Novel Symmetrical Benzazolyl Derivatives Endowed with Potent Anti-Heparanase Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b01497

Heparanase is the only mammalian endo-β-d-glucuronidase involved in a variety of major diseases. The up-regulation of heparanase expression increases tumor size, angiogenesis, and metastasis, representing a validated target in the anti-cancer field. To date, only a few small-molecule inhibitors have been described, but none have gotten through pre-clinical development. Previously, we explored 2-(4-(4-(bromo-methoxybenzamido)benzylamino)phenyl) benzazole derivatives as anti-heparanase agents, proposing this scaffold for development of broadly effective heparanase inhibitors. Herein, we report an extended investigation of new symmetrical 2-aminophenyl-benzazolyl-5-acetate derivatives, proving that symmetrical compounds are more effective than asymmetrical analogues, with the most-potent compound, 7g, being active at nanomolar concentration against heparanase. Molecular docking studies were performed on the best-acting compounds 5c and 7g to rationalize their interaction with the enzyme. Moreover, invasion assay confirmed the anti-metastatic potential of compounds 5c, 7a, and 7g, proving the inhibition of the expression of proangiogenic factors in tumor cells.

Full text of DOI:10.1021/acs.jmedchem.8b01497

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Article
Novel Symmetrical Benzazolyl Derivatives
Endowed with Potent anti-Heparanase Activity
Antonella Messore, Valentina Noemi Madia, Luca Pescatori, Francesco Saccoliti, Valeria Tudino,
Alessandro De Leo, Martina Bortolami, Daniela De Vita, Luigi Scipione, Federico Pepi, Roberta Costi,
Silvia Rivara, Laura Scalvini, Marco Mor, Fabiana Fosca Ferrara, Emiliano Pavoni, Giuseppe Roscilli,
Giuliana Cassinelli, Ferdinando M. Milazzo, Gianfranco Battistuzzi, Roberto Di Santo, and Giuseppe Giannini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01497 • Publication Date (Web): 09 Nov 2018
Downloaded from http://pubs.acs.org on November 17, 2018
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Novel Symmetrical Benzazolyl Derivatives
Endowed with Potent anti-Heparanase Activity
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a
a
a
Antonella Messore, Valentina Noemi Madia, Luca Pescatori, Francesco
a
a
a
a
Saccoliti, Valeria Tudino, Alessandro De Leo, Martina Bortolami, Daniela De
a
a
a
a
b
Vita, Luigi Scipione, Federico Pepi, Roberta Costi, Silvia Rivara, Laura
Scalvini, Marco Mor, Fabiana Fosca Ferrara, Emiliano Pavoni, Giuseppe
Roscilli, Giuliana Cassinelli, Ferdinando M. Milazzo,
b
b
c
c
c
d°
e°
e°
Gianfranco Battistuzzi,
a*
e°*
Roberto Di Santo, and Giuseppe Giannini
a
Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione
Cenci
Bolognetti, “Sapienza” Università di Roma, p.le Aldo Moro 5, I-00185,
Roma Italy
b
Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di
Parma, Parco Area delle Scienze 27/A, I- 43124 Parma, Italy
c
Takis s.r.l., Via Castel Romano 100, I-00128 Roma, Italy
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d
Dipartimento di Ricerca Applicata e Sviluppo Tecnologico, Unità di
Farmacologia Molecolare, Fondazione IRCCS Istituto Nazionale dei Tumori, via
Amadeo 42, I-20133 Milano, Italy
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e
R&D Alfasigma S.p.A., Via Pontina Km 30,400, Pomezia, I-00071Roma, Italy
KEYWORDS.
Heparanase
inhibitors,
Anticancer
agents,
Angiogenesis,
Inflammation, Benzimidazole and Benzoxazole derivatives.
°Note: Activity carried out within a collaborative research agreement with
Leadiant Biosciences S.A. (Formerly Sigma-Tau Research Switzerland, S.A.),
Mendrisio, CH.
ABSTRACT.
Heparanase is the only mammalian endo-β-D-glucuronidase involved in a variety
of major diseases. Up-regulation of heparanase expression increases tumor
size, angiogenesis and metastasis, representing a validated target in anticancer
field. To date only few small molecule inhibitors have been described but none
2
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has got through preclinical development. Previously we explored 2-(4-(4-(bromo-
methoxybenzamido)benzylamino)phenyl)
benzazole
derivatives
as
anti-
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heparanase agents, proposing this scaffold for development of broadly effective
heparanase inhibitors. Herein we report an extended investigation of new
symmetrical
2-aminophenyl-benzazolyl-5-acetate
derivatives,
proving
that
symmetrical compounds are more effective than asymmetrical analogues, with
the most potent compound 7g active at nanomolar concentration against
heparanase. Molecular docking studies were performed on the best acting
compounds 5c and 7g to rationalize their interaction with the enzyme.
