Matrix metalloproteinase-12 inhibitors: synthesis, structure-activity relationships and intestinal absorption of novel sugar-based biphenylsulfonamide carboxylates

Article abstract of DOI:10.1016/j.bmc.2018.10.024

MMP-12 is a validated target in pulmonary and cardiovascular diseases. The principal obstacles to clinical development of MMP-12 inhibitors are an inadequate selectivity for the target enzyme and a poor water solubility, with consequent poor oral bioavailability. We recently reported a new class of sugar-based arylsulfonamide carboxylates with a nanomolar activity for MMP-12, a good selectivity and an improved water solubility. In this study, we designed and synthesized new derivatives to characterize the structure-activity relationship (SAR) within this class of glycoconjugate inhibitors. All the new derivatives were tested on human recombinant MMP-12 and MMP-9 in order to evaluate their affinity and the selectivity for the target enzyme. Among them, the four most promising compounds were selected to assess their intestinal permeability using an ex vivo everted gut sac model. Given the high polarity and structural similarity to glucose, compound 3 was demonstrated to cross the intestinal membrane by using the facilitative GLUT2 transport.

Full text of DOI:10.1016/j.bmc.2018.10.024

Accepted Manuscript
Matrix Metalloproteinase-12 Inhibitors: Synthesis, Structure-Activity Relation-
ships and Intestinal Absorption of novel Sugar-Based Biphenylsulfonamide
Carboxylates
Doretta Cuffaro, Caterina Camodeca, Felicia D'Andrea, Eugenia Piragine, Lara
Testai, Vincenzo Calderone, Elisabetta Orlandini, Elisa Nuti, Armando Rossello
PII:
S0968-0896(18)31375-0
https://doi.org/10.1016/j.bmc.2018.10.024
BMC 14583
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 July 2018
24 October 2018
25 October 2018
Please cite this article as: Cuffaro, D., Camodeca, C., D'Andrea, F., Piragine, E., Testai, L., Calderone, V., Orlandini,
E., Nuti, E., Rossello, A., Matrix Metalloproteinase-12 Inhibitors: Synthesis, Structure-Activity Relationships and
Intestinal Absorption of novel Sugar-Based Biphenylsulfonamide Carboxylates, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.024
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Matrix Metalloproteinase-12 Inhibitors: Synthesis, Structure-Activity Relationships and
Intestinal Absorption of novel Sugar-Based Biphenylsulfonamide Carboxylates
Doretta Cuffaro,1 Caterina Camodeca,1 Felicia D’Andrea,1 Eugenia Piragine,1 Lara Testai,1 Vincenzo
Calderone,1 Elisabetta Orlandini,2 Elisa Nuti,1 and Armando Rossello1,*
1Dipartimento di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy.
*Corresponding author: Armando Rossello, phone, +39 050 2219562; fax, +39 050 2219605; e-mail:
armando.rossello@farm.unipi.it
Keywords: MMP-12 inhibitors, glycoconjugates, intestinal permeability, glucose transporters.
Abstract
MMP-12 is a validated target in pulmonary and cardiovascular diseases. The principal obstacles to
clinical development of MMP-12 inhibitors are an inadequate selectivity for the target enzyme and a
poor water solubility, with consequent poor oral bioavailability. We recently reported a new class of
sugar-based arylsulfonamide carboxylates with a nanomolar activity for MMP-12, a good selectivity
and an improved water solubility. In this study, we designed and synthesized new derivatives to
characterize the structure-activity relationship (SAR) within this class of glycoconjugate inhibitors. All
the new derivatives were tested on human recombinant MMP-12 and MMP-9 in order to evaluate their
affinity and the selectivity for the target enzyme. Among them, the four most promising compounds
were selected to assess their intestinal permeability using an ex vivo everted gut sac model. Given the

high polarity and structural similarity to glucose, compound 3 was demonstrated to cross the intestinal
membrane by using the facilitative GLUT2 transport.
1. Introduction
MMP-12, or macrophage metalloelastase, belongs to the matrix metalloproteinases (MMPs), a family
of endopeptidases able to degrade the structural components of the extracellular matrix (ECM).1 MMP-
12 can cleave a broad spectrum of extracellular matrix components including elastin, the major
constituent of alveolar walls, and other proteins such as type IV collagen, fibronectin, laminin, gelatin,
vitronectin, and chondroitin sulfates.2 This enzyme is mainly produced by macrophages and is
involved in acute and chronic pulmonary inflammatory diseases associated with intense airway
remodeling, such as chronic obstructive pulmonary disease (COPD) and emphysema.3 Moreover,
MMP-12 has been shown to be critical in the initiation and progression of atherosclerosis by
stimulating the transformation of fatty streaks into fibrous plaques.4 Given its upregulation in
atherosclerosis,5 aneurysms,6 and cardiovascular diseases,7 MMP-12 selective tracers for molecular
imaging have been recently developed.8 Similarly, several MMP-12 selective inhibitors have been
reported and are being tested in various preclinical trials for the treatment of lung9 and cardiovascular10
diseases. The principal obstacles to clinical development of MMP inhibitors (MMPIs) are an
inadequate selectivity for the target enzyme and a poor water solubility, with consequent poor oral
bioavailability.
To overcome these problems, in the last years some carbohydrate-containing MMPIs were developed
in order to obtain more bioavailable derivatives containing the classical saccharide moiety but also the
more specific azasugars.11,12,13 Unfortunately, these novel MMPIs were characterized by an increased
hydrophilicity but a low selectivity for a specific MMP.
In a previous work,14 we reported the synthesis and the biological activity of new sugar-based
arylsulfonamide carboxylates as MMP-12 inhibitors. These glycoconjugates were prepared starting
from the previously described carboxylate MMP-12 inhibitor 115 (Figure 1) through the insertion of a
β-N-acetyl-D-glucosamine (GlcNAc) moiety in P2'. In particular, compounds 2 and 3 (Figure 1) were
able to conjugate a nanomolar activity for MMP-12, with a good selectivity and an improved water
solubility with respect to derivative 1, devoid of the sugar moiety.

Figure 1.Chemical structure of selective MMP-12 inhibitors 1-3.
On the basis of these encouraging results, in the present study we undertook the synthesis of new
glycoconjugate inhibitors of MMP-12 (4-15, Figure 2) in order to perform a Structure-Activity
Relationship (SAR) analysis for this class of compounds. In fact, a SAR analysis could be useful to
further optimize the glycoconjugate inhibitors already developed. The modifications included the
introduction of a different glycosidic moiety, by replacing N-acetyl glucosamine with glucose, the
change of the conjugation position (from C-1 to C-6 sugar position) and the spacer length. In particular,
the conjugation of the sugar to the MMP inhibitor scaffold was obtained through two different
strategies: by Mitsunobu reaction and by thioureidic linker formation (Figure 2).

Figure 2. General structure of new glycoconjugates 4-15.
All the new derivatives were tested by fluorometric assay on human recombinant MMP-12 and MMP-9
in order to evaluate their affinity and the selectivity for the target enzyme. Among them, the most
promising compounds were selected to assess their intestinal permeability using an ex vivo everted gut
sac model. In fact, it is well known that intestinal absorption of a drug after oral administration relies
on both solubility and the ability to pass gastrointestinal (GI) membranes. At this regard, intestinal
permeability is referred to the ability of a compound to penetrate through the intestinal epithelium; then
it is a critical parameter of absorption. The quantification of permeated compounds has been performed
by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection, a commonly used
technique that has been successfully applied to the determination of the intestinal permeation of
drugs.16 Finally, we investigated the absorption mechanism of glycoconjugate inhibitors of MMP-12 in
the ex vivo model. On the basis of the well-known glucose transporter mechanisms present at the
intestinal level, inhibition studies were conducted using the pharmacological agents phloridzin, a potent
inhibitor of sodium-dependent glucose transporter 1 (SGLT1), or phloretin, an inhibitor of glucose
transporter 2 (GLUT2).17
2. Results and Discussion
2.1. Design of new glycoconjugate MMP-12 inhibitors

In order to develop a SAR study for this class of MMP-12 inhibitors, the previously described
thioureido derivatives 2 and 3 were selected as starting point. In particular, compound 3 showed a
nanomolar activity on MMP-12 (IC50= 40 nM) and a good selectivity over MMPs with improved
hydrophilicity (water solubility > 5 mM and clog P = 3.15).14 This hit compound presents a carboxylate
as zinc-binding group (ZBG) able to chelate the catalytic zinc ion and a biphenyl sulfonamide scaffold
able to interact with some fundamental aminoacid residues of the MMP-12 active site (PDB ID: 5I3M).
Moreover, in this compound a β-N-acetyl-D-glucosamine residue is linked to the sulfonamide nitrogen
atom (P2' substituent) through insertion of a thioureido group that is biocompatible and stable in most
biological systems.
In the present work, a substitution of N-acetyl glucosamine with glucose was undertaken in order to
promote intestinal absorption of the new compounds by exploiting the glucose transporters. Since
glucose is actively absorbed by sodium-dependent glucose cotransporter 1 (SGLT1) in the brush-border
membrane (BBM) of intestinal epithelial cells,18 SGLT1 could also be involved in the transport of
some sugar analogues. Sugar-protected analogues, acetylated on the hydroxy groups, were also
synthesized and tested on isolated enzymes. Moreover, the influence of the conjugation position on
activity was evaluated by linking the MMP inhibitor scaffold in C-6 or in C-1 position on the sugar
moiety with or without insertion of the thioureido group.
2.2. Chemistry
The new glycoconjugates 4-15 were synthesized as reported in Scheme 1, 2 and 3. These new
compounds were prepared following two different conjugation strategies: by Mitzsunobu reaction
(Scheme 1) or by the insertion of a thioureidic linker (Scheme 2 and 3).
The synthesis of glycoconjugates 19, 20, 23 and 24 is described in Scheme 1. A Mitsunobu
condensation of sulfonamide 1814 with alcohols 16,19 17,19 2120 and 2221 in the presence of diisopropyl
azodicarboxylate (DIAD) and triphenylphosphine (PPh3) gave glycoconjugates 19, 20, 23 and 24 (42-
55% yields).

