Monovalent mannose-based DC-SIGN antagonists: Targeting the hydrophobic groove of the receptor

European Journal of Medicinal Chemistry 75 (2014) 308e326

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Monovalent mannose-based DC-SIGN antagonists: Targeting the

hydrophobic groove of the receptor

a

b

a

a,c

ꢀ ꢁ ^a

ꢀ

Tihomir Tomasic , David Hajsek , Urban Svajger , Jernej Luzar , Natasa Obermajer

,

Isabelle Petit-Haertlein ^{d f}, Franck Fieschi ^{d f}, Marko Anderluh ^a

University of Ljubljana, Faculty of Pharmacy, Askerceva 7, SI-1000 Ljubljana, Slovenia

,

e

,

e,

,

*

a

ꢀ

b

c

ꢀ

Blood Transfusion Center of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Slovenia

ꢀ

Jozef Stefan Institute, Department of Biotechnology, Jamova 39, 1000 Ljubljana, Slovenia

^dUniv. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38000 Grenoble, France

^eCNRS, IBS, F-38000 Grenoble, France

^fCEA, IBS, F-38027 Grenoble, France

a r t i c l e i n f o

a b s t r a c t

Article history:

Dendritic cell-speciﬁc, intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a C-type

lectin expressed speciﬁcally on dendritic cells. It is a primary site for recognition and binding of various

pathogens and thus a promising therapeutic target for inhibition of pathogen entry and subsequent

Received 12 September 2013

Received in revised form

17 January 2014

Accepted 19 January 2014

Available online 31 January 2014

prevention of immune defense cell infection. We report the design and synthesis of D-mannose-based

DC-SIGN antagonists bearing diaryl substituted 1,3-diaminopropanol or glycerol moieties incorporated to

target the hydrophobic groove of the receptor. The designed glycomimetics were evaluated by in vitro

assay of the isolated DC-SIGN extracellular domain for their ability to compete with HIV-1 gp120 for

binding to the DC-SIGN carbohydrate recognition domain. Compounds 14d and 14e, that display IC₅₀

Keywords:

Anti-infectives

Carbohydrates

DC-SIGN

DC-SIGN antagonists

Glycomimetics

Lectins

values of 40 mM and 50 mM, are among the most potent monovalent DC-SIGN antagonists reported. The

antagonistic effect of all the synthesized compounds was further evaluated by a one-point in vitro assay

that measures DC adhesion. Compounds 14d, 14e, 18d and 18e were shown to act as functional antag-

onists of DC-SIGN-mediated DC adhesion. The binding mode of 14d was also studied by molecular

docking and molecular dynamics simulation, which revealed ﬂexibility of 14d in the binding site and

provides a basis for further optimization.

1. Introduction

promising approach for preventing sexually transmitted HIV-1 in-

fections [2].

In 2012, the Joint United Nations Programme on HIV/AIDS

(UNAIDS) reported, between 2005 and 2011, a signiﬁcant reduction

in the rate of new HIV infections and a 25% decline in AIDS-related

deaths e the result of increased access of patients to antiretroviral

therapy [1]. Despite this fact, HIV is still one of the leading causes of

morbidity and mortality worldwide. Since HIV-1 infections occur

mainly via sexual transmission through mucosal surfaces, micro-

bicides for topical vaginal or rectal administration constitute a

DC-SIGN (Dendritic Cell-Speciﬁc Intercellular adhesion mole-

cule-3-Grabbing Non-integrin) is a C-type lectin expressed specif-

ically on dendritic cells (DC). Its functions span from acting as an

adhesion molecule that enables DC migration, pathogen/antigen

recognition, internalization and processing, to binding of DC to

CD4^þT cells via ICAM-3 [3]. DC-SIGN recognizes and binds distinct

molecular patterns (PAMPs e Pathogen-Associated Molecular Pat-

terns) of a number of pathogens, including HIV-1 [4]. On binding,

DC-SIGNepathogen complexes are internalized, degraded into

smaller fragments in lysosomes and conjugated with MHC class-II

proteins to initiate a humoral immune response from T cells. DC-

SIGN also activates signal transduction pathways that modulate

DC-maturation status, and alters the DC-cytokine proﬁle by

tailoring toll-like receptor signaling [5].

Abbreviations: CRD, carbohydrate recognition domain; DBU, 1,8-diazabicyclo

[5.4.0]undec-7-ene; DC, dendritic cell; DC-SIGN, dendritic cell-speciﬁc intercel-

lular adhesion molecule-3-grabbing non-integrin; DMF, N,N-dimethylformamide;

ECD, extracellular domain; MD, molecular dynamics; PAMPs, pathogen-associated

molecular patterns; PI, propidium iodide; RMSD, root mean square distance;

TMSOTf, trimethylsilyl triﬂuoromethanesulfonate.

Besides being a gatekeeper of DCs, DC-SIGN is involved in the

initial step of HIV-1 infection, since primary contact of HIV-1 with

immature DCs proceeds through interaction of HIV-1 envelope

* Corresponding author. Tel.: þ386 1 476 96 39; fax: þ386 1 425 80 31.

E-mail address: marko.anderluh@ffa.uni-lj.si (M. Anderluh).

http://dx.doi.org/10.1016/j.ejmech.2014.01.047

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

309

glycoprotein gp120 and DC-SIGN. The DC-SIGNeHIV-1 complex is

quickly endocytosed and most of the virus degraded. However, a

portion of the HIV-1 avoids lytic degradation and escapes the host

immune system [6,7]. DC-SIGN-bound HIV-1 is then transmitted

effectively to recipient CD4^þT cells, which leads to their productive

infection [8]. Thus, inhibition of HIV-1 interaction with DC-SIGN

represents a promising therapeutic approach for inhibition of

viral entry and subsequent infection of immune defense cells [9].

DC-SIGN speciﬁcally recognizes highly mannosylated viral and

bacterial PAMPs (HIV-1 gp120, GP Ebola virus, ManLAM Mycobac-

terium tuberculosis) [7,10] and mannose and fucose-based oligo-

domain, for their ability to compete with HIV-1 gp120 for binding

to DC-SIGN extracellular domain (ECD) and to prevent DC adhesion

to mannan-coated plates, using our recently described assay [30].

2. Results and discussion

2.1. Design

DC-SIGN is a type II transmembrane protein that consists of an

extracellular domain composed of a neck repeat domain, a CRD,

and a transmembrane domain with a short cytosolic tail attached.

The neck region is involved in the tetramerization of the receptor,

which enables multivalent interaction with glycan ligands [13],

while the CRD, which deﬁnes the ligand speciﬁcity, is involved in

ligand binding. The primary carbohydrate-binding site of DC-SIGN

CRD contains octacoordinated Ca^2þthat forms six coordination

bonds with the protein and two with the sugar moiety of the

saccharides such as blood group antigen Lewis^x(Gal

b4[Fuca3]

GlcNAc). It has a higher speciﬁcity for fucose-containing than for

mannose-containing oligosaccharides [11]. It exhibits very weak

afﬁnities for

D

-mannose (K_i¼ 13.1 mM) and -fucose (K_i¼ 6.7 mM)

L

[12,13], but mannose- and fucose-based glycomimetics designed to

inhibit DC-SIGN are reported to have higher afﬁnity [14]. The

challenging task of the design of therapeutically useful DC-SIGN

antagonists can be divided into three approaches e (i) design of

monovalent glycomimetics [15e19], (ii) multimeric presentation of

monosaccharides/oligosaccharides and glycomimetics [9,20e25]

and (iii) discovery of non-carbohydrate ligands [26e29].

binding oligosaccharide. In the case of D-mannose, the equatorial 3-

and 4-hydroxyl groups not only form coordination bonds with

Ca^2þ, but also form hydrogen bonds with the side chains of Glu347

and Glu354, residues that also interact with a Ca^2þion [12]. The

natural disaccharide Mana1-2Man displays two different binding

modes of the core mannose residue in the Ca^2þbinding site [31]. In

the major binding mode, 3- and 4-hydroxyl groups of the reducing

end mannose ring interact with Ca^2þwhile, in the minor mode,

coordination of the Ca^2þoccurs through the same groups of the

non-reducing terminal mannose residue [32]. In contrast to the

multiple binding modes of natural ligands in the Ca^2þbinding site,

it has recently been shown that pseudo-1,2-mannobioside glyco-

mimetic possesses a unique binding mode [19].

Recently, we reported a focused library of pseudomannobioside-

based DC-SIGN antagonists bearing additional hydrophobic groups

designed to target the lipophilic pocket of the receptor. Compounds

inhibited DC-SIGN-mediated adhesion of immature DC to mannan-

coated plates, with IC₅₀values in the lower micromolar range [18].

In the present study, we describe the design, synthesis and bio-

logical evaluation of more ﬂexible

glycomimetics bearing either a 1,3-diaminopropan-2-ol or a glyc-

erol linker between -mannose and different aryl moieties. These

aromatic groups were incorporated in the structure to yield

possible hydrophobic and/or interactions in the hydrophobic

D-mannose-based monovalent

a-D

In the present study, we have designed ligands, based on the D-

mannose sugar that serves as an anchor for binding to the Ca^2þ

pep

binding site of the DC-SIGN CRD. To improve the binding afﬁnity of

groove of the DC-SIGN carbohydrate recognition domain (CRD) and

consequently to increase the binding afﬁnity of the compounds.

Selection of candidate molecules for the synthesis was supported

by molecular modeling. The designed glycomimetics were evalu-

ated, in an in vitro assay on the isolated DC-SIGN extracellular

D

-mannose we have incorporated variously substituted diaryl

moieties that target the hydrophobic groove of the receptor deﬁned

by the side chain of Phe313 (Fig. 1). This amino acid residue pre-

sumably contributes steric hindrance to the binding of linear oli-

gosaccharides, but enables binding and increases afﬁnity of the

Fig. 1. Design of potential D-mannose-based DC-SIGN antagonists bearing aromatic moieties designed to target the hydrophobic groove of DC-SIGN CRD.

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

310

Scheme 1. Reagents and conditions. a) Ac₂O, pyridine, r.t., 24 h; b) hydrazine hydrate, AcOH, N,N-dimethylformamide, 50 ^ꢀC, 24 h; c) Cl₃CCN, DBU, CH₂Cl₂, r.t., 3 h.

branched oligosaccharides. In the crystal structure of DC-SIGN CRD

diaminopropan-2-ol (5) and the corresponding acyl chlorides 6ae

d, while propanols 7eeg were obtained by EDC/HOBt-promoted

coupling of 5 with carboxylic acids 6eeg. Mannosylation of prop-

anols 7aeg with trichloroacetimidate 4 and with trimethylsilyl tri-

in complex with tetramannoside Man₄(PDB entry: 1SL4) Phe313

forms weak hydrophobic interactions with the Man2 residue of

Man₄[12]. Since there are two hydrophobic pockets formed by the

Phe313 phenyl ring, one facing the Ca^2þbinding site and the other

behind the Phe313 side chain, our ligands were designed to target

both of them with the two aryl moieties attached through the

ﬂuoromethanesulfonate (TMSOTf) as promoter yielded

mannosides 8aeg stereospeciﬁcally. Deacetylation using Zemplén

conditions gave the ﬁnal -mannosides 9aeg.

a-D-

a-D

linker to the

D

-mannose core. Aromatic rings have often been used

1,3-Diarylglycerols 12aef were obtained from epichlorohydrin

(10) and corresponding phenols 11aec or naphthols 11def using

sodium ethoxide in absolute ethanol as a base (Scheme 3). Sur-

prisingly, when 2-naphthol (11f) was used as an alcohol, only

compound 12f was isolated as a product, with no expected 1,3-

bis(naphthalene-2-yloxy)propan-2-ol, while in all other cases 1,3-

diaryl-substituted glycerols 12aee were obtained as the main

products.

to improve the binding afﬁnity of glycomimetics targeting DC-SIGN

[14,33] and other lectins [34e36]. Preliminary docking studies us-

ing FlexX [37,38], as available in LeadIT [39], identiﬁed 1,3-

diaminopropanol and glycerol as linkers of suitable length be-

tween D-mannose and aryl moieties, enabling the latter to interact

with the two hydrophobic pockets deﬁned by Phe313 (Fig. 1).

However, the results of docking a small set of the designed ligands

showed comparable binding poses and scoring function scores.

Hence, it was not possible to predict the binding afﬁnity of com-

pounds bearing variously substituted aryl rings. A small library of

diaryl-based 1,3-diaminopropan-2-ol (I, Fig. 1) and glycerol (II and

III, Fig. 1) derivatives was therefore prepared to evaluate our hy-

pothesis and to provide some insight into the preliminary struc-

tureeactivity relationship.

1,2-Diarylglycerols 16aef (Scheme 4) were synthesized from

2,3-dibromopropan-1-ol (15) and phenols 11aef using the same

reaction conditions as for 12aef. Further, glycerols 12aef and 16ae

f were mannosylated with 4 and TMSOTf, and the obtained a-D-

mannosides 13aef and 17aef deprotected using the Zemplén

method to give the ﬁnal compounds 14aef and 18aef. In contrast

to symmetric 1,3-diarylglycerols 12aee, alcohols 12f and 16aef

were obtained as racemic mixtures, which also gave protected

mannosides 13f and 17aef, as well as ﬁnal compounds 14f and

18aef as mixtures of two diastereomers that could not be separated

using the methodology we used for isolating and purifying of the

parent symmetric compounds.

2.2. Chemistry

Synthesis of the designed DC-SIGN antagonists 9aeg (Schemes 1

and 2) started from -mannose (1), which was ﬁrst per-O-acetylated

D

using acetic anhydride in dry pyridine to give compound 2. The

anomeric hydroxyl group of 2 was removed selectively by hydrazine

hydrate in N,N-dimethylformamide (DMF) and the compound 3 thus

obtained was activated with trichloroacetonitrile in the presence of

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base to yield tri-

2.3. Biological activity

The designed mannose-based glycomimetics 9aeg, 14aef and

18aef were evaluated in vitro on isolated DC-SIGN extracellular

domain for their ability to compete with biotin-labeled HIV-1

gp120 for binding to DC-SIGN CRD (Tables 1 and 2). All ﬁnal

chloroacetimidate 4 in the

a conformation only. 2-Hydroxypropan-

1,3-bisamides 7aed (Scheme 2) were synthesized from 1,3-

Scheme 2. Reagents and conditions. a) for 7aed: Et₃N, CH₂Cl₂, 0 ^ꢀC, 1 h; b) for 7eeg: EDC/HOBt, N,N-dimethylformamide, N-methylmorpholine, r.t., 24 h; c) 4, TMSOTf, CH₂Cl₂, 0 ^ꢀC,

then r.t., 24 h; d) NaOMe, MeOH, 30 min, then Amberlite^ÒIR120 H, 15 min.

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

311

Scheme 3. Reagents and conditions. a) NaOEt, EtOH, reﬂux, 24 h; b) 4, TMSOTf, CH₂Cl₂, 0 ^ꢀC, then r.t., 24 h; c) NaOMe, MeOH, 30 min, then Amberlite^ÒIR120 H, 15 min.

Scheme 4. Reagents and conditions. a) NaOEt, EtOH, reﬂux, 24 h; b) 4, TMSOTf, CH₂Cl₂, 0 ^ꢀC, then r.t., 24 h; c) NaOMe, MeOH, 30 min, then Amberlite^ÒIR120 H, 15 min.

compounds were tested in the presence of detergent Tween^Ò-20 to

avoid nonspeciﬁc inhibition due to aggregate formation [40].

extending the molecule by an additional methylene group between

L-

the linker and the phenyl ring, as in the benzyl-based compound 9f,

had a negative effect on afﬁnity (IC₅₀¼ 14.94 mM), although an

additional nitro group on the para position of the benzyl moiety

(9g) increased potency slightly (IC₅₀¼ 6.49 mM). Furthermore, DC-

SIGN ligands were evaluated by a one-point in vitro assay that

measures DC-SIGN-mediated immature dendritic cell adhesion to

mannan-coated plates [18,30]. The results demonstrate that the

synthesized compounds 9aeg inhibit DC adhesion (results repre-

sent DC adhesion percentage, Table 1) only weakly at a concen-

Fucose was used as a positive control in the assay and found to

inhibit the binding of gp120 to DC-SIGN CRD with an IC₅₀value of

2.95 mM (Table 1), in good agreement with its K_ivalue of 6.7 mM

[13]. We have also evaluated the activity of the two known

pseudomannobioside-based DC-SIGN antagonists, IV [17,19] and V

[41] (Table 1). Compounds V and IV have been reported to inhibit

DC-SIGN binding to mannosylated BSA in the SPR assay, with IC₅₀

values of 325

pounds showed comparable potencies of inhibition of gp120

binding to DC-SIGN ECD with IC₅₀values of 420 M and 2760 M,

mM and 1020 mM [17]. In our assay, these two com-

tration of 500 mM. The amide bond between the aromatic moiety

m

and the linker in compounds 9aeg is rigid and may not allow

optimal orientation of the aromatic moieties in the hydrophobic

pocket formed by Phe313. We therefore replaced the 1,3-

diaminopropan-2-ol by a glycerol linker to give more ﬂexible

ethers 14aef and 18aef (Table 2). Compounds 14aef possess a

symmetrical 1,3-diaryloxypropan-2-ol group, in contrast to com-

pounds 18aef with an asymmetric 2,3-diaryloxypropan-1-ol group

that makes them mixtures of two diastereomers. Phenyl-based

compound 14a showed weak activity, with an IC₅₀value of

2.41 mM, while compound 18a, like compound 9a, was devoid of

afﬁnity (IC₅₀¼ 8.64 mM). The effect of the electron donor methoxy

group on the para position of the phenyl ring depends on the

symmetry of the diaryl group. In the case of symmetrical com-

pound 14b (IC₅₀¼ 5.15 mM) there is a negative effect on the afﬁnity,

while compound 18b (IC₅₀¼ 1.01 mM) showed a more than 8-fold

greater potency than its parent compound 18a. The 4-nitrophenyl-

which shows the assay to be suitable for determining the potency

of the newly designed DC-SIGN ligands.

First, a series of amides bearing the 1,3-diaminopropan-2-ol

linker, 9aeg, were synthesized and their activity evaluated on the

isolated DC-SIGN extracellular domain of the receptor (Table 1).

Phenyl-based compound 9a showed a very weak afﬁnity for DC-

SIGN ECD, with an IC₅₀value of 8.79 mM. Incorporation of the

electron withdrawing cyano group (9b) and of the electron donor

methoxy group (9c) on the para position of the phenyl ring

increased binding afﬁnity by approximately 2-fold (IC₅₀values of

4.37 mM and 3.89 mM). The potency was further improved by

replacing the phenyl ring by a 2-naphthyl ring to give compound

9d, found to be the most potent DC-SIGN antagonist of this series

with an IC₅₀value of 1.34 mM. Compound 9e, carrying a pyrrole

moiety, had negligible potency (IC₅₀¼ 13.25 mM). Similarly,

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

312

Table 1

results correlate quite well with the results of the competitive

IC₅₀values of L-fucose, compounds IV, V and 9aeg determined in the isolated DC-

SIGN extracellular domain assay and DC adhesion assay.

gp120-displacement assay, and offer additional evidence that

compounds with a 1,3-diaryloxypropan-2-ol group and large hy-

drophobic moieties attached act as functional antagonists of the

DC-SIGN primary function of adhesion molecule.

The potential cytotoxicity of representative synthetic DC-SIGN

antagonists on target cells was evaluated with a propidium iodide

(PI) uptake assay (see Experimental section) of DC cultures treated

with compounds 9b, 9d, 14d, 14e, 18b or 18f for 24 h. All the

compounds are shown to be non-cytotoxic to DC at 50

Moderate cytotoxicity on DC was observed for compounds 14d and

14e at 100 M, while compounds 9d, 14d, 14e and 18f show cyto-

toxic effects on DC at the highest tested concentration of 250 M.

