Full text of DOI:10.1039/d0md00366b

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RESEARCH ARTICLE
The modulatory role of sulfated and non-sulfated
small molecule heparan sulfate-glycomimetics in
endothelial dysfunction: absolute structural
clarification, molecular docking and simulated
dynamics, SAR analyses and ADMET studies†
Cite this: DOI: 10.1039/
d0md00366b
Daniel M. Gill,a Ana Paula R. Povinelli,ab Gabriel Zazeri,ab Sabrina A. Shamir,c
def
Ayman M. Mahmoud,
Fiona L. Wilkinson,f M. Yvonne Alexander,f
Marinonio L. Corneliob and Alan M. Jones
a
*
The conceptual technology of small molecule glycomimetics, exemplified by compounds C1–4, has shown
promising protective effects against lipid-induced endothelial dysfunction, restorative effects on diabetic
endothelial colony forming cells, and preventative effects on downstream vascular calcification amongst
other important in vitro and ex vivo studies. We report the optimised synthesis of an array of 17 small
molecule glycomimetics, including the regio-, enantio- and diastereo-meric sulfated scaffolds of a hit
structure along with novel desulfated examples. For the first time, the absolute stereochemical
configurations of C1–4 have been clarified based on an identified and consistent anomaly with the
Sharpless asymmetric dihydroxylation reaction. We have investigated the role and importance of sulfation
pattern, location, regioisomers, and spatial orientation of distal sulfate groups on the modulation of
endothelial dysfunction through their interaction with hepatocyte growth factor (HGF). In silico studies
demonstrated the key interactions the persulfated glycomimetics make with HGF and revealed the
importance of both sulfate density and positioning (both point chirality and vector) to biological activity. In
vitro biological data of the most efficient binding motifs, along with desulfated comparators, support the
modulatory effects of sulfated small molecule glycomimetics in the downstream signaling cascade of
endothelial dysfunction. In vitro absorption, distribution, metabolism, elimination and toxicity (ADMET) data
demonstrate the glycomimetic approach to be a promising approach for hit-to-lead studies.
Received 29th October 2020,
Accepted 25th March 2021
DOI: 10.1039/d0md00366b
rsc.li/medchem
complications,2 characterized by the reduced bioavailability of
the vasodilator nitric oxide (NO) and increased levels of reactive
Introduction
The endothelium plays a crucial role in the regulation of vascular
haemostasis, vascular tone, thrombosis and thrombolysis.1
Endothelial dysfunction (ED) has been associated with the
increased risk of atherosclerosis and cardiovascular
oxygen species (ROS).3 Elevation in the level of free fatty acids
(FFA) in the blood plasma has been implicated as an associated
factor for the destruction of the endothelium, partly due to the
enhanced production of ROS, which in turn reduces the
availability of intracellular NO.4 Activation of NADPH oxidase by
FFA leads to the deactivation of endothelial nitric oxide synthase
(eNOS) and the down-regulation of the protective pathways.5
Examples of endothelial modulators are shown in Fig. 1,
including the polysaccharide-based glycomimetic Equitend,6
an analogue of prostacyclin Iloprost7 and chalcone derivative
1m-6.8 Our approach uses glycomimetics—small molecules
that are designed to mimic the function of polysaccharide
based biomolecules—and serve as an attractive therapeutic
technology in a wide range of diseases.9,10 Recent advances
of the glycomimetic approach have included the development
of selective carbonic anhydrase inhibitors;11 and the use of
highly sulfated glycomimetics as inhibitors of viral binding.12
a School of Pharmacy, University of Birmingham, Edgbaston, B15 2TT, UK.
E-mail: a.m.jones.2@bham.ac.uk; Tel: +44(0)121 414 7288
b Departamento de Física – IBILCE, Rua Cristovão Colombo, 2265 CEP 15054-000
São José do Rio Preto, São Paulo, Brazil
c Department of Natural Sciences, Manchester Metropolitan University, M1 5GD, UK
d Physiology Division, Department of Zoology, Faculty of Science, Beni-Suef
University, Egypt
e Department of Endocrinology, Diabetes & Nutrition, Center for Cardiovascular
Research (CCR), Charité – Universitätsmedizin Berlin, Berlin, Germany
f Centre for Biomedicine, Manchester Metropolitan University, M1 5GD, UK
† Electronic supplementary information (ESI) available: Compound
characterisation, 1H and 13C NMR spectra, docking studies, crystallographic data
for 22 and biological assays. CCDC 2038360. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/d0md00366b
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Fig. 1 Molecular scaffold approaches to modulating endothelial dysfunction.
The first generation glycomimetics (C1–C4, Fig. 2a), were
originally designed as inhibitors of hepatocyte growth factor/
scatter factor (HGF/SF)-induced hepatocyte growth factor
receptor (c-Met) activation, affecting tumour cell growth and
angiogenesis.13 It should be noted that the findings of the
present work indicate that the original discovery
experiments13 were likely performed with ambiguous
mixtures of these glycomimetics.