Moreover, invasion assay confirmed the anti-metastatic potential of compounds
5
c and 7a and 7g, proving to inhibit the expression of proangiogenic factors in
tumor cells.
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Introduction
Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules
associated with cell surface and extracellular matrix (ECM) of a wide range of
cells and have been found to play regulatory roles in many biological functions,
including both physiological and pathological processes.1-3
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This class of carbohydrate-modified proteins exerts its multiple functions via
several mechanisms combining structural, biochemical and regulatory aspects.
By interacting with other macromolecules such as laminin, fibronectin, and
collagens I and IV, HSPGs contribute to the structural integrity, self-assembly
and insolubility of the ECM and basement membrane, thus intimately
4
modulating cell-ECM interactions. Additionally, HSPGs regulate the transfer of
information from extracellular space to intracellular kinases and cytoskeletal
elements, thus affecting cell signaling, adhesion and motility.5
The key structural component of cell surface and matrix-associated HSPGs is
heparan sulfate (HS), whose function is to bind and interact with signaling
proteins, growth factors, plasma proteins, immune-modulators and other factors,
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thereby regulating protein distribution, bioavailability and action on target cells.
Moreover, the HS chains ensure that a wide variety of bioactive molecules bind
to the cell surface and ECM, thus regulating morphogenesis, tissue repair,
inflammation, vascularization and cancer metastasis.6
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The sole enzyme responsible for the degradation of mammalian HS is
heparanase (Hpse), an endo-β-D-glucuronidase that cleaves the HS side chains
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of HSPGs into segments of 10-20 sugar units, by hydrolyzing the β-glycosidic
bond at specific intrachain sites with retention of the anomeric configuration,
thus producing physiologically active saccharide fragments of 10-20 sugar units,
8
and allowing at the same time the release of HS-bound macromolecules. In
virtue of its activity on HSPGs, Hpse influences a multitude of both normal and
disease-related processes.9 Indeed, under physiological conditions, Hpse is
expressed at high levels in placenta and some blood-borne cells such as
platelets, neutrophils, mast cells and lymphocytes, while it is undetectable in
other human tissues. On the contrary, deregulated expression/function of Hpse
has been involved not only in several inflammatory-based diseases (as chronic
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inflammation, atherosclerosis, thrombosis, fibrosis, neurite outgrowth, diabetic
nephropathy and bone osteolysis), but also in a variety of human hematological
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and solid malignancies of both epithelial and mesenchymal origin.10,11
Clinically, Hpse up-regulation has been associated with growth/aggressiveness
of several cancer types being correlated with an increased tumor size,
angiogenesis, metastasis and poor prognosis.10 Pioneering and breakthrough
studies have highlighted the link between Hpse and cancer. In particular, the
significant impairment of tumor growth and progression observed in tumor-
bearing mice by Hpse gene expression knock down or by treatment with Hpse
inhibitors, has provided the preclinical evidence of the potential of Hpse as
target in anticancer therapy.12 Recently, Hpse up-regulation has also been
associated with the induction of resistance in response to anticancer
therapeutics, an effect attributed, at least in part, to Hpse-mediated promotion of
autophagy.13 On the other hand, chemotherapy itself induces the expression
and the release of Hpse by surviving tumor cells thus contributing to promote
an aggressive phenotype.14
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Overall, these studies provide a strong rationale to include Hpse inhibitors in
combination
regimens
with
chemotherapeutic
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to
overcome
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chemoresistance. Indeed, compounds able to specifically target Hpse are highly
desired to widen therapeutic options in the oncologic context as well as in
those clinical indications in which Hpse is pathologically involved. The relevant
role of Hpse in sustaining specific pathological conditions has led to the search
for molecules able to interfere with its biological activity. However, although in
the last two decades intensive efforts have been made in developing Hpse
inhibitors and huge progresses have been made in the knowledge of Hpse
functions, no drug able to inhibit or modulate Hpse activity and functions has
been registered so far.8,15,16
Several Hpse inhibitors have been described, spanning from nucleic acids,
proteins, monoclonal antibodies to polysulfated saccharides. Among them, only
four drugs have hitherto reached different phases of clinical trials. They are
polysaccharide derivatives of natural origin, such as roneparstat (SST0001),
muparfostat (PI-88) and necuparanib (M-402), or obtained by synthesis as
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pixatimod (PG545). These HS mimics show various limitations, mainly related to
their high molecular weight and heterogeneous nature which could affect
product characterization, standardization and interpretation of biological data,
and also related to their parenteral delivery route that might limit the patient
compliance.8 Moreover, despite expectancy from preclinical and early clinical
trials, after interim analysis muparfostat was discontinued as it failed to meet
the trial primary end point17 and, similarly, the necuparanib trial was interrupted
as a consequence of interim futility analysis.18 On the other hand, the pixatimod
trial was initially suspended due to local injection site reactions seen in patients
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and then the route of administration is currently being changed from sc. to
iv.19,20
Recent clinical reports showed that pixatimod and roneparstat displayed good
safety profile in advanced solid tumors and multiple myeloma in early clinical
trials.21,22
The development of small molecule inhibitors of Hpse has the potential to
overcome some of the limitations of polysaccharides. Small molecules are, in
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fact, particularly desirable since they offer the opportunity to be properly
designed to get favorable pharmacokinetic properties and oral availability.