Scheme 1. Reagents and conditions: i) 18, PPh3, DIAD or DMEAD, THF dry, rt, 42-55%; ii) TFA,
DCM dry, rt, 89-92%; iii) NH3-MeOH 7N, MeOH, rt, 85-90%.

Scheme 2. Reagents and conditions: i) NaN3, DMF, rt (quantitative); ii) PPh3.resin, THF-H2O 20:1, rt;
iii) DPT, DCM, rt (22% for two steps); iv) 18, Et3N, DCM, 73%; v) TFA, DCM, 42%; vi) NH3-MeOH
7N, MeOH, 75%.
The β-azido glucose derivative 25 was obtained by conversion of the commercial 2,3,4,6-tetra-O-
acetyl--D-glucopyranosyl bromide through SN2 reaction (quantitative yield). The β-glucose
isothiocyanate 27 (Scheme 2) was prepared starting from β-azido glucose 25. The azido derivative 2522
was reduced to the corresponding amine 26 by Staudinger reaction using PPh3 in THF dry and without
further purification was transformed into the corresponding isothiocyanate derivative 27 by treatment
with di-2-pyridyl thionocarbonate (DPT) (22% yield from 25).
Scheme 3. Reagents and conditions: i) DPT, DCM, rt, 78%; ii) H2/Pd, MeOH/DCM/H2O, rt, 50%; iii)
32, Et3N, DCM, 47%; iv) TFA, DCM, 52.5%; v) NH3-MeOH 7N, MeOH,73%.
The C-6 azido D-glucose 31 (Scheme 3) was prepared following the reported procedure.23 The azido
functionality of 31 was reduced to the corresponding amine 32 by Pd catalyzed hydrogenation (50%
yield). Thioureido derivatives 29 and 33 (Scheme 2 and 3) were obtained through direct coupling of
trifluoroacetate salt MMP inhibitor amino 2814 with the isothiocyanate β-glucose derivative 27
(Scheme 2) or by conjugation of the C-6 amino glucose 32 with the isothiocyanate MMP inhibitor 30
(78% yield, Scheme 3), obtained by treatment of 28 with DPT as described above for the

isothiocyanate 27. The condensations between isothiocyanates 27 or 30 and amines 28 or 32 were
performed in DCM and in presence of triethylamine (Et3N) at 60 °C (47-73% yields). Purification of
the crude products afforded pure esters 29 and 33.
Removal of the tert-butyl group of esters 19, 20, 23, 24, 29 and 33 by treatment with trifluoroacetic
acid (TFA) gave the desired carboxylic acids 4, 5, 8, 9, 12 and 14 after purification by flash
chromatography. Finally, the desacetylation of 4, 5, 8, 9, 12 and 14 obtained by basic hydrolysis with
3.5 N NH3-MeOH afforded deprotected derivatives 6, 7, 10, 11, 13 and 15 in good yields.
2.3. MMP inhibition studies
The series of new glycoconjugate arylsulfonamide carboxylates, the sulfonamides 4-11 (Table 1)
devoid of the ethylene thioureido spacer and the thioureido derivatives 12-15 and (Table 2), were
screened in vitro on human recombinant MMP-12 and MMP-9 by a fluorometric assay. An improved
selectivity for MMP-12 over MMP-9 could be indicative of the ability of the new derivatives to
discriminate among MMPs given the high homology between the catalytic domains of these two
enzymes both characterized by a “medium” S1’ pocket.24 The glycoconjugates 2 and 3 were used as
reference compounds.
Analyzing the sulfonamide series, those compounds functionalized on the anomeric position (4-7)
showed a decreased activity on MMP-12 (IC50 = 1.3 - 40 µM) with respect to the reference compounds
2 and 3 (IC50 = 18 - 40 nM). These results indicate that the insertion of a spacer in the anomeric
position is fundamental to have a nanomolar affinity for the target enzyme.
In the same series, it is noteworthy the difference of MMP-12 affinity due to the nature of the sugar
moiety, in fact the glucose glycoconjugates 5 and 7 presented a better activity than the corresponding
N-acetyl glucosamine derivatives 4 and 6, respectively. In particular, the protected glucose carboxylate
5 was 4-fold more active than the glucosamine derivative 4 and the deprotected glucose derivative 7
was 7-fold more active than 6. Finally, the modification of the conjugation position was fundamental,
considering the significant improvement of MMP-12 activity for C-6 derivatives. In fact, the C-6
glycoconjugates 9 and 11 presented a nanomolar activity for MMP-12 with a 114-fold improvement of
activity for carboxylic acid 9 with respect to its C-1 substituted analogue 5 and with a 25-fold improved
activity of the deprotected 11 compared to its C-1 substituted analogue 7. These results confirmed also
the need for a flexible linker between the MMP inhibitor scaffold and the sugar moiety in the
sulfonamide series. This condition is satisfied for compounds 8-11 showing a direct conjugation in C-6
position but not for compounds 4-7 with a direct conjugation in the anomeric position. The best

compound of this series was the C-6 GlcNAc derivative 10, with an activity on MMP-12 comparable to
3 (IC50 = 41 nM) but a lower selectivity over MMP-9.

Table 1. MMP-12 and MMP-9 inhibitory activity (IC50 nM values) of sulfonamido derivatives 4-11.
IC50 (nM)[a]
Compd
4
R
MMP-12
MMP-9
40000
nd
9600
9200
1300
83
nd
nd
5
6
>50000
1235
2600
890
7
8
84
9
41
10
11
51
2200
18
40
1200
5400
2
3
[a]IC50values are the average of three determinations
with a standard deviation of <10%; nd: not determined
In the thioureido series (Table 2) the C-1 glycoconjugates 12 and 13, glucose analogues of the
reference compounds 2 and 3, showed a nanomolar activity for MMP-12. In particular, their MMP-12
affinity was comparable to the one of the reference compounds. Moreover, the higher activity of the

acetylated derivative 12 with respect to the deprotected 13 confirmed the data already obtained for
compounds 2 and 3. These results indicated that in the thioureido series of compounds the nature of the
sugar moiety does not affect the affinity for MMP-12 (see 12 and 13 compared to 2 and 3). Moreover,
in this series of compounds all presenting a flexible ethylene thioureido linker, the conjugation position
does not have a strong impact on MMP-12 inhibitory activity (see 14 and 15 compared to 12 and 13).
However, the conjugation position was crucial for determining the selectivity of thioureidic derivatives.
In fact, the C-6 glucoconjugates 14 and 15 revealed a similar inhibitory activity with respect to 12 and
13 but a significantly decreased selectivity for MMP-12 over MMP-9. This decrease of selectivity,
namely due to an improved activity on MMP-9, could be explained on the basis of the crystal structure
previously obtained for compound 2 co-crystallized with the catalytic domain of MMP-12 and MMP-
9.14 The MMP-9 catalytic site did not allow a proper positioning of the sugar moiety of 2 but the same
moiety present in 14 conjugated in C-6 position instead of in C-1 could restore a good binding with the
molecular surface of MMP-9.

Table 2. MMP-12 and MMP-9 inhibitory activity (IC50 nM values) of thioureido derivatives 12-15 and
the reference compounds 2-3.
IC50 (nM)[a]
Compd
12
R
MMP-12 MMP-9
19
98
880
1120
13
14
20
65
32
520
15
18
40
1200
5400
2
3
[a]IC50 Values are the average of three determinations
with a standard deviation of <10%.
Overall, this SAR analysis allowed us to conclude that the most promising compounds obtained so far
were the C-1 thioureido derivatives 2 and 3 and their glucose analogues 12 and 13. Given their activity
and selectivity for MMP-12, these four compounds were selected for further studies to evaluate their
intestinal permeability.
The physicochemical properties, i.e. octanol/water partition coefficient (cLogP) and topological polar
surface area (TPSA), of the most promising new compounds 12 and 13 were predicted in silico in
comparison with 2 and 3 by using OSIRIS Property Explorer (Table 3). The deprotected derivatives 3

and 13 presented a particularly low cLogP value, probably due to the presence of the desacetylated
glycosidic residues. Futhermore, the permeability properties evaluated by the TPSA parameter,
presented quite high values for all compounds. Molecular polar surface area (PSA), is a descriptor that
was shown to correlate well with passive molecular transport through membranes and, therefore, it is
used to predict the transport properties of drugs.25 These results agree with our hypothesis that the
highly polar glycosylated derivatives could cross the GI membrane not by passive diffusion but through
an active transport mechanism.
Table 3. Predicted physicochemical properties of compounds 2, 3, 12 and 13.
Compd
cLogP[a]
TPSA[a]
253.4
238.2
256.4
229.3
1.99
12
13
2
0.05
1.51
0.06
3
[a] clogP (o/w) and TPSA values were calculated by using OSIRIS Property Explorer26
2.4. Permeability studies
Everted gut sac model is a efficient tool for studying ex vivo drug absorption mechanisms, intestinal
metabolism of drugs, role of transporter in drug absorption, and for investigating the role of intestinal
enzymes during drug transport through the intestine;27 therefore we here used it to evaluate the
intestinal permeability of the selected compounds. In these experimental conditions (see Experimental
section for details), the non-glycoconjugated compound 1, as well as the glycoconjugated 2, 12 and 13,
were poorly absorbed through intestinal membrane. Indeed, the maximal concentration detected for
these compounds used at 100 µM was lower than 10 µM after 60 min of incubation (Figure 3 and
Table 4).
On the other hand, compound 3 (100 µM) showed a better intestinal permeability. Indeed, after 60 min
of incubation its concentration inside the sacs was 19.51 ± 2.27 µM. Since compound 3 demonstrated
to be the most promising among new MMP-12 inhibitors, a further evaluation of its transport
mechanism was carried out.
Glucose and glucose analogues cross the cell membranes with the help of two transporters belonging
either to the family of facilitative glucose transporters (GLUTs) or to the family of sodium glucose
cotransporters (SGLUTs). In particular, at the intestinal level SGLUT1 is located in brush border

membranes, whereas in basolateral membranes are located GLUT2 transporters. A second series of
experiments showed that SGLUT1 is not involved in the transport of compound 3, since the addition of
phloridzin 10 µM as well as 100 µM did not inhibit the adsorption of 3 through intestinal membrane.
Instead, the addition of phloretin 100 µM, a known GLUT2 inhibitor, significantly reduced the passage
of compound 3 suggesting that it uses the facilitative GLUT2 transport to cross intestinal membrane
(Figure 4 and Table 5).
25
Compd 1 100 M
Compd 2 100 M
20
Compd 3 100 M
Compd 12 100 M
Compd 13 100 M
15
10
5
0
0
15
30
45
60
Time (min)
Figure 3. Intestinal permeability of selected MMP-12 inhibitors in Everted Gut Sac model.
Data shown are 6 ± 2.
Table 4. Amounts of compound permeated through intestinal membrane (µM). Data are shown ± SEM.
Compd 1
3.87 ± 0.71
4.63 ± 0.24
6.03 ± 1.27
Compd 2
1.25 ± 0.07
2.04 ± 0.30
2.23 ± 0.16
Compd 3
6.62 ± 1.18
7.14 ± 0.91
19.51 ± 2.27
Compd 12
0.97 ± 0.16
2.21 ± 0.17
3.37 ± 0.32
Compd 13
2.48 ± 0.04
5.0 ± 0.43
5.82 ± 0.26
15 min
30 min
60 min