It was subsequently found that C1–C4 are superior to
attenuate lipid-induced endothelial dysfunction and
regenerating eNOS activity.14 We have also recently disclosed
that our aryl templated, small molecule heparan sulfate-
glycomimetics modulate vascular calcification15 and diabetic
endothelial colony forming cells in vitro.16
Hepatocyte growth factor HGF/c-Met signalling has been
reported to affect vascular calcification, an end point of
endothelial dysfunction in the atherosclerotic spectrum.17
Heparan sulfate acts as a ligand for activation of the HGF/c-
Met signalling pathway, thus one can attenuate this pathway
using small molecule heparan sulfate-glycomimetics and
previously described chiral HPLC study, investigating the
tandem Sharpless asymmetric dihydroxylation (AD) on diene 1,
provided evidence for the stereochemical configurations of
glycomimetics C1 and C2 (Fig. 2b/Scheme 1).18 The chiral HPLC
results highlighted that the tetraol precursors (α-2 and β-2)
contain mixtures of diastereo- and enantio-meric pairs, and
importantly the stereochemistry of the major diastereomeric
species is reversed in comparison to the original literature
report.13 The stereochemistry of the originally assigned tetraols
and subsequent glycomimetics from the original report13 were
clarified (Fig. 2b and Scheme 1). The persulfation of all tetraol
intermediates was carried out using the TBSAB-sulfation
methodology19–21 (Scheme 1), specifically, the use of this
reagent afforded the heparan sulfate-glycomimetics α-4 (C1)
and β-5 (C2) in 88% and 86% yield, respectively.
The synthesis of diols R-10 and S-10 were adapted from
the original report13 (Scheme 2). With alkene 7 in hand, AD
reactions were performed with Sharpless formulations α and
β under standard conditions,22 affording diols S-8 and R-8 in
99% yield (Scheme 2). A chemoselective hydrolysis of the
acetate functionality using K2CO3 in methanol afforded triols
S-9 and R-9 in 99% yield. Persulfation of the triols with
TBSAB afforded the first-generation heparan sulfate-
glycomimetics R-10 (previously known as C3) and S-10
(previously known as C4) in 72% and 97% yield, respectively.
Asymmetric induction in the AD has been demonstrated to
work effectively at the meta position alkene in 7.18 Therefore this
investigation was confident that the desired enantiomers were
synthesized in a large enantiomeric excess. However, to further
confirm the stereochemistry of these enantiomers, the synthesis
of enantiomer S-9 was prepared using a chiral pool method
(Scheme 3), and the optical rotations of prepared S-9 (Scheme 2)
and authentic chiral pool S-9 were compared.
reduce vascular calcification as
a
risk factor for
cardiovascular diseases. The downstream signalling of HGF/
c-Met modulates the phosphoinositide 3-kinase/protein
kinase B (PI3K/Akt) pathway leading to the production of the
biomarker nitric oxide (NO) via eNOS which has restorative
effects on damaged endothelial cells.14
Herein, we report the structural clarification, and the
optimised modular synthesis of small molecule heparan
sulfate-glycomimetics, with molecular docking and dynamics
of their interaction with HGF. The biological evaluation of
small molecule glycomimetics as modulators for endothelial
dysfunction combined with ADMET results demonstrate the
concept of this new technology in glycomimetic design.
The optical rotations correlated with the predicted
stereochemical configuration, and the closely matching
values (−7.01 vs. −8.67, c 1.0 in CHCl3) confirmed that the
correct enantiomer was synthesised with a calculated 91 : 9 e.
Results and discussion
An improved synthesis of C1–C4
Initially, we sought to prepare the first generation
glycomimetics (C1–C4, Fig. 2a) as a route to access both
sulfated and non-sulfated analogues for a parallel biological
evaluation. The knowledge gained from the results of our
r
(S/R) (Scheme 3). Furthermore, the stereochemical
configurations of the triols 9 and resulting heparan sulfate-
glycomimetics 10 also opposed the assigned stereochemistry
from the original report13 (Fig. 2a).
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Fig. 2 (a) Originally proposed stereoisomers of C1–C4;13 and (b) clarification of the stereochemical outcomes for C1–C4 from this work.
Scheme 1 The synthesis of first-generation heparan sulfate-glycomimetics α-4 (C1) and β-5 (C2), including biologically relevant intermediates
t
considered in this investigation. Conditions: i) K2CO3, K3Fe(CN)6, K2OsO2(OH)4, BuOH/H2O, 0 °C, 12 h, 99% for both; ii) TBSAB, MeCN, 90 °C, 8 h
88% and 86% for α-4 and β-5, respectively; iii) NaOH, MeOH, 70 °C, 2 h 99% for both.
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Scheme 2 The synthesis of heparan sulfate-glycomimetics S-10 (C4) and R-10 (C3). Conditions: i) 4-bromobutylacetate, K2CO3, TBAI, acetone,
t
t
reflux, 12 h, 90%, ii) ADmix β, BuOH/H2O, 0 °C, 12 h, 99% for both; iii) ADmix α, BuOH/H2O, 0 °C, 12 h, 99% for both; iv) K2CO3, MeOH, rt, 12 h,
99% for both; v) TBSAB, MeCN, 90 °C, 12 h, 72% and 97% for R-10 and S-10, respectively.
Scheme 3 A chiral pool synthesis of S-9 for the comparison of optical rotation. Conditions: i) 4-bromobutylacetate, K2CO3, TBAI, acetone, reflux,
12 h, 90%, ii) 1) NaOMe, MeOH, 2) TFA, MeOH, 12 h, 99%.
Overall, this investigation has assigned the correct
stereochemistry to all previously reported heparan sulfate-
glycomimetics (C1–C4) and provided a practical and efficient
synthesis to the final target structures.
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In silico studies
in Table S3.† The exploration of detailed energy composition
obtained from MM/PBSA, revealed that the predominant
interaction of a negatively charged glycomimetic toward
N-domain of the protein is electrostatic in nature. Not
surprisingly, the microenvironment of interaction disclosed
by docking is largely composed by positively charged amino
acids residues of: Arg73 and Lys78, 58, 60, 62, 63, and 78
(Fig. S1†).
A computational approach was used to triage the key
modified compounds based on α-4 as the prototypical first-
generation small molecule glycomimetic, to take forward for
biological evaluation and in vitro ADMET studies.