Among synthetic small molecules, some benzimidazole and benzoxazole
derivatives have been described as Hpse inhibitors, such as 1-[4-(1H-
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benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea
1,23
or
benzoxazol-5-yl acetic acid 2,24,25 that displayed good Hpse inhibitory potency
(
Figure 1).
Very recently, our efforts to develop small molecule Hpse inhibitors led to the
identification
methoxybenzamido)benzylamino)phenyl)benzimidazoles 3 (SST0867AA1) and 4
SST0871AA1) as effective Hpse inhibitors, with IC50 of 2.86 and 0.64 µM,
of
2-(4-(4-(bromo-
(
respectively (Figure 1).26 These compounds did not affect cell proliferation, but
were able to decrease the in vitro invasive ability of human cancer cell lines
and to affect transcription of genes encoding for proangiogenic factors, effects
that can be referred to their good ability to inhibit of Hpse enzymatic activity.
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H C
3
CH3
O
Br
H C
3
NH
CH3
HN
O
N
O
NH
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O
N
HO
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N
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H
H
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O
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H
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NH
Br
HO
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O
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Figure 1. Chemical structures of benzazole-Hpse inhibitors.
Notably, the potency of these inhibitors against Hpse was enhanced by the
presence of i) fluorine atom on the inner phenyl ring and ii) amino acid residue
on the lateral chain.
Taking into account the Hpse good inhibitory activity of symmetrical inhibitors
like compound 1,23 and suggestions provided by our previous molecular docking
studies performed with compounds 3 and 4, we decided to design new
symmetrical derivatives, introducing a more rigid central portion in the structure
to allow a better fit in the enzyme pocket, thus giving the opportunity to the
lateral chains of further positive interactions with the target.
1
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Chart 1. Structures of the Newly Designed Benzazole Derivatives 5a-f, 6a,b,
7a-g, 8a-c, 9a,b, 10a,b, 11a-i.
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N
H
N
H
5
5
5
5
a X=O, Y=4-NHCONH
b X=O, Y=3-NHCONH
c X=O, Y=3-F; 4-NHCONH
d X=O, Y=4-NHCSNH
7a X=O,Y=O, R=H, R =COOH
1
7b X=O,Y=O, R=CH , R =COOH
3
1
5e X=O, Y=3-F; 4-NHCSNH
7c X=O,Y=O, R=CH Ph, R =COOH
2 1
5
6
6
f X=O, Y=4-C(NH)3
7d X=O,Y=O, R=H, R =CH CH CH(CH )
1 2 2 3 2
7e X=O,Y=O, R=CH CH(CH ) , R =B(OH)
2 3 2 1 2
7f X=O,Y=O, R=H, R =B(OH)2
1
a X=NH, Y=3-F; 4-NHCONH
b X=NH, Y=3-F; 4-NHCSNH
7
g X=O,Y=S, R=H, R =COOH
1
CH3
8a X=NH, Y=O, R=H, R =COOH
1
H C
3
8
b X=NH,Y=O, R=CH Ph, R =COOH
2 1
CH3
8c X=NH,Y=O, R=H, R =B(OH)2
H C
3
O
1
O
R
R
N
N
O
R
R
N
H
N
H
9
9
a R=H
b R=F
X
R1
R1
X
R2
N
N
H
R2
H
Z
Y
Y
R
R
1
1a X=CO, Y=NH, Z=CO, R=OH, R =H, R =CH COOH
1
2
2
R1
R1
O
11b X=CO, Y=NH, Z=CO, R=H, R1=H, R2=CH2COOH
1c X=SO , Y=NH, Z=CO, R=H, R =H, R =CH COOH
O
1
2
1
2
2
N
N
N
N
O
11d X=CO, Y=NH, Z=CS, R=H, R =F, R =CH COOH
1
2
2
1
1e X=CO, Y=CH , Z=CHOH, R=OH, R =H, R =CH COOH
2 1 2 2
N
N
H
H
11f X=CO, Y=CH2, Z=CHOH, R=H, R1=H, R2=CH2COOH
1g X=CO, Y=CH , Z=CO, R=H, R =H, R =CH COOH
1
2
1
2
2
1
0a R=H, R =CH COOH
11h X=CO, Y=NH, Z=CO, R=H, R1=H, R2=COOH
11i X=CO, Y=NH, Z=CO, R=H, R1=H, R2=B(OH)2
1
2
10b R=R =CH
1
3
Thus, we designed and synthesized the derivatives reported in Chart 1. In
detail, the new compounds are characterized by a rigid central core, typically a
ureidic group, and by a symmetrical shape. The new derivatives show one or
more of the following structural characteristics: (i) a benzoxazol-5-yl or
1
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benzimidazol-5-yl acetic acid scaffold; (ii) functionalization of the acetic acid
moiety with amino acidic groups or aminoalkyl boronic acids; (iii) replacement of
the benzoxazol-5-yl acetic acid scaffold with a 5,6-dimethylbenzoxazolyl one; (iv)
replacement of the urea with a thiourea, a guanidine, an isopropanol or
acetone; (v) introduction of a 1,3-di(piperidin-4-yl)urea replacing the 1,3-
diphenylurea; (vi) benzazole ring opening and introduction of an ortho-
hydroxyamide, amide or sulfonamide function; (vii) replacement of the 2-(3-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzamidophenyl)acetic acid with
a
3-benzamidobenzoic acid or (3-
benzamidophenyl)boronic acid; (viii) shift of the urea moiety from para to meta
position of the inner rings; (ix) introduction of a fluorine atom in ortho to urea
or thiourea moiety.