These results imply that their antagonistic effect on DC-SIGN DC

adhesion could be the consequence of the cytotoxic effect. How-

ever, the cytotoxicity is measured after 24 h while the dendritic cell

adhesion measurement takes no more than 90 min and, during this

period, only negligible cytotoxicity was observed, even at the

highest assayed concentration. We therefore conclude that the

compounds assayed, although cytotoxic at the highest assayed

concentration and during prolonged period, also serve as inhibitors

of DC adhesion.

mM (Fig. 2).

m

Compd

R₁

DC-SIGN IC₅₀[mM]^a

DC adh. [%]^b

L

^cIV

-fucose

/

2.95 ꢁ 0.24

2.76 ꢁ 0.34

/

OMe

^dV

0.42 ꢁ 0.07

/

9a

9b

9c

9d

8.79 ꢁ 0.81

4.37 ꢁ 0.24

3.89 ꢁ 0.18

1.34 ꢁ 0.08

100 ꢁ 0.13

89.6 ꢁ 0.11

95.5 ꢁ 0.24

95.2 ꢁ 0.10

2.4. Molecular modeling

Plausible binding modes of the most potent DC-SIGN antago-

nists to DC-SIGN CRD, 14d, 14e and 18f, were explored, initially

using the molecular docking tool FlexX [37,38] as available in LeadIT

[39]. In the crystal structure of DC-SIGN CRD in complex with Man₄

(PDB entry: 1SL4), the binding site was deﬁned as the area within

ꢂ

7 A from the ligand. In order to predict the binding orientation of

9e

9f

13.25 ꢁ 0.28

95.7 ꢁ 0.22

the discovered antagonists correctly, we have validated the docking

protocol by redocking Man₄in the deﬁned binding site under

pharmacophore type constraints. To place the core mannose res-

idue correctly, the side chain carboxylate groups of Glu347 and

Glu354 were deﬁned as hydrogen bond acceptors that form

hydrogen bonds with 3- and 4-hydroxyl groups of the core

mannose residue in the crystal structure. Further, to account

correctly for complex interactions between Ca^2þand mannose, a

pharmacophore with spherical coordination around Ca^2þwas

additionally deﬁned. The described docking protocol reproduced

the binding of Man₄with an all heavy atom root mean square

14.94 ꢁ 2.26

6.49 ꢁ 0.10

73.0 ꢁ 0.21

81.2 ꢁ 0.16

9g

a

IC₅₀of compounds in the DC-SIGN extracellular domain assay.

% DC adhesion at 0.5 mM of the tested compound.

DC-SIGN antagonist with an IC₅₀value of 1020

b

c

m

M in the SPR competition test

with immobilized Man-BSA [17,19].

ꢂ

d

distance (RMSD) of 3.9 A, which is usually not sufﬁcient for further

DC-SIGN antagonist with IC₅₀value of 325

mM in the SPR competition test with

docking studies. However, the binding mode of the Man1 and Man2

residues of Man₄was reproduced reasonably well with an RMSD

value of 1.6 A. The Man1 and Man2 residues of Man₄form several

immobilized Man-BSA [41].

ꢂ

based compound 18c (IC₅₀¼ 5.73 mM) showed weaker activity

than 18b; the activity of 14c could not be determined because of its

low solubility. The highest concentration of the stock solution that

could be prepared for 14c and for the poorly soluble 1-naphthyl-

based 18d and 7-methoxy-2-naphthyl-based 18e was 0.5 mM and

these compounds showed no activity at this concentration. In

contrast, their analogs 14d and 14e were found to be the most

potent DC-SIGN antagonists designed and synthesized in our li-

brary. Both compounds inhibited gp120 binding to DC-SIGN ECD

with IC₅₀values in the low micromolar range (IC₅₀s for 14d and 14e

interactions with the protein while, in contrast, Man3 and Man4

residues point towards the solvent and only interact weakly with

DC-SIGN CRD. This is why the program was not able to reproduce

the binding of the latter two residues, which resulted in a high

RMSD for Man₄. Since the designed ligands are composed of only

one mannose residue, reproduction of the core mannose binding

mode is the important one. Given that the RMSD value for Man1

ꢂ

was only 0.8 A, we concluded that the docking protocol is suitable

for predicting the binding poses of our novel DC-SIGN antagonists.

Indeed, in all our docking experiments this docking protocol

correctly placed the mannose moiety of the designed ligands in the

deﬁned Ca^2þbinding site.

were 40

mM and 50

mM). 2-Naphthyl-based compound 18f exhibi-

ted modest potency with IC₅₀value of 395

mM, while the mono-2-

naphthyl derivative 14f was inactive. Thus, the dinaphthyl-based

compounds 14d, 14e and 18f are the most potent compounds of

our focused library of mannosides, showing promising afﬁnity for

DC-SIGN CRD, and are among the most potent monovalent

carbohydrate-based DC-SIGN antagonists so far [14]. Compounds

14d, 14e, 18d, and 18e also inhibited DC adhesion at concentrations

The calculated binding modes of DC-SIGN antagonists 14d

(Fig. 2), 14e and 18f are as expected. The D-mannose residues of all

three antagonists bind in a manner consistent with the deﬁned

pharmacophore type constraints, forming hydrogen bonds with the

side chain carboxylates of Glu347 and Glu354 and coordination

bonds with Ca^2þ. In the case of antagonists 14d and (S)-enantiomer

of 500

m

M (presented as DC adhesion percentage, Table 2). These

of 18f, the D-mannose residue is predicted to bind in the same

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

313

Table 2

IC₅₀values of compounds 14aef and 18aef determined in the isolated DC-SIGN extracellular domain assay and the DC adhesion assay.

R₁

Compd

^aDC-SIGN IC₅₀[mM]

^bDC adh. [%]

Compd

^aDC-SIGN IC₅₀[mM]

^bDC adh. [%]

14a

2.41 ꢁ 0.15

102 ꢁ 0.07

18a

8.64 ꢁ 5.77

104 ꢁ 0.06

80.7 ꢁ 0.18

76.2 ꢁ 0.03

14b

14c

5.15 ꢁ 1.72

>0.5

83.3 ꢁ 0.15

59.0 ꢁ 0.14

18b

18c

1.01 ꢁ 0.19

5.73 ꢁ 1.32

14d

14e

0.04 ꢁ 0.03

0.05 ꢁ 0.01

0.904 ꢁ 0.23

7.55 ꢁ 0.11

18d

>0.5

3.73 ꢁ 0.54

18e

18f

>0.5

5.29 ꢁ 0.17

87.6 ꢁ 0.12

0.395 ꢁ 0.08

e

14f

7.16 ꢁ 0.73

80.4 ꢁ 0.09

a

IC₅₀of compounds in the DC-SIGN extracellular domain assay.

% DC adhesion at 0.5 mM of the tested compound.

b

manner as Man1 of Man₄, while the

D

-mannose moiety of 14e is

1-

the starting structure. This was ﬁrst solvated using the TIP3P water

model and then subjected to 8.5 ns molecular dynamics simulation.

There are several parameters that have to be monitored to conﬁrm

the physical stability of a MD simulation, including volume, tem-

perature and total energy. All these parameters of the system were

stable during the production run (Fig. 4aec). The next important

parameter is the RMSD of the backbone atoms that can provide

information about the structural stability of the system during the

MD simulation. It increased gradually during equilibration of the

system and then remained stable during the production run

(Fig. 4d). On the contrary, monitoring the RMSD of the ligand

showed signiﬁcant ﬂuctuations during the equilibration and in the

beginning of the production run, but then remained fairly stable for

rotated by 180^ꢀ, as observed in the case of dimannoside Man

a

2Man in complex with DC-SIGN CRD (PDB entry: 2IT6) [31]. Both

aromatic moieties of 14d, 14e and (S)-18f form hydrophobic and/or

p-p interactions with the Phe313 side chain, while (R)-18f cannot

interact with the protein in the expected binding mode.

Superposition of crystal structures of DC-SIGN CRD in complex

with mannose-based oligosaccharides (PDB entries: 1SL4, 1SL6,

2IT5, 2IT6) [12,31] or glycomimetics (PDB entry: 2XR5) [19] shows

that Phe313 is rather ﬂexible, which has to be considered in the

design of ligands and in molecular modeling studies. We have

therefore studied the binding mode of compound 14d in DC-SIGN

CRD by molecular dynamics simulation. The FlexX-calculated

pose of 14d in complex with DC-SIGN CRD (Fig. 3) was taken as

ꢂ

the last 3 ns of the simulation at around 2.3 A (Fig. 4d). Detailed

analysis of the trajectory revealed that the conformation, as well as

the binding mode, of 14d changed signiﬁcantly from the starting

FlexX-calculated binding pose. While the position and conforma-

tion of the mannose moiety of 14d remained nearly constant during

the simulation, the glycerol moiety with the two naphthalene rings

attached showed much more ﬂexibility and adopted a stable

binding mode towards the end of the simulation. One of the

naphthalene rings of 14d remained in contact with Phe313 most of

the simulation time, while the orientation of the other one changes

and extends towards the groove formed by the Arg345 and Asn362

side chains (Fig. 5). Thus, analysis of the MD trajectory shows that

the D-mannose residue indeed serves as an anchor for the binding

of 14d to DC-SIGN CRD, while the remaining part of the ligand,

designed to interact with Phe313, shows much more ﬂexibility. This

behavior of the ligand in DC-SIGN CRD is probably one of the

Fig. 2. Cytotoxicity of mannose-based DC-SIGN inhibitors 9b, 9d, 14d, 14e, 18b and 18e.

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

314

Fig. 3. FlexX-calculated binding mode of 14d in DC-SIGN CRD. a) Surface representation of DC-SIGN CRD in complex with 14d; b) 2D diagram showing the interactions of 14d in

complex with DC-SIGN CRD residues.

reasons for the afﬁnity being limited to the micromolar level, which

could be improved with the design of conformationally more

constricted analogs of 14d or by strengthening interaction forces by

forming the interactions other than hydrophobic/van der Waals

interactions.

tested in an in vitro assay on the isolated DC-SIGN extracellular

domain for their ability to compete with HIV-1 gp120 for binding to

DC-SIGN CRD. In the series of bisarylamides 9aeg, the 2-naphthyl-

based compound 9d showed the most potent activity, with an IC₅₀

of 1.34 mM. Replacement of the amide bond in 1,3-diaminopropan-

2-ols 9aeg by an ether bond in the more ﬂexible 1,3-diarylglycerols

(14aef) or 2,3-diarylglycerols (18aef) resulted in the most potent

DC-SIGN antagonists 14d and 14e. The 1-naphthyl-based 14d and

the 7-methoxy-2-naphthyl-based 14e inhibited the binding of HIV-

3. Conclusion

We have designed and synthesized a series of

DC-SIGN antagonists bearing two hydrophobic residues on 1,3-

diaminopropan-2-ol or a glycerol linker attached to -mannose in

the conﬁguration. Final compounds 9aeg, 14aef and 18aef were

D-mannose-based

1 gp120 with IC₅₀s of 40 and 50

most potent -mannose-based monovalent DC-SIGN antagonists

reported to date. Of the 2,3-diarylglycerols 18aef, compound 18e,

mM, respectively, and are thus the

D

a

Fig. 4. a) Temperature, b) volume and c) total energy vs time plots during the MD simulation of DC-SIGN CRD in complex with 14d; d) Backbone RMSD (in red) and ligand (14d)

RMSD (in green) vs time plots during the MD simulation of DC-SIGN CRD in complex with 14d. (For interpretation of the references to color in this ﬁgure legend, the reader is

referred to the web version of this article.)

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

315

Fig. 5. Binding mode of the molecular dynamics average structure of 14d to DC-SIGN CRD. a) Surface representation of DC-SIGN CRD in complex with 14d; b) 2D diagram showing

the interactions of 14d in complex with DC-SIGN CRD residues.

bearing a 2-naphthyl moiety, displayed the highest afﬁnity, with an

IC₅₀of 395 M.

warm to room temperature and stirred overnight. Ethyl acetate

(100 mL) was added and the organic phase successively washed

with saturated aqueous NaHCO₃solution (2 ꢂ 80 mL), 1 M HCl

(2 ꢂ 80 mL) and brine (80 mL). The organic phase was dried over

Na₂SO₄, ﬁltered, and the solvent evaporated under reduced pres-

m

The antagonistic effect of all the synthesized compounds was

further evaluated by a one-point in vitro assay that measures DC

adhesion. Compounds 14d, 14e, 18d and 18e were shown to act as

functional antagonists of DC-SIGN-mediated DC adhesion. The

binding modes of antagonists 14d, 14e and 18f, studied by molec-

ular docking, predicted hydrophobic interactions of both aryl sub-

stituents with the side chain of Phe313 and their embedment in the

groove occupied by 3 out of the 4 mannose residues in Man₄tet-

rasaccharide. However, the molecular dynamics simulation of 14d

in complex with DC-SIGN CRD revealed the ﬂexibility of the ligand

in the binding site as well as a ﬁnal binding mode different from

that predicted by docking. The more reliable binding mode of 14d

to DC-SIGN CRD determined by MD simulation provides a starting

point for further optimization towards monovalent glycomimetics

with improved afﬁnity for DC-SIGN.

sure. The compound was obtained (mixture of

¼ 33:67) as a white solid. Yield: 12.699 g (79.0%); colorless oil;

_Dþ24.9 (c 0.23, MeOH); IR (NaCl) 2991, 1740, 1434, 1368, 1207,

1147, 1086, 1048, 971, 786, 685, 600 cm^ꢃ^{1 1}H NMR (400 MHz,

CDCl₃), signals of -anomer, 1.98 (s, 3H, COCH₃), 2.03 (s, 3H,

a and b anomer: a/

b

[a

]

n

;

a

d

COCH₃), 2.07 (s, 3H, COCH₃), 2.15 (s, 3H, COCH₃), 2.16 (s, 3H, COCH₃),

0

3.79 (ddd, 1H, J_5,6¼ 2.4 Hz, J_5,6¼ 5.3 Hz, J_4,5¼ 9.9 Hz, H-5), 4.11 (dd,

0

1H, J_5,6¼ 2.4 Hz, J_6,6¼ 12.4 Hz, H-6), 4.28 (dd, 1H, J_5,6¼ 5.3 Hz,

J_6,6¼ 12.4 Hz, H-6⁰), 5.11 (dd, 1H, J_2,3¼ 3.3 Hz, J_3,4¼ 10.0 Hz, H-3),

0

5.27 (t, 1H, J_3,4;

¼ 10.0 Hz, H-4), 5.46 (dd, 1H, J_1,2¼ 1.2 Hz,

4,5

J_2,3¼ 3.3 Hz, H-2), 5.84 (d, 1H, J_1,2¼ 1.2 Hz, H-1); signals of

b-

anomer, 1.98 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃), 2.08 (s, 3H,

d

COCH₃), 2.15 (s, 3H, COCH₃), 2.19 (s, 3H, COCH₃), 3.99e4.05 (m, 1H,

0

H-5), 4.07 (dd, 1H, J_5,6¼ 2.4 Hz, J_6,6¼ 12.4 Hz, H-6), 4.26 (dd, 1H,

4. Experimental protocols

J_5,6¼ 4.9 Hz, J_6,6¼ 12.4 Hz, H-6⁰), 5.23 (dd, 1H, J_1,2¼ 2.0 Hz,

J_2,3¼ 3.1 Hz, H-2), 5.31e5.34 (m, 2H, H-3, H-4), 6.06 (d, 1H,

J_1,2¼ 2.0 Hz, H-1) ppm.

0

4.1. Chemistry

Dichloromethane was dried over calcium hydride and N,N-

dimethylformamide over activated molecular sieves. All reagents

were used as received from commercial sources without further

puriﬁcation unless otherwise indicated. Analytical TLC was per-

formed on Merck silica gel (60F₂₅₄) plates (0.25 mm) and compo-

nents visualized with staining reagents or ultraviolet light. Flash

column chromatography was carried out on silica gel 60 (particle

size 240e400 mesh). Melting points were determined on a Reichert

hot stage microscope and are uncorrected. ¹H NMR and ¹³C NMR

spectra were recorded at 400 MHz and 100 MHz, respectively, on a

Bruker AVANCE III spectrometer in DMSO-d₆, CDCl₃or CD₃OD so-

lution, with TMS as internal standard at 25 ^ꢀC. Spectra were assigned

using gradient COSY, HSQC and DEPT experiments. IR spectra were

recorded on a PerkineElmer Spectrum BX FT-IR spectrometer. Mass

spectra were obtained using a VGAnalytical Autospec Q mass

spectrometer. All reported yields refer to puriﬁed products. Optical

rotation was measured on a PerkineElmer 241 MC polarimeter. The

reported values for speciﬁc rotation are the average of 5 successive

measurements using an integration time of 5 s.

4.1.2. 2,3,4,6-Tetra-O-acetyl-a-D-mannopyranose (3)

To a solution of hydrazine hydrate (5.03 mL, 56.2 mmol) in N,N-

dimethylformamide (20 mL) acetic acid was added (3.75 mL,

65.5 mmol). A solution of 2 (18.277 g, 46.9 mmol) in N,N-dime-

thylformamide (10 mL) was added and the reaction mixture stirred

at 50 ^ꢀC overnight. The mixture was concentrated in vacuo, then

water (70 mL) was added and extracted with ethyl acetate

(3 ꢂ 100 mL). The organic phase was washed successively with 1 M

HCl (3 ꢂ 100 mL), saturated aqueous NaHCO₃solution (2 ꢂ 100 mL)

and brine (100 mL), dried over Na₂SO₄, ﬁltered and evaporated

under reduced pressure. Yield: 8.801 g (54.0%); colorless oil;

[

a

]

_Dþ13.1 (c 0.25, MeOH); IR (NaCl)

1214, 1163, 1125, 1043, 977, 907, 792, 600 cm^ꢃ1; ¹H NMR (400 MHz,

CDCl₃), 2.02 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃), 2.13 (s, 3H,

n 3446, 2961, 1739, 1434, 1369,

d

COCH₃), 2.19 (s, 3H, COCH₃), 3.35 (d, 1H, J ¼ 4.2 Hz, OH), 4.16 (dt, 1H,

0

J_5,6¼ 2.6 Hz, J_6,6¼ 4.4 Hz, H-6), 4.24e4.30 (m, 2H, H-5, H-6 ), 5.27e

5.35 (m, 3H, H-1, H-2, H-4), 5.45 (dd, 1H, J_2,3¼ 3.4 Hz, J_3,4¼ 10.0 Hz,

H-3) ppm.

4.1.1. 1,2,3,4,6-Penta-O-acetyl-

Acetic anhydride (20 mL, 0.21 mol) was added drop-wise to a

stirred solution of -mannose (4.981 g, 41.5 mmol) in anhydrous

pyridine (25 mL) at 0 ^ꢀC under argon. The mixture was allowed to

D

-mannopyranose (2)

4.1.3. 2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl

trichloroacetimidate (4)

To a stirred solution of 3 (3.600 g, 12.8 mmol) in dry dichloro-

methane (50 mL) were added trichloroacetonitrile (15.4 mL,

D

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

316

0.15 mol) and DBU (0.534 mL, 3.58 mmol). The reaction mixture

was stirred for 3 h at room temperature. The crude product was

then concentrated in vacuo and puriﬁed by ﬂash column chroma-

tography using ethyl acetate/hexane (1:3) as eluent. Yield: 3.518 g

4.1.5. General procedure B. EDC/HOBt-promoted coupling

A solution of 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol),

carboxylic acid (10.56 mmol) and HOBt (1.80 g, 13.34 mmol) in DMF

(8 mL) was prepared and the pH adjusted to 8 with N-methyl-

morpholine. The solution was cooled to 0 ^ꢀC, then EDC$HCl (2.60 g,

13.34 mmol) and dichloromethane (2 mL) were added and the

reaction mixture stirred overnight at room temperature. The sol-

vent was evaporated in vacuo and the oily residue dissolved in ethyl

acetate (50 mL) and washed successively with saturated aqueous

NaHCO3 solution (3 ꢂ 20 mL) and brine (20 mL). The organic phase

was dried over Na₂SO₄, ﬁltered and the solvent evaporated under

reduced pressure.