Compd 3 100 M
25
20
15
10
5
Compd 3 + phloridzin 10 M
Compd 3 + phloridzin 100 M
Compd 3 + phloretin 100 M
*
0
0
15
30
45
60
Time (min)
Figure 4. Intestinal permeability of compound 3 in the presence of phloridzin (10 µM and 100 µM),
phloretin (100 µM), or in their absence. Data shown are 8 ± 3 and they were analyzed with two-way
ANOVA test. The asterisk indicates a statistically significant difference vs pre-incubation with
inhibitor (*p<0.05).
Table 5. Amounts of compound permeated through intestinal membrane (µM). Data are shown ± SEM.
Compd 3
Compd 3 +
phloridizin 10 µM
Compd 3 +
phloridzin 100
µM
Compd 3 +
phloretin 100 µM
15 min
30 min
3.87 ± 0.71
4.63 ± 0.24
3.0 ± 0.06
3.1 ± 0.15
3.47 ± 0.74
2.47 ± 0.12
3.48 ± 0.41
4.33 ± 0.09
60 min
6.03 ± 1.27
19.45 ± 0.15
16.05 ± 2.95
8.37 ± 0.9
To quantify the intestinal permeability of the selected compounds 2, 3, 12 and 13, in comparison with
reference compound 1, analytical HPLC methods have been developed. For each compound, a
calibration curve at different concentration in the range of 500-1 µM has been prepared. Good linearity
between the peak-area and the respective concentration of compounds was observed, and the
correlation coefficients (r2) for the selected compounds were in the range 0.984 ≤ r2 ≤ 0.999.

The selectivity of the method has been determined by testing the presence in the medium of other
substances that could interfere with the peak of the compound in the chromatogram. Therefore,
chromatograms were compared, and in the chromatographic conditions used for the analysis no other
interfering peaks were observed. The lower limit of quantification was 0.7 µM. The methods have been
demonstrated to be sensitive and accurate for the determination of the compounds 1, 2, 3, 12 and 13.
The developed methods have been used to analyze samples derived from ex vivo everted gut sac
permeation experiments. The samples of serosal fluid at the selected time point (0, 15, 30, 60 min)
were injected in the HPLC system and the concentration of the compounds has been directly
determined from the linear regression equation derived from the calibration curve.
3.
Conclusions
Oral administration of selective MMP-12 inhibitors might be useful to treat pulmonary and
cardiovascular diseases. To achieve this purpose, compounds selective for the target enzyme with
adequate hydrophilicity should be developed. In this study, we designed and synthesized new
glycoconjugates belonging to the class of sugar-based arylsulfonamide carboxylates previously
reported as MMP-12 inhibitors. In this class, compounds 2 and 3 were able to conjugate a nanomolar
activity for MMP-12, with a good selectivity and an improved water solubility with respect to the
parent compound 1, devoid of the sugar moiety. All new derivatives were tested in vitro on human
recombinant MMP-12 and MMP-9 by fluorometric assay. The SAR results confirmed that the most
promising compounds obtained so far were the C-1 thioureido N-acetylglucosamino-based derivatives
2 and 3 and their glucose analogues 12 and 13. Given their activity and selectivity for MMP-12, these
four compounds were selected to assess their intestinal permeability using an ex vivo everted gut sac
model as a first step to evaluate their intestinal absorption. In fact, it is well known that intestinal
absorption of a drug after oral administration relies on both solubility and the ability to pass
gastrointestinal membranes. Among the four glycoconjugates, compound 3 showed the highest
intestinal permeability and further experiments were conducted to determine the absorption
mechanism. Considering its polarity and structural similarity to glucose an active transport was
hypothesized and our results showed that compound 3 crossed the intestinal membrane by using the
facilitative GLUT2 transport. Further studies to determine the pharmacokinetic characteristics of this
lead compound before in vivo testing are ongoing.
4.
Experimental Section

4.1.
Chemistry. Melting points were determined with a Kofler hot-stage apparatus and are
1
uncorrected. Optical rotations were measured with a Perkin-Elmer 241 polarimeter at 20±2 °C. H
NMR spectra were recorded in appropriate solvents with a Bruker Avance II operating at 250.12 MHz
13
or a Bruker Avance III HD 400 spectrometer operating at 400 MHz. C NMR spectra were recorded
with the spectrometers operating at 62.9 MHz or 100.57 MHz. The assignments were made, when
possible, with the aid of DEPT, COSY, HSQC experiments. The first order proton chemical shifts are
referenced to residual solvents and J-values are given in Hz. All reactions were followed by TLC on
Kieselgel 60 F254 with detection by UV light and/or with ethanolic 10% phosphomolybdic or sulfuric
acid, and heating. Kieselgel 60 (Merck, 70-230 and 230-400 mesh, respectively) was used for column
and flash chromatography. Some of flash chromatography were conducted by the automated system
Isolera Four SVTM (Biotage®), equipped with UV detector with variable wavelength (200-400 nm) or
using prepacked ISOLUTE Flash Si II cartridges (Biotage). Microwave-assisted reactions were run in a
CEM Discover LabMate microwave synthesizer. Unless otherwise noted, solvents and reagents were
obtained from commercial suppliers and used without further purification. All reactions involving air-
or moisture-sensitive reagents were performed under an argon atmosphere using anhydrous solvents.
Anhydrous dimethylformamide (DMF), dichloromethane (CH2Cl2), 1,2-dichloethane (DCE) and THF
were purchased from Sigma-Aldrich. MgSO4 or Na2SO4 were used as the drying agents for solutions.
Elemental analysis was used to determine the purity of target compounds. Analytical results are within
± 0.40% of the theoretical values.
4.1.1. General procedure for the synthesis of tert-butyl derivatives 19, 20, 23 and 24
To a solution of the appropriate sugar (16, 17, 21 or 22, 1 equiv) in dry THF, sulfonamide 18 (1.3
equiv) was added under Argon atmosphere. The mixture was cooled down to 0 °C, then dry PPh3 (3.5
equiv) and di-2-methoxyethylazodicarboxylate (DMEAD) or di-isopropylazodicarboxylate (DIAD)
(3.5 equiv) were added. The reaction mixture was stirred at room temperature until TLC analysis
revealed a complete disappearance of the saccharide starting material. Subsequently, the solvent was
evaporated and the residue was taken up with CH2Cl2 and washed with saturated solution of NaHCO3
(1 × 20 mL). The aqueous phase was extracted with CH2Cl2 (2× 20 mL). The combined organic phases
were dried with Na2SO4, filtered and evaporated under reduced pressure. The crude was purified by
flash chromatography.
4.1.1.1.
tert-Butyl
(2R)-2-(N-((2R,3R,4S,5S,6R)-3-acetamido-4,5-diacetoxy-6-
(19).
(acetoxymethyl)tetrahydro-2H-pyran-2-yl)biphenyl-4-ylsulfonamido)-3-methylbutanoate

