hexane/EtOAc 1:1) showed
complete consumption of the starting material. The reaction
mixture was washed with saturated sodium bicarbonate solution
and water. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo to afford the mesylate as a yellow to orange
colored oil. To this crude mesylate, dissolved in DMF (12 mL), was
added sodium azide (0.722 g, 11.1 mmol). After overnight reaction
at 60 ꢀC, TLC analysis (hexane/EtOAc 1:1) showed the presence of
one major product. The solvent was evaporated and the residue was
taken up in EtOAc. The resulting solution was washed with satu-
rated NaHCO3 solution and water. The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. This crude material was
purified by flash column chromatography (hexane/EtOAc
10:0 / 5:5) to afford azide 25 as a colorless liquid (91% over two
6.1.24. (R)-1-(benzyloxy)-3-chloropropan-2-ol (22)
To a flame-dried, three neck round bottomed flask was added
dry 1,2-dichloroethane (45 mL). R-(ꢁ)-epichlorohydrin (3.92 mL,
50.0 mmol) and benzyl alcohol (10.4 mL, 100 mmol) were added
and the flask was purged with N2 gas. The solution was cooled on
ice to 0 ꢀC under N2. The stirring solution was then treated with
boron trifluoro etherate (0.272 mL, 2.20 mmol) and again purged
with N2. The reaction mixture was allowed to attain room tem-
perature overnight (16 h). Subsequently, the mixture was heated to
reflux for 2 h and then allowed to cool and washed with NaHCO3
(sat. aq. soln.). The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. Excess benzyl alcohol was removed under
reduced pressure overnight in an oil bath at 50 ꢀC. FCC (hexane/
EtOAc 10:0 / 7:3) of the residue afforded the title compound as a
colorless liquid in 48% yield. 1H NMR (300 MHz, CDCl3)
d ppm 2.54
steps). 1H NMR (300 MHz, CDCl3)
1 H) 3.47e3.85 (m, 9 H) 4.55 (s, 2 H) 7.21e7.39 (m, 5 H). 13C NMR
d
ppm 3.26 (dd, J ¼ 12.9, 5.0 Hz,
(d, J ¼ 5.9 Hz, 1 H) 3.55e3.69 (m, 4 H) 3.94e4.06 (m, 1 H) 4.56 (s,
2 H) 7.26e7.40 (m, 5 H). 13C NMR (75 MHz, CDCl3)
d ppm 46.2, 70.4,
(75 MHz, CDCl3) d ppm 49.9, 64.0, 64.7, 68.2, 71.9, 72.5, 73.4, 127.71,
127.74, 128.4, 137.8. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
70.9, 73.7, 127.9, 128.1, 128.6, 137.7.
C
13H18N3Oþ4 264.13427; Found 264.1349.
6.1.25. (S)-3-(((R)-1-(benzyloxy)-3-chloropropan-2-yl)oxy)-2-
hydroxypropyl 4-methylbenzenesulfonate (23)
A solution of compound 22 (1.87 g, 9.32 mmol) in dry 1,2-
dichloroethane (40 mL) was purged with N2. S-glycidyl tosylate
(0.710 g, 3.11 mmol) was added and the resulting solution was back-
flushed and cooled on ice to 0 ꢀC. Boron trifluoro etherate (catalytic
6.1.28. N-(((2R,5R)-5-((benzyloxy)methyl)-1,4-dioxan-2-yl)methyl)
benzamide (26)
Compound 25 was subjected to general procedure 2. Colorless
liquid, 90%. 1H NMR (300 MHz, CDCl3)
d
ppm 3.52e3.88 (m, 10 H)
4.56 (s, 2 H) 6.53 (br. s., 1 H) 7.27e7.55 (m, 8 H) 7.72e7.82 (m, 2 H).
13C NMR (75 MHz, CDCl3)
ppm 39.5, 64.2, 65.2, 68.4, 72.0, 72.4,
quantity, 100 mL) was added and the reaction mixture was allowed
to attain room temperature overnight. The mixture was washed
with NaHCO3 (sat. aq. soln.) and brine. The organic layer was dried
over Na2SO4, filtered and concentrated under reduced pressure.
FCC (hexane/EtOAc 10:0 / 4:6) afforded compound 23 as a
d
73.6, 127.1, 127.87, 127.93, 128.6, 128.7, 131.7, 134.5, 138.0, 167.8.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C20H24NOþ4 342.16998;
Found 342.1703.