(65.0%); yellow oil; [

1744, 1678, 1637, 1534, 1434, 1368, 1211, 1155, 1087, 1043, 973, 938,

835, 795, 643, 599, 578 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

2.01 (s,

a

]_Dþ9.3 (c 0.26, MeOH); IR (NaCl)

n 3321, 2941,

d

3H, COCH₃), 2.07 (s, 3H, COCH₃), 2.09 (s, 3H, COCH₃), 2.20 (s, 3H,

0

COCH₃), 4.15e4.22 (m, 2H, H-5, H-6), 4.28 (dd, 1H, J_5,6¼ 4.7 Hz,

0

J_6,6¼ 12.0 Hz, H-6 ), 5.40e5.42 (m, 2H, H-3, H-4), 5.47e5.48 (m, 1H,

H-2), 6.29 (d, 1H, J_1,2¼ 1.8 Hz, H-1), 8.79 (s, 1H, NH) ppm.

4.1.4. General procedure A. Synthesis of bisarylamides

A solution of 1,3-diaminopropan-2-ol (5.55 mmol) and trie-

thylamine (15.8 mmol) in dry dichloromethane (10 mL) was cooled

to 0 ^ꢀC and a solution of acyl chloride (10.56 mmol) added drop-

wise. The reaction mixture was stirred for 1 h and the precipitate

was ﬁltered off.

4.1.5.1. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(1H-pyrrole-2-

carboxamide)

(7e). Prepared from 1,3-diaminopropan-2-ol

(0.500 g, 5.55 mmol) and 1H-pyrrole-2-carboxylic acid (1.173 g,

10.56 mmol) according to the general procedure B. Yield: 1.234 g

(81.2%); yellowish amorphous powder; IR (KBr)

1527, 1407, 1327, 1208, 1130, 1103, 1085, 1040, 996, 837, 772, 743,

604 cm^ꢃ1; ¹H NMR (400 MHz, DMSO-d₆)

n 3300, 1619, 1570,

4.1.4.1. N,N⁰-(2-Hydroxypropane-1,3-diyl)dibenzamide

(7a).

d

3.26 (t, 4H, J ¼ 5.9 Hz, 2ꢂ

Prepared from 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol) and

benzoyl chloride (1.29 mL, 11.1 mmol) according to the general

procedure A. Yield: 0.576 g (34.8%); white powder; mp 160 ^ꢀC; IR

CH₂), 3.69e3.75 (m, 1H, CH), 5.15 (br s, 1H, OH), 6.09 (dd, 2H,

J₁¼ 2.5 Hz, J₂¼ 3.6 Hz, AreH), 6.80 (dd, 2H, J₁¼ 1.4 Hz, J₂¼ 3.6 Hz,

AreH), 6.86 (dd, 2H, J₁¼ 1.4 Hz, J₂¼ 2.5 Hz, AreH), 8.05 (t, 2H,

J ¼ 5.9 Hz, CONH), 11.47 (s, 2H, NH) ppm; MS (ESIþ): m/z (%) ¼ 277

([M þ H]^þ, 100).

(KBr)

1315, 1219, 1182, 1091, 1029, 984, 933, 845, 802, 696, 672 cm^ꢃ1; ¹H

NMR (400 MHz, CDCl₃)

n 3284, 2932, 2874, 1750, 1648, 1622, 1602, 1541, 1489, 1423,

d

3.60e3.73 (m, 4H, 2ꢂ CH₂), 4.02e4.08 (m,

1H, CH), 4.38 (d, 1H, J ¼ 4.2 Hz, OH), 7.33 (t, 2H, J ¼ 6.2 Hz, 2ꢂ

CONH), 7.44e7.57 (m, 6H, AreH), 7.86e7.89 (m, 4H, AreH) ppm;

HRMS (ESIþ): m/z for C₁₇H₁₉N₂O₃([M þ H]^þ): calcd 299.1396;

found 299.1391.

4.1.5.2. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(2-phenylacetamide)

(7f). Prepared from 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol)

and 2-phenylacetic acid (1.513 g, 10.56 mmol) according to the

general procedure B. Yield: 1.454 g (81.2%); white amorphous

powder; IR (KBr)

1356, 1268, 1160, 1124, 1028, 866, 768, 715, 694, 621, 548 cm^ꢃ1; ¹H

NMR (400 MHz, DMSO-d₆) 2.96e3.02 (m, 2H, CHCH₂), 3.07e3.13

(m, 2H, CHCH₂), 3.43 (s, 4H, 2ꢂ COCH₂), 3.51e3.56 (m, 1H, CH), 5.07

(br s, 1H, OH), 7.19e7.31 (m, 10H, AreH), 8.06 (t, 2H, J ¼ 5.7 Hz,

CONH) ppm; MS (ESIþ): m/z (%) ¼ 327 ([M þ H]^þ, 100).

n 3391, 3253, 3083, 1621, 1561, 1493, 1454, 1428,

4.1.4.2. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(4-cyanobenzamide)

(7b). Prepared from 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol)

and 4-cyanobenzoyl chloride (1.785 g,10.56 mmol) according to the

general procedure A. Yield: 0.677 g (36.8%); white amorphous

d

powder; IR (KBr)

1069, 1018, 984, 853, 761, 685, 636, 565, 545 cm^ꢃ1

(400 MHz, DMSO-d₆)

n

3378, 2232, 1636, 1546, 1499, 1288, 1220, 1096,

;

¹H NMR

d

3.25e3.42 (m, 4H, 2ꢂ CH₂), 3.82e3.90 (m,

4.1.5.3. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(2-(4-nitrophenyl)acet-

amide) (7g). Prepared from 1,3-diaminopropan-2-ol (0.500 g,

5.55 mmol) and 2-(4-nitrophenyl)acetic acid (1.913 g, 10.56 mmol)

according to the general procedure B. Yield: 2.260 g (98.0%);

1H, CH), 5.18 (d, 1H, J ¼ 5.2 Hz, OH), 7.95e8.03 (m, 8H, AreH), 8.77

(t, 2H, J ¼ 5.8 Hz, CONH) ppm; MS (ESIþ): m/z (%) ¼ 349 ([M þ H]^þ,

100).

yellowish amorphous powder; IR (KBr)

1255, 1204, 1121, 856, 816, 710, 578 cm^ꢃ1

DMSO-d₆) 2.96e3.02 (m, 2H, CHCH₂), 3.10e3.16 (m, 2H, CHCH₂),

3.51e3.58 (m, 1H, CH), 3.62 (s, 4H, 2ꢂ COCH₂), 7.54 (d, 4H,

J ¼ 8.9 Hz, AreH), 8.16 (d, 4H, J ¼ 8.9 Hz, AreH), 8.26 (t, 2H,

J ¼ 5.6 Hz, CONH) ppm; MS (ESIþ): m/z (%) ¼ 417 ([M þ H]^þ, 100).

n

3291, 1630, 1539, 1352,

;

4.1.4.3. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(4-methoxybenzamide)

(7c). Prepared from 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol)

and 4-methoxybenzoyl chloride (1.802 g, 10.56 mmol) according to

the general procedure A. Crude product was recrystallized from

¹H NMR (400 MHz,

d

ethanol. Yield: 1.715 g (90.0%); white amorphous powder; IR (KBr)

3475, 3304, 2962, 1636, 1551, 1508, 1329, 1305, 1253, 1185, 1114,

1089, 1036, 848, 766, 687 cm^ꢃ^{1 1}H NMR (400 MHz, DMSO-d₆)

n

;

4.1.6. General procedure C. Synthesis of diarylglycerols

d

3.25e3.36 (m, 4H, 2ꢂ CH₂), 3.78e3.81 (m, 7H, CH, 2ꢂ OCH₃), 5.11

To a freshly prepared solution of sodium ethoxide (20 mmol) in

absolute ethanol (50 mL) phenol or naphthol (20 mmol) was added.

After stirring at room temperature for 15 min epichlorohydrin

(10 mmol) was added and the reaction mixture heated under reﬂux

overnight. The solvent was evaporated under reduced pressure, the

crude residue suspended in 2 M NaOH (50 mL) and extracted with

ethyl acetate (3 ꢂ 50 mL). Combined organic phases were washed

with brine (50 mL), dried over Na₂SO₄, ﬁltered and the solvent

removed in vacuo.

(d, 1H, J ¼ 5.0 Hz, OH), 7.00 (d, 4H, J ¼ 8.9 Hz, AreH), 7.85 (d, 4H,

J ¼ 8.9 Hz, AreH), 8.36 (t, 2H, J ¼ 5.7 Hz, CONH) ppm; MS (ESIþ): m/

z (%) ¼ 359 ([M þ H]^þ, 100).

4.1.4.4. N,N⁰-(2-Hydroxypropane-1,3-diyl)bis(2-naphthamide) (7d).

Prepared from 1,3-diaminopropan-2-ol (0.500 g, 5.55 mmol) and 2-

naphthoyl chloride (2.013 g, 10.56 mmol) according to the general

procedure A. Yield: 1.715 g (90.0%); off-white amorphous powder;

IR (KBr)

1205, 1147, 1125, 1015, 955, 915, 871, 839, 809, 780, 764, 734,

670 cm^ꢃ^{1 1}H NMR (400 MHz, DMSO-d₆)

CH₂), 3.91e3.98 (m, 1H, CH), 5.21 (d, 1H, J ¼ 5.1 Hz, OH), 7.57e7.64

(m, 4H, AreH), 7.96e8.03 (m, 8H, AreH), 8.50 (s, 2H, AreH), 8.70 (t,

2H, J ¼ 5.8 Hz, CONH) ppm; MS (ESIþ): m/z (%) ¼ 399 ([M þ H]^þ,

100).

n 3448, 3277, 3056, 1619, 1558, 1503, 1458, 1432, 1338, 1251,

4.1.6.1. 1,3-Diphenoxypropan-2-ol (12a). Prepared from phenol

(0.941 g, 10 mmol) and epichlorohydrin (0.392 mL, 5 mmol) ac-

cording to the general procedure C. The crude product was puriﬁed

by ﬂash column chromatography using dichloromethane/methanol

(60:1) as eluent. Yield: 0.420 g (34.4%); white crystalline solid; mp

;

d

3.37e3.51 (m, 4H, 2ꢂ

79e80 ^ꢀC; IR (KBr)

n 3515, 3058, 2944, 1599, 1587, 1497, 1457, 1443,

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T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

317

1323, 1294, 1244, 1176, 1117, 1084, 1058, 1035, 1013, 992, 904, 829,

815, 755, 691, 637, 575, 509 cm^ꢃ^{1 1}H NMR (400 MHz, DMSO-d₆)

4.1.6.6. 1-Ethoxy-3-(naphthalen-2-yloxy)propan-2-ol

(12f).

Prepared from 2-naphthol (2.880 g, 20.0 mmol) and epichlorohy-

drin (0.783 mL, 10.0 mmol) according to the general procedure C.

The crude product was puriﬁed by ﬂash column chromatography

using dichloromethane as eluent. Yield: 0.150 g (6.2%); brown oil;

;

d

2.68 (d, 1H, J ¼ 5.2 Hz, OH), 4.18 (dd, 2H, J₁¼ 5.0 Hz, J₂¼ 9.2 Hz, 2ꢂ

CH₂eH_A), 4.21 (dd, 2H, J₁¼ 5.0 Hz, J₂¼ 9.2 Hz, 2ꢂ CH₂eH_B), 4.40e

4.47 (m, 1H, CH), 6.96e7.03 (m, 6H, AreH), 7.31e7.36 (m, 4H, AreH)

ppm; ¹³C NMR (100 MHz, CDCl₃)

d

68.6 (2ꢂ CH₂), 68.8 (CH), 114.5,

IR (NaCl)

1183, 1090, 1050, 881, 838, 805, 748 cm^ꢃ1

DMSO-d₆)

n

3340, 2975, 2885, 1925, 1631, 1602, 1381, 1272, 1218,

121.2, 129.5, 158.3 (12ꢂ AreC) ppm. HRMS (ESIþ): m/z for C₁₅H₁₇O₃

;

¹H NMR (400 MHz,

([M þ H]^þ): calcd 245.1178; found 245.1174.

d

1.31 (t, 3H, J ¼ 7.0 Hz, CH₂CH₃), 3.44e3.53 (m, 4H, 2ꢂ

CH₂), 3.97e4.04 (m, 2H, CH₂CH₃), 4.07e4.11 (m, 1H, CH), 5.16 (d, 1H,

J ¼ 5.0 Hz, OH), 7.18 (dd, 1H, J₁¼ 2.6 Hz, J₂¼ 9.0 Hz, AreH), 7.33 (d,

1H, J ¼ 1.5 Hz, AreH), 7.34e7.37 (m, 1H, AreH), 7.46 (ddd, 1H,

J₁¼1.3 Hz, J₂¼ 6.9 Hz, J₃¼ 8.2 Hz, AreH), 7.80e7.84 (m, 3H, AreH)

4.1.6.2. 1,3-Bis(4-methoxyphenoxy)propan-2-ol

(12b). Prepared

from 4-methoxyphenol (2.480 g, 20 mmol) and epichlorohydrin

(0.784 mL, 10 mmol) according to the general procedure C. The

crude product was recrystallized from ethyl acetate/hexane. Yield:

ppm; ¹³C NMR (100 MHz, CDCl₃)

d 15.2 (CH₂CH₃), 67.1 (CH₂CH₃),

0.900 g (14.8%); white crystalline solid; mp 80e82 ^ꢀC; IR (KBr)

n

69.1, 69.2 (2ꢂ CH₂), 71.4 (CH), 106.9, 118.8, 123.8, 126.5, 126.8, 127.7,

3576, 3004, 2947, 1628, 1609, 1516, 1466, 1445, 1389, 1253, 1211,

1166, 1119, 1030, 998, 860, 839, 630 cm^ꢃ^{1 1}H NMR (400 MHz,

CDCl₃)

129.1, 129.5, 134.5, 156.6 (10ꢂ AreC) ppm. HRMS (ESIþ): m/z for

;

C

₁₅H₁₈O₃([M þ H]^þ): calcd 247.1334; found 247.1339.

d

3.69 (s, 6H, 2ꢂ CH₃), 3.94 (dd, 2H, J₁¼ 5.3 Hz, J₂¼ 9.8 Hz,

2ꢂ CH₂-H_A), 4.00 (dd, 2H, J₁¼5.3 Hz, J₂¼ 9.2 Hz, 2ꢂ CH₂-H_B), 4.07e

4.1.7. General procedure D. Synthesis of diarylglycerols

4.13 (m,1H, CH), 6.84e6.91 (m, 8H, AreH) ppm; ¹³C NMR (100 MHz,

To a freshly prepared solution of sodium ethoxide (20 mmol) in

absolute ethanol (50 mL) phenol or naphthol (20 mmol) was added.

After stirring at room temperature for 15 min 2,3-dibromopropan-

1-ol (10 mmol) was added and the reaction mixture heated under

reﬂux overnight. The solvent was evaporated under reduced pres-

sure, the crude residue suspended in 2 M NaOH (50 mL) and

extracted with ethyl acetate (3 ꢂ 50 mL). The combined organic

phases were washed with brine (50 mL), dried over Na₂SO₄, ﬁltered

and the solvent removed in vacuo.

CDCl₃)

d

55.8 (2ꢂ OCH₃), 69.0 (2ꢂ CH₂), 69.5 (CH), 114.7, 115.6,

152.6, 154.2 (12ꢂ AreC) ppm. HRMS (ESIþ): m/z for C₁₇H₂₁O₅

([M þ H]^þ): calcd 305.1389; found 305.1391.

4.1.6.3. 1,3-Bis(4-nitrophenoxy)propan-2-ol (12c). Prepared from 4-

nitroxyphenol (2.783 g, 20 mmol) and epichlorohydrin (0.784 mL,

10 mmol) according to the general procedure C. The crude product

was recrystallized from ethyl acetate/hexane. Yield: 0.233 g (7.0%);

white crystalline solid; mp 122e123 ^ꢀC; IR (KBr)

n

3572, 3115, 1610,

1592, 1509, 1466, 1337, 1301, 1258, 1174, 1027, 942, 848, 751, 690,

668, 652, 629 cm^ꢃ1; ¹H NMR (400 MHz, DMSO-d₆)

4.18e4.72 (m,

5H, CH, 2ꢂ CH₂), 5.63 (d, 1H, J ¼ 4.6 Hz, OH), 7.18e7.22 (m, 4H, Are

H), 8.20e8.24 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

68.3

4.1.7.1. 2,3-Diphenoxypropan-1-ol (16a). Prepared from phenol

(1.881 g, 20.0 mmol) and 2,3-dibromopropan-1-ol (1.03 mL,

10.0 mmol) according to the general procedure D. The crude

product was puriﬁed by ﬂash column chromatography using

dichloromethane/methanol (70:1) as eluent. Yield: 1.187 g (48.6%);

d

(2ꢂ CH₂), 69.2 (CH), 114.6, 126.0, 142.1, 163.1 (12ꢂ AreC) ppm.

HRMS (ESIþ): m/z for C₁₅H₁₅N₂O₇([M þ H]^þ): calcd 335.0879;

found 335.0880.

white crystalline solid; mp 80e82 ^ꢀC; IR (KBr)

n 3515, 3058, 2944,

1598, 1587, 1497, 1456, 1443, 1322, 1294, 1244, 1175, 1117, 1084,

1058, 1035, 1013, 992, 904, 828, 815, 755, 691, 637, 575, 541,

4.1.6.4. 1,3-Bis(naphthalen-1-yloxy)propan-2-ol

(12d).

509 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃)

d

2.66 (d, 1H, J ¼ 5.2 Hz, OH),

Prepared from 1-naphthol (2.516 g,17.5 mmol) and epichlorohydrin

(0.684 mL, 8.73 mmol) according to the general procedure C. The

crude product was puriﬁed by ﬂash column chromatography using

dichloromethane as eluent. Yield: 1.918 g (64.0%); white crystalline

4.15e4.23 (m, 4H, 2ꢂ CH₂), 4.40e4.47 (m, 1H, CH), 6.96e7.03 (m,

6H, AreH), 7.29e7.35 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

CDCl₃)

d

68.7 (2ꢂ CH₂), 68.8 (CH), 114.6,121.3,129.6,158.4 (12ꢂ Are

C) ppm. HRMS (ESIþ): m/z for C₁₅H₁₇O₃([M þ H]^þ): calcd 245.1178;

solid; mp 85e86 ^ꢀC; IR (KBr)

n

3432, 2932, 2880, 1595, 1518, 1456,

1430, 1268, 1239, 1178, 1112, 1070, 947, 899, 826, 770 cm^ꢃ1; ¹H NMR

(400 MHz, CDCl₃)

found 245.1173.

d

4.47 (dd, 2H, J₁¼ 4.5 Hz, J₂¼ 8.8 Hz, 2ꢂ CH₂e

4.1.7.2. 2,3-Bis(4-methoxyphenoxy)propan-1-ol

(16b). Prepared

H_A), 4.51 (dd, 2H, J₁¼ 4.5 Hz, J₂¼ 8.8 Hz, 2ꢂ CH₂eH_B), 4.73e4.80

(m, 1H, CH), 6.93 (dd, 2H, J₁¼ 0.8 Hz, J₂¼ 7.6 Hz, AreH), 7.42 (t, 2H,

J ¼ 7.6 Hz, AreH), 7.48e7.56 (m, 6H, AreH), 7.83e7.86 (m, 2H, Are

H), 8.29e8.32 (m, 2H, AreH) ppm; HRMS (ESIþ): m/z for C₂₃H₂₁O₃

([M þ H]^þ): calcd 345.1491; found 345.1495.

from 4-methoxyphenol (2.483 g, 20.0 mmol) and 2,3-

dibromopropan-1-ol (1.03 mL, 10.0 mmol) according to the gen-

eral procedure D. The crude product was recrystallized from ethyl

acetate/hexane. Yield: 2.756 g (90.7%); white crystalline solid; mp

92e93 ^ꢀC; IR (KBr)

n

3493, 3104, 3098, 2880, 1879, 1512, 1456, 1387,

1294, 1229, 1178, 1116, 1049, 983, 951, 863, 827, 781, 745 cm^ꢃ^{1 1}H

NMR (400 MHz, CDCl₃)

2.64 (d, 1H, J ¼ 5.0 Hz, OH), 3.80 (s, 6H, 2ꢂ

CH₃), 4.08e4.16 (m, 4H, 2ꢂ CH₂), 4.34e4.40 (m, 1H, CH), 6.84e6.92

(m, 8H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

;

4.1.6.5. 1,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-2-ol (12e).