The Mitsunobu reaction was conducted following the general procedure, starting from a solution of
compound 16 (40.0 mg, 0.116 mmol) in dry THF (2.36 mL), the biphenylsulfonamide 18 (58.6 mg,
0.150 mmol), PPh3 (106.0 mg), and DMEAD (94.9 mg, 0.405 mmol). The reaction mixture was stirred
at room temperature for 22 h. After work-up, the crude (284 mg) was purified by flash chromatography
(n-hexane-EtOAc 4:6 + 0.01% Et3N) affording derivative 19 (39.5 mg, 47% yield) as a clear syrup. 1H
NMR (250.12 MHz, CD3CN): δ 7.97 (m, 2H, 2×Ar-H), 7.79 (m, 2H, 2×Ar-H), 7.67 (m, 2H, 2×Ar-H),
7.54-7.45 (m, 3H, 3×Ar-H), 6.59 (d, 1H, J2,NH=8.2 Hz, NHAc), 5.15 (m, 1H, H-3), 5.12 (d, 1H, J1,2=9.3
Hz, H-1), 5.04 (m, 1H, H-2), 4.99 (m, 1H, H-4), 4.10 (m, 1H, H-6a), 3.97 (m, 1H, H-6b), 3.88 (d, 1H,
J=10.2 Hz, CHNSO2), 3.83 (m, 1H, H-5), 2.20 (m, 1H, Me2CH), 1.99, 1.97, 1.94 (3s, each 3H,
3×MeCOO), 1.72 (s, 3H, MeCON), 1.31 (s, 9H, CMe3), 0.92 (d, 3H, Jvic=6.5 Hz, CHMe2), 0.79 (d, 3H,
13
Jvic=6.5 Hz, CHMe2). C NMR (62.9 MHz, CD3CN): δ 171.2-170.4 (4× C=O), 146.5 (Ar-C-SO2),
139.8, 138.7 (2×Ar-C), 130.1-128.2 (Ar-CH), 85.7 (C-1), 83.8 (CMe3), 76.3 (C-3), 74.6 (C-5), 69.3 (C-
4), 67.6 (CHNSO2), 63.3 (C-6), 53.1 (C-2), 30.1 (CHMe2), 28.0 (CMe3), 23.5 (MeCON), 20.9
(3×MeCO), 20.5, 19.5 (CHMe2).
4.1.1.2. tert-Butyl (2R)-2-(N-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-
pyran-2-yl)biphenyl-4-ylsulfonamido)-3-methylbutanoate (20). The Mitsunobu reaction was
conducted following the general procedure, starting from a solution of compound 17 (50.0 mg, 0.144
mmol) in dry THF (2.90 mL), the biphenylsulfonamide 18 (83.9 mg, 0.215 mmol), PPh3 (118.0 mg,
0.502 mmol), and DMEAD (118 mg, 0.502 mmol). The reaction mixture was stirred at room
temperature for 46 h. After work-up, the crude residue (380 mg) was purified by flash chromatography
1
(n-hexane-EtOAc 6:4 + 0.01% Et3N) affording derivative 20 (41 mg, 40% yield) as white foam. H
NMR (250.12 MHz, CDCl3 δ 8.01 (m, 2H, 2×Ar-H), 7.68 (m, 2H, 2×Ar-H), 7.57 (m, 2H, 2×Ar-H),
7.51-7.26 (m, 3H, 3×Ar-H), 5.85 (dd, 1H, J2,3=9.3 Hz, J3,4=8.6 Hz, H-3), 5.42 (d, 1H, J1,2=9.7 Hz, H-
1), 5.09 (dd, 1H, H-2), 5.14 (dd, 1H, J4,5=9.7 Hz, H-4), 4.13 (dd, 1H, J6a,6b=12.0 Hz, J5,6b=5.6 Hz, H-
6b), 3.96 (m, 1H, H-6a), 3.64 (ddd, 1H, J5,6a=2.6 Hz, H-5), 3.94 (d, 1H, J=10.2 Hz, CHNSO2), 2.03,
2.01, 1.98, 1.80 (4s, each 3H, 4×MeCOO), 1.81 (m, 1H, CHMe2), 1.35 (s, 9H, CMe3), 0.97 (d, 3H,
13
Jvic=6.7 Hz, CHMe2), 0.92 (d, 3H, Jvi =6.7 Hz, CHMe2). C NMR (62.9 MHz, CDCl3): δ 171.1-169.8
(4×C=O), 146.4 (Ar-C-SO2), 140.0, 139.9 (2×Ar-C), 129.1-128.3 (Ar-CH), 85.6 (C-1), 82.7 (CMe3),
75.7 (C-2), 74.6 (C-3), 70.2 (C-5), 68.8 (C-4), 67.3 (CHNSO2), 63.0 (C-6), 31.3 (CHMe2), 28.5
(CMe3), 21.2 (4×MeCO), 20.6, 20.4 (CHMe2).
4.1.1.3.
tert-Butyl
(2R)-3-methyl-2-(N-(((2R,3R,4S,5R,6R)-3,4-diacetoxy-5-acetamido-6-
(23). The
methoxytetrahydro-2H-pyran-2-yl)methyl)biphenyl-4-ylsulfonamido)butanoate

Mitsunobu reaction was conducted following the general procedure, starting from a solution of
compound 21 (100 mg, 0.3122 mmol) in dry THF (6 mL), the biphenylsulfonamide 18 (122 mg,
0.3122 mmol), PPh3 (287 mg, 1.09 mmol), and DIAD (0.21 mL). The reaction mixture was stirred at
room temperature for 48 h. After work-up, the crude (925 mg) was purified by flash chromatography
using an Isolute Flash Si II cartridge (Petroleum Ether- EtOAc in gradient from 2:1 to 1:2) affording
derivative 23 (79 mg, 40% yield), as syrup. 1H NMR (400 MHz, CDCl3) δ 7.84-7.81 (m, 2H, 2× Ar-H),
7.69-64 (m, 2H, 2× Ar-H), 7.56-7.53 (m, 2H, 2× Ar-H), 7.49-7.44 (m, 3H, 3× Ar-H), 5.48 (d, 1H, J2,NH
= 8.2 Hz, NHAc), 5.18 (dd, 1H, J2,3=10.8 Hz, J3,4 = 9.2 Hz, H-3), 4.84 (dd, 1H, J4,5 = 9.6 Hz, H-4), 4.42
(d, 1H, J1,2=8.4 Hz, H-1),) 4.15 (ddd, J5,6a=1.2 Hz, J5,6b=8.4 Hz, 1H, H-5), 4.02 (dd, 1H, H-2), 3.96 (d,
1H, J=10.0 Hz, CHNSO2 ), 3.74 (dd, J6a,6b=16.1 Hz, 1H, H-6a), 3.49 (s, 3H, OMe), 3.41 (dd, 1H, H-6b)
2.15, 2.03, 1.96 (3s, each 3H, 2×MeCOO, MeCON), 2.04-2.02 (m, 1H, Me2CH), 1.19 (s, 9H, Me3C),
1.09 (d, 3H, Jvic=6.4 Hz, Me2CH), 0.93 (d, 3H, Jvic = 6.4 Hz, Me2CH).
4.1.1.4. tert-Butyl (2R)-3-methyl-2-(N-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)biphenyl-4-ylsulfonamido)butanoate (24). The Mitsunobu reaction was
conducted following the general procedure, starting from a solution of compound 22 (50.0 mg, 0.144
mmol) in dry THF (2.90 mL), the biphenylsulfonamide 18 (83.9 mg, 0.215 mmol), PPh3 (118.0 mg,
0.502 mmol), and DMEAD (118 mg, 0.502 mmol). The reaction mixture was stirred at room
temperature for 46 h. After the treatment, the crude residue (380 mg), was purified by flash
chromatography (n-hexane-EtOAc 6:4 + 0.01% Et3N) affording derivative 24 (41 mg, 40% yield) as a
white foam. 1H NMR (250.12 MHz, CDCl3): δ 8.01 (m, 2H, 2×Ar-H), 7.68 (m, 2H, 2×Ar-H), 7.57 (m,
2H, 2×Ar-H), 7.51-7.26 (m, 3H, 3×Ar-H), 5.85 (dd, 1H, J2,3=9.3 Hz, J3,4=8.6 Hz, H-3), 5.42 (d, 1H,
J1,2=9.7 Hz, H-1), 5.09 (dd, 1H, H-2), 5.14 (dd, 1H, J4,5=9.7 Hz, H-4), 4.13 (dd, 1H, J6a,6b=12.0 Hz,
J5,6b=5.6 Hz, H-6b), 3.96 (m, 1H, H-6a), 3.64 (ddd, 1H, J5,6a=2.6 Hz, H-5), 3.94 (d, 1H, J=10.2 Hz,
CHNSO2), 2.03, 2.01, 1.98, 1.80 (4s, each 3H, 4×MeCO), 1.81 (m, 1H, CHMe2), 1.35 (s, 9H, CMe3),
0.97 (d, 3H, Jvic=6.7 Hz, CHMe2), 0.92 (d, 3H, Jvic=6.7 Hz, CHMe2). 13C NMR (62.9 MHz, CDCl3): δ
171.1-169.8 (4×C=O), 146.4 (Ar-C-SO2), 140.0, 139.9 (2×Ar-C), 129.1-128.3 (Ar-CH), 85.6 (C-1),
82.7 (CMe3), 75.7 (C-2), 74.6 (C-3), 70.2 (C-5), 68.8 (C-4), 67.3 (CHNSO2), 63.0 (C-6), 31.3
(CHMe2), 28.5 (CMe3), 21.2 (4×MeCO), 20.6, 20.4 (CHMe2).
4.1.2. 2,3,4,6-Tri-O-acetyl-β-D-glucopyranosyl azide (25).
To a solution of the commercial 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl bromide (1.00 g, 2.43
mmol) in dry DMF (3 mL) 205 mg (3.16 mmol) of NaN3 were added and the mixture was stirred for