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
11
6.1.29. N-(((2R,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)
benzamide (27)
6.1.33. Tert-butyl (R)-2-(azidomethyl)morpholine-4-carboxylate
(31)
A solution of benzamide 26 (440 mg, 1.29 mmol) in MeOH
(50 mL) was placed under an N2 atmosphere. Palladium black
(catalytic amount) was added and the reaction vessel was purged
again with N2. Hydrogen gas was bubbled through the solution for
3 h (MS analysis for conversion). The vessel was purged with ni-
trogen gas and the reaction mixture was filtered over a Whatman
fiberglass filter. The filtrate was concentrated in vacuo and the
residue (i.e. the product) was obtained as a colorless oil, quantita-
A solution of compound 30 (1.56 g, 5.07 mmol) in MeOH (50 mL)
was placed under an N2 atmosphere. Palladium black (catalytic
amount) was added and the reaction vessel was purged again with
N2. Hydrogen gas was bubbled through the solution for 3 h. The
vessel was purged with nitrogen gas and the reaction mixture was
filtered over a Whatman fiberglass filter. The filtrate was concen-
trated in vacuo and the residue was subjected to a procedure that is
similar to the one described for 25 and 28. Colorless oil, 81% from
tively. 1H NMR (300 MHz, CDCl3)
d
ppm 3.45e3.84 (m, 11 H) 7.17 (br.
30. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 1.41 (s, 9 H) 2.69 (dd,
s., 1 H) 7.32e7.53 (m, 3 H) 7.70e7.86 (m, 2 H). 13C NMR (75 MHz,
J ¼ 12.9, 10.3 Hz, 1 H) 2.89 (ddd, J ¼ 13.3, 11.3, 3.7 Hz, 1 H) 3.28e3.37
CDCl3)
d ppm 39.2, 60.7, 63.3, 64.9, 71.6, 73.9, 127.0, 128.5, 131.6,
(m, 2 H) 3.37e3.56 (m, 2 H) 3.72 (ddt, J ¼ 13.3, 3.0, 1.7 Hz, 1 H)
134.1, 168.1. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C13H18NOþ4
3.74e3.87 (m, 2 H). 13C NMR (75 MHz, 80 ꢀC, DMSO-d6)
d ppm 27.7,
252.12303; Found 252.1236.
42.8, 45.1, 51.6, 65.2, 73.4, 78.8, 153.6. HRMS (ESI-TOF) m/z: [MþH]þ
Calcd for C10H19N4Oþ3 243.14517; Found 243.1452.
6.1.30. N-(((2R,5R)-5-(azidomethyl)-1,4-dioxan-2-yl)methyl)
benzamide (28)
6.1.34. (R)-(2-(azidomethyl)morpholino)(phenyl)methanone (32)
Compound 31 was subjected to general procedure 4. Pale yellow
oil, 82%. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C12H15N4Oþ2
247.11895; Found 247.1201.
Following a similar procedure described for compound 25,
compound 27 (325 mg,1.29 mmol) gave azide 28 as a pale oil in 69%
yield. 1H NMR (300 MHz, CDCl3)
d ppm 3.27e3.35 (m, 1 H)
3.48e3.59 (m, 1 H) 3.60e3.89 (m, 8 H) 6.76 (t, J ¼ 4.8 Hz, 1 H)
6.1.35. (R)-(2-(azidomethyl)morpholino)(2-chlorophenyl)
methanone (33)
Compound 31 was subjected to general procedure 4. Pale yellow
oil, 79%. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C12H14ClN4Oþ2
281.07998; Found 281.0803.
7.37e7.54 (m, 3 H) 7.75e7.84 (m, 2 H). 13C NMR (75 MHz, CDCl3)
d
ppm 39.4, 50.0, 64.2, 64.7, 72.0, 72.1, 127.0, 128.6, 131.6, 134.2,
167.8. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C13H17N4Oþ3
277.12952; Found 277.1299.
6.1.36. (S)-N-((4-benzoylmorpholin-2-yl)methyl)-2-
chlorobenzamide (34)
6.1.31. N-(((2R,5R)-5-(benzamidomethyl)-1,4-dioxan-2-yl)methyl)-
2-chlorobenzamide (29)
Compound 32 was subjected to general procedure 2. White
Compound 28 was subjected to general procedure 2. White
foam, 62%. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 2.89 (dd,
foam, 74%. 1H NMR (300 MHz, MeOD)
d
ppm 3.47e3.58 (m, 2 H)
3.64e3.89 (m, 8 H) 7.33e7.56 (m, 7 H) 7.79e7.85 (m, 2 H). 13C NMR
(75 MHz, MeOD) ppm 40.5, 40.6, 65.8, 66.0, 73.3, 73.4,128.1,128.3,
J ¼ 12.9, 10.5 Hz, 1 H) 3.01e3.15 (m, 1 H) 3.23e3.43 (m, 2 H)
3.44e3.64 (m, 2 H) 3.78e3.93 (m, 2 H) 3.96e4.23 (br. s., 1 H)
7.17e7.53 (m, 9 H) 8.24 (br. s., 1 H). 13C NMR (75 MHz, 80 ꢀC, DMSO-
d
129.6, 129.9, 131.0, 131.9, 132.2, 132.7, 135.6, 137.6, 170.3, 170.5.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C20H22ClN2Oþ4 389.12626;
Found 389.1264.
d6)
d ppm 40.9, 45.0 (weak), 48.2 (weak), 65.4, 73.6, 126.47, 126.52,
128.0, 128.4, 129.08, 129.13, 129.6, 130.2, 135.4, 136.4, 166.1, 168.9.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C19H20ClN2Oþ3 359.11570;
Found 359.1168.