Prepared from 7-methoxy-2-naphthol (2.521 g, 14.5 mmol) and

epichlorohydrin (0.567 mL, 7.24 mmol) according to the general

procedure C. The crude product was recrystallized from ethyl ace-

tate. Yield: 0.816 g (27.9%); white crystalline solid; mp 143e144 ^ꢀC;

d

55.7 (2ꢂ OCH₃),

68.9 (2ꢂ CH₂), 69.5 (CH), 114.7, 115.6, 152.6, 154.2 (12ꢂ AreC) ppm.

HRMS (ESIþ): m/z for C₁₇H₂₁O₅([M þ H]^þ): calcd 305.1389; found

305.1383.

IR (KBr)

1240, 1179, 1122, 1050, 984, 828, 746 cm^ꢃ1

CDCl₃)

n

3493, 2939, 2882, 2837,1513,1467,1456,1442,1388,1294,

;

¹H NMR (400 MHz,

d

3.92 (s, 6H, 2ꢂ OCH₃), 4.33 (dd, 2H, J₁¼ 4.7 Hz, J₂¼ 9.0 Hz,

4.1.7.3. 2,3-Bis(4-nitrophenoxy)propan-1-ol (16c). Prepared from 4-

nitrophenol (2.780 g, 20.0 mmol) and 2,3-dibromopropan-1-ol

(1.03 mL, 10.0 mmol) according to the general procedure D. The

crude product was recrystallized from ethyl acetate/hexane. Yield:

1.543 g (44.8%); off-white crystalline solid; mp 135e136 ^ꢀC; IR (KBr)

2ꢂ CH₂eH_A), 4.36 (dd, 2H, J₁¼ 4.7 Hz, J₂¼ 9.0 Hz, 2ꢂ CH₂eH_B),

4.52e4.59 (m, 1H, CH), 7.02e7.08 (m, 6H, AreH), 7.15 (d, 2H,

J ¼ 2.5 Hz, AreH), 7.69 (d, 2H, J ¼ 5.8 Hz, AreH), 7.71 (d, 2H,

J ¼ 5.8 Hz, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

d

55.3 (2ꢂ

OCH₃), 68.8 (2ꢂ CH₂), 68.9 (CH), 105.3, 106.4, 116.0, 116.4, 124.5,

129.2, 129.3, 135.8, 157.0, 158.3 (20ꢂ AreC) ppm. HRMS (ESIþ): m/z

for C₂₅H₂₅O₅([M þ H]^þ): calcd 405.1702; found 405.1705.

n

3513, 3111, 2936, 1607, 1590, 1506, 1449, 1340, 1299, 1259, 1173,

1112, 1048, 1032, 948, 900, 865, 838, 753, 698, 690, 656, 632,

592 cm^ꢃ^{1 1}H NMR (400 MHz, CDCl₃)

2.69 (s, 1H, OH), 4.29 (dd,

;

d

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

318

2H, J₁¼ 5.0 Hz, J₂¼ 8.9 Hz, 2ꢂ CH₂eH_A), 4.32 (dd, 2H, J₁¼ 4.0 Hz,

J₂¼ 8.9 Hz, 2ꢂ CH₂eH_B), 4.50e4.54 (m, 1H, CH), 7.02e7.06 (m, 4H,

AreH), 8.23e8.27 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

4.1.8.1. 1,3-Dibenzamidopropan-2-yl

2,3,4,6-tetra-O-acetyl-a-D-

mannopyranoside (8a). Prepared from 7a (0.177 g, 0.59 mmol) and

4 (0.449 g, 0.86 mmol) according to the general procedure E. The

crude product was puriﬁed by ﬂash column chromatography using

dichloromethane/methanol (20:1) as eluent. Yield: 0.228 g (61.1%);

d

66.2 (2ꢂ CH₂), 68.5 (CH), 113.6, 124.7, 140.3, 162.6 (12ꢂ AreC)

ppm. HRMS (ESIþ): m/z for C₁₅H₁₅N₂O₇([M þ H]^þ): calcd 335.0879;

found 335.0874.

white crystalline solid; mp 68e70 ^ꢀC; [

(KBr)

1132,1043, 977, 694, 598 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

a

]

_Dþ35.6 (c 0.15, MeOH); IR

3323, 1743, 1643, 1579, 1533, 1489, 1432, 1377, 1217, 1163,

2.04 (s,

n

d

4.1.7.4. 2,3-Bis(naphthalen-1-yloxy)propan-1-ol

(16d).

3H, COCH₃), 2.06 (s, 3H, COCH₃), 2.09 (s, 3H, COCH₃), 2.20 (s, 3H,

COCH₃), 3.27e3.35 (m, 2H, CH₂), 4.04e4.21 (m, 5H, H-5, H-6, CH,

Prepared from 1-naphthol (2.883 g, 20.0 mmol) and 2,3-

dibromopropan-1-ol (1.03 mL, 10.0 mmol) according to the gen-

eral procedure D. The crude product was puriﬁed by ﬂash column

chromatography using dichloromethane/methanol (100:1) as

CH₂), 4.32 (dd, 1H, J_5,6¼ 6.7 Hz, J_6,6¼ 12.8 Hz, H-6⁰), 5.12 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 5.27e5.32 (m, 2H, H-2, H-4), 5.39 (dd, 1H,

J_2,3¼ 3.3 Hz, J_3,4¼ 10.1 Hz, H-3), 7.19e7.22 (m,1H, CONH), 7.40e7.43

(m, 1H, CONH), 7.49e7.59 (m, 6H, AreH), 7.92e7.94 (m, 4H, AreH)

0

eluent. Yield: 1.215 g (35.3%); brown solidiﬁed oil; IR (KBr)

n 3422,

3053, 2931, 1595, 1578, 1508, 1456, 1400, 1267, 1239, 1178, 1102,

ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.9 (4ꢂ COCH₃),

1072, 945, 897, 796, 770, 731, 572 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃)

38.9, 40.24 (2ꢂ CH₂), 62.7 (C-6), 66.2 (C-4), 68.9 (C-3), 69.2 (C-2),

69.65 (C-5), 77.2 (CH), 96.7 (C-1), 127.1, 127.2, 128.79, 131.8, 131.9,

133.6, 133.8 (12ꢂ AreC), 168.1, 168.4 (2ꢂ CONH), 169.9, 170.1, 170.2,

170.7 (4ꢂ CO) ppm; HRMS (ESIꢃ): m/z for C₃₁H₃₅N₂O₁₂([M ꢃ H]^ꢃ):

calcd 627.2190; found 627.2198.

d

2.90 (d, 1H, J ¼ 5.3 Hz, OH), 4.47 (dd, 2H, J₁¼5.0 Hz, J₂¼ 8.9 Hz, 2ꢂ

CH₂eH_A), 4.51 (dd, 2H, J₁¼ 4.3 Hz, J₂¼ 8.9 Hz, 2ꢂ CH₂eH_B), 4.72e

4.79 (m, 1H, CH), 6.93 (dd, 2H, J₁¼ 0.8 Hz, J₂¼ 7.6 Hz, AreH), 7.42 (t,

2H, J ¼ 7.6 Hz, AreH), 7.50e7.56 (m, 6H, AreH), 7.84e7.86 (m, 2H,

AreH), 8.30e8.33 (m, 2H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

d

69.1 (CH), 69.2 (2ꢂ CH₂), 105.1, 121.0, 121.7, 125.4, 125.5, 125.9,

126.6, 127.7, 134.6, 154.1 (20ꢂ AreC) ppm. HRMS (ESIþ): m/z for

4.1.8.2. 1,3-Bis(4-cyanobenzamido)propan-2-yl

2,3,4,6-tetra-O-

C

₂₃H₂₁O₃([M þ H]^þ): calcd 345.1491; found 345.1498.

acetyl- -mannopyranoside (8b). Prepared from 7b (0.423 g,

a-D

1.22 mmol) and 4 (0.500 g, 1.02 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (3:1) as eluent. Yield:

4.1.7.5. 2,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-1-ol (16e).

Prepared from 7-methoxy-2-naphthol (3.480 g, 20.0 mmol) and

2,3-dibromopropan-1-ol (1.03 mL, 10.0 mmol) according to the

general procedure D. The crude product was recrystallized from

ethyl acetate/hexane. Yield: 1.844 g (45.6%); white crystalline solid;

0.510 g (78.5%); white crystalline solid; mp 110 ^ꢀC; [

a

]

þ55.0 (c

D

0.17, MeOH); IR (KBr)

n

3422, 2233, 1752, 1638, 1543, 1374, 1227,

1.99 (s,

1135, 1046, 858, 766, 600 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

d

mp 147e149 ^ꢀC; IR (KBr)

1445, 1389, 1254, 1226, 1187, 1166, 1120, 1039, 998, 860, 839, 816,

629 cm^ꢃ^{1 1}H NMR (400 MHz, DMSO-d₆)

n

3576, 3004, 2947, 1628, 1609, 1516, 1466,

3H, COCH₃), 2.08 (s, 6H, 2ꢂ COCH₃), 2.18 (s, 3H, COCH₃), 3.27e3.37

(m, 2H, CH₂), 3.96e4.08 (m, 3H, H-5, CH₂), 4.12e4.19 (m, 2H, CH, H-

0

;

d

3.84 (s, 6H, 2ꢂ OCH₃),

6), 4.29 (dd, 1H, J_5,6¼ 5.8 Hz, J_6,6¼ 12.1 Hz, H-6⁰), 5.07 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 5.25 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.4 Hz, H-2), 5.29

(d, 1H, J_{3,4; 4,5}¼ 9.8 Hz, H-4), 5.34 (dd, 1H, J_2,3¼ 3.4 Hz, J_3,4¼ 9.8 Hz,

H-3), 7.32e7.36 (m, 1H, CONH), 7.55e7.58 (m, 1H, CONH), 7.79 (d,

4H, J ¼ 7.9 Hz, AreH), 8.02 (dd, 4H, J₁¼ 1.5 Hz, J₂¼ 8.6 Hz, AreH)

4.19 (dd, 2H, J₁¼ 5.8 Hz, J₂¼ 9.9 Hz, 2ꢂ CH₂eH_A), 4.24 (dd, 2H,

J₁¼ 4.7 Hz, J₂¼ 9.9 Hz, 2ꢂ CH₂eH_B), 4.29e4.34 (m, 1H, CH), 5.54 (d,

1H, J ¼ 4.5 Hz, OH), 6.99 (dd, 2H, J₁¼ 2.6 Hz, J₂¼ 8.9 Hz, AreH), 7.04

(dd, 2H, J₁¼ 2.6 Hz, J₂¼ 8.9 Hz, AreH), 7.23 (d, 2H, J ¼ 2.5 Hz, AreH),

7.29 (d, 2H, J ¼ 2.5 Hz, AreH), 7.72 (d, 2H, J ¼ 5.0 Hz, AreH), 7.75 (d,

ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.8, 20.9 (4ꢂ

2H, J ¼ 5.0 Hz, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃)

d

55.3 (2ꢂ

COCH₃), 39.2, 40.4 (2ꢂ CH₂), 62.8 (C-6), 66.2 (C-4), 68.8 (C-3), 69.3

(C-2), 69.5 (C-5), 75.3 (CH), 96.8 (C-1), 115.4, 115.5, 118.0, 127.9,

128.0, 132.5, 137.3, 137.5 (12ꢂ AreC), 166.3, 166.8 (2ꢂ CONH), 169.8,

170.3, 170.4, 170.8 (4ꢂ CO) ppm; HRMS (ESIþ): m/z for C₃₃H₃₅N₄O₁₂

([M þ H]^þ): calcd 679.2251; found 679.2268.

OCH₃), 68.8 (CH, 2ꢂ CH₂), 105.3, 106.5, 116.0, 116.4, 124.5, 129.2,

129.3, 135.8, 157.0, 158.3 (20ꢂ AreC) ppm. HRMS (ESIþ): m/z for

C

₂₅H₂₅O₅([M þ H]^þ): calcd 405.1702; found 405.1699.

4.1.7.6. 2,3-Bis(naphthalen-2-yloxy)propan-1-ol

(16f). Prepared

from 2-naphthol (0.971 g, 6.73 mmol) and 2,3-dibromopropan-1-ol

(0.364 mL, 3.37 mmol) according to the general procedure D. The

crude product was puriﬁed by ﬂash column chromatography using

dichloromethane as eluent. Yield: 0.381 g (32.9%); brown solidiﬁed

4.1.8.3. 1,3-Bis(4-methoxybenzamido)propan-2-yl

2,3,4,6-tetra-O-

acetyl- -mannopyranoside (8c). Prepared from 7c (0.456 g,

a-D

1.22 mmol) and 4 (0.500 g, 1.02 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (3:1) as eluent. Yield:

0.250 g (35.6%); white amorphous powder; mp 103e104 ^ꢀC;

oil; IR (KBr)

1217, 1182, 1118, 1036, 992, 960, 910, 837, 815, 743, 622 cm^ꢃ1

NMR (400 MHz, CDCl₃)

n

3421, 3056, 2930, 1630, 1600, 1510, 1456, 1390, 1258,

;

¹H

d

2.69 (br s, 1H, OH), 4.29e4.40 (m, 4H, 2ꢂ

[

a

]

þ12.0 (c 0.19, MeOH); IR (KBr)

n

3448, 2965, 1752, 1627, 1551,

1508, 1375, 1256, 1178, 1117, 1053, 978, 837, 745, 687, 604 cm^ꢃ1; ¹H

NMR (400 MHz, CDCl₃), 2.04 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃),

D

CH₂), 4.55e4.61 (m,1H, CH), 7.18e7.25 (m, 4H, AreH), 7.37e7.41 (m,

2H, AreH), 7.46e7.50 (m, 2H, AreH), 7.76e7.82 (m, 6H, AreH) ppm;

d

¹³C NMR (100 MHz, CDCl₃)

d

69.0 (CH, 2ꢂ CH₂), 107.0, 118.7, 124.0,

2.08 (s, 3H, COCH₃), 2.20 (s, 3H, COCH₃), 3.22e3.31 (m, 2H, CH₂),

3.89 (2ꢂ s, 6H, 2ꢂ OCH₃), 4.02e4.19 (m, 5H, H-5, H-6, CH, CH₂), 4.31

126.6, 126.9, 127.7, 129.2, 129.7, 134.5, 156.4 (20ꢂ AreC) ppm. HRMS

for C₂₃H₂₁O₃([M þ H]^þ): calcd 345.1491; found 345.1496.

(dd, 1H, J_5,6¼ 6.5 Hz, J_6,6¼ 12.6 Hz, H-6⁰), 5.11 (d, 1H, J_1,2¼ 1.8 Hz,

H-1), 5.27e5.32 (m, 2H, H-2, H-4), 5.38 (dd, 1H, J_2,3¼ 3.4 Hz,

J_3,4¼ 10.0 Hz, H-3), 6.97e7.00 (m, 4H, AreH), 7.17e7.20 (m, 1H,

CONH), 7.36e7.40 (m, 1H, CONH), 7.88e7.91 (m, 4H, AreH) ppm;

0

4.1.8. General procedure E. Glycosylation

To a solution of diarylalcohol (1.2 mmol) and 4 (1 mmol) in dry

dichloromethane (20 mL) under argon trimethylsilyl tri-

ﬂuoromethanesulfonate (TMSOTf) (1.3 mmol) was added at 0 ^ꢀC.

After stirring at 0 ^ꢀC for 30 min the reaction mixture was allowed to

warm to room temperature and stirred overnight. Et₃N (2.6 mmol)

was then added, the solvent removed under reduced pressure and

crude product puriﬁed by ﬂash column chromatography.

¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.8, 20.9 (4ꢂ COCH₃), 41.9

(2ꢂ CH₂), 55.4 (2ꢂ OCH₃), 62.5 (C-6), 66.1 (C-4), 68.4 (C-3), 68.7 (C-

2), 69.0 (C-5), 69.9 (CH), 92.2 (C-1), 113.8, 126.2, 128.8, 128.9 (12ꢂ

AreC), 162.4, 167.8 (2ꢂ CONH), 169.7, 169.9, 170.1, 170.7 (4ꢂ CO)

ppm; HRMS (ESIþ): m/z for C₃₃H₄₁N₂O₁₄([M þ H]^þ): calcd

689.2558; found 689.2545.

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

319

4.1.8.4. 1,3-Bis(2-naphthamido)propan-2-yl 2,3,4,6-tetra-O-acetyl-

a

-

column chromatography using ethyl acetate/hexane (3:1) as eluent.

Yield: 0.190

(25.0%); yellowish powder; mp 140e143 ^ꢀC;

_Dþ17.2 (c 0.03, MeOH); IR (KBr) 3428, 1750, 1655, 1508, 1262,

1172,1039, 859, 763, 645 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

2.05 (s,

3H, COCH₃), 2.09 (s, 3H, COCH₃), 2.10 (s, 3H, COCH₃), 2.17 (s, 3H,

COCH₃), 3.02e3.16 (m, 2H, CH₂), 3.47e3.62 (m, 2H, CH₂), 3.69 (2ꢂ s,

4H, 2ꢂ CH₂Ph), 3.76e3.80 (m, 1H, H-5), 3.98e4.02 (m, 1H, CH), 4.14

D-mannopyranoside (8d). Prepared from 7d (0.485 g, 1.22 mmol)

g

and 4 (0.500 g, 1.02 mmol) according to the general procedure E.

The crude product was puriﬁed by ﬂash column chromatography

using ethyl acetate/hexane (3:1) as eluent. Yield: 0.278 g (38.0%);

[a

]

n

d

white powder; mp 119 ^ꢀC; [

a

]

D

þ46.4 (c 0.13, MeOH); IR (KBr)

n

3443, 1752, 1624, 1543, 1370, 1302, 1223, 1131, 1047, 972, 867, 825,

780 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

d

2.05 (s, 3H, COCH₃), 2.06 (s,

(dd, 1H, J_5,6¼ 2.6 Hz, J_6,6¼ 12.3 Hz, H-6), 4.24 (dd, 1H, J_5,6¼ 5.6 Hz,

0

J_6,6¼ 12.3 Hz, H-6⁰), 4.90 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 5.11 (dd, 1H,

0

3H, COCH₃), 2.07 (s, 3H, COCH₃), 2.21 (s, 3H, COCH₃), 3.39e3.47 (m,

2H, CH₂), 4.17e4.26 (m, 5H, H-5, H-6, CH, CH₂), 4.34 (dd, 1H,

J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2), 5.20 (dd, 1H, J_2,3¼ 3.2 Hz,

J_3,4¼ 10.1 Hz, H-3), 5.26 (t, 1H, J_{3,4; 4,5}¼ 10.1 Hz, H-4), 6.39e6.45 (m,

1H, CONH), 6.57e6.63 (m, 1H, CONH), 7.50 (dd, 4H, J₁¼ 2.0 Hz,

J₂¼ 8.8 Hz, AreH), 8.23 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

J_5,6¼ 5.9 Hz, J_6,6¼ 11.9 Hz, H-6⁰), 5.18 (d, 1H, J_1,2¼ 1.7 Hz, H-1),

0

5.29e5.36 (m, 2H, H-2, H-4), 5.44 (dd, 1H, J_2,3

¼

3.4 Hz,

J_3,4¼ 10.0 Hz, H-3), 7.38e7.42 (m,1H, CONH), 7.56e7.64 (m, 5H, Are

H, CONH), 7.91e7.93 (m, 2H, AreH), 7.96e8.04 (m, 6H, AreH), 8.49

CDCl₃),

d

20.7, 20.8, 20.9 (4ꢂ COCH₃), 39.0, 40.2 (2ꢂ CH₂), 43.1 (2ꢂ

(s, 2H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.8,

CH₂), 62.8 (C-6), 66.2 (C-4), 68.7 (C-3), 69.2 (C-2), 69.5 (C-5), 75.9

(CH), 97.0 (C-1), 124.0, 124.1, 130.1, 130.2, 142.0, 142.1 (12ꢂ AreC),

169.7, 170.1,170.2,170.4,170.5,170.8 (2ꢂ CONH, 4ꢂ CO) ppm; HRMS

(ESIþ): m/z for C₃₃H₃₉N₄O₁₆([M þ H]^þ): calcd 747.2361; found

747.2350.