The computational model was built based on the X-ray
crystal structures of NK1–HGF (PDB: 1BHT, 2.0
Å
resolution).23 Each heparan sulfate-glycomimetic and non-
sulfated intermediate was docked individually against 1BHT
under matching conditions and each minimum docking
energy was calculated.24
Molecular docking ranked the glycomimetics according to
their affinities toward the NK1–heparin binding site (Table
S3†). MM/PBSA calculations were performed along with
molecular dynamics to obtain a more accurate prediction of
binding free energy of the active candidates. Such techniques
Interestingly, the docked structure of the α-4–HGF
complex (Fig. 3A) shows that the methyl ester is orientated
directly into a hydrophobic pocket on the protein's accessible
surface (Fig. 3B). Comparing this interaction to the β-5–HGF
complex (Fig. S1†) indicates that this mode of binding is
specific to the methyl ester group (present in heparan sulfate-
glycomimetics α-4, R-10, and S-10), and the anionic
carboxylate group (present in heparan sulfate-glycomimetic
β-5) is orientated differently inside the heparan sulfate-
binding site of HGF. This interaction is not gained through
further electrostatic forces and is likely driven by enhanced
hydrogen bonding and van der Waals forces. Consequently,
the methyl ester is considered important to the design of a
second generation of heparan sulfate-glycomimetics.
incorporate
conformational
fluctuations,
entropic
contributions, and solvent molecules interactions, which are
not considered by molecular docking that is based on simple
energetic and geometric criteria. The glycomimetic binding
energies were rescored based on MM/PBSA, as summarised
Fig. 3 A) The minimum energy docked structure of heparan sulfate-glycomimetic α-4 into the heparan sulfate-binding site of NK1 (HGF), Table 1,
entry 3; B) zoomed in view of the docked structure. Key: amino acids are represented as green ribbons and polar surface area is opaque white; C)
Ligplot representation of individual binding interactions displaying vicinal amino acid residues and hydrogen bonds.
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Computational SAR around
prototypical glycomimetic 4
4/2R,5S-4 have closely matched energies ( 1.6 kJ mol−1). A
single point change in stereochemistry at the 2-position had
an associated decrease in hydrogen bonding, suggesting a
non-optimal orientation of the substituent.
Overall, the results gained by this docking study propose
that the heparan sulfate-glycomimetic's binding to HGF are
not substantially determined by their inherent asymmetry,
which is in agreement with the near-equipotent biological
results gained from the first generation of asymmetric
heparan sulfate-glycomimetics, C1–C4.14
The first generation glycomimetics included a modified
linear linker at the 2-position, and comparison with α-4
showed a modest loss of affinity (R-10 −41.0 or S-10 −42.0 vs.
α-4 −47.4 kJ mol−1 (Table 1)). The molecules α-4, β-5 and 26
presented the greatest binding energy values and therefore
the highest affinity for the HGF/NK1 binding site. Moreover,
the results showed that the binding sites are composed
mainly by charged and polar amino acids and that
electrostatic interaction and hydrogen bonding interactions
dominate. Both α-4 and β-5 presented a considerable number
of hydrogen bonds with the amino acids at Lys60, Lys62, and
Arg73, (β-5 also had further interactions with Gln69 and
Thr61) (Fig. S1†). These results indicated that α-4 and β-5 are
the most promising molecules to bind HGF–NK1, as they
have both high affinity to the binding site and provided high
stability to the resulting complex formed. In summary, it was
Furthermore, from the results of our initial in silico study,
this investigation decided that a stereocontrolled synthesis
was not imperative in the design of further generations of
heparan sulfate-glycomimetics targeting HGF. Therefore, the
design of
a
second generation of heparan sulfate-
glycomimetics focussed on the regioisomeric orientation of
anionic sulfate groups around an aromatic core structure.
shown that the sulfate groups present in α-4/β-5 are essential Computational SAR of second
to high affinity of the glycomimetic scaffold to the binding
site from an energetic perspective.
generation glycomimetics
In the case of the desulfated α-2, four hydrogen bonds
were observed with Lys58 and Ala56, such interactions likely
play an important role in the stabilization of the complex
formed by α-2 and unlike the other molecules α-2 had a
stronger influence through van der Waals interactions than
electrostatic.
For synthetic details of the preparation of racemic second
generation regioisomeric glycomimetics see Schemes S1–S3.†
To streamline the docking study of all the following second
generation sulfated glycomimetics, the nS,nS or S-enantiomers
from the racemates were docked against NK1 (HGF) using a
globally parametrized training set of structures. A series of
point changes were made to the core structure of 4, as follows:
converting the methyl ester to the carboxylic acid (β-5), resulted
in an increased affinity (β-5 −51.4 vs. α-4 −47.4 kJ mol−1,
Table 1); deletion of the methyl ester, led to a modest loss of
affinity (17f −45.8 vs. α-4 −47.4 kJ mol−1). Removal of the sulfate
units from either the methyl ester (α-4) or the carboxylic acid
(β-5), led to a dramatic loss of affinity for NK1 (α-2 −9.2 vs. α-4
−47.4 kJ mol−1 and β-3 −19.2 or α-3 −15.8 vs. β-5 −51.4 kJ
mol−1).
We next considered whether the 2,5-regioisomer of the
sulfated glycols around the benzoate core was optimal
(Table 1). The results of this study suggest that altering the
orientation of sulfate groups via the investigation of
regioisomers around an aromatic framework have little effect
on the binding interactions with HGF and thus the
2,5-regioisomers were progressed to further experimental
evaluation.
Is the absolute stereochemistry
critical to the binding of HGF?
As heparan sulfate is a larger, linear polysaccharide chain
containing numerous chiral centres, with some degree of
flexibility of iduronic acid motifs, we considered whether the
stereochemical positioning of the sulfate vectors on our more
rigid aromatic core influenced the binding of the heparan
sulfate-glycomimetics to HGF in silico.