6.1.32. Tert-butyl (R)-2-((benzyloxy)methyl)morpholine-4-
carboxylate (30)
6.1.37. (S)-N-((4-(2-chlorobenzoyl)morpholin-2-yl)methyl)
benzamide (35)
Benzyl-(R)-glycidyl ether (2.00 g, 12.2 mmol) and NaOH (4.00 g,
100 mmol) in H2O (9.2 mL) and MeOH (3.6 mL) were treated with
2-aminoethyl hydrogen sulphate (7.00 g, 49.59 mmol). The reaction
mixture was stirred for 90 min at 40 ꢀC. The mixture was allowed to
cool to room temperature, toluene (14 mL) and NaOH (2.00 g,
50.0 mmol) were added and then it was stirred overnight at 65 ꢀC.
Toluene (5 mL) and H2O (20 mL) were added and the organic layer
was separated. The water layer was extracted with CH2Cl2
(2 ꢂ 10 mL). The combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo to give crude amine which was
taken up in acetone (20 mL) and H2O (6 mL) at 0 ꢀC. Di-tert-butyl
dicarbonate (2.60 g, 11.9 mmol) was added and the resulting
mixture was stirred vigorously for 2 h. The acetone was removed
under reduced pressure and the aqueous solution was extracted
with CH2Cl2. The organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated in vacuo. FCC (hexane/EtOAc
10:0 / 7:3) afforded the title compound as a colorless oil in 43%
Compound 33 was subjected to general procedure 2. White
foam, 91%. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d
ppm 2.69e4.00
(m, 8 H) 4.26e5.53 (m, 1 H) 7.27e7.95 (m, 9 H) 8.10e8.44 (m, 1 H).
13C NMR (75 MHz, 80 ꢀC, DMSO-d6)
ppm 41.1, 44.0, 48.1 (weak),
d
65.3, 73.9, 126.7, 127.1, 127.6, 127.7, 128.9, 129.0, 130.0, 130.6, 134.2,
135.2, 165.4, 166.3. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
C
19H20ClN2Oþ3 359.11570; Found 359.1158.
6.1.38. Tert-butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)
pyrrolidine-1-carboxylate (37)
4-hydroxyproline 36 (5.00 g, 21.6 mmol) was purged with N2
and cooled on ice to 0 ꢀC. BH3$THF (1 M solution, 64.9 mL,
64.9 mmol) was added dropwise and the solution was stirred for
1 h at 0 ꢀC. When complete, the excess BH3$THF was quenched by
careful addition of water (60 mL). The mixture was extracted with
brine and EtOAc (3 ꢂ 100 mL), the combined organic layers were
dried over Na2SO4, filtered an concentrated in vacuo. FCC (CH2Cl2/
MeOH 92:8) gave the title compound as a thick colorless oil in 96%
(over 2 steps). 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 1.41 (s,
9 H) 2.69 (dd, J ¼ 12.9, 9.4 Hz, 1 H) 2.87 (ddd, J ¼ 13.2, 11.4, 3.5 Hz,
1 H) 3.28e3.56 (m, 4 H) 3.68 (ddt, J ¼ 13.2, 3.0, 1.6 Hz, 1 H)
3.75e3.86 (m, 2 H) 4.50 (s, 2 H) 7.22e7.38 (m, 5 H). 13C NMR
yield. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 1.40 (s, 9 H)
1.74e1.87 (m, 1 H) 1.89e2.01 (m, 1 H) 3.09 (br. s, 1 H) 3.17e3.31 (m,
2 H) 3.40 (app. dd, J ¼ 10.5, 5.9 Hz, 1 H) 3.48 (app. dd, J ¼ 10.5,
3.8 Hz,1 H) 3.74e3.84 (m, 1 H) 4.22 (app. dt, J ¼ 9.2, 4.5 Hz,1 H) 4.46
(75 MHz, 80 ꢀC, DMSO-d6)
d ppm 27.7, 43.0, 45.3, 65.1, 70.2, 72.2,
73.6, 78.7, 126.9, 127.0, 127.7, 138.0, 153.6. HRMS (ESI-TOF) m/z:
[MþH]þ Calcd for C17H26NOþ4 308.18563; Found 308.1867.
(br. s, 1 H). 13C NMR (75 MHz, 80 ꢀC, DMSO-d6)
d ppm 27.9, 36.7,
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12
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
54.6, 57.2, 62.2, 67.7, 77.8, carbonyl peak missing, even at 80 ꢀC.