The newly synthesized compounds have been evaluated in vitro for their
capacity to inhibit Hpse enzymatic activity, to affect proliferation and invasive
potential of malignant cells. In addition, the effect of selected compounds on
the expression of genes encoding for proangiogenic factors has been assessed
in a tumor cell line. Moreover, based on docking studies in the crystal structure
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of human Hpse, a rationalization of the interaction between the best acting
compounds and the biological target has been proposed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results and discussion
Chemistry. The benzazole derivatives 5a-e, 6a,b, 7a-c,g and 8a,b were
synthesized as reported in Scheme 1. Urea and thiourea derivatives 16a-d and
1
7a were obtained from the corresponding amines 12a-c27 and 1326 after
reaction with triphosgene or thiocarbonyldiimidazole (thio-CDI) as condensing
agent, as. Differently, thioureas 16e and 17b have been obtained by
microwave-assisted reaction of the amines 12c and 13 with isothiocyanates 14
and 15, which have been generated by reaction of the same amines 12c and
1
3 with thiophosgene.
Then the esters 16a-e, 17 a,b were hydrolized in alkaline medium (LiOH or
NaOH) to give the corresponding acid derivatives 5a-e and 6a,b. The latter
underwent to coupling reactions with the proper amino ester in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-
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dimethylaminopyridine (DMAP) using N,N-diisopropylethylamine (DIPEA) as base
to give esters 18a-c,g and 19a,b that were in turn hydrolyzed in alkaline
medium to give the corresponding amino acid derivatives 7a-c,g and 8a,b.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthetic Route to 5a-e, 6a,b, 7a-c,g and 8a,b derivativesa
O
O
X
N
R
H3CO
i or ii
O
OCH3
O
v or vi
X
X
H3CO
N
Z
Z
N
1
2a-c
13
O
HO
OH
Y
X
X
iii
16a-e
17a,b
N
Z
Z
N
F
iv
X
Y
O
16a X=O, Y=O, Z=4-NH
16b X=O, Y=O, Z=3-NH
16c X=O, Y=O, Z=3-F; 4-NH
NCS
5a X=O, Y=O, Z=4-NH
5b X=O, Y=O, Z=3-NH
5c X=O, Y=O, Z=3-F; 4-NH
5d X=O, Y=S, Z=4-NH
5e X=O, Y=S, Z=3-F; 4-NH
6a X=NH, Y=O, Z=3-F; 4-NH
6b X=NH, Y=S, Z=3-F; 4-NH
N
5a-e
H3CO
6a,b
16d X=O, Y=S, Z=4-NH
1
1
4
5
16e X=O, Y=S, Z=3-F; 4-NH
17a X=NH, Y=O, Z=3-F; 4-NH
17b X=NH, Y=S, Z=3-F; 4-NH
1
1
1
2a X=O,R=4-NH2
2b X=O,R=3-NH2
13 X=NH,R=3-F; 4-NH2
14 X=O
vii
2c X=O,R=3-F; 4-NH2 15 X=NH
R2O
O
R1
R1
OR
2
O
O
HN
NH
O
X
X
F
F
N
Y
N
HO
O
R1
R1
O
OH
N
N
O
H
H
HN
NH
O
X
X
18a-c,g
19a,b
F
F
v
N
Y
N
N
H
N
H
18a X=O, Y=O, R1=H, R2=Et
18b X=O, Y=O, R1=CH3, R2=Et
18c X=O, Y=O, R =CH Ph, R =CH
3
7a X=O, Y=O, R1=H
7a-d,g
8a,b
1
2
2
7b X=O, Y=O, R1=CH3
1
8g X=O, Y=S, R1=H, R2=Et
19a X=NH, Y=O, R1=H, R2=Et
9b X=NH, Y=O, R1=CH2Ph, R2=CH3
7
7
c X=O, Y=O, R1=CH2Ph
g X=O, Y=S, R1=H
1
8a X=NH, Y=O, R1=H
b X=NH, Y=O, R1=CH2Ph
8
a
Reagents and conditions: (i) triphosgene, Et N, CH Cl dry or THF dry, N , 0
3
2
2
2
°
6
C to room temp, 24-44 h, 40-100% yield; (ii) thio-CDI, CH Cl , 40 °C, 12 h,
0% yield; (iii) thiophosgene, chlorobenzene, DMF cat., reflux, 2-4 h, 100%
2
2
yield; (iv) 12c or 13, DMSO dry, 200 W, N , 80 °C, 3 min, 30-70% yield; (v)
2
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
LiOH, 5:1 THF/H O, room temp, 3.5-48 h, 49-99% yield; (vi) NaOH, 5:1
2
THF/H O, room temp, overnight, 44-100% yield; (vii) appropriate amino ester
2
detailed in the experimental section, EDCI, DMAP, DIPEA, THF dry or DMF
dry, N , room temp, overnight, 64-85% yield.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Guanidine derivative 5f was synthesized according to Scheme 2. Compound
16d was reacted with hexamethyldisilazane in the presence of EDCI to furnish
guanidine 20 that subsequently underwent to alkaline hydrolysis with LiOH to
give the corresponding acid derivative 5f.