two days. The solvent was removed in vacuo and the residue was taken up with EtOAc and water and
the aqueous phase was extracted with EtOAc (3×20 mL). The organic phases were combined, dried
(Na2SO4), filtered and concentrated under reduced pressure affording the pure azide 25 (762 mg,
1
quantitative yield) as a white foam. H NMR (400 MHz, CDCl3): δ 5.22 (dd, J2,3=9.4 Hz, J3,4=9.6 Hz,
1H, H-3), 5.10 (dd, J4,5=10.4 Hz, 1H, H-4), 4.94 (dd, J1,2=8.9 Hz, 1H, H-2), 4.64 (d, 1H, H-1), 4.28 (dd,
J5,6b=4.8 Hz, J6a,6b=12.4 Hz, 1H, H-6b), 4.17 (dd, J5,6a=2.3 Hz, 1H, H-6a), 3.79 (ddd, 1H, H-5), 2.10,
2.07, 2.03, 2.01 (3s, each 3H, 4 × MeCO).
4.1.3. 2,3,4,6-Tri-O-acetyl-β-D-glucopyranosyl isothiocyanate (27).
To solution of azide 25 (762 mg, 2.04 mmol) in amixture 20:1 THF-H2O (6.45 mL), commercial PPh3
(1.08 g) supported on resin (3 mmol/g) was added and the mixture was stirred at room temperature
until the starting compound was completely reacted. After filtration over a small layer of Celite the
solution was concentrated under reduced pressure and the crude residue was constituted exclusively by
amine 26 (254.5 mg) as clear syrup. 1H NMR (400 MHz, CDCl3): δ 5.24 (dd, J2,3=9.4 Hz, J3,4=9.6 Hz,
1H, H-3), 5.05 (dd, J4,5=10.0 Hz, 1H, H-4), 4.90 (dd, J1,2=9.2 Hz, 1H, H-2), 4.30 (bd, 1H, H-1), 4.23
(dd, J5,6b=4.4 Hz, J6a,6b=12.0 Hz, 1H, H-6b), 4.11 (dd, J5,6a=2.4 Hz, 1H, H-6a), 3.73 (ddd, 1H, H-5),
2.10, 2.09, 2.03, 2.01 (4s, each 3H, 4 × MeCO).
Without further purification, a solution of amine 26 (254 mg) in dry CH2Cl2 (10 mL) was treated with
2-pyridyl-thiocarbonate (DPT, 473 mg, 2.04 mmol). After stirring at room temperature overnight, the
mixture was diluted with CH2Cl2 (20 mL) and water (20 mL) and then extracted with CH2Cl2 (4×20
mL). The combined organic extracts were dried (Na2SO4), filtered and concentrated under reduced
pressure. Purification of the residue by flash chromatography on silica gel (n-hexane/EtOAc 10:1) and
trituration of the crude with hexane geve the isothiocianate 27 (119 mg, 15% yield from 25) as a white
1
foam. H NMR (400 MHz, CDCl3): δ 5.20 (dd, J2,3=9.2 Hz, J3,4=9.4 Hz, 1H, H-3), 5.11 (dd, 1H,
J4,5=10.1 Hz, H-4), 5.09 (dd, J1,2=8.9 Hz, 1H, H-2), 5.01 (d, 1H, H-1), 4.24 (J5,6b=4.8 Hz, J6a,6b=12.5
Hz, 1H, H-6b), 4.14 (J5,6a=2.3 Hz, 1H, H-6a), 3.74 (ddd, 1H, H-5), 2.11, 2.10, 2.03, 2.02, (4s, each 3H,
4×MeCO).
4.1.4.
tert-Butyl
(2R)-3-methyl-2-(N-(2-(3-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-yl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoate
(29).
The glucose isothiocyanate 27 (135 mg, 0.3467 mmol) was dissolved in dry 4:1 CH2Cl2/DMF mixture
(10 mL) and the solution of amine 18 (173 mg, 0.3152 mmol) in dry CH2Cl2/DMF 4:1 (10 mL)

containing Et3N (0.043 mL) was added dropwise. The reaction mixture was stirred at 60 °C until TLC
analysis (EtOAc) revealed the complete disappearance of the amines (12 h) and concentrated under
reduced pressure. The purification of crude product by flash chromatography (Petroleum Ether/EtOAc
1
3:1) using an Isolute Flash Si II cartridge afforded pure 29 (145 mg, 83% yield) as a white foam. H
NMR (CD3CN, 400 MHz): δ 7.91(m, 2H, 2×Ar-H), 7.81(m, 2H, 2×Ar-H), 7.70 (m, 2H, 2×Ar-H), 7.55-
7.47 (m, 3H, 2×Ar-H), 7.03 (bs, 1H, NHC=S), 6.84 (bs, 1H, NHC=S), 5.36 (dd, J2,3=9.4 Hz, J3,4=9.6
Hz, 1H, H-3), 5.04 (dd, J4,5=9.8 Hz, 1H, H-4), 5.02 (dd, J1,2=7.2 Hz, 1H, H-2), 4.21 (dd, J6a,6b=12.3 Hz,
J5,6b=4.9Hz 1H, H-6b), 4.21 (dd, J5,6a=2.7 Hz 1H, H-6a), 4.08 (d, 1H, H-1), 3.92 (ddd, 1H, H-5), 3.90
(d, Jvic=10.6 Hz, 1H, CHNSO2), 3.89-3.80 (m, 3H, CH2NHCS, CH2NSO2), 3.40 (m, 1H, CH2NSO2),
2.22 (m, 1H, CHMe2), 2.02, 2.01, 2.00, 1.98 (4s, each 3H, 4×MeCO). 1.25 (s, 9H, CMe3), 1.03 (d, 3H,
Jvic= 6.6 Hz, CHMe2), 0.96 (d, 3H, Jvic= 6.8 Hz, CHMe2).
4.1.5
tert-Butyl
(2R)-(N-(2-isothiocyanatoethyl)-[1,1'-biphenyl]-4-ylsulfonamido)-3-
methylbutanoate (30).
To a solution of trifluoroacetate salt 28 (300 mg, 0.549 mmol) in dry CH2Cl2 (9.0 mL) 2-pyridyl-
thiocarbonate (DPT, 127 mg, 0.549 mmol) was added. The reaction mixture was stirred at room
temperature overnight under nitrogen atmosphere. The mixture was diluted with CH2Cl2 (20 mL) and
water (20 mL) and then extracted with CH2Cl2 (4×20 mL). The combined organic extracts were dried
(Na2SO4), filtered and concentrated under reduced pressure. Purification of the crude by flash
chromatography using an Isolute Flash Si II cartridge (n-hexane/EtOAc 5:1) afforded isothyocianate
derivative 30 (202 mg, 78% yield) as a white foam. 1H NMR (CDCl3, 400 MHz): δ 7.90 (m, 2H, 2×Ar-
H), 7.72 (m, 2H, 2×Ar-H), 7.57 (m, 2H, 2×Ar-H), 7.50-7.40 (m, 3H, 3×Ar-H), 3.97 (d, Jvic=10.3 Hz,
1H, CHNSO2), 4.03-3.96, 3.94-3.76 (2m, 3H, CH2NHCS, CH2NSO2), 3.54 (ddd, Jgem=14.6 Hz, Jvic=8.8
Hz, Jvic=5.3 Hz, 1H, CH2NSO2), 2.03 (m, 1H, CHMe2), 1.26 (s, 9H, CMe3), 1.03 (d, 3H, Jvic=6.6 Hz,
CHMe2), 0.98 (d, 3H, Jvic=6.6 Hz, CHMe2).
4.1.6. Methyl 2,3,4-tri-O-acetyl-6-amino-6-deoxy-β-D-glucopyranoside (32).
To a solution of the azide 31 (50 mg, 0.145 mmol) in a mixture of DCM:H2O:MeOH 3:1:0.5 (17 mL),
109 mg di Pd/C 10%.were added. The reaction was stirred overnight under hydrogen atmosphere. After
filtration over a small layer of Celite, the solution was concentrated under reduced pressure and the
residue was constituted exclusively by amine 32 as clear syrup. 1H NMR (400 MHz, CD3OD): δ 5.31
(dd, J2,3=9.4 Hz, J3,4=9.6 Hz, 1H, H-3), 4.95 (m, 2H, H-2, H-4), 4.65 (d, J1,2=8.0 Hz, 1H, H-1), 3.94

(ddd, 1H, J4,5=10.3 Hz J5,6a=3.0 Hz, J5,6b=8.4 Hz H-5), 3.55 (s, 3H, OMe); 3.24 (dd, J6a,6b=13.6 Hz 1H,
H-6a), 3.05 (dd, 1H, H-6b), 2.06, 2.02, 1.98 (3s, each 3H, 3 × MeCO). 13C NMR (100.62 MHz,
CD3OD) δ: 171.7, 171.4, 171.1 (3×C=O), 102.8 (C-1), 73.6 (C-3), 72.5 (C-5), 71.3, 71.1 (C-2, C-4),
57.8 (OMe), 41.3 (C-6), 20.5 (3×MeCO).
4.1.7.
tert-Butyl
(2R)-3-methyl-2-(N-(2-(3-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-
methoxytetrahydro-2H-pyran-2-yl)methyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoate
(33).
The isothiocyanate 30 (40 mg, 0.08 mmol) was dissolved in dry 4:1 CH2Cl2/DMF mixture (1 mL) and
the solution of the C-6 glucosamine 32 (25 mg, 0.07 mmol) in dry CH2Cl2/DMF 4:1 (1 mL) containing
Et3N (0.01 mL) was added dropwise. The reaction mixture was stirred at 60 °C until TLC analysis
(EtOAc) revealed the complete disappearance of the amines (12 h) and concentrated under reduced
pressure. Flash chromatographic purification over silica gel of the crude product (n-hexane/EtOAc 2:1)
1
using an Isolute Flash Si II cartridge afforded pure 33 (29 mg, 47 % yield) as a clear syrup. H NMR
(CDCl3, 400 MHz): δ 7.88 (m, 2H, 2×Ar-H); 7.71 (m, 2H, 2×Ar-H); 7.58 (m, 2H, 2×Ar-H); 7.56-7.41
(m, 3H, 3×Ar-H); 5.19 (dd, J2,3=9.6 Hz, J3,4=9.2 Hz, 1H, H-3), 5.00-4.94 (m, 2H, H-2, H-4), 4.44 (d,
J1,2=8.0 Hz, 1H, H-1), 3.90 (d, J=10.0 Hz, 1H, CHNSO2), 4.10-3.67 (m, 5H, H-5, H-6b, CH2NHCS,
CH2NSO2), 3.49 (s, 3H, OMe); 3.55-3.42 (m, 2H, H-6a, CH2NSO2), 2.11 (m, 1H, CHMe2), 2.06, 2.04,
1.98 (3s, each 3H, 3×MeCO), 1.25 (s, 9H, CMe3), 0.99 (d, 3H, Jvic= 6.4 Hz, CHMe2), 0.95 (d, 3H,
Jvic=6.5 Hz, CHMe2). 13C NMR (100 MHz, CDCl3): δ 182.3 (C=S), 170.4, 170.3, 170.0, 169.5
(4×C=O), 146.2 (Ar-C-SO2), 139.3, 138.0 (2×Ar-C), 129.2.-127.4 (Ar-CH), 101.7 (C-1), 82.6
(Me3CO), 72.8 (C-3), 71.4, 71.3 (C-5, C-2), 70.0 (C-4), 57.3 (OMe), 66.8 (CHNSO2), 42.1-43.5 (C-6,
CH2NHCS, CH2NSO2), 29.8 (CHMe2), 27.9 (CMe3), 20.9, 20.8, 20.7 (3×MeCO), 20.2, 19.3 (CHMe2).
4.1.8 General procedure for the synthesis of carboxylic acids 4, 5, 8, 9, 12 and 14. The appropriate
ester (19, 20, 23, 24, 29 or 33, 0.1 mmol) was dissolved in CH2Cl2 (1.7 mL), treated with CF3COOH
(0.6 mL) and stirred at 0 °C for 1 h. After 5-24 h at room temperature, TLC analysis (EtOAc) revealed
the complete disappearance of the starting material and the formation of a more retained product. The
solution was coevaporated with toluene (4×10 mL) under reduced pressure and trituration of the crude
product with Et2O afforded pure carboxylic acids 4, 5, 8, 9, 12 and 14.
4.1.8.1. (2R)-2-(N-((2R,3R,4S,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-
pyran-2-yl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid (4). The title compound was prepared