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C10H20NOþ4 218.13868;
Found 218.1390.
(m, 4 H) 7.80e7.87 (m, 2 H) 8.30 (t, J ¼ 5.3 Hz, 1 H). 13C NMR
(75 MHz, 80 ꢀC, DMSO-d6)
d ppm 18.4, 26.3, 27.7, 32.0, 37.4, 41.3,
50.7, 57.8, 63.3, 77.9, 126.7, 127.26, 127.31, 127.7, 129.2, 130.4, 132.9,
134.6, 153.0, 159.1. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
6.1.39. Tert-butyl (2S,4R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-
4-hydroxypyrrolidine-1-carboxylate (38)
C
34H45N2O4Siþ 573.31431; Found 573.3157.
A solution of hydroxyprolinol 37 (3.00 g, 13.8 mmol) in DMF
(40 mL) was cooled on ice to 0 ꢀC and purged with N2. Imidazole
(1.88 g, 27.6 mmol) and tert-butyl(chloro)diphenyl silane (3.95 mL,
15.2 mmol) were added and the resulting solution was flushed
again. After 16 h, volatile organics were removed under reduced
pressure, the residue was taken up in EtOAc and washed with water
and brine. The organic phase was dried over Na2SO4, filtered and
concentrated in vacuo. FCC (toluene/EtOAc 1:0 / 0:1) yielded the
title product as a colorless oil in 63% yield. 1H NMR (300 MHz, 80 ꢀC,
6.1.42. Tert-butyl (2S,4R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-
4-((2-chlorobenzamido)methyl)pyrrolidine-1-carboxylate (41)
Following a similar procedure described for compound 40,
compound 39 (0.153 g, 0.329 mmol) gave benzamide 41 as a
colorless oil in 60% yield. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 1.03 (s, 9 H) 1.32 (s, 9 H) 1.72e1.89 (m, 1 H) 2.16e2.43 (m,
2 H) 2.92 (app. t, J ¼ 10.3 Hz, 1 H) 3.19e3.41 (m, 2 H) 3.72e3.90 (m,
4 H) 7.28e7.52 (m, 10 H) 7.56e7.67 (m, 4 H) 8.27 (t, J ¼ 5.3 Hz, 1 H).
13C NMR (75 MHz, 80 ꢀC, DMSO-d6)
d ppm 18.4, 26.3, 27.8, 32.0,
DMSO-d6)
d
ppm 1.02 (s, 9 H) 1.33 (s, 9 H) 1.88e2.02 (m, 1 H)
37.3, 41.3, 50.7, 57.8, 63.5, 77.9, 126.5, 127.32, 127.34, 128.4, 129.1,
129.3, 130.1, 132.95, 133.02,134.6,153.0,159.2. HRMS (ESI-TOF) m/z:
[MþH]þ Calcd for C34H44ClN2O4Siþ 607.27534; Found 607.2766.
2.08e2.22 (m, 1 H) 3.33 (app. d, J ¼ 4.1 Hz, 2 H) 3.66e4.00 (m, 3 H)
4.32 (m, 1 H) 4.66 (d, J ¼ 4.1 Hz, 1 H) 7.33e7.67 (m, 10 H). 13C NMR
(75 MHz, 80 ꢀC, DMSO-d6)
d ppm 18.4, 26.3, 27.8, 36.7 (weak), 54.7,
56.7, 64.2 (weak), 67.7, 77.8, 127.28, 127.31, 129.3 (2 C), 132.9, 133.0,
6.1.43. Tert-butyl (2S,4R)-2-(azidomethyl)-4-(benzamidomethyl)
pyrrolidine-1-carboxylate (42)
134.5, 134.6, 153.4. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
C
26H38NO4Siþ 456.25646; Found 456.2375.
To a solution of protected alcohol 40 (0.211 g, 0.368 mmol) in
THF (3.5 mL) was added tetrabutylammonium fluoride solution
(1 M in THF, 0.405 mL, 0.405 mmol). After 3 h, the reaction mixture
was taken up in EtOAc and washed with water (2 ꢂ 10 mL) and the
combined water layers were re-extracted with EtOAc. The organic
layers were combined, dried over Na2SO4, filtered and concentrated
in vacuo. The crude residue was dissolved in CH2Cl2 (3.5 mL) and
cooled on ice to 0 ꢀC. Triethylamine (0.103 mL, 0.736 mmol) and
6.1.40. Tert-butyl (2S,4S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-
4-cyanopyrrolidine-1-carboxylate (39)
To an ice-cold solution of alcohol 38 (0.630 g, 1.38 mmol) in
CH2Cl2 (14 mL) was added triethylamine (0.385 mL, 2.76 mmol) and
mesyl chloride (0.128 mL, 1.66 mmol). The reaction mixture was
allowed to attain room temperature overnight and when finished,
it was washed with a saturated solution of NaHCO3. The organic
phase was dried over Na2SO4, filtered and concentrated in vacuo.