Scheme 2. Synthetic Route to 5f Derivativea
1
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O
O
H CO
OCH3
3
O
O
N
N
S
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
1
6d
O
O
i
H CO
3
OCH3
O
O
N
N
NH
N
H
N
H
20
O
O
ii
HO
OH
O
O
N
N
NH
N
H
N
H
5f
a
Reagents and conditions: (i) hexamethyldisilazane, EDCI, MeCN, 0 °C to
room temp, 5 d, 70% yield; (ii) LiOH, 5:1 DMSO/H O, room temp, 3 d, 70%
2
yield.
Boronic acids 7e,f and 8c and isopentyl derivative 7d were synthesized as
described in Scheme 3. The appropriate alkyl boronic ester as ammonium salts
were coupled with benzazol-5yl acetic acid derivatives 5c and 6a in the presence of 2-(1H-
Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and DIPEA at -80 °C
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8
for 3.5 h, giving the pinanediol boronic ester derivative 21 as previously described in literature
or boronic acids 7f and 8c.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthetic Route to 7d-f and 8c Derivativesa
O
O
HN
NH
O
O
F
F
N
O
N
N
H
N
H
7d
HO
OH
B
O
O
B
O
O
HO HN
NH OH
HO
i
OH
X
X
X
X
F
F
ii
N
O
N
F
F
N
O
N
N
H
N
H
N
H
N
H
7f X=O
8c X=NH
5
c X=O
7f
5
c
6a X=NH
8c
6a
H
H
iii
O
O
B
O
O
B
H
H
O
CH3
HN
NH
O
H3C
H
H
O
O
F
F
N
O
N
N
H
N
H
HO
OH
B
NH OH
21
B
O
O
HO HN
O
O
iv
F
F
N
O
N
N
N
H
H
7e
a
Reagents and conditions: (i) (R)-boroLeu-(+)-pinanediol HCl, EDCI, DMAP,
DIPEA, THF dry, N , room temp, overnight, 87% yield; (ii) (4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)methanaminium chloride, TBTU, DIPEA, DMF dry, N , -80
C to room temp, 5 h, 30-35 yield; (iii) (R)-boroLeu-(+)-pinanediol HCl, TBTU,
2
1
2
°
DIPEA, DMF dry, N , -80 °C to room temp, 5 h, 96.2% yield; (iv) NH OAc
2
4
aq
0
.1 N, NaIO , THF, room temp, 47 h, 60% yield.
4
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We speculated that pinanediol esters are more resistant to hydrolysis and, therefore, an
oxidative cleavage of the pinanediol was carried out by using sodium
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
metaperiodate in the presence of NH OAc
0.1 N, according to a known
4
aq
procedure reported for boronic esters.30 The use of such electrophilic oxidant
was due to the awareness of the susceptibility of boronic acids to nucleophilic
oxidative agents.
Interestingly, by modifying both coupling reaction conditions and coupling
agent, the isopentylacetamide derivative 7d was obtained. In fact this compound
was obtained from the benzoxazol-5-yl acetic acid 5c that was reacted with (R)-
boroLeu-(+)-pinanediol HCl in the presence of EDCI and DMAP, using DIPEA
as a base, at room temperature for 24 h.