20.9 (4ꢂ COCH₃), 39.3, 40.5 (2ꢂ CH₂), 62.8 (C-6), 66.2 (C-4), 69.0 (C-

3), 69.3 (C-2), 69.7 (C-5), 75.7 (CH), 96.8 (C-1), 123.6, 123.7, 126.8,

127.7, 127.8, 128.0, 128.6, 129.1, 129.2, 130.9, 131.0, 132.7, 134.9, 135.0

(20ꢂ AreC), 168.2, 168.68 (2ꢂ CONH), 169.7, 170.3, 170.8 (4ꢂ CO)

ppm; HRMS (ESIþ): m/z for C₃₉H₄₁N₂O₁₂([M þ H]^þ): calcd

729.2660; found 729.2662.

4.1.8.8. 1,3-Diphenoxypropan-2-yl 2,3,4,6-tetra-O-acetyl-a-D-man-

nopyranoside (13a). Prepared from 12a (0.222 g, 0.91 mmol) and 4

(0.395 g, 0.76 mmol) according to the general procedure E. The

crude product was puriﬁed by ﬂash column chromatography using

ethyl acetate/hexane (1:2) as eluent. Yield: 0.196 g (42.0%); yellow

4.1.8.5. 1,3-Bis(1H-pyrrole-2-carboxamido)propan-2-yl 2,3,4,6-tetra-

O-acetyl-a-D-mannopyranoside (8e). Prepared from 7e (0.337 g,

1.22 mmol) and 4 (0.500 g, 1.02 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (3:1) as eluent. Yield:

oil; [a

]

_Dþ36.3 (c 0.20, MeOH); ¹H NMR (400 MHz, CDCl₃),

d 2.01 (s,

3H, COCH₃), 2.06 (s, 3H, COCH₃), 2.07 (s, 3H, COCH₃), 2.20 (s, 3H,

0

0.155 g (25.6%); yellowish amorphous powder; IR (KBr)

2957, 1751, 1636, 1560, 1526, 1371, 1325, 1230, 1133, 1046, 980, 747,

602 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

2.04 (s, 3H, COCH₃), 2.07 (s,

3H, COCH₃), 2.08 (s, 3H, COCH₃), 2.19 (s, 3H, COCH₃), 3.30e3.39 (m,

2H, CH₂), 3.89e3.99 (m, 3H, H-5, CH₂), 4.12e4.20 (m, 3H, H-6, CH),

n

3399,

COCH₃), 4.06 (dt, 1H, J_5,6¼ 2.0 Hz, J_6,6¼ 3.9 Hz, H-6), 4.16e4.25 (m,

4H, CH₂CHCH₂), 4.31e4.37 (m, 2H, H-5, H-6⁰), 4.45e4.51 (m, 1H,

CH₂CHCH₂), 5.23 (d, 1H, J_1,2¼ 1.8 Hz, H-1), 5.32 (dd, 1H, J_1,2¼ 1.8 Hz,

J_2,3¼ 3.2 Hz, H-2), 5.35 (d, 1H, J_3,4¼ 10.0 Hz, H-4), 5.39 (dd, 1H,

J_2,3¼ 3.2 Hz, J_3,4¼ 10.0 Hz, H-3), 6.92e6.95 (m, 4H, AreH), 6.98e

7.03 (m, 2H, AreH), 7.30e7.35 (m, 4H, AreH) ppm; ¹³C NMR

d

4.30 (dd, 1H, J_5,6¼ 5.8 Hz, J_6,6¼ 11.8 Hz, H-6⁰), 5.08 (d, 1H,

J_1,2¼ 1.8 Hz, H-1), 5.26e5.30 (m, 2H, H-2, H-4), 5.38 (dd, 1H,

J_2,3¼ 3.4 Hz, J_3,4¼ 10.0 Hz, H-3), 6.27e6.30 (m, 2H, AreH), 6.78e

6.83 (m, 2H, AreH), 6.95e6.98 (m, 3H, 2ꢂ AreH, CONH), 7.08e7.11

(m,1H, CONH) ppm; HRMS (ESIþ): m/z for C₂₇H₃₅N₄O₁₂([M þ H]^þ):

calcd 607.2251; found 607.2237.

0

(100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 62.3 (C-6), 66.0 (C-4),

67.1, 67.5 (2ꢂ CH₂), 68.7 (C-3), 69.0 (C-2), 69.7 (C-5), 75.0 (CH), 97.3

(C-1), 114.4, 114.6, 121.3, 121.4, 129.6, 129.7, 158.2, 158.3 (12ꢂ AreC),

169.8, 169.9, 170.0, 170.7 (4ꢂ CO) ppm; HRMS (ESIþ): m/z for

C

₂₉H₃₄O₁₂Na ([M þ Na^þ]^þ): calcd 597.1948; found 597.1946.

4.1.8.6. 1,3-Bis(2-phenylacetamido)propan-2-yl

2,3,4,6-tetra-O-

4.1.8.9. 1,3-Bis(4-methoxyphenoxy)propan-2-yl

2,3,4,6-tetra-O-

acetyl- -mannopyranoside (8f). Prepared from 7f (0.397 g,

a

-

D

acetyl- -mannopyranoside (13b). Prepared from 12b (0.350 g,

a-D

1.22 mmol) and 4 (0.500 g, 1.02 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (3:1) as eluent. Yield:

1.15 mmol) and 4 (0.500 g, 0.96 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

0.400 g (60.0%); white powder; mp 112e113 ^ꢀC; [

a]

_Dþ22.7 (c 0.15,

3422, 1752, 1662, 1261, 1172, 1037, 849, 763, 732,

642, 518 cm^ꢃ1; ¹H NMR (400 MHz, CDCl₃),

0.244 g (33.4%); yellow oil; [

3349, 2974, 1756, 1508, 1379, 1231, 1089, 1049, 881 cm^ꢃ1

(400 MHz, DMSO-d₆), 1.94 (s, 3H, COCH₃),1.97 (s, 3H, COCH₃), 2.02

a]

þ35.9 (c 0.20, MeOH); IR (NaCl)

n

D

MeOH); IR (KBr)

n

;

¹H NMR

d

2.04 (s, 6H, 2ꢂ COCH₃),

d

2.10 (s, 3H, COCH₃), 2.17 (s, 3H, COCH₃), 3.03e3.13 (m, 2H, CH₂),

3.43e3.53 (m, 2H, CH₂), 3.60 (2ꢂ s, 4H, 2ꢂ CH₂Ph), 3.74 (td, 1H,

(s, 3H, COCH₃), 2.13 (s, 3H, COCH₃), 3.70 (s, 6H, 2ꢂ OCH₃), 3.98 (dd,

0

1H, J_5,6¼ 2.6 Hz, J_6,6¼ 12.2 Hz, H-6), 4.09e4.24 (m, 6H, CH₂CHCH₂,

J_5,6¼ 4.9 Hz, J_4,5¼ 10.0 Hz, H-5), 3.86e3.91 (m, 1H, CH), 4.04 (dd,

H-5, H-6⁰), 4.29e4.34 (m,1H, CH₂CHCH₂), 5.09e5.17 (m, 3H, H-2, H-

3, H-4), 5.24 (d, 1H, J_1,2¼ 1.1 Hz, H-1), 6.85e6.94 (m, 8H, AreH)

0

1H, J_5,6¼ 2.6 Hz, J_6,6¼ 12.1 Hz, H-6), 4.15 (dd, 1H, J_5,6¼ 4.9 Hz,

J_6,6¼ 12.1 Hz, H-6⁰), 4.87 (d, 1H, J_1,2¼ 1.8 Hz, H-1), 5.10 (dd, 1H,

ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 55.7 (2ꢂ

0

J_1,2¼ 1.8 Hz, J_2,3¼ 3.0 Hz, H-2), 5.17e5.23 (m, 2H, H-3, H-4), 6.17e

6.23 (m, 1H, CONH), 6.31e6.35 (m, 1H, CONH), 7.29e7.40 (m, 10H,

OCH₃), 62.3 (C-6), 66.0 (C-4), 68.1, 68.3 (2ꢂ CH₂), 68.7 (C-2), 69.0 (C-

3), 69.7 (C-5), 75.2 (CH), 97.3 (C-1), 114.7, 114.8, 115.4, 115.7, 152.4,

152.5, 154.2, 154.3 (12ꢂ AreC), 169.8, 169.9, 170.0, 170.7 (4ꢂ CO)

ppm; HRMS (ESIþ): m/z for C₃₁H₃₉O₁₄([M þ H]^þ): calcd 635.2340;

found 635.2352.

AreH) ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.9, 21.1 (4ꢂ

COCH₃), 39.0, 40.1 (2ꢂ CH₂), 43.6, 43.7 (2ꢂ CH₂), 62.7 (C-6), 66.2 (C-

4), 68.8 (C-3), 69.1 (C-2), 69.5 (C-5), 76.2 (CH), 96.9 (C-1), 127.3,

127.4, 129.0, 129.1, 129.3, 134.7 (12ꢂ AreC), 169.7, 170.1, 170.6, 171.2,

172.0, 172.2 (2ꢂ CONH, 4ꢂ CO) ppm; HRMS (ESIþ): m/z for

4.1.8.10. 1,3-Bis(4-nitrophenoxy)propan-2-yl 2,3,4,6-tetra-O-acetyl-

a-D-mannopyranoside (13c). Prepared from 12c (0.200 g,

C

₃₃H₄₁N₂O₁₂([M þ H]^þ): calcd 657.2660; found 657.2678.

0.60 mmol) and 4 (0.284 g, 0.54 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

4.1.8.7. 1,3-Bis(2-(4-nitrophenyl)acetamido)propan-2-yl 2,3,4,6-

tetra-O-acetyl- -mannopyranoside (8g). Prepared from 7g

a-D

(0.507 g, 1.22 mmol) and 4 (0.500 g, 1.02 mmol) according to the

general procedure E. The crude product was puriﬁed by ﬂash

0.260 g (72.0%); %); white solid; mp 110e111 ^ꢀC; [

a

]

_Dþ28.0 (c 0.23,

MeOH); IR (KBr)

n 3375, 2938, 1756, 1594, 1508, 1457, 1369, 1340,

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

320

1256, 1175, 1113, 1041, 1011, 979, 862, 848, 754, 692, 676, 650 cm^ꢃ1

¹H NMR (400 MHz, DMSO-d₆),

1.92 (s, 3H, COCH₃), 1.98 (s, 3H,

COCH₃), 2.01 (s, 3H, COCH₃), 2.12 (s, 3H, COCH₃), 3.99 (dd, 1H,

;

chromatography using ethyl acetate/hexane (1:3) as eluent. Com-

pound was obtained as a mixture of two diastereomers in 1:1

d

ration. Yield: 0.250 g (41.7%); yellow solid; mp 74e75 ^ꢀC; [

a

]

_Dþ55.7

3460, 2934, 1751, 1628, 1601, 1507, 1375,

1219, 1184, 1054, 983, 851, 750, 694, 624, 597 cm^ꢃ^{1 1}H NMR

(400 MHz, CDCl₃)

1.21e1.26 (m, 6H, 2ꢂ CH₂CH₃), 2.01e2.02 (2ꢂ s,

0

J_5,6¼ 2.6 Hz, J_6,6¼ 12.2 Hz, H-6), 4.13e4.17 (m, 2H, H-5, H-6 ), 4.41e

4.49 (m, 5H, CH₂CHCH₂, CH₂CHCH₂), 5.05 (dd, 1H, J_2,3¼ 3.4 Hz,

J_3,4¼ 10.2 Hz, H-3), 5.09e5.12 (m, 2H, H-2, H-4), 5.29 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 7.22e7.24 (m, 4H, AreH), 8.21e8.26 (m, 4H, Are

(c 0.30, MeOH); IR (KBr) n

;

d

9H, 3ꢂ CH₃), 2.04 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.13 (s, 3H, CH₃),

2.20 (s, 6H, 2ꢂ CH₃), 3.54e3.60 (m, 4H, 2ꢂ CH₂CH₃), 3.62e3.69 (m,

4H), 4.06e4.24 (m, 6H), 4.29e4.43 (m, 6H, 2ꢂ CH), 5.20 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 5.25 (d, 1H, J_1,2¼ 1.7 Hz, H-1⁰), 5.32e5.45 (m, 6H),

7.12e7.19 (m, 4H, AreH), 7.34e7.39 (m, 2H, AreH), 7.44e7.49 (m,

2H, AreH), 7.74e7.80 (m, 6H, AreH) ppm; HRMS (ESIþ): m/z for

H) ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.7, 20.8, 20.9 (4ꢂ COCH₃),

62.4 (C-6), 66.0 (C-4), 67.5, 67.9 (2ꢂ CH₂), 68.6 (C-3), 69.1 (C-2), 69.6

(C-5), 74.5 (CH), 97.4 (C-1), 114.5, 114.7, 126.0, 126.1, 142.2, 162.9

(12ꢂ AreC), 169.7, 170.0, 170.1, 170.6 (4ꢂ CO) ppm; HRMS (ESIþ):

m/z for C₂₉H₃₂N₂O₁₆Na ([M þ Na^þ]^þ): calcd 687.1650; found

687.1652.

C

₂₉H₃₇O₁₂([M þ H]^þ): calcd 577.2285; found 577.2275.

4.1.8.11. 1,3-Bis(naphthalen-1-yloxy)propan-2-yl

2,3,4,6-tetra-O-

4.1.8.14. 2,3-Diphenoxypropan-1-yl 2,3,4,6-tetra-O-acetyl-a-D-man-

acetyl- -mannopyranoside (13d). Prepared from 12d (0.643 g,

a

-

D

nopyranoside (17a). Prepared from 16a (0.332 g, 1.36 mmol) and 4

(0.592 g, 1.13 mmol) according to the general procedure E. The

crude product was puriﬁed by ﬂash column chromatography using

ethyl acetate/hexane (1:2) as eluent. Yield: 0.154 g (20.0%); yellow

1.87 mmol) and 4 (0.815 g, 1.56 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

0.377 g (34.0%); yellow solid; mp 78 ^ꢀC; [

IR (KBr)

1226, 1137, 1046, 980, 793, 772, 599, 571 cm^ꢃ1; ¹H NMR (400 MHz,

a

]

_Dþ26.6 (c 0.23, MeOH);

oil; [

1587, 1496, 1456, 1369, 1218, 1174, 1218, 1174, 1134, 1042, 980, 910,

825, 754, 691, 598 cm^ꢃ^{1 1}H NMR (400 MHz, DMSO-d₆),

1.93 (s,

a]

þ7.3 (c 0.30, MeOH); IR (NaCl)

n 3446, 2936, 1742, 1599,

D

n

3463, 3054, 2937, 1751, 1596, 1581, 1508, 1458, 1395, 1369,

;

d

CDCl₃),

d

1.99 (s, 6H, 2ꢂ COCH₃), 2.01 (s, 3H, COCH₃), 2.17 (s, 3H,

3H, COCH₃), 1.96 (s, 3H, COCH₃), 2.02 (s, 3H, COCH₃), 2.13 (s, 3H,

0

COCH₃), 3.89 (dd, 1H, J_5,6¼ 2.4 Hz, J_6,6¼ 12.4 Hz, H-6), 4.10 (dd, 1H,

COCH₃), 3.98 (dd, 1H, J_5,6¼ 2.6 Hz, J_6,6¼ 12.2 Hz, H-6), 4.12e4.31

J_5,6¼ 5.0 Hz, J_6,6¼ 12.4 Hz, H-6⁰), 4.28 (ddd, 1H, J_5,6¼ 2.3 Hz,

J_5,6’¼ 5.0 Hz, J_4,5¼ 10.1 Hz, H-5), 4.46e4.60 (m, 4H, CH₂CHCH₂),

4.77e4.83 (m, 1H, CH₂CHCH₂), 5.31 (t, 1H, J_{3,4; 4,5}¼ 10.0 Hz, H-4),

5.35 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 5.40 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.4 Hz,

H-2), 5.44 (dd, 1H, J_2,3¼ 3.4 Hz, J_3,4¼ 10.0 Hz, H-3), 6.90e6.96 (m,

2H, AreH), 7.39e7.44 (m, 2H, AreH), 7.48e7.56 (m, 6H, AreH),

7.81e7.87 (m, 2H, AreH), 8.23e8.31 (m, 2H, AreH) ppm; ¹³C NMR

(m, 6H, H-5, H-6⁰, CH₂CHCH₂), 4.36e4.41 (m, 1H, CH₂CHCH₂), 5.13e

5.15 (m, 3H, H-2, H-3, H-4), 5.27 (d,1H, J_1,2¼ 0.9 Hz, H-1), 6.93e7.00

(m, 6H, AreH), 7.28e7.34 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

0

CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 62.3 (C-6), 66.0 (C-4), 67.2, 67.5 (2ꢂ

CH₂), 68.7 (C-3), 69.0 (C-2), 69.7 (C-5), 75.0 (CH), 97.3 (C-1), 114.4,

114.6, 121.3, 121.4, 129.6, 129.7, 1582, 158.3 (12ꢂ AreC), 169.8, 169.9,

170.0, 170.8 (4ꢂ CO) ppm; HRMS (ESIþ): m/z for C₂₉H₃₄O₁₂Na

([M þ Na^þ]^þ): calcd 597.1948; found 597.1946.

(100 MHz, CDCl₃),

d

20.6, 20.7, 20.8, 20.9 (4ꢂ COCH₃), 62.2 (C-6),

65.9 (C-4), 67.5, 67.9 (2ꢂ CH₂), 68.9 (C-3), 69.0 (C-2), 69.7 (C-5), 75.2

(CH), 97.5 (C-1), 105.0, 105.1, 121.1, 121.2, 121.7, 121.8, 125.4, 125.5,

125.7, 125.8, 126.5, 126.6, 127.6, 127.7, 134.5, 134.6, 153.9 (20ꢂ Are

C), 169,7, 169.9, 170.0, 170.6 (4ꢂ CO) ppm; HRMS (ESIþ): m/z for

4.1.8.15. 2,3-Bis(4-methoxyphenoxy)propan-1-yl

2,3,4,6-tetra-O-

acetyl- -mannopyranoside (17b). Prepared from 16b (0.405 g,

a-D

1.33 mmol) and 4 (0.633 g, 1.21 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

C

₃₇H₃₉O₁₂([M þ H]^þ): calcd 675.2442; found 675.2426.