The obtained crude mesylate was taken up in acetonitrile (14 mL)
and tetrabutylammonium cyanide (1.85 g, 6.90 mmol) was added
carefully. The reaction mixture was heated to 65 ꢀC overnight and
then poured into a separating funnel that contained an EtOAc/
hexane 1:4 mixture. This organic phase was washed with a satu-
rated aquatic solution of NaHCO3 and brine, then dried over
Na2SO4, filtered and concentrated in vacuo. FCC of the residue
(hexane/EtOAc 1:0 / 1:1) gave nitrile 39 as a pale yellow oil in 26%
mesyl chloride (34.2 mL, 0.442 mmol) were added. After 3 h, the
reaction mixture was washed with NaHCO3 (sat. aq. soln.) and
brine, then dried over Na2SO4, filtered and concentrated in vacuo.
This residue was taken up in DMF (3.5 mL) and sodium azide
(0.120 g, 1.84 mmol) was added. The reaction mixture was stirred
overnight at 60 ꢀC. Volatile organics were evaporated under
reduced pressure and the residue was taken up in EtOAc, which was
washed with NaHCO3 (sat. aq. soln.) and brine. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. FCC
(toluene/EtOAc 1:0 / 0:1) gave the title azide as a colorless oil in
55% yield. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C18H26N5Oþ3
360.20302; Found 360.2024.
yield. 1H NMR (300 MHz, 80 ꢀC, DMSO-d6)
d ppm 1.03 (s, 9 H) 1.32
(s, 9 H) 2.23e2.34 (m, 1 H) 2.50e2.58 (m, 1 H) 3.27e3.39 (m, 2 H)
3.76e3.95 (m, 4 H) 7.36e7.49 (m, 6 H) 7.58e7.64 (m, 4 H). 13C NMR
(75 MHz, 80 ꢀC, DMSO-d6)
d
ppm 18.4, 25.2, 26.3, 27.6, 31.3, 49.3,
6.1.44. Tert-butyl (2S,4R)-2-(azidomethyl)-4-((2-chlorobenzamido)
methyl)pyrrolidine-1-carboxylate (43)
57.2, 63.5, 78.8, 120.4, 127.4, 129.4, 132.7, 134.6, 152.6. HRMS (ESI-
TOF) m/z: [MþH]þ Calcd for C27H37N2O3Siþ 465.25680; Found
465.2574.
Following a similar procedure described for compound 42,
compound 41 (95.0 g, 0.156 mmol) gave benzamide 43 as a color-
less oil in 55% yield. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
6.1.41. Tert-butyl (2S,4R)-4-(benzamidomethyl)-2-(((tert-
C
18H25ClN5Oþ3 394.16404; Found 394.1632.
butyldiphenylsilyl)oxy)methyl)pyrrolidine-1-carboxylate (40)
To a cooled (0 ꢀC) solution of nitrile 39 (280 mg, 0.603 mmol) in
MeOH (6 mL) was added cobalt(II) chloride (78.0 mg, 0.603 mmol)
and the resulting pink solution was stirred for 10 min. To this
mixture was added NaBH4 (57.0 mg, 1.51 mmol) in portions as to
maintain a black precipitate that appeared following each addition.
After overnight reaction, a saturated solution of NH4Cl (3.5 mL) was
added to quench the reaction and this mixture was stirred for
another hour. The mixture was made alkaline by the addition of
NaHCO3 (sat. aq. soln.), after which the black precipitate was
filtered off. A clear pink solution was obtained, which was extracted
with CH2Cl2. The combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo. The crude amine was subjected
to general procedure 1. Colorless oil, 68%. 1H NMR (300 MHz, 80 ꢀC,
6.1.45. Tert-butyl (2S,4R)-4-(benzamidomethyl)-2-((2-
chlorobenzamido)methyl)pyrrolidine-1-carboxylate (44)
Compound 42 was subjected to general procedure 2. White
foam, 38%. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C25H31ClN3Oþ4
472.19976; Found 472.1992.
6.1.46. Tert-butyl (2S,4R)-2-(benzamidomethyl)-4-((2-
chlorobenzamido)methyl)pyrrolidine-1-carboxylate (45)
Compound 43 was subjected to general procedure 2. White
foam, 66%. HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C25H31ClN3Oþ4
472.19976; Found 472.1996.