A chiral pool method18 was employed to access all four
possible stereochemical combinations of α-4. Each stereo-
defined tetraol (Scheme 4) was in turn sulfated with TBSAB,
affording the corresponding persulfated glycomimetic (α-4)
in 49–82% isolated yield as their tetrakis sodium salts.
A computational docking study was performed on each
potential stereoisomer of 4 to determine if the binding
interactions of the heparan sulfate-glycomimetics are affected
by single point changes in chirality. The results of the
computational docking study suggested that each
stereoisomer binds to HGF with similar energies (within 2.9
kJ mol−1). Docking results calculated that the enantiomer
2S,5S-4 to have the strongest energy binding interaction with
HGF. Furthermore, the enantiomer 2R,5R-4 was calculated to
have the weakest binding energy. A difference of 2.9 kJ
mol−1 suggests that non-electrostatic intermolecular forces,
such as van der Waals interactions, have minimal effects on
the free energy binding interaction. The diastereomers 2S,5S-
We next considered what is driving the interaction of α-4
with HGF, is it the 2 or 5-sulfated glycol motif (Table 1). The
removal of an entire sulfated glycol side chain afforded
disulfates 17g–i (Table 1). The structurally smaller disulfates
17g–i (−40% by molecular mass compared to the
tetrasulfates), however only an on average −16.5% drop in
binding energy, suggesting the that the disulfates are likely
to be more efficient at binding HGF than the tetrasulfates
(17a–f).
A
rationale for this difference between the
tetrasulfates and disulfates, is that an optimal interaction
with the first disulfate chain may compromise an efficient
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Table 1 Docking scores for sulfated and non-sulfated glycomimetic analogues. Key: sulfate groups (red); alcohol groups (blue)
Entry
1
Code
Docked structure
Docking score (kJ mol−1
)
R-10
−41.0
2
3
S-10
α-4
−42.0
−47.4
4
5
β-5
−51.4
−47.7
26
6
7
α-2
−9.2
17f
−45.8
8
β-3
α-3
17g
−19.2
−15.8
−36.9
9
10
11
12
17h
17i
−43.6
−38.3
13
17a
−51.2
14
15
17b
17c
−47.3
−42.8
16
17
17d
17e
−47.4
−45.4
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Scheme 4 A chiral pool synthesis of α-4, synthesising each stereochemical combination. Conditions: i) 1) Bu3N·SO3, MeCN, 90 °C, 2) sodium
t
2-ethylhexanoate, BuOMe/EtOH.
binding event for the second disulfate chain at an orthosteric
position.
Considering heparan sulfate has many non-sulfated
regions in its macrostructure, and non-ionic interaction can
account for up to 70% of the binding free energy,25 it is
proposed that the interactions of endogenous heparan sulfate
with HGF do not rely predominantly on anionic charge
density from multiple sulfate functionalities. Taken together
Fig. 4 (a) RMSD of the protein backbone along 50 ns of molecular dynamics in the presence of ligands: α-4; β-5; R-10; S-10; α-2; α-3; and β-3;
(b) distance between COG of domain 1 and COG of ligands: α-4; β-5; R-10; S-10; α-2; α-3; and β-3.
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these results elucidate
glycomimetic manifold.
a
valuable SAR profile for the
action of the triaged glycomimetics α-4, R-10, S-10 and β-5, as
well as 3 non-sulfated intermediates α-2, α-3 and β-3, on free
fatty acid (FFA) induced, oxidatively stressed endothelial cell
cultures (Table 2).
The results of the biological assays demonstrate similar
trends across each compound (Table 2). Administration of the
heparan sulfate-glycomimetics and non-sulfated intermediates
generates an increase in eNOS phosphorylation with an
associated output of NO. Furthermore, each compound
Overall, the results of the computational study propose
that the heparan sulfate-glycomimetics bind HGF
predominantly through electrostatic interactions. However,
the results also highlight the potential significance of the
methyl ester and point chirality to the binding interaction of
HGF.
Molecular dynamic binding simulation demonstrated a correlation in the reduction of ROS and
NADPH oxidase activity. Specifically, for the heparan sulfate-
glycomimetic α-4 (Table 2, entry 1), the concentration of NO
increase by +110% compared to the control. However, the
studies of key sulfated and desulfated
glycomimetics
increased phosphorylation of eNOS is comparable to the other
compounds tested, with the exception of R-10, which is shown
to increase eNOS phosphorylation significantly greater than all
Root-mean-squared deviation (RMSD) analyses showed the
stability of the protein structure along 50 ns of molecular
dynamics in the presence of each triaged 2,5-regioisomeric
the other compounds tested (+613%, Table 2, entry 4). All the
glycomimetic scaffold investigated. It is notable that the
remaining compounds demonstrated similar biological activity.
glycomimetics did not cause any abrupt movements in the
protein, indicating that NK1 remained stable in the presence
of the glycomimetics. The protein remained stable after 10 ns ADMET data on hit glycomimetics
of simulation in the presence of the molecules with small
The triaged glycomimetics have been independently analysed
fluctuations (Fig. 4a). The distance between the centers of
for their ADMET properties (Table 3). All showed high kinetic
geometry (COG) of the N-domain with each glycomimetic
solubility, no cytotoxic effect in Hep G2 cells, long half-life in
structure remained stable with small fluctuations, indicating
mouse liver microsomes (MLMs) and rat liver microsomes
that the molecules remained in the binding site along 50 ns
(RLMs) and due to the compound stability, low levels of
of molecular dynamics (Fig. 4b).