from ester 19 following the general procedure. Purification by trituration with n-hexane afforded pure 4
1
(52 mg, 89 % yield) as a white solid; Rf 0.46 (n-hexane-EtOAc 1:9); mp. 86-90 °C; H NMR (250
MHz, CD3CN-CD3OD): δ 8.01 (m, 2H, 2×Ar-H), 7.77 (m, 2H, 2×Ar-H), 7.58-7.44 (m, 6H, 5×Ar-H,
NHAc), 5.24-5.14 (m, 2H, H-1, H-3), 5.07-4.89 (m, 2H, H-2, H-4), 4.16 (m, 1H, H-6b), 4.03-3.89 (m,
2H, H-6a, CHNSO2), 3.82 (m, 1H, H-5), 2.15 (m, 1H, CHMe2), 1.95, 1.94, 1.93 (3s, each 3H,
3×MeCO), 1.85 (s, 3H, MeCON), 0.90 (d, 3H, Jvic=6.7 Hz, Me2CH), 0.67 (d, 3H, Jvic=6.7 Hz, CHMe2).
13C NMR (62.9 MHz , CD3CN-CD3OD): δ 171.9, 171.8, 171.7, 171.6, 171.1 (5×C=O), 146.7 (Ar-C-
SO2), 140.1, 139.9 (2×Ar-C), 130.3-128.2 (Ar-CH), 85.7 (C-1), 76.0 (C-3), 74.8 (C-5), 69.6 (C-4), 64.5
(CHNSO2), 63.6 (C-6), 54.0 (C-2), 30.5 (CHMe2), 23.3 (MeCON), 21.3 (3×MeCOO), 20.3, 20.2
(CHMe2). Elemental Analysis for C31H38N2O12S calculated: % C, 56.18; % H, 5.78; % N 4.23; found:
% C, 56.23; % H, 5.81; % N 4.20.
4.1.8.2. (2R)-2-(N-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-
yl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid (5). The title compound was prepared from
ester 20 following the general procedure as a white solid (11 mg, 78 % yield). Rf 0.53 (n-hexane-
EtOAc 1:9); mp. 88-90 °C; 1H NMR (250 MHz, CD3CN-CD3OD): δ 7.99 (m, 2H, 2×Ar-H), 7.69-7.58
(m, 4H, 4×Ar-H), 7.51-7.31 (m, 3H, 3×Ar-H), 5.65-5.50 (m, 2H, H-1, H-3), 5.10-4.91 (m, 2H, H-2, H-
4), 4.13 (m, 1H, H-6b), 4.05-3.70 (m, 3H, H-6a, H-5, CHNSO2), 2.01, 1.99, 1.95, 1.90 (4s, each 3H,
4×MeCO), 1.85 (m, 1H, CHMe2), 0.95 (d, 3H, Jvic=6.7 Hz, CHMe2), 0.71 (d, 3H, Jvic=6.7 Hz, CHMe2).
13C NMR (CD3CN-CD3OD 62.9 MHz): δ 171.4, 171.2, 170.5, 170.3, 170.2 (5×C=O), 146.4 (Ar-C-
SO2), 139.5, 138.2 (2×Ar-C), 129.5-127.6 (Ar-CH), 85.3 (C-1), 75.2 (C-2), 74.4 (C-3), 70.5 (C-5), 68.6
(C-4), 67.4 (CHNSO2), 62.8 (C-6), 30.1 (CHMe2), 20.9 (4×MeCO), 21.7, 21.0 (CHMe2). Elemental
Analysis for C31H37NO13S calculated: % C, 56.10; % H, 5.62; % N 2.11; found: % C, 56.15; % H,
5.68; % N 2.10.
4.1.8.3. (2R)-3-methyl-2-(N-(((2R,3R,4S,5R,6R)-3,4-diacetoxy-5-acetamido-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)biphenyl-4-ylsulfonamido)butanoic acid (8). The title compound was
prepared from ester 23 following the general procedure. Purification by flash cromathography using an
Isolute Silica II cartridge (CHCl3-MeOH 50:1) afforded pure 8 (60 mg, 52% yield) as a white solid; Rf
0.37 (CHCl3-MeOH 20:1); mp. 100-102 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.87 (bs, 1H, COOH),
7.91 (m, 2H, 2× Ar-H), 7.81-7.57 (m, 4H, 3×Ar-H, NHAc), 7.61-7.50 (m, 3H, 3×Ar-H), 7.45 (m, 1H,
Ar-H), 5.10 (dd, 1H, J2,3=10.3 Hz, J3,4=9.6 Hz, H-3), 4.64 (dd, J4,5=9.7 Hz, 1H, H-4), 4.47 (d, J1,2=8.4
Hz, 1H, H-1), 3.99 (m, 1H, H-2), 3.93 (d, J=10 Hz, CHNSO2), 3.74 (ddd, J5,6a=1.2 Hz, J5,6b=8.6 Hz,

1H, H-5), 3.52 (dd, 1H, J6a,6b=16.4 Hz, H6b), 3.32 (s, 3H, OMe), 3.21 (dd, 1H, H-6a), 1.93 (m, 1H,
Me2CH), 2.11, 1.93 (2s, each 3H, 2×MeCO), 1.76 (s, 3H, MeCONH), 1.01 (d, 3H, Jvic=6.5 Hz,
13
CHMe2), 0.92 (d, 3H, Jvic=6.5 Hz, CHMe2). C NMR (100 MHz, DMSO-d6): δ 171.7, 170.3, 170.1,
169.6 (4×C=O), 145.0 (Ar-C-SO2), 138.6, 137.1 (2×Ar-C), 132.0-127.6 (Ar-CH), 101.7 (C-1), 73.8,
73.1 (C-2, C-3), 71.1 (C-4), 66.4 (CHNSO2), 56.8 (OMe), 53.3 (C-2), 47.2 (C-6), 28.7 (CHMe2), 23.2
(MeCON), 21.0,19.6 (CHMe2), 20.8, 20.9 (2×MeCO). Elemental Analysis for C30H38N2O11S
calculated: % C, 56.77; % H, 6.03; % N 4.41; found: % C, 56.81; % H, 5.10; % N 4.40.
4.1.8.4. (2R)-3-methyl-2-(N-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-methoxytetrahydro-2H-pyran-
2-yl)methyl)biphenyl-4-ylsulfonamido)butanoic acid (9). The title compound was prepared from
ester 24 following the general procedure. Purification by flash cromathography (n-hexane-EtOAc
45:55) followed by trituration with Et2O afforded pure 9 (56 mg, 89% yield) as a white solid; Rf 0.51
1
(n-hexane-EtOAc 3:7); mp. 77-80 °C; H NMR (250 MHz, CD3CN-D2O): δ 7.85-7.70 (m, 4H, 4×Ar-
H), 7.66 (m, 2H, 2×Ar-H), 7.55-7.39 (m, 3H, 3×Ar-H), 5.24 (dd, 1H, J2,3=9.9 Hz, J3,4=9.3 Hz, H-3),
4.83 (dd, 1H, J1,2=8.0 Hz, H-2), 4.80 (dd, 1H, J4,5=10.0 Hz, H-4), 4.49 (d, 1H, H-1), 4.12 (ddd, 1H,
J5,6a=2.0 Hz, J5,6b=8.5 Hz, H-5), 3.98 (d, 1H, J=10.2 Hz, CHNSO2), 3.57 (dd, 1H, J6a,6b=16.3 Hz, H-
6b), 3.40 (s, 3H, OMe), 3.33 (dd, 1H, H-6a), 2.15 (m, 1H, CHMe2), 2.09, 1.99, 1.95 (3s, each 3H,
3×MeCO), 1.01 (d, 3H, Jvic=6.5 Hz, CHMe2), 0.95 (d, 3H, Jvic=6.5 Hz, CHMe2). 13C NMR (62.9 MHz,
CD3CN-D2O): δ 171.7, 170.7, 170.6, 170.1 (4×C=O), 146.2 (Ar-C-SO2), 139.6, 139.7 (2×Ar-C), 129.8-
127.9 (Ar-CH), 102.0 (C-1), 74.4 (C-5), 73.1 (C-3), 71.6 (C-2), 71.3 (C-4), 66.6 (CHNSO2), 57.8
(OMe), 47.7 (C-6), 29.3 (CHMe2), 20.8 (3×MeCOO), 21.0, 19.4 (CHMe2). Elemental Analysis for
C30H37NO12S calculated: % C, 56.68; % H, 5.87; % N 2.20; found: % C, 56.72; % H, 5.90; % N 2.17.
4.1.8.5.
(2R)-3-methyl-2-(N-(2-(3-((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-yl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoic
acid (12). The title compound was prepared from ester 29 following the general procedure. Purification
by flash cromathography using an Isolute Flash Si II cartridge (CHCl3-MeOH 20:1) afforded pure 12
1
(41 mg, 32% yield) as a white solid; mp. 140-143 °C. H NMR (250 MHz, CD3CN): δ 7.85 (m, 2H,
2×Ar-H), 7.79 (m, 2H, 2×Ar-H), 7.68 (m, 2H, 2×Ar-H), 7.64-7.51 (m,3H, 3×Ar-H), 7.13 (bs, 1H,
CH2NHCS), (bd, J2,3=8.8 Hz, 1H, CHNHCS), 5.61-5.68 (m, 1H, H-1), 5.34 (dd, J2,3=9.6 Hz, J3,4=9.5
Hz ,1H, H-3), 5.02-4.91 (m, 2H, H-2, H-4), 4.20 (dd, J5,6b=4.8 Hz 1H, J6a,6b=12.4 Hz, H-6b), 4.06 (dd,
J5,6a=2.1 Hz 1H, H-6a), 3.99 (d, J=10.4 Hz 1H, CHNSO2), 4.00-3.70 (m, 4H, H-5,
CH2NHCS,CH2NSO2), 3.34 (m, 1H, CH2NHCS), 2.02 (m, 1H, CH(Me)2), 1.98, 1.97, 1.96, 1.95 (4s,