6.1.47. N-(((2S,4S)-4-(benzamidomethyl)pyrrolidin-2-yl)methyl)-
2-chlorobenzamide (46)
Compound 44 (29.0 mg, 0.0610 mmol) was dissolved in a 20%
trifluoroacetic acid in CH2Cl2 mixture (2.5 mL) and cooled on ice to
DMSO-d6)
d
ppm 1.00 (s, 9 H) 1.31 (s, 9 H) 1.84 (ddd, J ¼ 12.4, 10.9,
7.8 Hz, 1 H) 2.11e2.44 (m, 2 H) 2.93 (app. t, J ¼ 10.3 Hz, 1 H)
3.24e3.47 (m, 2 H) 3.62e3.90 (m, 4 H) 7.24e7.53 (m, 9 H) 7.55e7.65
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A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
13
0
ꢀC. The mixture was stirred for 3 h and allowed to attain room
Control wells received 50
m
l MilliQ. Wells used to evaluate pre-
temperature. Volatile organics were evaporated under reduced
pressure and the residue was purified via FCC (CH2Cl2/MeOH/
NH4OH 100:0:0.1 / 90:10:0.1). White foam, q. 1H NMR (300 MHz,
treatment received 50 ml of HAM-analogue solution. Bacteria
were allowed to adhere and grow without agitation for 4 h at 37 ꢀC.
After 4 h, medium was removed, and the adhered cells were
washed with sterile physiological saline (0.9% NaCl; PS). After this
DMSO-d6)
d
ppm 1.19e1.41 (m, 1 H) 2.15 (app. dt, J ¼ 13.1, 6.5 Hz,
1 H) 2.42e2.60 (m, 2 H) 2.88 (dd, J ¼ 11.0, 7.5 Hz, 1 H) 3.14 (dd,
washing step, control wells were filled with 50
m
l 2 ꢂ MH and 50
ml
J ¼ 11.0, 8.1 Hz,1 H) 3.25e3.58 (m, 5 H) 7.33e7.57 (m, 7 H) 7.80e7.88
MilliQ. Other wells were filled with 50
ml 2 ꢂ MH and 50 l of HAM
m
(m, 2 H) 8.54e8.65 (m, 2 H). 13C NMR (75 MHz, DMSO-d6)
d
ppm
analogue solution, and the plate was incubated for 20 h at 37 ꢀC. To
evaluate the effect of co-treatment on mature biofilms, control
biofilms were formed in the absence of HAM analogues, as
described above. After 24 h of biofilm formation, the medium was
removed and the wells were rinsed with PS. Control wells were
33.1, 38.3, 42.0, 42.1, 48.8, 58.7,127.1,127.2,128.2,129.0,129.6,129.9,
130.9, 131.1, 134.5, 136.6, 166.5, 166.8. HRMS (ESI-TOF) m/z: [MþH]þ
Calcd for C20H23ClN3Oþ2 372.14733; Found 372.1471.
6.1.48. N-(((3S,5S)-5-(benzamidomethyl)pyrrolidin-3-yl)methyl)-
2-chlorobenzamide (47)
Following a similar procedure described for compound 46,
compound 45 (24.0 g, 0.0508 mmol) gave benzamide 47 as a
colorless oil in quantitative yield. 1H NMR (300 MHz, DMSO-d6)
either filled with 100
50 l antibiotic solution. Wells used to evaluate the effect of pre-
treatment were also filled with 50 l PS and 50 l antibiotic solu-
tion while wells used to evaluate combination treatment were filled
with 50 l of a HAM analogue solution and 50 l antibiotic solution.
ml PS (untreated controls) or with 50 ml PS and
m
m
m
m
m
d
ppm 1.40e1.60 (m, 1 H) 2.25 (app. dt, J ¼ 13.0, 6.6 Hz, 1 H)
The plates were then incubated for an additional 24 h at 37 ꢀC. After
biofilm formation and treatment of the biofilms, the number colony
forming units (CFU) per biofilm were determined by conventional
plating. To collect the cells for plating, plates were rinsed with PS,
sessile cells were removed from the microtiter plate by two cycles
of vortexing (5 min) and sonication (5 min) and the number of CFU/
biofilm was determined by plating the resulting suspensions. The
number of CFU/biofilm (for plating) of the control biofilms was set
to 100% and the results of the treated biofilms were compared to
this. Each condition was tested in at least three wells in each assay,
and each assay was carried out at least in triplicate (n ꢃ 9).
2.54e2.69 (m, 1 H) 3.06 (dd, J ¼ 11.4, 8.8 Hz, 1 H) 3.28e3.49 (m, 4 H)
3.55e3.66 (m, 2 H) 3.70e3.85 (m, 1 H) 7.30e7.64 (m, 7 H) 7.84e7.94
(m, 2 H) 8.64 (t, J ¼ 5.7 Hz, 1 H) 8.89 (t, J ¼ 5.7 Hz, 1 H). 13C NMR
(75 MHz, DMSO-d6)
d ppm 32.6, 37.8, 40.8, 41.4, 48.1, 60.1, 127.5,
127.8, 128.8, 129.2, 130.0, 130.2, 131.2, 132.0, 134.3, 136.6, 167.60
(2 C). HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C20H23ClN3Oþ2
372.14733; Found 372.1473.