intrinsic clearance (CLint). Furthermore, no inhibition of the
cardiac ion channel, hERG was observed. As expected, Caco-2
cell permeability levels were low or not detected due to the
Biological evaluation
The assays focused on the production of nitric oxide (NO)
and the upregulation of nitric oxide synthase (eNOS), the
production of reactive oxygen species (ROS) and the activity
of nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase (Table 2). From the hypothesised model of heparan
sulfate-glycomimetic activity,17 the glycomimetics are
proposed to attenuate the binding of HGF to the cMET
receptor. This disruption to MET-receptor activation is
proposed to modulate intracellular phosphorylation leading
to an upregulation of eNOS and increased output of
extracellular NO. Furthermore, the mechanism of action has
also been demonstrated to modulate the enzymes: superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidase
(GPx), which quench intracellular ROS using NADPH as
cofactor. Overall, the cross examination of NO output and
reduction of ROS was used to determine the therapeutic
highly polar nature of β-5 and R-10 lending support to the
cell receptor interaction proposed. A potential concern is
CYP-mediated
metabolic
oxidation
due
to
the
para-arrangement of the phenolic groups in the
2,5-substituted glycomimetics, potentially leading to
quinone-type reactive intermediates or loss of the sulfate
groups, was not observed in the examples tested.
Conclusions
We have identified a promising series of persulfated and
non-sulfated small molecule heparan sulfate-glycomimetics
that elicit a protective effect in our simulated lipid-induced
endothelial dysfunction model. All examples tested
demonstrate an increase in NO production after oxidative
stress through the eNOS phosphorylation biomarker, and
Table 2 Biological data for eNOS and ROS levels. All data are averaged % of increase (+) or decrease (−) compared to control (FFA induced oxidatively
stressed endothelial cells) (n = 3)
Entry
Compound number
NO production
eNOS phosphorylation
ROS production
NADPH oxidase activity
1
2
3
4
5
6
7
α-4
β-5
S-10
R-10
α-3
β-3
+110
+62
+63
+52
+41
+52
+46
+342
+318
+397
+613
+312
+294
+286
−28
−29
−39
−23
−23
−19
−36
−29
−31
−27
−31
−21
−27
−26
α-2
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and IV (5.0 mL, 20.0 μmol).27 Methyl 2,5-bis(allyloxy)benzoate
(1) (158 mg, 0.50 mmol) was added and the reaction mixture
was stirred at 0 °C for 6 h with monitoring (EtOH/EtOAc 1 : 4,
Rf = 0.20). The solvent was removed under reduced pressure
and the crude mixture was directly purified by
chromatography (SiO2, EtOAc/hexane 1 : 4) to afford the title
Table
glycomimetics. NR
3
Physicochemical and ADMET properties of representative
no response, ND not determined, MEC
=
=
=
minimum effective concentration that significantly crosses vehicle control
threshold, AC50 = the concentration at which 50% maximum effect is
observed for each cell health parameter. Kinetic solubility measured with
a turbidimetric assay (controls: nicardipine and pyrene). MTT (cytotoxicity)
study using HepG2 cells (Cyprotex, Macclesfield, UK; controls: carbonyl
cyanide 3-chlorophenylhydrazone and chlorpromazine). MLM assay
(controls: diphenhydramine and diazepam). hERG assay (controls:
quinidine and dimethylsulfoxide). Caco-2 assay (ATCC, Virginia, USA;
controls: atenolol, propranolol, and talinolol)
25
compound as a clear oil (156 mg, 99%). [α]D −13.15 (c. 1.0,
MeOH, 36 : 6 : 4 : 54 e.r/d.r (2R,5R; 2S,5S; 2R,5S; 2S,5R)); IR
νmax cm−1 3268 w, 2939 w, 2890 w, 1699 s (CO), 1601 w,
1
1499 s, 1435 w; H-NMR (400 MHz, (CD3)2SO) δH 7.18 (d, J =
Compounds
2.7 Hz, 1H, C6–H_), 7.15–7.04 (m, 2H, C3–H_ & C4–H_), 4.94 (d,
J = 5.0 Hz, 1H, CH–OH_), 4.84 (d, J = 5.0 Hz, 1H, CH–OH_),
4.66 (t, J = 5.7 Hz, 1H, CH2–OH_), 4.59 (t, J = 5.7 Hz, 1H, CH2–
OH_), 4.01–3.85 (m, 3H), 3.86–3.69 (m, 6H, Me), 3.55–3.35 (m,
Parameters
α-4
β-5
R-10
S-10
Molecular weight (Da)
tPSA (Å2)
clog P
Number of HBAs
Number of HBDs
Chemical stability
723.87
299
−1.44
20
731.83
310
−1.96
20
619.95
235
−0.13
16
619.95
235
−0.13
16
4H); 13C-NMR (101 MHz, (CD3)2SO) δC 166.0 (CO Me), 152.2
_
2
(C5), 152.0 (C2), 121.0 (C1), 119.8 (C3/C4), 115.94 (C3/C4),
4
5
3
3
Yes
Yes
Yes
Yes
115.89 (C6), 71.3 (ArO–CH ), 70.3 (ArO–CH ), 69.9 (2C, CH–
_
_
_
2
2
OH), 62.72 (CH_2–OH), 62.66 (CH_2–OH), 52.0 (Me); LRMS m/z
(ESI+) 339.12 (100%, [M + Na]+); HRMS m/z (ESI+) C14H20O8-
Na requires: 339.1056, found: 339.1057 ([M + Na]+).
Assay results
Kinetic solubility (μM) >100 >100
HUVEC cell viability
(1–500 μM)
>100
NR
>100
NR
NR
NR
(α-3)
sodium
5-((R)-2,3-dihydroxypropoxy)-2-(( )-2,3-
dihydroxypropoxy)benzoate. A solution of methyl 5-((R)-2,3-
dihydroxypropoxy)-2-(( )-2,3-dihydroxypropoxy)benzoate (α-2)
(100 mg, 0.32 mmol) in MeOH (10 mL) was charged with
NaOH (13 mg, 0.32 mmol). The reaction mixture was heated
under reflux for 2 h, then cooled to room temperature. The
solvent was removed under reduced pressure to afford the
title compound as a hygroscopic white solid (103 mg, 99%).