4.1.8.12. 1,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-2-yl

2,3,4,6-tetra-O-acetyl- -mannopyranoside (13e). Prepared from

12e (0.776 g,1.92 mmol) and 4 (0.835 g,1.60 mmol) according to the

general procedure E. The crude product was puriﬁed by ﬂash col-

umn chromatography using ethyl acetate/hexane (1:3) as eluent.

0.405 g (52.7%); yellow oil; [

3350, 2975, 2887, 1752, 1381, 1089, 1050, 881, 462 cm^ꢃ1

(400 MHz, CDCl₃), 2.01 (s, 3H, COCH₃), 2.05 (s, 3H, COCH₃), 2.07 (s,

a

]

þ36.9 (c 0.20, MeOH); IR (NaCl)

n

D

a-D

;

¹H NMR

d

3H, COCH₃), 2.19 (s, 3H, COCH₃), 3.78 (s, 3H, OCH₃), 3.79 (s, 3H,

0

OCH₃), 4.05 (dd, 1H, J_5,6¼ 3.2 Hz, J_6,6¼ 10.8 Hz, H-6), 4.07e4.16 (m,

Yield: 0.410 g (33.0%); yellow solid; mp 88e89 ^ꢀC; [

a

]

D

þ33.8 (c

6H, H-5, H-6⁰, CH₂CHCH₂), 4.39e4.44 (m, 1H, CH₂CHCH₂), 5.20 (d,

1H, J_1,2¼ 1.7 Hz, H-1), 5.31 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2),

5.34 (d, 1H, J_3,4¼ 9.5 Hz, H-4), 5.38 (dd, 1H, J_2,3¼ 3.2 Hz,

J_3,4¼ 9.5 Hz, H-3), 6.83e6.89 (m, 8H, AreH) ppm; ¹³C NMR

0.21, MeOH); IR (KBr)

1222, 1162, 1137, 1042, 980, 860, 833, 599 cm^ꢃ1; ¹H NMR (400 MHz,

CDCl₃), 2.01 (s, 3H, COCH₃), 2.02 (s, 3H, COCH₃), 2.05 (s, 3H,

n 3448, 2940, 1751, 1633, 1516, 1466, 1388,

d

COCH₃), 2.21 (s, 3H, COCH₃), 3.93 (s, 6H, 2ꢂ OCH₃), 4.09 (dd, 1H,

(100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 55.7 (2ꢂ OCH₃), 62.3 (C-

J_5,6¼ 2.0 Hz, J_6,6¼ 12.0 Hz, H-6), 4.29e4.42 (m, 6H, H-5, H-6⁰,

6), 66.0 (C-4), 68.1, 68.3 (2ꢂ CH₂), 68.7 (C-3), 69.0 (C-2), 69.7 (C-5),

75.2 (CH), 97.3 (C-1), 114.7, 114.8, 115.4, 115.7, 152.5, 152.6, 154.2,

154.3 (12ꢂ AreC), 169.8, 169.9, 170.0, 170.7 (4ꢂ CO) ppm; HRMS

(ESIþ): m/z for C₃₁H₃₉O₁₄([M þ H]^þ): calcd 635.2340; found

635.2330.

0

CH₂CHCH₂), 4.58e4.63 (m, 1H, CH₂CHCH₂), 5.28 (d, 1H, J_1,2¼ 1.7 Hz,

H-1), 5.32e5.38 (m, 2H, H-2, H-4), 5.42 (dd, 1H, J_2,3¼ 3.2 Hz,

J_3,4¼ 10.0 Hz, H-3), 7.00e7.08 (m, 6H, AreH), 7.14 (dd, 2H,

J₁¼ 2.5 Hz, J₂¼ 7.1 Hz, AreH), 7.67e7.72 (m, 4H, AreH) ppm; ¹³C

NMR (100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 55.3 (2ꢂ OCH₃),

62.3 (C-6), 66.0 (C-4), 67.2, 67.7 (2ꢂ CH₂), 68.8 (C-3), 69.0 (C-2), 69.7

(C-5), 74.9 (CH), 97.3 (C-1), 105.3, 106.3, 106.4, 115.8, 116.0, 116.4,

116.5, 124.5, 124.6, 129.2, 129.4, 129.5, 135.8, 135.9, 156.8, 156.9,

158.2, 158.3 (20ꢂ AreC), 169.8, 169.9, 170.1, 170.7 (4ꢂ CO) ppm;

HRMS (ESIþ): m/z for C₃₉H₄₃O₁₄([M þ H]^þ): calcd 735.2653; found

735.2661.

4.1.8.16. 2,3-Bis(4-nitrophenoxy)propan-1-yl 2,3,4,6-tetra-O-acetyl-

a-D-mannopyranoside (17c). Prepared from 16c (0.425 g,

1.27 mmol) and 4 (0.554 g, 1.06 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

0.389 g (46.0%); white solid; mp 128e129 ^ꢀC; [

a

]

þ30.7 (c 0.19,

D

MeOH); IR (KBr)

1370, 1341, 1256, 1175, 1113, 1041, 1010, 979, 862, 848, 753, 691, 675,

597 cm^ꢃ1; ¹H NMR (400 MHz, DMSO-d₆),

1.92 (s, 3H, COCH₃), 1.98

(s, 3H, COCH₃), 2.01 (s, 3H, COCH₃), 2.13 (s, 3H, COCH₃), 3.98 (dd,1H,

n 3511, 3086, 2940, 1756, 1594, 1509, 1498, 1458,

4.1.8.13. 1-Ethoxy-3-(naphthalen-2-yloxy)propan-2-yl 2,3,4,6-tetra-

O-acetyl-

a-D

-mannopyranoside (13f). Prepared from 12f (0.282 g,

d

1.14 mmol) and 4 (0.543 g, 1.04 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

0

J_5,6¼ 2.6 Hz, J_6,6¼ 12.0 Hz, H-6), 4.13e4.17 (m, 2H, H-5, H-6 ), 4.38e

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

321

4.52 (m, 5H, CH₂CHCH₂, CH₂CHCH₂), 5.05 (dd, 1H, J_2,3¼ 3.4 Hz,

0.378 g (75.6%); yellow oil; [

3463, 3057, 1751, 1629, 1601, 1512, 1465, 1369, 1217, 1181, 1136, 1045,

979, 838, 749, 623 cm^ꢃ^{1 1}H NMR (400 MHz, CDCl₃),

2.02 (s, 3H,

a

]

þ43.4 (c 0.22, MeOH); IR (KBr)

n

D

J_3,4¼ 10.2 Hz, H-3), 5.09e5.15 (m, 2H, H-2, H-4), 5.29 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 7.21e7.26 (m, 4H, AreH), 8.22e8.26 (m, 4H, Are

;

d

H) ppm; ¹³C NMR (100 MHz, CDCl₃),

d

20.6, 20.7, 20.9 (4ꢂ COCH₃),

COCH₃), 2.03 (s, 6H, 2ꢂ COCH₃), 2.21 (s, 3H, COCH₃), 4.01 (dd, 1H,

J_5,6¼ 2.1 Hz, J_6,6¼ 12.0 Hz, H-6), 4.31e4.42 (m, 6H, H-5, H-6⁰,

0

62.4 (C-6), 66.0 (C-4), 67.5, 67.9 (2ꢂ CH₂), 68.6 (C-3), 69.1 (C-2), 69.6

(C-5), 74.5 (CH), 97.4 (C-1), 114.5, 114.6, 126.0, 126.1, 142.1, 163.0

(12ꢂ AreC), 169.7, 170.0, 170.1, 170.6 (4ꢂ CO) ppm; HRMS (ESIþ):

m/z for C₂₉H₃₂N₂O₁₆Na ([M þ Na^þ]^þ): calcd 687.1650; found

687.1652.

CH₂CHCH₂), 4.60e4.65 (m, 1H, CH₂CHCH₂), 5.30 (d, 1H, J_1,2¼ 1.7 Hz,

H-1), 5.33e5.38 (m, 2H, H-2, H-4), 5.44 (dd, 1H, J_2,3¼ 3.4 Hz,

J_3,4¼ 10.0 Hz, H-3), 7.16e7.24 (m, 4H, AreH), 7.36e7.41 (m, 2H, Are

H), 7.46e7.50 (m, 2H, AreH), 7.76e7.82 (m, 6H, AreH) ppm; ¹³C

NMR (100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 62.4 (C-6), 66.0 (C-

4.1.8.17. 2,3-Bis(naphthalen-1-yloxy)propan-1-yl

2,3,4,6-tetra-O-

4), 67.2, 67.7 (2ꢂ CH₂), 68.8 (C-3), 69.0 (C-2), 69.7 (C-5), 74.9 (CH),

97.3 (C-1), 106.8, 106.9, 118.5, 118.7, 123.9, 124.0, 126.5, 126.6, 126.8,

127.7, 129.2, 129.3, 129.7, 129.8, 134.4, 134.5, 156.2, 156.3 (20ꢂ Are

C), 169.8, 170.0, 170.1, 170.7 (4ꢂ CO) ppm; HRMS (ESIþ): m/z for

acetyl- -mannopyranoside (17d). Prepared from 16d (0.280 g,

a-D

0.81 mmol) and 4 (0.386 g, 0.74 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

C

₃₇H₃₉O₁₂([M þ H]^þ): calcd 675.2442; found 675.2440.

0.403 g (80.1%); brown oil; [

3375, 3246, 1697, 1617, 1508, 1387, 1240, 1109, 930, 836, 771, 649,

619 cm^ꢃ1; ¹H NMR (400 MHz, DMSO-d₆),

1.90 (s, 3H, COCH₃), 1.92

(s, 3H, COCH₃), 1.96 (s, 3H, COCH₃), 2.12 (s, 3H, COCH₃), 3.79 (dd, 1H,

a

]

þ4.5 (c 0.23, MeOH); IR (KBr)

n

D

4.1.9. General procedure F. Zemplén deacetylation

d

The protected mannopyranoside (1 mmol) was dissolved in dry

methanol and sodium methanolate solution (30 wt. % in methanol,

0.01 mmol) was added. The reaction mixture was stirred at room

temperature for 30 min and then the acidic ion exchange resin

Amberlite^ÒIR120 H was added for neutralization. After stirring the

mixture for 10 min, the resin was ﬁltered off, washed with meth-

anol and the solvent removed in vacuo. Final compounds were

obtained in quantitative yield and no further puriﬁcation was car-

ried out.

0

J_5,6¼ 2.5 Hz, J_6,6¼ 12.4 Hz, H-6), 3.94 (dd, 1H, J_5,6¼ 4.9 Hz,

J_6,6¼ 12.3 Hz, H-6⁰), 4.28 (ddd, 1H, J_5,6¼ 2.5 Hz, J_5,6’¼ 4.9 Hz,

0

J_4,5¼ 9.9 Hz, H-5), 4.52e4.64 (m, 4H, CH₂CHCH₂), 4.71e4.75 (m, 1H,

CH₂CHCH₂), 5.12 (t, 1H, J_3,4¼ 9.9 Hz, H-4), 5.20 (dd, 1H, J_2,3¼ 3.4 Hz,

J_3,4¼ 9.9 Hz, H-3), 5.25 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.4 Hz, H-2), 5.45

(d,1H, J_1,2¼ 1.7 Hz, H-1), 7.09e7.14 (m, 2H, AreH), 7.42e7.47 (m, 2H,

AreH), 7.48e7.57 (m, 6H, AreH), 7.88e7.91 (m, 2H, AreH), 8.17e

8.23 (m, 2H, AreH) ppm; ¹³C NMR (100 MHz, CDCl₃),

d 20.7, 20.8,

20.9 (4ꢂ COCH₃), 62.2 (C-6), 65.9 (C-4), 67.5, 67.9 (2ꢂ CH₂), 68.9 (C-

3), 69.0 (C-2), 69.6 (C-5), 75.2 (CH), 97.5 (C-1), 105.0, 105.1, 121.1,

121.2, 121.7, 121.8, 125.4, 125.5, 125.7, 125.8, 126.6, 126.7, 127.6, 127.7,

134.5, 134.6, 153.9, 163.8 (20ꢂ AreC), 169.8, 170.0, 170.1, 170.6 (4ꢂ

CO) ppm; HRMS (ESIþ): m/z for C₃₇H₃₉O₁₂([M þ H]^þ): calcd

675.2442; found 675.2448.

4.1.9.1. 1,3-Dibenzamidopropan-2-yl

Prepared from 8a (0.164 g, 0.27 mmol) according to the general

procedure F. Yield: 0.124 g (100%); white crystalline solid; mp 46e

a-D-mannopyranoside (9a).

48 ^ꢀC; [

a

]

_Dþ33.0 (c 0.17, MeOH); IR (KBr)

n 3398, 3069, 2933, 1635,

1576, 1534, 1489, 1435, 1292, 1125, 1096, 1023, 978, 929, 804,

692 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

d

3.53e3.80 (m, 9H, 2ꢂ CH₂,

H-3, H-4, H-5, H-6, H-6⁰), 3.86 (dd, 1H, J_1,2¼ 1.8 Hz, J_2,3¼ 3.3 Hz, H-

2), 4.07e4.13 (m, 1H, CH), 5.05 (d, 1H, J_1,2¼ 1.5 Hz, H-1), 7.47e7.52

(m, 4H, AreH), 7.54e7.59 (m, 2H, AreH), 7.87e7.91 (m, 4H, AreH)

4.1.8.18. 2,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-1-yl

2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside (17e). Prepared from

16e (0.382 g, 0.94 mmol) and 4 (0.449 g, 0.86 mmol) according to

the general procedure E. The crude product was puriﬁed by ﬂash

column chromatography using ethyl acetate/hexane (1:2) as eluent.

ppm; ¹³C NMR (100 MHz, CD₃OD),

d

41.4, 43.0 (2ꢂ CH₂), 62.8 (C-6),

68.6 (C-4), 72.4 (C-3), 72.5 (C-2), 75.3 (C-5), 76.9 (CH), 101.3 (C-1),

128.4, 128.5, 129.6, 129.7, 135.4, 135.6 (12ꢂ AreC), 170.5, 170.6 (2ꢂ

CO) ppm; HRMS (ESIþ): m/z for C₂₃H₂₇N₂O₈([M þ H]^þ): calcd

459.1767; found 459.1773.

Yield: 0.410 g (65.0%); brown oil; [

a

]

_Dþ28.4 (c 0.17, MeOH); IR (KBr)

n

3421, 2946, 1627, 1516, 1465, 1388, 1253, 1226, 1210, 1187, 1166,

1119, 1030, 860, 840, 801 cm^ꢃ1

;

¹H NMR (400 MHz, DMSO-d₆),

d

1.93 (s, 3H, COCH₃), 1.94 (s, 3H, COCH₃), 2.01 (s, 3H, COCH₃), 2.14

4.1.9.2. 1,3-Bis(4-cyanobenzamido)propan-2-yl

a-D-mannopyrano-

(s, 3H, COCH₃), 3.85 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.00 (dd, 1H,

side (9b). Prepared from 8b (0.350 g, 0.52 mmol) according to the

0

J_5,6¼ 2.6 Hz, J_6,6¼ 12.2 Hz, H-6), 4.17 (dd, 1H, J_5,6¼ 4.8 Hz,

general procedure F. Yield: 0.215 g (81.2%); white crystalline solid;

J_6,6¼ 12.2 Hz, H-6⁰), 4.31 (ddd, 1H, J_5,6¼ 2.6 Hz, J_5,6’¼ 4.8 Hz,

mp 122e124 ^ꢀC; [

a

]

þ37.0 (c 0.15, MeOH); IR (KBr)

n

3392, 2933,

0

D

J_4,5¼ 9.9 Hz, H-5), 4.34e4.47 (m, 4H, CH₂CHCH₂), 4.51e4.56 (m, 1H,

CH₂CHCH₂), 5.15e5.17 (m, 2H, H-3, H-4), 5.21 (dd, 1H, J_1,2¼ 1.7 Hz,

J_2,3¼ 3.2 Hz, H-2), 5.35 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 7.00 (dd, 2H,

J₁¼ 2.6 Hz, J₂¼ 8.9 Hz, AreH), 7.06 (ddd, 2H, J₁¼ 2.6 Hz, J₂¼ 8.9 Hz,

J₃¼ 14.1 Hz, AreH), 7.22 (t, 2H, J ¼ 2.8 Hz, AreH), 7.34 (t, 2H,

J ¼ 2.8 Hz, AreH), 7.74e7.79 (m, 4H, AreH) ppm; ¹³C NMR

2232, 1648, 1543, 1498, 1438, 1287, 1127, 1099, 1054, 977, 858, 765,

679 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

d

3.54e3.75 (m, 8H, 2ꢂ CH₂,

H-3, H-4, H-5, H-6), 3.78 (dd, 1H, J_1,2¼ 2.1 Hz, J_2,3¼ 11.3 Hz, H-6⁰),

3.84 (dd, 1H, J_1,2¼ 1.8 Hz, J_2,3¼ 3.3 Hz, H-2), 4.08e4.14 (m, 1H, CH),

5.02 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 7.85e7.90 (m, 4H, AreH), 8.01e8.06

(m, 4H, AreH) ppm; ¹³C NMR (100 MHz, CD₃OD),

d

41.9, 43.3 (2ꢂ

(100 MHz, CDCl₃),

d

20.7, 21.0 (4ꢂ COCH₃), 55.3 (2ꢂ OCH₃), 62.3 (C-

CH₂), 62.9 (C-6), 68.6 (C-4), 72.3 (C-3), 72.4 (C-2), 75.3 (C-5), 77.0

(CH), 101.3 (C-1), 116.1, 116.2, 119.0, 119.1, 129.3, 129.4, 133.5, 133.6,

139.6, 139.8 (12ꢂ AreC), 168.7, 168.8 (2ꢂ CO) ppm; HRMS (ESIþ):

m/z for C₂₅H₂₅N₄O₈([M ꢃ H]^ꢃ): calcd 509.1672; found 509.1680.

6), 66.0 (C-4), 67.2, 67.7 (2ꢂ CH₂), 68.8 (C-3), 69.0 (C-2), 69.7 (C-5),

74.9 (CH), 97.3 (C-1), 105.3, 106.3, 106.4, 115.8, 116.0, 116.4, 116.5,

124.5, 124.6, 129.2, 129.4, 129.5, 135.8, 135.9, 156.8, 156.9, 158.3,

158.4 (20ꢂ AreC), 169.8, 170.0, 170.1, 170.8 (4ꢂ CO) ppm; HRMS

(ESIþ): m/z for C₃₉H₄₃O₁₄([M þ H]^þ): calcd 735.2653; found

735.2632.

4.1.9.3. 1,3-Bis(4-methoxybenzamido)propan-2-yl

a-D-mannopyr-

anoside (9c). Prepared from 8c (0.205 g, 0.30 mmol) according to

the general procedure F. Yield: 0.133 g (85.7%); white crystalline

4.1.8.19. 2,3-Bis(naphthalen-2-yloxy)propan-1-yl

2,3,4,6-tetra-O-

solid; mp 110e111 ^ꢀC; [

a]

þ34.6 (c 0.10, MeOH); IR (KBr)

n

3403,

D

acetyl- -mannopyranoside (17f). Prepared from 16f (0.281 g,

a-D

2934, 1607, 1542, 1458, 1300, 1257, 1182, 1027, 845, 767, 608 cm^ꢃ1

;

0.82 mmol) and 4 (0.387 g, 0.74 mmol) according to the general

procedure E. The crude product was puriﬁed by ﬂash column

chromatography using ethyl acetate/hexane (1:2) as eluent. Yield:

¹H NMR (400 MHz, CD₃OD),

d

3.50e3.79 (m, 9H, 2ꢂ CH₂, H-3, H-4,

H-5, H-6, H-6⁰), 3.85 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2), 3.87

(2ꢂ s, 6H, 2ꢂ OCH₃), 4.04e4.09 (m,1H, CH), 5.04 (d,1H, J_1,2¼ 1.7 Hz,

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

322

H-1), 7.00e7.03 (m, 4H, AreH), 7.85e7.89 (m, 4H, AreH) ppm; ¹³C

NMR (100 MHz, CD₃OD),

(C-6), 68.5 (C-4), 72.3 (C-3), 72.4 (C-2), 75.3 (C-5), 76.8 (CH), 101.2

(C-1), 114.7, 114.8, 127.4, 127.6, 130.2, 130.3 (12ꢂ AreC), 164.1, 170.2

(2ꢂ CO) ppm; HRMS (ESIþ): m/z for C₂₅H₃₃N₂O₁₀([M þ H]^þ): calcd

521.2135; found 521.2142.