6.2. Virtual screening
The open source platform KNIME (version 2.11.0) was used as
interface. The SDF files from the ZINC database Drugs Now were
downloaded in February 2014 and used: http://zinc.docking.org/
subsets/drugs-now.
A substructure search was performed using 2-chloro-N-(2-
methoxyethyl)benzamide as reference fragment. The CDK Sub-
structure search Community Node was used.
6.3.5. Statistical evaluation
The normal distribution of the data was checked by using the
Shapiro-Wilk test. Normally distributed data were analyzed using a
one-way ANOVA. Non-normally distributed data were analyzed
using the Kruskal-Wallis test. Statistics were determined using
SPSS software, version 22.0.
2D-fingerprints were generated with the CDK Fingerprints
community node in KNIME. Lead compound 2 was used as refer-
ence molecule. MACCS Fingerprints were generated and for each
fingerprint, a Tanimoto coefficient (Tc) was generated.
Acknowledgements
The authors gratefully acknowledge funding by the Research
Fund Flanders (FWO) and the Institute for the Promotion of Inno-
vation through Science and Technology in Flanders (IWT-Vlaan-
deren, SBO programme).
The authors would like to thank Izet Karalic,Jolien Claeys,
Jeremy Dierickx and Jolien Scheerlinck for excellent technical
assistance. We thank Alexander Alex for advice and discussion.
6.3. Microbiology
6.3.1. Reagents used
Hamamelitannin (HAM) and vancomycin (VAN) were purchased
from Sigma Aldrich (Bornem, Belgium). HAM was stored in DMSO
at ꢁ20 ꢀC. VAN was dissolved in ultrapure water and stored at 4 ꢀC.
Appendix A. Supplementary data
6.3.2. Strains and culture conditions
Methicillin-resistant Staphylococcus aureus Mu50 (MRSA Mu50)
was cultured in Mueller-Hinton broth (MH, Oxoid, Basingstoke,
England) at 37 ꢀC under aerobic conditions.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.10.056.
References
6.3.3. Determination of the MIC
MICs of HAM analogues used against S. aureus Mu50 were
determined in triplicate using flat-bottom 96-well microtiter plates
(TPP, Trasadingen, Switzerland) as previously described [21].
[1] Tackling drug-resistant infections globally: final report and recommendations
(2016). Available at:http://amr-review.org/sites/default/files/160525_Final%
20paper_with%20cover.pdf.
[2] S.Y.C. Tong, J.S. Davis, E. Eichenberger, T.L. Holland, V.G. Fowler, Staphylococcus
aureus infections: epidemiology, pathophysiology, clinical manifestations, and
management, Clin. Microbiol. Rev. 28 (2015) 603e661.
[3] J.L. Lister, A.R. Horswill, Staphylococcus aureus biofilms: recent developments
in biofilm dispersal, Front. Cell Infect. Microbiol. 4 (2014) 178.
[4] L.J.V. Piddock, The crisis of no new antibiotics - what is the way forward?
Lancet Infect. Dis. 12 (2011) 249e253.
[5] M.D. Kiran, N.V. Adikesavan, O. Cirioni, A. Giacometti, C. Silvestri, G. Scalise,
R. Ghiselli, V. Saba, F. Orlando, M. Shoham, N. Balaban, Discovery of a quorum-
sensing inhibitor of drug-resistant staphylococcal infections by structure-
based virtual screening, Mol. Pharmacol. 73 (2008) 1578e1586.
[6] N. Balaban, T. Goldkorn, Y. Gov, M. Hirshberg, N. Koyfman, H.R. Matthews,
6.3.4. Effect of pretreatment and co-treatment on biofilm
susceptibility
S. aureus Mu50 biofilms were formed and HAM analogues were
evaluated as previously described [7e9]. In brief, overnight cultures
in MH were centrifuged, the pellet was resuspended in double-
concentrated MH (2 ꢂ MH) and diluted to an OD590
of 0.2.
nm
Fifty microliter of the diluted bacterial suspension was transferred
to the wells of a round-bottom 96-well microtiter plate (TPP).
Please cite this article in press as: A. Vermote, et al., Novel hamamelitannin analogues for the treatment of biofilm related MRSA infectionseA
scaffold hopping approach, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.056

14
A. Vermote et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
R.T. Nhan, B. Singh, O. Uziel, Regulation of Staphylococcus aureus pathogenesis
via target of RNAIII-activating protein (TRAP), J. Biol. Chem. 276 (2001)
2658e2667.
A. Offidani, Efficacy of the quorum sensing inhibitor FS10 alone and in com-
bination with tigecycline in an animal model of staphylococcal infected
wound, PLoS One 11 (2016) e0151956.