MTT (MEC, μM)
MTT (AC50, μM)
MLM t1/2 (min)
Mouse CLint
NR
NR
1840
0.755 2.88
NR
NR
482
NR
NR
3450
0.402
NR
NR
4160
0.333
(μL min−1 mg−1
RLM t1/2 (min)
Rat CLint
)
145
9.56
2140
0.649
338
4.11
109
12.8
(μL min−1 mg−1
hERG
)
ND
ND
>25 μM
>25 μM
ND
25
[α]D −10.48 (c. 1.0, MeOH, 36 : 6 : 4 : 54 e.r/d.r (RR,SS,RS,SR));
Caco-2
Not detected Not detected ND
IR νmax cm−1 3246 br s (O–H), 2934 w, 2877 w, 1559 s (CO),
1491 s, 1455 w, 1420 s, 1374 s; 1H-NMR (400 MHz, CD3OD)
δH 7.36 (d, J = 3.1 Hz, 1H, C6–H_), 7.15 (dd, J = 9.0, 3.1 Hz,
1H, C4–H_), 7.12–7.05 (m, 2H, C6–H_ & C3–H_), 6.93 (d, J = 9.0
Hz, 1H, C3–H_), 6.87 (dd, J = 9.0, 3.0 Hz, 1H, C4–H_), 4.16–3.86
(m, 2 × 6H), 3.79–3.57 (m, 2 × 4H); 13C-NMR (101 MHz, CD3-
OD) δC 175.9, 154.4, 154.34, 154.25, 151.7, 132.8, 122.0, 121.5,
117.8, 117.2, 116.7, 116.6, 116.2, 73.2, 72.8, 71.9, 71.8 (2C),
71.6, 71.1, 70.9, 64.3, 64.2, 64.1, 63.9; LRMS m/z (ESI+) 325.09
(30%, [M + H]+); HRMS m/z (ESI+) C13H18NaO8 requires:
325.0894, found: 325.0895 ([M + H]+).
(B2A Papp 10−6 cm s−1
Caco-2
)
ND
Not detected 0.931 ND
(A2B Papp 10−6 cm s−1
)
significant decrease in reactive oxygen species (ROS)
production through NADPH oxidase activity knock-down.
Significantly higher protective effects were observed in the
sulfated glycomimetic series compared to the desulfated
precursors.
Therefore, coupled with these heparan sulfate-
glycomimetics encouraging ADMET profiles (chemically
stable, non-toxic against HUVECs, MTT and hERG; excellent
water solubility; and long t1/2), modelled interaction profile
through molecular docking to HGF and simulated dynamics
studies demonstrate these heparan sulfate-glycomimetics as
a new source of bespoke mimics for ongoing hit-to-lead
studies targeting inflammation and cardiovascular disease.
(β-3)
sodium
5-((S)-2,3-dihydroxypropoxy)-2-(( )-2,3-
dihydroxypropoxy)benzoate. A solution of methyl 5-((S)-2,3-
dihydroxypropoxy)-2-(( )-2,3-dihydroxypropoxy)benzoate (β-2)
(100 mg, 0.32 mmol) in MeOH (10 mL) was charged with
NaOH (13 mg, 0.32 mmol). The reaction mixture was heated
under reflux for 2 h, then cooled to room temperature. The
solvent was removed under reduced pressure to afford the
title compound as a hygroscopic white solid (103 mg, 99%).
25
[α]D −8.31 (c. 1.0, MeOH, 1 : 51 : 46 : 2 e.r/d.r (RR,SS,RS,SR));
Experimental
Selected compound characterisation
IR νmax cm−1 3249 br s (O–H), 2934 w, 2879 w, 1559 s (CO),
1491 s, 1455 w, 1420 s, 1374 s; 1H-NMR (400 MHz, CD3OD)
δH 7.36 (d, J = 3.1 Hz), 7.15 (dd, J = 8.9, 3.1 Hz), 7.12–7.07 (m,
1H, C6–H_), 6.93 (d, J = 8.9 Hz, 1H, C3–H_), 6.87 (dd, J = 8.9,
3.1 Hz, 1H, C4–H_), 4.20–3.84 (m, 2 × 6H), 3.78–3.55 (m, 2 ×
All general procedures can be located in the ESI.†
(α-2)
methyl
5-((R)-2,3-dihydroxypropoxy)-2-((S)-2,3-
dihydroxypropoxy)benzoate
Adapted from general procedure D26 using stock solutions. I
(2 mL, 3.0 mmol), II (2 mL, 3.0 mmol), III (1.0 mL, 2.0 μmol)
4H); 13C-NMR (101 MHz, CD3OD) δC 176.4 (CO Me), 154.9,
_
2
154.9, 154.8, 152.3, 133.3, 122.0, 118.4, 117.7, 117.3, 117.12
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(2C), 116.8, 116.7, 73.7, 73.3, 72.4, 72.3, 72.1, 71.6, 71.4, 64.8,
64.7, 64.6, 64.5; LRMS m/z (ESI+) 325.09 (30%, [M + H]+);
HRMS m/z (ESI+) C13H18NaO8 requires: 325.0894, found:
325.0890 ([M + H]+).
LRMS m/z (ESI+) 732.84 (10%, [M + H]+); HRMS m/z (ESI+)
C13H14Na5O20S4 requires: 732.8444, found: 732.8439 ([M +
H]+).