(2ꢂ CH₂), 43.2, 43.4 (2ꢂ COCH₂), 63.1 (C-6), 68.8 (C-4), 72.2 (C-3),

72.4 (C-2), 75.4 (C-5), 76.9 (CH),101.0 (C-1),124.6,124.7,131.4,131.5,

144.6, 144.7, 148.4, 148.5 (12ꢂ AreC), 172.9, 173.0 (2ꢂ CO) ppm;

HRMS (ESIþ): m/z for C₂₅H₃₁N₄O₁₂([M þ H]^þ): calcd 579.1938;

found 579.1939.

d

41.3, 42.8 (2ꢂ CH₂), 55.9 (2ꢂ OCH₃), 62.8

4.1.9.8. 1,3-Diphenoxypropan-2-yl

a-D-mannopyranoside (14a).

4.1.9.4. 1,3-Bis(2-naphthamido)propan-2-yl

a-D

-mannopyranoside

Prepared from 13a (0.141 g, 0.23 mmol) according to the general

(9d). Prepared from 8d (0.250 g, 0.343 mmol) according to the

procedure F. Yield: 0.134 g (100%); white crystalline solid; mp

general procedure F. Yield: 0.144 g (75.0%); white crystalline solid;

128 ^ꢀC; [

a]

_Dþ32.8 (c 0.23, MeOH); IR (KBr)

n 3421, 2928, 1599, 1457,

mp 118e119 ^ꢀC; [

a

]

þ21.0 (c 0.18, MeOH); IR (KBr)

n

3369, 2930,

1241, 1173, 1134, 1068, 977, 865, 753, 690 cm^ꢃ1; ¹H NMR (400 MHz,

D

1638, 1542, 1432, 1300, 1237, 1053, 907, 864, 824, 777, 760 cm^ꢃ1; ¹H

CD₃OD),

d

3.66e3.78 (m, 4H, H-3, H-4, H-6, H-6⁰), 3.82 (ddd, 1H,

0

NMR (400 MHz, CD₃OD),

d

3.63e3.83 (m, 9H, 2ꢂ CH₂, H-3, H-4, H-5,

J_5,6¼ 2.7 Hz, J_5,6¼ 4.8 Hz, J_4,5¼ 10.6 Hz, H-5), 3.86 (dd, 1H,

J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2), 4.19e4.32 (m, 4H, CH₂CHCH₂), 4.44e

4.49 (m, 1H, CH₂CHCH₂), 5.16 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 6.92e7.00

(m, 6H, AreH), 7.27e7.31 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

H-6, H-6⁰), 3.91 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2), 4.18e4.23

(m, 1H, CH), 5.12 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 7.56e7.63 (m, 4H, AreH),

7.93e8.02 (m, 8H, AreH), 8.46 (d, 2H, J ¼ 11.3 Hz, AreH) ppm; ¹³

C

NMR (100 MHz, CD₃OD),

d

41.6, 43.2 (2ꢂ CH₂), 62.8 (C-6), 68.6 (C-

CD₃OD),

d

62.7 (C-6), 68.4 (C-4), 68.8, 68.9 (2ꢂ CH₂), 72.3 (C-3), 72.4

4), 72.4 (C-3), 72.5 (C-2), 75.3 (C-5), 76.8 (CH), 101.3 (C-1), 124.9,

125.0, 127.8, 127.9, 128.7, 128.8, 128.9, 129.0, 129.1, 129.4, 129.5,

130.1, 130.2, 132.6, 132.7, 134.0, 134.1, 136.4 (20ꢂ AreC), 170.6, 170.7

(2ꢂ CO) ppm; HRMS (ESIþ): m/z for C₃₁H₃₃N₂O₈([M þ H]^þ): calcd

561.2237; found 561.2245.

(C-2), 74.9 (C-5), 75.6 (CH), 101.7 (C-1), 115.6, 115.7, 122.0, 122.2,

130.5, 130.6, 160.1, 160.2 (12ꢂ AreC) ppm; HRMS (ESIꢃ): m/z for

C

₂₁H₂₅O₈([M ꢃ H]^ꢃ): calcd 405.1549; found 405.1542.

4.1.9.9. 1,3-Bis(4-methoxyphenoxy)propan-2-yl

a-D-mannopyrano-

side (14b). Prepared from 13b (0.190 g, 0.30 mmol) according to the

general procedure F. Yield: 0.140 g (100%); orange solid; mp 69e

4.1.9.5. 1,3-Bis(1H-pyrrole-2-carboxamido)propan-2-yl

nopyranoside (9e). Prepared from 8e (0.097 g, 0.16 mmol) accord-

a-D-man-

71 ^ꢀC; [

a

]

_Dþ35.0 (c 0.20, MeOH); IR (KBr)

1458, 1289, 1232, 1133, 1107, 1064, 1044, 978, 824, 754, 670,

520 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

3.65e3.74 (m, 3H, H-3, H-

n 3378, 2925, 2832, 1507,

ing to the general procedure F. Yield: 0.065 g (92.0%); white

crystalline solid; mp 135e137 ^ꢀC; [

a

]

þ39.5 (c 0.07, MeOH); IR

d

D

(KBr)

n

3400, 2930, 1629, 1560, 1523, 1408, 1328, 1205, 1128, 1044,

4, H-6), 3.75 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.77e3.84 (m, 2H, H-

5, H-6⁰), 3.86 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2), 4.11e4.25 (m,

4H, CH₂CHCH₂), 4.37e4.42 (m, 1H, CH₂CHCH₂), 5.14 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 6.84e6.87 (m, 4H, AreH), 6.89e6.93 (m, 4H, Are

884, 745, 605 cm^ꢃ1

;

¹H NMR (400 MHz, CD₃OD),

d

3.45e3.67 (m,

6H, 2ꢂ CH₂, H-4, H-6), 3.71e3.81 (m, 3H, H-3, H-5, H-6⁰), 3.85 (dd,

1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2), 3.97e4.02 (m, 1H, CH), 5.01 (d,

1H, J_1,2¼ 1.7 Hz, H-1), 6.19 (ddd, 2H, J₁¼ 2.0 Hz, J₂¼ 2.5 Hz,

J₃¼ 3.8 Hz, AreH), 6.86 (ddd, 2H, J₁¼1.4 Hz, J₂¼ 3.8 Hz, J₃¼ 4.6 Hz,

H) ppm; ¹³C NMR (100 MHz, CD₃OD),

d

56.1 (2ꢂ OCH₃), 62.8 (C-6),

68.4 (C-4), 69.5, 69.7 (2ꢂ CH₂), 72.3 (C-3), 72.5 (C-2), 74.8 (C-5),

AreH), 6.93e6.94 (dd, 2H, J₁¼ 1.4 Hz, J₂¼ 2.5 Hz, AreH) ppm; ¹³

C

75.7 (CH), 101.7 (C-1), 115.6, 115.8, 116.6, 116.7, 154.2, 154.4, 155.6,

NMR (100 MHz, CD₃OD),

d

40.5, 42.0 (2ꢂ CH₂), 62.8 (C-6), 68.56 (C-

155.7 (12ꢂ AreC) ppm; HRMS (ESIꢃ): m/z for

C₂₃H₂₉O₁₀

4), 72.3 (C-3), 72.4 (C-2), 75.2 (C-5), 77.2 (CH), 101.2 (C-1), 110.2,

110.3, 111.8, 112.2, 123.0, 123.1, 126.7, 126.8 (8ꢂ AreC), 164.0, 164.1

(2ꢂ CO) ppm; HRMS (ESIþ): m/z for C₁₉H₂₇N₄O₈([M þ H]^þ): calcd

439.1829; found 439.1822.

([M ꢃ H]^ꢃ): calcd 465.1761; found 465.1757.

4.1.9.10. 1,3-Bis(4-nitrophenoxy)propan-2-yl

a-D-mannopyranoside

(14c). Prepared from 13c (0.107 g, 0.16 mmol) according to the

general procedure F. Yield: 0.080 g (100%); yellow solid; mp 98e

4.1.9.6. 1,3-Bis(2-phenylacetamido)propan-2-yl

side (9f). Prepared from 8f (0.310 g, 0.47 mmol) according to the

general procedure F. Yield: 0.231 (100.0%); colorless oil;

þ27.3 (c 0.12, MeOH); IR (KBr)

cm^{ꢃ1 1}H NMR (400 MHz,

a

-D-mannopyrano-

99 ^ꢀC; [

a

]

_Dþ18.7 (c 0.25, MeOH); IR (KBr)

n 3420, 2928, 1592, 1507,

1340, 1299, 1256, 1175, 1111, 1028, 863, 844, 751, 689 cm^ꢃ1; ¹H NMR

g

(400 MHz, CD₃OD), d 3.64e3.74 (m, 3H, H-3, H-4, H-6), 3.78 (dd, 1H,

[

a]

n

;

J_5,6¼ 2.7 Hz, J_4,5¼ 8.8 Hz, H-5), 3.81e3.85 (m, 2H, H-2, H-6⁰), 4.38e

4.48 (m, 4H, CH₂CHCH₂), 4.52e4.56 (m, 1H, CH₂CHCH₂), 5.15 (d, 1H,

J_1,2¼ 1.7 Hz, H-1), 7.17e7.19 (m, 4H, AreH), 8.23e8.26 (m, 4H, Are

D

CD₃OD),

d

3.23 (dd,1H, J₁¼5.6 Hz, J₂¼ 14.3 Hz, CH₂eH_A), 3.34e3.39

(m, 3H, CH₂, CH₂eH_B), 3.55 (s, 2H, COCH₂), 3.56 (s, 2H, COCH₂),

3.59e3.72 (m, 4H, H-3, H-5, H-6, H-6⁰), 3.77 (dd, 1H, J_1,2¼ 1.8 Hz,

J_2,3¼ 3.3 Hz, H-2), 3.78e3.82 (m,1H, CH), 3.88 (d, 1H, J ¼ 10.0 Hz, H-

H) ppm; ¹³C NMR (100 MHz, CD₃OD),

d 63.0 (C-6), 68.5 (C-4), 69.3,

69.4 (2ꢂ CH₂), 72.2 (C-2), 72.4 (C-3), 75.3 (C-5, CH), 101.9 (C-1),

115.9, 116.0, 126.8, 126.9, 143.1, 143.2, 165.1, 165.2 (12ꢂ AreC) ppm;

HRMS (ESIꢃ): m/z for C₂₁H₂₃N₂O₁₂([M ꢃ H]^ꢃ): calcd 495.1251;

found 495.1264.

4), 4.89 (d,1H, J_1,2¼ 1.8 Hz, H-1), 7.24e7.35 (m,10H, AreH) ppm; ¹³

C

NMR (100 MHz, CD₃OD),

d

40.9, 42.3 (2ꢂ CH₂), 43.8, 43.9 (2ꢂ

CH₂CO), 63.0 (C-6), 68.8 (C-4), 72.2 (C-3), 72.4 (C-2), 75.3 (C-5), 76.7

(CH), 101.0 (C-1), 127.9, 128.0, 129.6, 129.7, 130.1, 130.2, 136.8, 136.9

(12ꢂ AreC), 174.5, 174.6 (2ꢂ CO) ppm; HRMS (ESIþ): m/z for

4.1.9.11. 1,3-Bis(naphthalen-1-yloxy)propan-2-yl

a-D-mannopyrano-

C

₂₅H₃₃N₂O₈([M þ H]^þ): calcd 489.2237; found 489.2227.

side (14d). Prepared from 13d (0.327 g, 0.45 mmol) according to

the general procedure F. Yield: 0.251 g (100%); white crystalline

4.1.9.7. 1,3-Bis(2-(4-nitrophenyl)acetamido)propan-2-yl

nopyranoside (9g). Prepared from 8g (0.143 g, 0.19 mmol) accord-

a-D

-man-

solid; mp 192e194 ^ꢀC; [

a

]

D

þ8.5 (c 0.23, MeOH); IR (KBr)

n

3421,

2927, 1580, 1457, 1267, 1238, 1102, 1069, 865, 792, 771, 572 cm^ꢃ1; ¹H

ing to the general procedure F. Yield: 0.111 g (100%); brown oil;

NMR (400 MHz, CD₃OD), d 3.65e3.72 (m, 2H, H-4, H-6), 3.75 (dd,

[

a

]

þ25.3 (c 0.08, MeOH); IR (KBr)

n

cm^ꢃ1

;

¹H NMR (400 MHz,

1H, J_5,6¼ 2.7 Hz, J_6,6¼ 11.3 Hz, H-6⁰), 3.81 (dd, 1H, J_2,3¼ 3.3 Hz,

0

D

0

CD₃OD),

d

3.27 (dd,1H, J₁¼5.6 Hz, J₂¼ 14.2 Hz, CH₂eH_A), 3.35e3.42

J_3,4¼ 9.4 Hz, H-3), 3.88 (ddd, 1H, J_5,6¼ 2.7 Hz, J_5,6¼ 5.4 Hz,

(m, 3H, CH₂, CH₂eH_B), 3.56e3.68 (m, 4H, H-3, H-5, H-6, H-6⁰), 3.69

(s, 2H, COCH₂), 3.71 (s, 2H, COCH₂), 3.76 (dd, 1H, J_1,2¼ 1.8 Hz,

J_2,3¼ 3.3 Hz, H-2), 3.81e3.84 (m, 1H, CH), 3.89 (d, 1H, J ¼ 9.3 Hz, H-

4), 4.88 (d, 1H, J_1,2¼ 1.8 Hz, H-1), 7.54e7.58 (m, 4H, AreH), 8.19e

J_4,5¼ 8.2 Hz, H-5), 3.94 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2),

4.53e4.65 (m, 4H, CH₂CHCH₂), 4.79e4.82 (m, 1H, CH₂CHCH₂), 5.34

(d, 1H, J_1,2¼ 1.7 Hz, H-1), 7.01e7.04 (m, 2H, AreH), 7.38e7.42 (m,

2H, AreH), 7.45e7.52 (m, 6H, AreH), 7.80 (m, 2H, AreH), 8.26e8.30

8.23 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz, CD₃OD),

d

41.2, 42.5

(m, 2H, AreH) ppm; ¹³C NMR (100 MHz, CD₃OD),

d 62.7 (C-6), 68.3

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

323

(C-4), 69.3, 69.4 (2ꢂ CH₂), 72.4 (C-2), 72.5 (C-3), 75.1 (C-5), 75.9

(CH), 101.9 (C-1), 106.2, 106.3, 121.2, 121.7, 126.3, 126.4, 126.9, 127.0,

127.1, 127.4, 128.5, 128.6, 136.0, 136.1, 155.6, 155.7 (20ꢂ AreC) ppm;

HRMS (ESIꢃ): m/z for C₂₉H₂₉O₈([M ꢃ H]^ꢃ): calcd 505.1862; found

505.1869.

J_{3,4; 4,5}¼ 9.5 Hz, H-4), 3.71e3.76 (m, 8H, H-3, H-6, 2ꢂ OCH₃), 3.78e

3.84 (m, 2H, H-5, H-6⁰), 3.86 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2),

4.11e4.25 (m, 4H, CH₂CHCH₂), 4.37e4.42 (m, 1H, CH₂CHCH₂), 5.14

(d, 1H, J_1,2¼ 1.7 Hz, H-1), 6.84e6.88 (m, 4H, AreH), 6.89e6.93 (m,

4H, AreH) ppm; ¹³C NMR (100 MHz, CD₃OD),

d

56.1 (2ꢂ OCH₃), 62.8

(C-6), 68.4 (C-4), 69.5, 69.7 (2ꢂ CH₂), 72.3 (C-2), 72.5 (C-3), 74.9 (C-

5), 75.7 (CH), 101.7 (C-1), 115.7, 116.6, 116.7, 154.2, 154.3, 155.6, 155.7

(12ꢂ AreC) ppm; HRMS (ESIꢃ): m/z for C₂₃H₂₉O₁₀([M ꢃ H]^ꢃ):

calcd 465.1761; found 465.1751.

4.1.9.12. 1,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-2-yl

a-D-

mannopyranoside (14e). Prepared from 13e (0.359 g, 0.46 mmol)

according to the general procedure F. Yield: 0.281 g (100%); white

crystalline solid; mp 178e179 ^ꢀC; [

n

a

]_Dþ19.0 (c 0.25, MeOH); IR (KBr)

3398, 2933, 1630, 1516, 1465, 1387, 1256, 1210, 1185, 1160, 1137,

1023, 975, 839, 806 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

3.70 (t, 1H,

4.1.9.16. 2,3-Bis(4-nitrophenoxy)propan-1-yl a-D-mannopyranoside

d

(18c). Prepared from 17c (0.324 g, 0.49 mmol) according to the

general procedure F. Yield: 0.242 g (100%); brown solid; mp 176e

J_{3,4; 4,5}¼ 9.6 Hz, H-4), 3.73e3.85 (m, 3H, H-3, H-6, H-6⁰), 3.88e3.91

(m, 7H, H-5, 2ꢂ OCH₃), 3.92 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2),

4.36e4.49 (m, 4H, CH₂CHCH₂), 4.58e4.64 (m, 1H, CH₂CHCH₂), 5.24

(d, 1H, J_1,2¼ 1.7 Hz, H-1), 6.97 (ddd, 2H, J₁¼ 0.95 Hz, J₂¼ 2.5 Hz,

J₃¼ 8.8 Hz, AreH), 7.05 (ddd, 2H, J₁¼1.7 Hz, J₂¼ 2.5 Hz, J₃¼ 8.8 Hz,

AreH), 7.15 (dd, 2H, J₁¼ 2.3 Hz, J₂¼ 8.8 Hz, AreH), 7.25 (d, 2H,

J ¼ 2.6 Hz, AreH), 7.65e7.70 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

179 ^ꢀC; [

a

]

_Dþ22.3 (c 0.22, MeOH); IR (KBr)

n 3421, 1593, 1507, 1340,

1254, 1174, 1111, 1024, 863, 844, 751, 690 cm^ꢃ1; ¹H NMR (400 MHz,

CD₃OD),

d

3.64 (t, 1H, J_{3,4; 4,5}¼ 9.3 Hz, H-4), 3.66e3.74 (m, 2H, H-3,

H-6), 3.77e3.85 (m, 3H, H-2, H-5, H-6⁰), 4.38e4.48 (m, 4H,

CH₂CHCH₂), 4.53e4.58 (m, 1H, CH₂CHCH₂), 5.15 (d, 1H, J_1,2¼ 1.7 Hz,

H-1), 7.16e7.20 (m, 4H, AreH), 8.23e8.26 (m, 4H, AreH) ppm; ¹³C

CD₃OD),

d

55.9 (2ꢂ OCH₃), 62.8 (C-6), 68.5 (C-4), 68.9, 69.0 (2ꢂ CH₂),

NMR (100 MHz, CD₃OD),

CH₂), 72.2 (C-2), 72.4 (C-3), 75.3 (C-5, CH), 101.9 (C-1), 116.0, 126.9,

d

62.9 (C-6), 68.5 (C-4), 69.3, 69.4 (2ꢂ

72.4 (C-2), 72.6 (C-3), 75.2 (C-5), 75.3 (CH), 101.8 (C-1), 106.5, 106.6,

107.7, 107.8, 117.2, 117.3, 117.4, 117.5, 125.9, 126.0, 130.2, 130.4, 130.5,

137.6, 158.6, 158.7, 159.8, 159.9 (20ꢂ AreC) ppm; HRMS (ESIꢃ): m/z

for C₃₁H₃₃O₁₀([M ꢃ H]^ꢃ): calcd 565.2074; found 565.2073.