Scheme 4. Synthetic Route to 9a,b Derivativesa
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H C
OH
O
ii
R
3
H C
OH
3
H C
i
3
O
N
R
NO2
H C
3
N
H C
3
NH2
H
H C
3
NO2
22a,b
23a,b
23a R=H
3b R=F
22a R=H
2
22b R=F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
iii
CH3
CH3
H C
3
H C
O
3
O
R
H C
3
R
R
O
N
N
N
O
iv
NH2
H C
3
N
H
N
H
24a,b
9a R=H
b R=F
24a R=H
24b R=F
9
a,b
9
a
Reagents and conditions: (i) proper acyl chloride, Et N, THF dry, N , 0 °C to
room temp, 3 h, 100% yield; (ii) PTSA, toluene, reflux, overnight, 40-68% yield;
(iii) H , Pd/C, AcOEt, 40 psi, room temp, 3-6 h, 75-95% yield; (iv) triphosgene,
Et N, CH Cl dry, N , 0 °C to room temp, overnight, 32% yield.
3
2
2
3
2
2
2
5,6-Dimethylbenzoxazolyl derivatives 9a,b were synthesized as shown in
Scheme 4. 2-Amino-4,5-dimethylphenol31 was acylated with the proper acyl
chloride in the presence of triethylamine to furnish amides 22a,b that
subsequently underwent to ring closure using p-toluenesulfonic acid (PTSA) as
a catalyst, obtaining 5,6-dimethylbenzoxazolyl nitro derivatives 23a,b. After
catalytic reduction, the amines 24a,b were reacted with triphosgene to give the
corresponding urea derivatives 9a,b.
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Scheme 5. Synthetic Route to 10a,b Derivativesa
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H3C
H3C
OH
NH2
R
R
O
N
O
N
or
i
ii
N
NHBoc
N
NH2
R1
R1
OH
26a R=H, R1=CH2COOCH3
26b R=CH3, R1=CH3
O
25a,b
26a,b
H3CO
NH2
25a R=H, R1=CH2COOCH3
25b R=CH3, R1=CH3
iii
O
O
R
R
HO
OH
R1
R1
O
O
O
O
iv
N
N
O
N
N
N
N
O
N
N
N
N
N
N
H
H
H
H
27a R=H, R1=CH2COOCH3
10a
27a
0b
10b R=CH3, R1=CH3
1
a
Reagents and conditions: (i) 1) potassium ethyl xanthogenate, pyridine, N2,
reflux, 2h, 2) 4-(N-Boc-amino)piperidine, m-xylene, N , 150°C, 1.5 h, 62-67%
2
yield; (ii) CF COOH, CH Cl dry, room temp, 45 min, 100% yield; (iii)
3
2
2
triphosgene, Et N, CH Cl dry, 0°C to room temp, overnight, 11-68% yield; (iv)
3
2
2
LiOH, 5:1 THF/H O, room temp, overnight, 100% yield.
2
Alicyclic urea derivatives 10a,b were achieved as depicted in Scheme 5. 2-
Amino-4,5-dimethylphenol31 or methyl 2-(3-amino-4-hydroxyphenyl)acetate27 were
reacted with potassium O-ethyl xanthogenate that can act as a double
electrophilic reagent, thus providing a convenient synthetic equivalent of
thiophosgene.32 The resulting unstable 2-mercaptobenzoxazole intermediates
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
immediately underwent to nucleophilic substitution by treating the sulfur moiety
with 4-(N-Boc-amino)piperidine,33 resulting in derivatives 25a,b. Following
deprotection of the Boc-protected amine by means of trifluoroacetic acid, amine
derivatives 26a,b were obtained and then condensed with triphosgene furnishing
ureas 27a and 10b. Finally, the acetic acid derivative 10a was obtained through
alkaline hydrolysis with LiOH of the corresponding ester 27a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Syntheses of amide derivatives 11a-h were performed as reported in Schemes
6
and 7. More in detail, compounds 11b,d-h were synthesized by applying a
divergent synthetic approach, first building the central diphenyl urea, thiourea,
isopropanol or acetone moiety, while derivatives 11a,c were obtained by
synthesizing the amide function first.
In particular, derivatives 11b,d-h have been synthesized as shown in Scheme 6. The key
intermediates for these syntheses are the acids 33b,d,e,g.
The (thio)ureas 33b,d, were obtained by alkaline hydrolysis of the corresponding esters 29b,d.
The urea 29b was synthesized by reaction of methyl 4-aminobenzoate with triphosgene using
triethylamine as a base, while the thiourea 29d was obtained via microwave-assisted reaction of
methyl 4-amino-3-fluorobenzoate with the 28. This isothiocyanate has been achieved by reaction
of the same methyl 4-amino-3-fluorobenzoate with thiophosgene.
2
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The isopropanol and ketal derivatives 33e,g were synthesized by reaction with aqueous base of
the corresponding esters 32 and 31, respectively. Both compounds have been obtained starting
form the joint intermediate 30, which reacted with ethylene glycol to furnish ketal 31, or
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
underwent reduction by NaBH to give the corresponding alcohol 32. The key 1,3-
4
diphenylacetone derivative 30 was obtained via a TosMIC-modified reaction following a
procedure previously reported in literature.34
Thus, acids 33b,d,e,g were then transformed into the corresponding amides 34b,d-h by
reaction with the proper amine (methyl 2-(3-aminophenyl)acetate for 34b,d,f,g; methyl 2-(3-
amino-4-hydroxyphenyl)acetate for 34e, ethyl 3-aminobenzoate for 34h) using EDCI and 1-
hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM) as base, or via acyl chloride.