[7] G. Brackman, K. Breyne, R. De Rycke, A. Vermote, F. Van Nieuwerburgh,
E. Meyer, S. Van Calenbergh, T. Coenye, The quorum sensing inhibitor
hamamelitannin increases antibiotic susceptibility of Staphylococcus aureus
biofilms by affecting peptidoglycan biosynthesis and eDNA release, Sci. Rep. 6
(2016) 20321.
[14] K. Henrick, M. Hirshberg, Structure of the signal transduction protein TRAP
(target of RNAIII-activating protein), Acta Crystallogr. F. 68 (2012) 744e750.
[15] J.J. Irwin, T. Sterling, M.M. Mysinger, E.S. Bolstad, R.G. Coleman, ZINC: a free
tool to discover chemistry for biology, J. Chem. Inf. Model 52 (2012)
1757e1768.
[8] A. Vermote, G. Brackman, M.D.P. Risseeuw, B. Vanhoutte, P. Cos, K. Van Hecke,
K. Breyne, E. Meyer, T. Coenye, S. Van Calenbergh, Hamamelitannin analogues
that modulate quorum sensing as potentiators of antibiotics against Staphy-
lococcus aureus, Angew. Chem. Int. Ed. 55 (2016) 6551e6555.
[9] A. Vermote, G. Brackman, M.D.P. Risseeuw, T. Coenye, S. Van Calenbergh,
Design, synthesis and biological evaluation of novel hamamelitannin ana-
logues as potentiators for vancomycin in the treatment of biofilm related
Staphylococcus aureus infections, Bioorg. Med. Chem. 24 (2016) 4563e4575.
[10] A. Vermote, G. Brackman, M.D.P. Risseeuw, T. Coenye, S. Van Calenbergh,
Novel potentiators for vancomycin in the treatment of biofilm related
Staphylococcus aureus infections via a mix and match approach, ACS Med.
Chem. Lett. Submitt. (2016).
[16] M.I. Simone, A.A. Edwards, G.E. Tranter, G.W.J. Fleet, Carbon-branched d-
tetrahydrofuran sugar amino acids (SAAs) as dipeptide isostere scaffolds,
Tetrahedron Asymmetry 19 (2008) 2887e2894.
[17] Renate M.v. Well, L. Marinelli, K. Erkelens, Gijsbert A. van d. Marel,
A. Lavecchia, Herman S. Overkleeft, Jacques H.v. Boom, H. Kessler,
M. Overhand, Synthesis and structural analysis of cyclic oligomers consisting
of furanoid and pyranoid ε-sugar amino acids, Eur. J. Org. Chem. 2003 (2003)
2303e2313.
[18] K. Fuji, S. Nakano, E. Fujita, An improved method for methoxymethylation of
alcohols under mild acidic conditions, Synthesis 1975 (1975) 276e277.
[19] Boris D. Zlatopolskiy, H.-P. Kroll, E. Melotto, A. de Meijere, Convergent syn-
theses of N-Boc-Protected (2S,4R)-4-(Z)-Propenylproline and 5-Chloro-1-
(methoxymethoxy)pyrrol-2-carboxylic acid ꢁ two essential building blocks
for the signal metabolite hormaomycin, Eur. J. Org. Chem. 2004 (2004)
4492e4502.
[11] Y. Gov, A. Bitler, G. Dell’Acqua, J.V. Torres, N. Balaban, RNAIII inhibiting peptide
(RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and
function analysis, Peptides 22 (2001) 1609e1620.
[12] L. Baldassarre, E. Fornasari, C. Cornacchia, O. Cirioni, C. Silvestri, P. Castelli,
A. Giocometti, I. Cacciatore, Discovery of novel RIP derivatives by alanine
scanning for the treatment of S. aureus infections, MedChemComm 4 (2013)
1114e1117.
[13] O. Simonetti, O. Cirioni, I. Cacciatore, L. Baldassarre, F. Orlando, E. Pierpaoli,
G. Lucarini, E. Orsetti, M. Provinciali, E. Fornasari, A. Di Stefano, A. Giacometti,
[20] A.-S. Messiaen, K. Forier, H. Nelis, K. Braeckmans, T. Coenye, Transport of
nanoparticles and tobramycin-loaded liposomes in Burkholderia cepacia
complex biofilms, PLoS One 8 (2013) e79220.
[21] G. Brackman, P. Cos, L. Maes, H.J. Nelis, T. Coenye, Quorum sensing inhibitors
increase the susceptibility of bacterial biofilms to antibiotics in vitro and
in vivo, Antimicrob. Agents Chemother. 55 (2011) 2655e2661.
Please cite this article in press as: A. Vermote, et al., Novel hamamelitannin analogues for the treatment of biofilm related MRSA infectionseA
scaffold hopping approach, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.056