(R-10)
sodium
(R)-3-(3-(methoxycarbonyl)-4-(4-
(α-4) sodium 3-(4-((S)-2,3-bis(sulfonatooxy)propoxy)-2-
(methoxycarbonyl)phenoxy)propane-1,2-diyl bis(sulfate)
Adapted from general procedure E28. A 25 mL Schlenk tube
was charged with α-5 (81 mg, 0.25 mmol) and Bu3N·SO3 (530
mg, 2.00 mmol) under Ar and MeCN was added (0.5 mL).
The flask was heated at 80 °C for 12 h with monitoring
(TLC). The flask was cooled to room temperature and the
solvent removed under reduced pressure to give a clear
(sulfonatooxy)butoxy)phenoxy)propane-1,2-diyl bis(sulfate)
Adapted from general procedure E28. A 25 mL Schlenk tube
was charged with S-9 (135 mg, 0.45 mmol) and Bu3N·SO3
(722 mg, 2.72 mmol) under Ar(g) and MeCN was added (1.5
mL). The flask was heated at 80 °C for 12 h with monitoring
(TLC). The flask was cooled to room temperature and the
solvent removed under reduced pressure to give a clear
i
viscous oil. The crude oil was dissolved in PrOH (5 mL) and
i
viscous oil. The crude oil was dissolved in PrOH (5 mL) and
transferred to a flask containing tBuOMe (35 mL). With
transferred to a flask containing tBuOMe (35 mL). With
vigorous stirring a solution of NEH (5.0 mL, 1.0 M) in
-
t
t
vigorous stirring a solution of NEH (5.0 mL, 1.0 M) in
-
BuOMe/iPrOH (1 : 7) was added dropwise over 10 min. The
precipitate that formed was collected by filtration, washed
BuOMe/iPrOH (1 : 7) was added dropwise over 10 min. The
precipitate that formed was collected by filtration, washed
with iPrOH (2
× 10 mL) and dried under vacuum.
with iPrOH (2
×
10 mL) and dried under vacuum.
Recrystallization from H2O/iPrOH afforded the title
25
Recrystallization from H2O/iPrOH afforded the title
compound as a white solid (260 mg, 94%). [α]D −3.11 (c.
compound as a white solid (160 mg, 88%). [α]D −3.35 (c.
1.0, CHCl3); M.P 207–210 °C (dec.); IR νmax cm−1 2972 w, 2166
w, 1703 w (CO), 1500 w, 1441 w, 1103 s (SO); 1H-NMR
(400 MHz, D2O) δH 7.42 (d, J = 3.2 Hz, 1H, C6–H_), 7.25 (dd, J
= 9.2, 3.2 Hz, 1H, C4–H_), 7.14 (d, J = 9.2 Hz, 1H, C3–H_), 4.86
(p, J = 4.8 Hz, 1H, CH_–OSO3Na), 4.42–4.29 (m, 3H), 4.26 (dd, J
= 11.0, 5.2 Hz, 1H), 4.16–4.05 (m, 4H), 3.90 (s, 3H, Me), 1.86
25
1.0, H2O, 36 : 6 : 4 : 54 e.r/d.r (SS,RR,SR,RS)); M.P 198–200 °C
(dec.); IR νmax cm−1 2988 w, 2164 w, 1711 w (CO), 1500 w,
1443 w, 1221 s (SO), 1131 s (SO); 1H-NMR (400 MHz,
D2O) δH 7.45 (d, J = 3.1 Hz, 1H, C6–H_), 7.27 (dd, J = 9.1, 3.1
Hz, 1H, C4–H_), 7.19 (d, J = 9.1 Hz, 1H, C3–H_), 4.87 (td, J =
4.8, 1.8 Hz, 2H, CH_–OSO3Na), 4.45–4.25 (m, 8H, Ar–OCH_2,
CH_2–OSO3Na), 3.93 (s, 3H, Me); 13C-NMR (101 MHz, D2O) δ
(m, 4H, (CH_2)2); 13C-NMR (101 MHz, D2O) δC 168.4 (CO Me),
_
2
152.6 (C2), 151.7 (C5), 121.6 (C4), 120.1 (C1), 117.3 (C6),
168.5 (CO Me), 152.2 (C2), 152.1 (C5), 121.4 (C4), 120.8 (C1),
116.4 (C3), 75.0 (CH–OSO Na), 69.7, 69.0, 67.3, 66.4, 52.7
_
3
_
2
117.4 (C6), 116.9 (C3), 75.03 (CH–OSO Na), 74.98 (CH–OSO -
(Me), 25.2 ((CH ) ), 24.9 ((CH ) ); LRMS m/z (ESI+) 642.94
_ _
2 2 2 2
_
_
3
3
Na), 68.2 (O–CH ), 67.3 (O–CH ), 66.44 (CH –OSO Na), 66.41
(40%, [M + Na]+); HRMS m/z (ESI+) C15H19Na4O16S3 requires:
_
_
_
2
2
2
3
(CH –OSO Na), 52.8 (Me); LRMS m/z (ESI+) 746.86 (20%, [M +
642.9421, found: 642.9426 ([M + Na]+).
_
2
3
Na]+); HRMS m/z (ESI+) C14H16Na5O20S4 requires: 746.8601,
(S-10)
sodium
(S)-3-(3-(methoxycarbonyl)-4-(4-
found: 746.8591 ([M + Na]+).