Scheme 6. Synthetic Route to 11b,d-g Derivativesa
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H3COOC
H3COOC
R (H,F)
NH2
H3COOC
R
R
COOCH3
H COOC
H3COOC
iv
COOCH3
i
Y
3
O
Br
N
H
2
N
H
9b,d
30
29b Y=O, R=H
9d Y=S, R=F
ii
vi
2
v
F
iii
H3COOC
COOCH3
H3COOC
COOCH3
OH
NCS
O
O
vii
or
viii
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28
32
31
viii
R1 R1
HOOC
COOH
vii
Z
Y
Y
R
R
33b,d,e,g
ix or x
33b Y=NH, Z=CO, R1=H
33d Y=NH, Z=CS, R1=F
33e Y=CH , Z=CHOH, R =H
2 1
O
O
O
O
R1 R1
H3CO
N
H
N
OCH3
n
H
n
2 2 2 2 1
33g Y=CH , Z=CO (CH ) , R =H
Z
Y
Y
vii
xii
3
3
3
3
3
3
4b Y=NH, Z=CO, R=R1=H, n=1
34b,d-h
4d Y=NH, Z=CS, R=H, R1=F, n=1
4e Y=CH, Z=CHOH, R=OH, R1=H, n=1
4f Y=CH, Z=CHOH, R=R1=H, n=1
4g Y=CH2, Z=CO2(CH2)2, R=R1=H, n=1
4h Y=NH, Z=CO, R=R1=H, n=0
O
O
O
O
HO
N
N
OH
vii or
viii or xi
O
O
H
H
35
R
R
O
O
O
O
1
1b Y=NH, Z=CO, R=R1=H, n=1
1d Y=NH, Z=CS, R=H, R1=F, n=1
11e Y=CH, Z=CHOH, R=OH, R1=H, n=1
R1 R1
Z
1
HO
n
N
N
OH
H
H
n
Y
Y
11f Y=CH, Z=CHOH, R=R =H, n=1
1
1
1g Y=CH2, Z=CO, R=R1=H, n=1
11h Y=NH, Z=CO, R=R1=H, n=0
1
1b,d-h
a
Reagents and conditions: (i) triphosgene, Et N, CH Cl dry, N , 0 °C to room
3
2
2
2
temp, overnight, 23% yield; (ii) thiophosgene, chlorobenzene, DMF cat., room
temp to reflux, 1 h, 61% yield; (iii) methyl 4-amino-3-fluorobenzoate, DMSO dry,
2
00 W, N , 80 °C, 1 min, 30% yield; (iv) 1) TosMIC, NaOH 40%, TBAI,
2
CH Cl , N , room temp, 3 h; 2) HCl 37%, 64% yield; (v) ethylene glycol, PTSA
2
2
2
monohydrate, toluene, reflux, overnight, 80% yield; (vi) NaBH , 1:1 MeOH/THF,
4
0
°C to room temp, overnight, 70% yield; (vii) LiOH, 5:1 THF/H O, room temp,
2
24-96 h, 45-82% yield; (viii) NaOH 20%, 5:1 THF/H O, reflux or room temp, 24-
2
7
2
h, 63-71% yield; (ix) methyl 2-(3-aminophenyl)acetate or ethyl 3-
aminobenzoate, EDCI, HOBt, NMM, DMF dry, N , room temp, 4-48 h, 51-85%
2
yield; (x) 1) oxalyl chloride, THF dry, DMF dry cat., N , room temp, 4-24 h; 2)
2
methyl 2-(3-aminophenyl)acetate or methyl 2-(3-amino-4-hydroxyphenyl)acetate,
Et N, CH Cl dry or THF dry, N , 0 °C to room temp, 0.5-24 h, 20-100% yield;
3
2
2
2
2
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(
xi) LiOH, 10:1 DMSO/H O, room temp, 0.5-72 h, 70-90% yield; (xii) CF COOH,
2
2
3
N , room temp, overnight, 80% yield.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, the ester function of 34b,d-h were hydrolyzed in alkaline medium to give the
corresponding acid derivatives 11b,d-f,h and 35. Notably, ketal 35 was subsequently deprotected
by reaction with trifluoroacetic acid to give the corresponding ketone 11g.