13
each 3H, CH3CO), 0.92 (bd, 6H, CH(Me)2). C NMR (69.12 MHz, CD3CN): δ 184.9 (C=S), 172.2,
171.3, 170.8, 170.5 (4 × C=O) 146.2 (Ar-C-SO2), 139.8, 138.3, (2×Ar-C) 130.0-128.1 (Ar-CH), 82.7
(C-1), 73.6, 73.5, 71.4, (C-2, C-3, C-5), 69.8 (C-4), 66.7 (CHNSO2), 62.7 (C-6), 45.5, 44.3 (CH2NHCS,
CH2NSO2), 29.17 (CHMe2), 20.9, (4 × MeCO), 20.4, 19.5 (CHMe2). Elemental Analysis for
C34H43N3O13S2 calculated: % C, 53.32; % H, 5.66; % N 5.49; found: % C, 53.42; % H, 5.70; % N 5.51.
4.1.8.6. (2R)-3-methyl-2-(N-(2-(3-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-methoxytetrahydro-2H-
pyran-2-yl)methyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoic acid (14). The title
compound was prepared from ester 33 following the general procedure. Purification by flash
cromathography using Isolute Flash Si II cartridge (CHCl3) afforded pure 14 (19.5 mg, 52.5% yield) as
1
a white solid; mp. 112-115 °C. H NMR (400 MHz, CD3OD): δ 7.95 (m, 2H, 2×Ar-H), 7.81 (m, 2H,
2×Ar-H), 7.68 (m, 2H, 2×Ar-H), 7.49 (m, 2H, 2×Ar-H), 7.41 (m, 1H, Ar-H), 5.22 (dd, J2,3=9.6 Hz,
J3,4=9.4 Hz, 1H, H-3), 4.92-4.86 (m, 2H, H-2, H-4), 4.53 (d, J1,2=8.0 Hz, 1H, H-1), 4.02 (d, J=10.4 Hz,
1H, CHNSO2), 3.82-3.76 (m, 5H, H-5, H-6a, H-6b, CH2NSO2,CH2NHCS), 3.64 (m, 1H, CH2NSO2),
3.47 (s, 3H, OMe); 2.25 (m, 1H, CHMe2), 2.04, 2.00, 1.94 (3s, each 3H, MeCO), 1.04 (d, Jvic=6.4 Hz,
13
3H, CHMe2); 0.99 (d, Jvic=6.8 Hz, 3H, CHMe2). C NMR (100 MHz, CD3OD): δ 183.1(C=S), 173.7,
171.6, 171.5, 171.2 (4×C=O), 147.0, (Ar-C-SO2), 140.6, 138.9 (2×Ar-C), 130.1-128.3 (Ar-CH), 102.6
(C-1), 74.4 (C-3), 73.7 (C-5), 72.8, (C-2), 71.1 (C-4), 67.9 (CHNSO2), 57.4 (OMe), 44.1-46.5 (C-6,
CH2NHCS, CH2NSO2), 29.8 (CHMe2), 20.9, 20.7, 20.3 (3×MeCO), 20.6, 19.8 (CHMe2). Elemental
Analysis for C33H43N3O12S2 calculated: % C, 53.72; % H, 5.87; % N 5.70; found: % C, 53.80; % H,
5.90; % N 5.65.
4.1.9. General procedure for the synthesis of deprotected carboxylic acids 6, 7, 10, 11, 13 and 15.
A solution of the appropriate protected carboxylic acid (4, 5, 8, 9, 12 and 14, 0.1 mmol) in MeOH (1
mL) was treated with NH3-MeOH 7N (1 mL) and the solution was stirred at room temperature until the
starting compound was completely reacted (TLC, CHCl3/MeOH 8:2, 20-24 h). The solution was
coevaporated with toluene (4×10 mL) under reduced pressure and the crystallization (MeOH/EtOAc)
or trituration of crude product with Et2O afforded pure carboxylic acids 6, 7, 10, 11, 13 and 15.
4.1.9.1. (2R)-2-(N-((2R,3R,4S,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-
2H-pyran-2-yl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid (6). The title compound was
prepared from the protected carboxylic acid 4 following the general procedure. Trituration with Et2O
1
afforded pure 6 (94% yield) as a white solid; mp. 151-155 °C; H NMR (250 MHz, CD3OD): δ 7.94-

7.31 (m, 7H, 7×Ar-H), 7.25-7.08 (m, 3H, 2×Ar-H, NHAc), 5.08 (m, H, H-1), 4.10-3.12 (m, 7H, H-2,
H-3, H-4, H-5, H-6a, H-6b, CHNSO2), 1.95 (m, 1H, Me2CH), 1.83 (s, 3H, MeCON), 1.05-0.81 (m, 6H,
13
Me2CH). C NMR (62.9 MHz, CD3OD): δ 172.1 (C=O), 146.6 (Ar-C-SO2), 141.3, 140.5 (2×Ar-C),
130.1-128.2 (Ar-CH), 86.3 (C-1), 80.4, 77.7 (C-3, C-5), 71.7 (C-4), 62.6 (C-6), 56.5 (C-2), 65.4
(CHNSO2), 30.7 (CHMe2), 21.9 (MeCON), 20.7, 20.2 (Me2CH). Elemental Analysis for C25H32N2O9S
calculated: % C, 55.96; % H, 6.01; % N 5.22; found: % C, 55.98; % H, 6.07; % N 5.20.
4.1.9.2. (2R)-2-(N-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid (7). The title compound was prepared from the
protected carboxylic acid 5 following the general procedure. Trituration with Et2O afforded pure 7
(94% yield) as a white solid; mp. 127-130 °C; 1H NMR (250 MHz, CD3OD): δ 8.08 (m, 2H, 2×Ar-H),
7.91-7.67 (m, 4H, 4×Ar-H), 7.79-7.39 (m, 3H, 3×Ar-H), 4.20-3.21 (m, 12H, H-1, H-2, H-3, H-4, H-5,
13
H-6a, H-6b, CHNSO2, 4×OH), 2.15 (m, 1H, CHMe2), 0.99-0.75 (m, 6H, CHMe2). C NMR (62.9
MHz, CD3OD): δ 174.1 (C=O), 146.1 (Ar-C-SO2), 140.6, 140.3 (2×Ar-C), 129.6-127.7 (Ar-CH), 88.6
(C-1), 80.2, 79.7, 72.6, 708 (C-2, C-3, C-4, C-5), 67.9 (CHNSO2), 62.4 (C-6), 30.3 (CHMe2), 21.7,
20.9 (CHMe2). Elemental Analysis for C23H29NO9S calculated: % C, 55.75; % H, 5.90; % N 2.83;
found: % C, 55.81; % H, 5.95; % N 2.85.
4.1.9.3. (2R)-3-methyl-2-(N-(((2R,3R,4S,5R,6R)-3,4-dihydroxy-5-acetamido-6-methoxytetrahydro-
2H-pyran-2-yl)methyl)biphenyl-4-ylsulfonamido)butanoic acid (10). The title compound was
prepared from the protected carboxylic acid 8 following the general procedure. Purification by flash
cromathography using Isolute Flash Si II cartridge (CHCl3-MeOH 20:1) afforded pure 10 (12 mg, 35%
1
yield) as a white solid. Mp. 116-117 °C; H NMR (400 MHz, CD3OD): δ 8.05 (d, J2,NH=9.2 Hz,
NHAc), 7.96 (m, 2H, 2×Ar-H), 7.80 (m, 2H, 2×Ar-H), 7.68 (m, 2H, 2×Ar-H), 7.48 (m, 2H, 2×Ar-H),
7.43-7.39 (m, 3H, 3×Ar-H), 4.27 (d, 1H, J1,2=8.4 Hz, H-1), 4.10 (bd, 1H, J=10.1 Hz, CHNSO2), 3.89
(bt, 1H, H-5), 3.77 (bd, 1H, H-6b), 3.65 (dd, 1H, J2,3=9.2 Hz, 1H, H-2), 3.53 (dd, J6a,6b=15.8 Hz,
J5,6a=9.0 Hz, 1H, H-6b), 3.48 (dd, J3,4=9.5 Hz, 1H, H-3), 3.41 (s, 3H, OMe), 3.05 (dd, 1H, J4,5=9.0 Hz,
1H, H-4), 2.19 (m, 1H, CHMe2), 1.97 (s, 3H, NHAc), 1.11 (d, Jvic= 6.4 Hz, 3H, Me2CH), 0.98 (d, 3H,
13
Jvic=7.2 Hz, Me2CH). C NMR (100 MHz, DMSO-d6): δ 176.3 (C=O), 149.4 (Ar-C-SO2), 143.2,
140.2, (2×Ar-C), 132.7-130.8 (Ar-CH), 106.1 (C-1), 79.3, 78.6, 77.1, (C-3, C-4, C-5), 59.9, 59.6 (C-2,
CHNSO2), 48.4 (C-6), 32.3 (CHMe2), 25.4, 22.9 (CHMe2), 23.9 (MeCO). Elemental Analysis for
C26H34N2O9S calculated: % C, 56.71; % H, 6.22; % N 5.09; found: % C, 56.79; % H, 6.27; % N 5.08.