(sulfonatooxy)butoxy)phenoxy)propane-1,2-diyl bis(sulfate)
Adapted from general procedure E28. A 25 mL Schlenk tube
was charged with R-9 (215 mg, 0.72 mmol) and Bu3N·SO3
(1.145 g, 2.56 mmol) under Ar(g) and MeCN was added (1.5
mL). The flask was heated at 80 °C for 12 h with monitoring
(TLC). The flask was cooled to room temperature and the
solvent removed under reduced pressure to give a clear
(β-5) sodium 5-((R)-2,3-bis(sulfonatooxy)propoxy)-2-(2,3-
bis(sulfonatooxy)propoxy)benzoate
Adapted from general procedure E28. A 25 mL Schlenk tube
was charged with β-3 (100 mg, 0.32 mmol) and Bu3N·SO3
(678 mg, 2.56 mmol) under Ar(g) and MeCN was added (0.6
mL). The flask was heated at 80 °C for 12 h and with
monitoring (TLC). The flask was cooled to room temperature
and the solvent removed under reduced pressure to give a
i
viscous oil. The crude oil was dissolved in PrOH (5 mL) and
transferred to a flask containing tBuOMe (35 mL). With
i
t
clear viscous oil. The crude oil was dissolved in PrOH (5 mL)
vigorous stirring a solution of NEH (5.0 mL, 1.0 M) in
-
t
and transferred to a flask containing BuOMe (35 mL). With
BuOMe/iPrOH (1 : 7) was added dropwise over 10 min. The
precipitate that formed was collected by filtration, washed
t
vigorous stirring a solution of NEH (5.0 mL, 1.0 M) in
-
BuOMe/iPrOH (1 : 7) was added dropwise over 10 min. The
precipitate that formed was collected by filtration, washed
with iPrOH (2
× 10 mL) and dried under vacuum.
Recrystallization from H2O/iPrOH afforded the title
25
with iPrOH (2
×
10 mL) and dried under vacuum.
compound as a white solid (430 mg, 97%). [α]D +3.01 (c.
Recrystallization from H2O/iPrOH afforded the title
1.0, H2O); M.P 207–210 °C (dec.); IR νmax cm−1 2971 w, 2164
w, 1710 w (CO), 1500 w, 1441 w, 1103 s (SO); 1H-NMR
(400 MHz, D2O) δH 7.43 (d, J = 3.2 Hz, 1H, C6–H_), 7.25 (dd, J
= 9.2, 3.2 Hz, 1H, C4–H_), 7.14 (d, J = 9.2 Hz, 1H, C3–H_), 4.87
(p, J = 4.7 Hz, 1H, CH_–OSO3Na), 4.44–4.29 (m, 3H), 4.26 (dd, J
= 11.0, 5.2 Hz, 1H), 4.12 (m, 4H), 3.90 (s, 3H, Me), 1.94–1.79
25
compound as a white solid (200 mg, 86%). [α]D −1.66 (c.
1.0, H2O, 1 : 51 : 46 : 2 e.r/d.r (SS,RR,SR,RS)); M.P 200–202 °C
(dec.); IR νmax cm−1 2988 w, 1712 w (CO), 1499 w, 1435 w,
1
1100 s (SO); H-NMR (400 MHz, D2O) δ 7.47–6.94 (m, 3H),
5.00–4.73 (m, 2H), 4.46–3.99 (m, 8H); 13C-NMR (101 MHz,
D O) δ 169.6 (CO Na), 152.3 (C–O), 151.8 (C–O), 121.5 (C–CO -
(m, 4H, (CH_2)2); 13C-NMR (101 MHz, D2O) δC 168.4 (CO Me),
_
_
_
_
_
2
2
2
2
Na), 120.7 (C–H), 117.4 (C–H), 116.2 (C–H), 75.0 (C–H), 74.9
152.6 (C2), 151.7 (C5), 121.6 (C4), 120.1 (C1), 117.3 (C6),
_
_
_
_
(C–H), 68.3 (C–H ), 67.3 (C–H ), 66.6 (C–H ), 66.4 (C–H );
116.4 (C3), 75.0 (CH–OSO Na), 69.7, 69.0, 67.4, 66.4, 52.7
_
_
_
_
_
_
2
2
2
2
3
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(Me), 25.2 ((CH ) ), 24.9 ((CH ) ); LRMS m/z (ESI+) 642.94
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_
_
2 2
2 2
(50%, [M + Na]+); HRMS m/z (ESI+) C15H19Na4O16S3 requires:
642.9421, found: 642.9417 ([M + Na]+).
Conflicts of interest
A. M. J. and M. Y. A. have a commercial interest in
glycomimetics (UK Patent Application No. 1515850.4 and PCT
Patent Application No. PCT/GB2016/052764).
Acknowledgements
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–
Finance Code 001 in the form of research scholarships for A.
P. R. P. and G. Z. D. M. G. thanks the University of
Birmingham for a Ph.D scholarship. S. A. S. and A. M. J. thank
the Wellcome Trust for the award of a Biomedical Vacation
Scholarship (202151/Z/16/Z). A. M. M. thanks the Alexander
Von Humboldt Foundation for funding his postdoctoral
fellowship (ID 1158232) and the International Atherosclerosis
Society (IAS; USA) for a travel grant. A. M. J., M. Y. A. and F. L.
W. thank the Healthcare Science Research Centre for a Support
of Excellence Award and Cyprotex (Macclesfield, UK) for
ADMET studies. The authors thank the Centre for Chemical
and Materials Analysis in the School of Chemistry, University
of Birmingham for analytical services and Dr Louise Male for
crystallographic analysis of 22. The authors thank Dr J.
Wilkinson and Dr N. I. Ziedan for helpful discussions. The
computations described in this paper were performed using
the University of Birmingham's BlueBEAR HPC service, which
provides
a high-performance computing service to the
University's research community. See http://www.birmingham.
ac.uk/bear for more details.
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22 H. C. Kolb, M. S. Van Nieuwenhze and K. B. Sharpless,
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26 See ESI† page S6.
27 See ESI† page S7.
28 See ESI† page S8.
RSC Med. Chem.
This journal is © The Royal Society of Chemistry 2021