143.1, 143.2, 165.1, 165.2 (12ꢂ AreC) ppm; HRMS (ESIþ): m/z for

C

₂₁H₂₄N₂O₁₂Na ([M þ Na^þ]^þ): calcd 519.1227; found 519.1218.

4.1.9.17. 2,3-Bis(naphthalen-1-yloxy)propan-1-yl a-D-mannopyrano-

4.1.9.13. 1-Ethoxy-3-(naphthalen-2-yloxy)propan-2-yl

a-D-mannopyr-

side (18d). Prepared from 17d (0.183 g, 0.27 mmol) according to the

anoside (14f). Prepared from 13f (0.200 g, 0.35 mmol) according to

the general procedure F. Compound was obtained as a mixture of

two diastereomers in 1:1 ratio. Yield: 0.142 g (100%); yellow solid;

general procedure F. Yield: 0.137 g (100%); light brown solid; mp

151e153 ^ꢀC; [

a

]

_Dþ23.4 (c 0.24, MeOH); IR (KBr)

n 3380, 2929, 1670,

1595,1579,1507,1460,1395,1340,1267,1238,1178,1136,1102,1069,

mp 188e190 ^ꢀC; [

a

]

þ33.9 (c 0.27, MeOH); IR (KBr)

n

3404, 2929,

1602, 1458, 1258, 1218, 1183, 1119, 1046, 978, 837, 747 cm^ꢃ1; ¹H NMR

(400 MHz, CD₃OD),

977, 833, 793, 769, 680 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

d

3.65e

D

0

3.72 (m, 2H, H-4, H-6), 3.76 (dd,1H, J_5,6¼ 2.3 Hz, J_6,6¼11.8 Hz, H-6 ),

3.81 (dd, 1H, J_2,3¼ 3.3 Hz, J_3,4¼ 9.5 Hz, H-3), 3.88 (ddd, 1H,

J_5,6¼ 2.3 Hz, J_5,6’¼ 5.5 Hz, J_4,5¼ 9.9 Hz, H-5), 3.94 (dd,1H, J_1,2¼1.7 Hz,

J_2,3¼ 3.3 Hz, H-2), 4.53e4.64 (m, 4H, CH₂CHCH₂), 4.78e4.83 (m, 1H,

CH₂CHCH₂), 5.34 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 7.02 (ddd, 2H, J₁¼ 0.9 Hz,

J₂¼ 2.8 Hz, J₃¼ 7.6 Hz, AreH), 7.38e7.51 (m, 8H, AreH), 7.80e7.83

(m, 2H, AreH), 8.26e8.30 (m, 2H, AreH) ppm; ¹³C NMR (100 MHz,

d

121e1.25 (2ꢂ t, 6H, 2ꢂ CH₂CH₃), 3.56e3.64 (m,

4H, 2ꢂ CH₂CH₃), 3.67e3.81 (m, 12H, 2ꢂ CH₂CHCH₂), 3.85e3.89 (m,

4H), 4.18e4.34 (m, 6H, 2ꢂ CH₂CHCH₂), 5.11 (d, 1H, J_1,2¼ 1.7 Hz, H-1),

5.16 (d,1H, J_1,2¼ 1.7 Hz, H-1⁰), 7.15e7.19 (m, 2H, AreH), 7.26e7.28 (m,

2H, AreH), 7.31e7.36 (m, 2H, AreH), 7.41e7.46 (m, 2H, AreH), 7.75e

7.79 (m, 6H, AreH) ppm; ¹³C NMR (100 MHz, CD₃OD),

d

15.5 (2ꢂ

CH₂CH₃), 62.6, 62.7 (2ꢂ C-6), 68.0, 68.1 (2ꢂ CH₂CH₃), 68.4, 68.5 (2ꢂ

C-4), 69.1, 69.4, 71.2, 71.5 (4ꢂ CH₂), 72.3, 72.4 (2ꢂ C-2), 72.5, 72.6 (2ꢂ

C-3), 74.7, 74.9 (2ꢂ C-5), 75.8, 75.9 (2ꢂ CH), 101.4, 101.7 (2ꢂ C-1),

107.7, 107.9, 119.7, 119.8, 124.7, 124.8, 127.3, 127.4, 127.9, 128.6, 130.4,

130.5, 130.6, 130.7, 136.1, 158.0, 158.1 (20ꢂ AreC) ppm; HRMS (ESIꢃ):

m/z for C₂₁H₂₇O₈([M ꢃ H]^ꢃ): calcd 407.1706; found 407.1702.

CDCl₃),

d

62.7 (C-6), 68.3 (C-4), 69.3, 69.4 (2ꢂ CH₂), 72.4 (C-3), 72.5

(C-2), 75.2 (C-5), 75.8 (CH), 101.9 (C-1), 106.2, 106.3, 121.6, 121.7,

122.8,123.0,126.3,126.4,126.9,127.0,127.1,127.4,128.5,128.6,136.0,

136.1, 155.6, 155.7 (20ꢂ AreC) ppm; HRMS (ESIꢃ): m/z for C₂₉H₂₉O₈

([M ꢃ H]^ꢃ): calcd 505.1862; found 505.1872.

4.1.9.18. 2,3-Bis((7-methoxynaphthalen-2-yl)oxy)propan-1-yl

a-D-

4.1.9.14. 2,3-Diphenoxypropan-1-yl

Prepared from 17a (0.154 g, 1.36 mmol) according to the general

procedure F. Yield: 0.109 g (100.0%); orange oil; [

_Dþ45.2 (c 0.20,

MeOH); IR (KBr) 3421, 2929, 1599, 1496, 1457, 1301, 1243, 1174,

1134, 1058, 976, 882, 816, 753, 690, 508 cm^ꢃ1; ¹H NMR (400 MHz,

CD₃OD),

3.67e3.78 (m, 4H, H-3, H-4, H-6, H-6⁰), 3.83 (ddd, 1H,

J_5,6¼ 2.5 Hz, J_5,6’¼ 5.1 Hz, J_4,5¼ 8.2 Hz, H-5), 3.87 (dd, 1H,

J_1,2¼ 1.7 Hz, J_2,3¼ 3.2 Hz, H-2), 4.18e4.30 (m, 4H, CH₂CHCH₂), 4.43e

4.48 (m, 1H, CH₂CHCH₂), 5.16 (d, 1H, J_1,2¼ 1.7 Hz, H-1), 6.93e6.99

(m, 6H, AreH), 7.26e7.31 (m, 4H, AreH) ppm; ¹³C NMR (100 MHz,

a

-

D

-mannopyranoside (18a).

mannopyranoside (18e). Prepared from 17e (0.200 g, 0.27 mmol)

according to the general procedure F. Yield: 0.154 g (100%); white

a

]

solid; mp 135 ^ꢀC; [

a

]

_Dþ31.1 (c 0.22, MeOH); IR (KBr)

1630, 1609, 1516, 1448, 1387, 1257, 1209, 1185, 1165, 1137, 1023, 974,

842, 831, 808, 631 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

3.70 (t, 1H,

n 3445, 2932,

n

d

J_{3,4; 4,5}¼ 9.7 Hz, H-4), 3.73e3.84 (m, 3H, H-3, H-6, H-6⁰), 3.89e3.90

(m, 7H, H-5, 2ꢂ OCH₃), 3.92 (dd, 1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2),

4.36e4.49 (m, 4H, CH₂CHCH₂), 4.59e4.64 (m, 1H, CH₂CHCH₂), 5.24

(d, 1H, J_1,2¼ 1.7 Hz, H-1), 6.97 (ddd, 2H, J₁¼ 0.97 Hz, J₂¼ 2.5 Hz,

J₃¼ 8.8 Hz, AreH), 7.05 (m, 2H, AreH), 7.15 (dd, 2H, J₁¼ 2.4 Hz,

J₂¼ 8.9 Hz, AreH), 7.25 (d, 2H, J ¼ 2.6 Hz, AreH), 7.65e7.70 (m, 4H,

CD₃OD),

d

62.8 (C-6), 68.4 (C-4), 68.7, 68.9 (2ꢂ CH₂), 72.3 (C-2), 72.5

(C-3), 74.9 (C-5), 75.6 (CH), 101.7 (C-1), 115.6, 115.7, 122.0, 122.1,

AreH) ppm; ¹³C NMR (100 MHz, CDCl₃),

d

55.9 (2ꢂ OCH₃), 62.9 (C-

130.5, 130.6, 160.1, 160.2 (12ꢂ AreC) ppm; HRMS (ESIþ): m/z for

6), 68.5 (C-4), 68.9, 69.0 (2ꢂ CH₂), 72.4 (C-3), 72.5 (C-2), 75.3 (C-5),

75.8 (CH), 101.8 (C-1), 106.5, 106.6, 107.7, 107.8, 117.2, 117.3, 117.4,

117.5, 125.9, 126.0, 130.0, 130.4, 130.5, 137.6, 158.6. 158.7, 159.8 (20ꢂ

AreC) ppm; HRMS (ESIꢃ): m/z for C₃₁H₃₃O₁₀([M ꢃ H]^ꢃ): calcd

565.2074; found 565.2073.

C

₂₁H₂₆O₈Na ([M þ Na^þ]^þ): calcd 429.1525; found 429.1528.

4.1.9.15. 2,3-Bis(4-methoxyphenoxy)propan-1-yl

a-D-mannopyrano-

side (18b). Prepared from 17b (0.310 g, 0.49 mmol) according to the

general procedure F. Yield: 0.228 g (100%); white solid; mp 90e

91 ^ꢀC; [

a

]

_Dþ39.3 (c 0.22, MeOH); IR (KBr)

1508, 1458, 1289, 1231, 1181, 1133, 1107, 1064, 1044, 978, 932, 883,

824, 753, 676, 520 cm^ꢃ1; ¹H NMR (400 MHz, CD₃OD),

3.68 (t, 1H,

n

3366, 2917, 2065, 1594,

4.1.9.19. 2,3-Bis(naphthalen-2-yloxy)propan-1-yl a-D-mannopyrano-

side (18f). Prepared from 17f (0.237 g, 0.35 mmol) according to the

general procedure F. Yield: 0.178 g (100%); yellow solid; mp 188e

d

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

324

189 ^ꢀC; [

a

]

_Dþ31.9 (c 0.25, MeOH); IR (KBr)

n

3430, 1629, 1600, 1511,

The Maximum number of solutions per iteration and the Maximum

number of solutions per fragmentation parameter values were

increased to 1000, while other parameters were set at their default

values.

1456, 1256, 1216, 1181, 1120, 1059, 975, 835, 745, 623 cm^ꢃ1; ¹H NMR

(400 MHz, CD₃OD),

d

3.71 (t,1H, J_{3,4; 4,5}¼ 9.6 Hz, H-4), 3.73e3.84 (m,

4H, H-3, H-5, H-6, H-6⁰), 3.92 (dd,1H, J_1,2¼ 1.7 Hz, J_2,3¼ 3.3 Hz, H-2),

4.38e4.51 (m, 4H, CH₂CHCH₂), 4.61e4.65 (m, 1H, CH₂CHCH₂), 5.25

(d,1H, J_1,2¼ 1.7 Hz, H-1), 7.17e7.23 (m, 2H, AreH), 7.32e7.36 (m, 4H,

In order to validate our docking protocol, crystal structure ligand

Man₄was docked into the deﬁned Ca^2þbinding site of the receptor

using the above described docking parameters and pharmacophore

constraints. The protocol was able to reproduce the binding of the

Man1 residue interacting with the Ca^2þand both Glu residues very

AreH), 7.41e7.45 (m, 2H, AreH), 7.76e7.80 (m, 6H, AreH) ppm; ¹³

C

NMR (100 MHz, CDCl₃),

d

62.6 (C-6), 68.4 (C-4), 68.9, 69.0 (2ꢂ CH₂),

72.4 (C-3), 72.5 (C-2), 74.9 (C-5), 75.5 (CH), 101.8 (C-1), 107.9, 108.0,

119.7,119.8,124.7,124.8,127.4,127.5,128.0,128.6,130.4,130.5,130.6,

130.7, 136.1, 158.0, 158.1 (20ꢂ AreC) ppm; HRMS (ESIꢃ): m/z for

ꢂ

well (RMSD value of 0.8 A). The RMSD value for Man1 and Man2

ꢂ

residues of Man₄was 1.6 A, while RMSD value for the complete

₂₉H₃₀O₈([M ꢃ H]^ꢃ): calcd 505.1862; found 505.1854.

Man₄was 3.9 A. Such a result is expected, since Man2, Man3 and

ꢂ

C

Man4 residues point towards the solvent and form only weak in-

teractions with the protein. Nevertheless, we concluded that our

docking protocol is suitable for binding mode studies of the

designed DC-SIGN ligands, which were docked using the same

settings as were used for docking protocol validation. Proposed

binding modes and scoring function scores of the top ﬁve highest

scored docking poses per ligand were evaluated and the highest

ranked binding pose was used for graphical representation in DS.

4.2. Molecular modeling

4.2.1. Computer hardware and software

All the computational work was performed on two workstations.

Accelrys Discovery Studio 3.0 (DS) [42] was running on a worksta-

tion with Intel core i7 860 CPU processor, 8 GB RAM, two 750 GB

hard drives and a Nvidia GT220 GPU graphic card, running Centos

5.5. LeadIT (version 2.1.2.) [39] and NAMD (version 2.9) [43] were

running on four octal core AMD Opteron CPU processors, 16 GB

RAM, two 750 GB hard drives, running 64-bit Scientiﬁc Linux 6.0.

4.2.4. Molecular dynamics

The molecular dynamics package NAMD (version 2.9) [43] and

CHARMM22 force ﬁeld [44] were used for molecular dynamics

simulations of DC-SIGNe14d complex. The best scored docking pose

of compound 14d in complex with DC-SIGN CRD was used as the

initial structure for MD simulation. Molecular mechanics parame-

ters for compound 14d were estimated using ParamChem tool [47e

49]. Steepest descent (10 000 steps) and adopted basis Newtone

Raphson (10 000 steps) energy minimizations were ﬁrst performed

to remove atomic clashes and to optimize the atomic coordinates of

the proteineligand complex. The structure of the energy minimized

complex for MD simulation was prepared using psfgen in VMD

(version 1.9.1.) [50]. The complex was then embedded in a box of

water, which was modeled explicitly by a TIP3P model [51]. The

system was neutralized by addition of KCl at 0.4 M concentration.

The MD simulation was carried out in the NPT ensemble employing

periodic boundary conditions. Langevin dynamics and Langevin

piston methods were used for temperature (300 K) and pressure

(1 atm) control, respectively. Short- and long-range forces were

calculated every 1 and 2 timesteps, respectively, with a time step of

2.0 ps. The smooth particle mesh Ewald method [52] was used to

calculate electrostatic interactions. The short-range interactions

4.2.2. Ligand and protein preparation

The tridimensional models of target compounds (Tables 1 and 2)

were built from a standard fragment library in Accelrys Discovery

Studio 3.0 (DS) [42]. The geometries of the molecules were opti-

mized using CHARMM force ﬁeld [44] with MMFF94 [45] partial

atomic charges. The energy was minimized using the Smart Mini-

mizer algorithm in DS until the gradient value was smaller than

ꢂ

0.001 kcal/(mol A). The optimized structure was further reﬁned

with GAMESS interface in ChemBio3D Ultra 13.0 using semi-

empirical PM3 method, QA optimization algorithm and Gasteiger

Hückel charges for all atoms for 100 steps [46].

The crystal structure of DC-SIGN CRD in complex with tetra-

mannoside Man₄(PDB entry: 1SL4) was taken as a receptor for

docking calculations using FlexX [37,38] as available in LeadIT [39].

Receptor was prepared in LeadIT graphical user interface using the

ꢂ

Receptor wizard. Amino acid residues within a radius of 7 A around

Man₄were deﬁned as the binding site. Hydrogen atoms were added

to the binding site residues and correct tautomers and protonation

states were assigned. Water molecules and the ligand were deleted

from the crystal structure, while Ca^2þion in the Ca^2þbinding site

was deﬁned as part of the receptor. Pharmacophore type constraints

in FlexX-Pharm were deﬁned in order to correctly place the core

mannose residue during the docking validation procedure (see

below). First, PharmMetal pharmacophore with spherical coordi-

nation around Ca^2þwas deﬁned to account correctly for complex

interactions between Ca^2þ(406) and Man1 residue of Man₄(in the

validation procedure) and mannose residue of the designed ligands

(in the docking procedure). Secondly, side chain carboxylate groups

of Glu347 and Glu354 were deﬁned as hydrogen bond acceptors,

which form hydrogen bonds with 3- and 4-hydroxyl groups of

mannose residue in the crystal structure. All three selected phar-

macophore type constraints were speciﬁed as essential. Addition-

ally, we have deﬁned Phe313 side chain as a phenyl ring center in

FlexX-Pharm, but the docking poses were comparable to those

without this pharmacophore constraint. Hence, this constraint was

not used in docking validation and ligand docking.

ꢂ

were cut off at 12 A. All chemical bonds between hydrogen and

heavy atoms were held ﬁxed using SHAKE algorithm [53]. The

simulation consisted of three consecutive steps: (i) solvent equili-

bration for 0.5 ns with ligand and protein constrained harmonically

around the initial structure; (ii) equilibration of the complete system

for 1 ns with ligand and protein released; (iii) an unconstrained 7 ns

production run to allow the protein and the ligand to position

themselves according to physical forces between them. The trajec-

tory of the equilibration and production run was used for analysis in

VMD. The average structure of the last ns of the production run was

calculated and used for analysis of binding mode of 14d.

4.3. Biological assays

4.3.1. Production of soluble DC-SIGN ECD

DC-SIGN ECD protein (residues 66-404) were overexpressed

and puriﬁed according to the already published procedure

[15,22,54].

4.2.3. Validation of the docking protocol and ligand docking

The FlexX molecular docking program, as available in LeadIT,

was used for ligand docking. Hybrid algorithm (enthalpy and en-

tropy driven ligand binding) was used to place the ‘base fragment’.

4.3.2. Competitive gp120-displacement assay

The binding afﬁnities of the synthesized compounds to DC-SIGN

ECD were characterized by a solid-phase competitive displacement

ꢀ ꢁ

T. Tomasic et al. / European Journal of Medicinal Chemistry 75 (2014) 308e326

325

assay. gp120 (Human Immunodeﬁciency Virus type 1/HIV-1 gp120

Protein, 100 mcg, 11233-V08H, Sino Biological Inc., China) was

dissolved in aqueous solution of NaCl (0.05 M, 0.300 mL) at 30 ^ꢀC,

and then further diluted with 0.600 mL of water, and 0.100 mL of

1 M NaHCO_3(aq). Biotin N-hydroxysuccinimide ester (1 mg) was

dissolved in N,N-dimethylformamide (1 mL) and 0.100 mL of the

solution was added to the solution of gp120. The reaction mixture

was incubated for 90 min at 30 ^ꢀC and dialyzed with a cut-off value

of 12.000e14.000 for 3 h at RT against buffer 1 (3 L, 20 mM Tris,

150 mM NaCl, pH ¼ 7.4). After dialysis, the solution was centrifuged

for 5 min at 5400 rpm and diluted with buffer 2 (20 mM Tris,

150 mM NaCl,1 mM CaCl₂, pH ¼ 7.4) to a volume of 5 mL to yield the

Institut Universitaire de France for ﬁnancial support. The authors

thank Prof. Roger Pain for critical reading of the manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2014.01.047.

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Article Doi

Monovalent mannose-based DC-SIGN antagonists: Targeting the hydrophobic groove of the receptor

DOI: 10.1016/j.ejmech.2014.01.047

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