4.1.9.4. (2R)-3-methyl-2-(N-(((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-
2-yl)methyl)biphenyl-4-ylsulfonamido)butanoic acid (11). The title compound was prepared from
the protected carboxylic acid 9 following the general procedure. The trituration with Et2O afforded
1
pure 11 (98% yield) as a white solid; mp. 95-98 °C; H NMR (250 MHz, CD3CN): δ 8.01 (m, 2H,
2×Ar-H), 7.89-7.61 (m, 4H, 4×Ar-H), 7.58-7.38 (m, 3H, 3×Ar-H), 4.11 (d, 1H, J1,2=7.8 Hz, H-1), 3.97
(bs, 1H, CHNSO2), 3.70 (m, 1H, H-5), 3.58-3.23 (m, 5H, H-3, H-4, H-6b, 2×OH), 3.49 (s, 3H, OMe),
3.19 (m, 1H, H-6a), 3.01 (m, 1H, H-2), 2.13 (m, 2H, CHMe2, OH), 1.14 (bs, 3H, CHMe2), 0.96 (d, 3H,
Jvic=6.5 Hz, CHMe2). 13C NMR (62.9 MHz, CD3CN): δ 171.3 (C=O), 146.5 (Ar-C-SO2), 140.7, 138.8
(2×Ar-C), 130.1-128.3 (Ar-CH), 105.4 (C-1), 77.8, 77.1, 75.0, 74.2 (C-2, C-3, C-4, C-5), 66.9
(CHNSO2), 57.8 (OMe), 48.1 (C-6), 28.3 (CHMe2), 21.7, 19.9 (CHMe2). Elemental Analysis for
C24H31NO9S calculated: % C, 56.57; % H, 6.13; % N 2.75; found: % C, 56.60; % H, 6.18; % N 2.70.
4.1.9.5.
(2R)-3-methyl-2-(N-(2-(3-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoic
acid (13). The title compound was prepared from the protected carboxylic acid 12 following the
general procedure. The trituration with Et2O afforded pure 13 (85.5% yield) as a white solid; mp. 165-
1
168 °C; H NMR (400 MHz, CD3OD): δ 7.97 (m, 2H, 2×Ar-H), 7.77 (m, 2H, 2×Ar-H), 7.68 (m, 2H,
2×Ar-H), 7.47 (m, 2H, 2×Ar-H), 7.39 (m, 2H, 2×Ar-H), 4.20-3.80 (m, 6H, H-1, H-6b, CHNSO2,
CH2NSO2, CH2NHCS), 3.78-3.35 (m, 4 H, H-3, H-4, H-5, H-6a), 3.25 (m, 1H, CH2NSO2), 2.12 (m,
1H, CHMe2), 1.00 (d, 3H, Jvic=6.4 Hz, CHMe2), 0.95 (bd, 3H, CHMe2). 13C NMR (100 MHz, CD3OD):
δ 186.2 (C=S), 175.0 (C=O), 145.3 (Ar-C-SO2), 139.4, 139.3 (2×Ar-C), 128.7-126.9 (Ar-CH), 83.1 (C-
1), 77.6, 77.4, 72.7, 70.1 (C-2, C-3, C-4, C-5), 66.5 (CHNSO2), 61.4. (C-6), 45.0, 43.2 (CH2NSO2,
CH2NHCS), 28.4 (CHMe2); 20.7, 18.8 (CHMe2). Elemental Analysis for C26H35N3O9S2 calculated: %
C, 52.25; % H, 5.90; % N 7.03; found: % C, 52.31; % H, 5.96; % N 7.00.
4.1.9.6. (2R)-3-methyl-2-(N-(2-(3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-
pyran-2-yl)methyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)butanoic acid (15). The title
compound was prepared from the protected carboxylic acid 14 following the general procedure. The
trituration with Et2O followed by a flash cromathography using an Isolute Flash Si II cartridge
1
(CH2Cl2-MeOH in gradient from 20:1 to 6:1) afforded pure 15 (73% yield) as a white foam. H NMR
(400 MHz, CD3OD): δ 7.94 (m, 2H, 2×Ar-H), 7.80 (m, 2H, 2×Ar-H), 7.69 (m, 2H, 2×Ar-H), 7.48 (m,
1H, 2×Ar-H), 7.40 (m, 1H, Ar-H), 4.17 (d, J1,2=7.8 Hz, 1H, H-1), 4.02 (d, J=10.4 Hz, 1H, CHNSO2),
3.95-3.78 (m, 4H, H-6b, CH2NSO2, CH2NCS), 3.51 (s, 3H, OMe), 3.41-3.02 (m, 4H, H-3, H-4, H-5,

CH2NSO2), 3.23-3.14 (m, 2H, H-2, H-6a), 2.24 (m, 1H, CHMe2), 1.31 (d, Jvic=6.5 Hz, 3H, CHMe2),
1.01 (d, Jvic=6.6 Hz, 3H, CHMe2). 13C NMR (100 MHz, CD3OD): δ 183.2 (C=S), 173.8 (C=O), 147.0
(Ar-C-SO2), 140.6, 138.9 (2×Ar-C), 130.1-128.3 (Ar-CH), 105.4 (C-1), 77.3, 76.1 (C-4, C-5), 75.1,
72.4 (C-2, C-4), 67.9 (CHNSO2), 57.4 (OMe), 47.1 (C-6), 45.3, 45.2 (CH2NSO2, CH2NHCS), 29.8
(CHMe2), 20.7, 19.8 (CHMe2). Elemental Analysis for C27H37N3O9S2 calculated: % C, 53.01; % H,
6.10; % N 6.87; found: % C, 53.10; % H, 6.17; % N 6.83.
4.2. MMP inhibition assays
Pro-MMP-12 was purchased by R&D Systems and pro-MMP-9 was purchased from Calbiochem
(Merck-Millipore). Pro-MMP-9 was activated immediately prior to use with p-aminophenylmercuric
acetate (1 mM APMA for 1 h at 37 °C). Pro-MMP-12 was autoactivated by incubating in Fluorometric
Assay Buffer (FAB: Tris 50 mM, pH 7.5, NaCl 150 mM, CaCl2 10 mM, Brij-35 0.05%, and DMSO
1%) for 30 h at 37 °C. For assay measurements, each inhibitor stock solution (DMSO, 10 mM) was
further diluted in FAB at seven different concentrations. Activated enzyme (final concentrations of 1.3
nM for MMP-9 and 2.3 nM for MMP-12) and inhibitor solutions were incubated in the assay buffer for
3 h at 25 °C. After the addition of 200 μM solution of the fluorogenic substrate Mca-Lys-Pro-Leu-Gly-
Leu-Dap(Dnp)-Ala-Arg-NH2 (Bachem) for all the enzymes in DMSO (final concentration of 2 μM for
all enzymes), the hydrolysis was monitored every 10 s for 15 min, recording the increase in
fluorescence (λex = 325 nm, λem = 400 nm) with a Molecular Devices SpectraMax Gemini XPS plate
reader. The assays were performed in triplicate in a total volume of 200 μL per well in 96-well
microtiter plates (Corning black, NBS). Control wells lack inhibitor. The MMP inhibition activity was
expressed in relative fluorescent units (RFU). Percent of inhibition was calculated from control
reactions without the inhibitor. IC50 was determined using the formula vi/v0 = 1/(1 + [I]/IC50), where vi
is the initial velocity of substrate cleavage in the presence of the inhibitor at concentration [I] and vo is
the initial velocity in the absence of the inhibitor. Results were analyzed using SoftMax Pro software
(version 5.4.3, Molecular Devices, Sunnyvale, CA) and Prism Software version 5.0 (GraphPad
Software, Inc., La Jolla, CA).
4.3. Evaluation of intestinal permeability by using an ex vivo everted gut sac model
Male Wistar rats (300-350 g) were taken from ENVIGO (Milan, Italy). All the experimental procedures
were performed according to european (EEC Directive 2010/63) and italian (D.L. 4 March 2014 n.26)

legislation. Rats were housed in cages with free access to standard food pellets and water on a 12h
light/dark cycle.
Animals were fasted overnight and anesthetized with an intraperitoneal injection of sodium tiopenthal
(100 mg/Kg); then, small intestine of each animal was quickly excised and placed in Krebs solution
(composition of saline: NaHCO3 0.025M; Glucose 0.012M; NaCl 0.118M; KCl 0.005M;
MgSO4×7H2O 0.0012M; CaCl2×2H2O 0.0025M; KH2PO4 0.0012M, pH 7.4) continuously gassed with
Clioxicarb, a mixture of O2 (95%) and CO2 (5%). According with Everted Gut Sac model, small
intestine was gently everted, filled with Krebs solution at 37 °C and divided into sacs approximately 3
cm length, using suture silk. Sacs were then placed in triplicate in tubes containing 30 mL of Krebs
solution, medicated with compound 1, 2, 3, 12, 13 (100 µM) or with their vehicle (DMSO 0.1%) and
gassed with Clioxicarb continuously. Then, the sacs were incubated at 37 °C in an oscillating water
bath, and at the selected time points (0, 15, 30 and 60 min), they were removed, washed in Krebs
solution and blotted dry. Finally, they were cut open and serosal fluid was drained into small tubes. The
remaining external solution was also collected. Serosal fluid and a sample of external solution were
analysed by HPLC, in order to quantify the intestinal absorption of tested compounds.
In a second series of experiments, the possible role of two different glucose transporters in the
intestinal permeability of the most promising derivative (compound 3) has been investigated. In
particular, phloridzin dihydrate 10 µM and 100 µM (Sigma-Aldrich, a selective inhibitor of sodium-
glucose transport protein 1, SGLT1) or the GLUT2 inhibitor phloretin 100 µM (Sigma-Aldrich) were
incubated for 10 min before adding compound 3.
4.3.1. Quantification of intestinal permeability by HPLC
Preparation of calibration standards
Standard stock solutions of compounds 1, 2, 3, 12 and 13, were prepared by dissolving an accurately
weighted amount of compound in DMSO with a final concentration of 10 mM. To build-up the
calibration curves for each compound, aliquots from these stock solutions were diluted with Krebs
solution to prepare samples with final concentration of 500, 300, 100, 50, 10, 1 µM. The peak area (Y)
and the concentrations of compounds (X) were used to plot the calibration curves. The linear
correlation coefficients (r2) for the tested compounds were: 0.998 (compound 1), 0.984 (compound 2),
0.999 (compound 3), 0.995 (compound 12), and 0.999 (compound 13).
Chromatographic conditions