Scheme 7. Synthetic Route to 11a,c Derivativesa
OH
OCOCH3
O
OH
O
O
O
iii
O
i
H3CO
H3CO
NH2
H3CO
H3CO
N
H CO
N
O
3
H
3
6a
NO2
37 H3C
NO2
O
O
S
ii
iv
O
N
H O
NH2
NO2
3
6c
R
38a X=CO, R=OCOCH , R =COCH
8c X=SO2, R=R1=H
3
1
3
O
3
v
X
N
H3CO
R1
38a,c
NH2
vi
R
R
R
R
O
O
vii
O
O
X
X
X
X
O
N
OH
O
N
OCH3
HO
N
H3CO
N
R1
R1
R1
R1
N
N
N
N
H
H
H
H
11a,c
34a,c
1
1a X=CO, R=OH, R1=H
3
4a X=CO, R=OCOCH3, R1=COCH3
11c X=SO2, R=R1=H
34c X=SO2, R=R1=H
a
Reagents and conditions: (i) p-nitrobenzoyl chloride, Et N, THF dry, N , 0 °C
3
2
to room temp, 3 h 100% yield; (ii) p-nitrobenzenesulfonyl chloride, pyridine,
dichloroethane dry, N , 0 °C to room temp, overnight, 88.5% yield; (iii) acetic
anhydride, Et N, room temp, 48 h, 52% yield; (iv) H , Pd/C, AcOEt, 40 psi,
room temp, 1 h, 100% yield; (v) ammonium formate, Et N, Pd/C, dioxane, N ,
2
3
2
3
2
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
0 °C, 2 h, 67% yield; (vi) triphosgene, Et N, CH Cl dry or 1:1 CH Cl /THF
3
2
2
2
2
dry, N , 0 °C to room temp, 24-48 h, 20-22% yield; (vii) LiOH, 5:1 THF/H O,
room temp, overnight, 13-49% yield.
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7 describes the synthetic route to obtain derivatives 11a,c.
Commercially available methyl 2-(3-aminophenyl)acetate or methyl 2-(3-amino-4-
hydroxyphenyl)acetate were acylated with p-nitrobenzoyl chloride or p-
nitrobenzenesulfonyl chloride, respectively, to give amides 36a27 and 36c. Nitro
derivatives 36c and 37, obtained after protection via acetylation with acetic
anhydride of 36a, underwent to Leuckart or catalytic reduction of nitro group,
giving the corresponding amines 38a,c that were reacted with triphosgene to
obtain urea compounds 34a,c. Finally, acid derivatives 11a,c were obtained
after an alkaline hydrolysis with LiOH.
The aryl-boronic acid derivative 11i was synthesized from the corresponding
bromo derivative 39 via boronic ester as depicted in Scheme 8. 3-Bromoaniline
was acylated with p-nitrobenzoyl chloride to give amide 39 that underwent to a
cross-coupling reaction with bis(pinacolate)diboron in the presence of a catalytic
2
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amount of PdCl (PPh ) , obtaining boronic ester derivative 40. Followed by a
2
3 2
catalytic reduction of nitro group, giving the corresponding amine 41 that was
reacted with triphosgene to obtain urea derivative 34i. Finally, the oxidative
cleavage of the pinacol ester group furnished the corresponding boronic acid
compound 11i.30
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 8. Synthetic Route to 11i Derivativea
O
O
O
ii
iii
O
i
O
B
N
H
Br
N
B
N
H
H
O
O
Br
NH2
NH2
NO2
NO2
39
40
41
iv
O
O
O
O
v
HO
OH
O
O
B
N
O
N
B
B
N
O
N
B
H
H
H
H
HO
OH
O
O
N
N
N
N
H
H
H
H
11i
34i
a
Reagents and conditions: (i) p-nitrobenzoyl chloride, Et N, CH Cl dry, N , 0
°C to room temp, 3 h, 92% yield; (ii) KOAc, pinacol diborane, PdCl (PPh ) ,
dioxane dry, N , 100 °C, 2 h, 75% yield; (iii) H , Pd/C, AcOEt, 40 psi, room
3
2
2
2
2
3 2
2
2
temp, 1.5 h, 95% yield; (iv) triphosgene, Et N, THF dry, N , 0 °C to room
3
2
temp, overnight, 43% yield; (v) 0.1 N NH OAc , NaIO , acetone, room temp,
4
aq
4
2
7 h, 95% yield.
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. Hpse inhibitory activities (Fondaparinux Hpse assay) of the newly synthesized compounds 5a-f, 6a,b,
7
a-g, 8a-c, 9a,b, 10a,b and 11a-i versus reference compounds (1-4 and SST0001).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
IC50a
µg/mL
1.80
Cpd
X
Y
Z
-
R
-
R1
-
R2
-
µM
5
a
O
4-NHCONH
3.20
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5
b
O
O
3-NHCONH
-
-
-
-
-
-
-
>10
5c
3-F,
NHCONH
4- -
0.18
0.10
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
5
5
5
6
6
7
7
d
e
f
O
O
O
4-NHCSNH
-
-
-
-
-
-
-
-
-
-
-
-
1.81
1.40
6.02
0.98
0.99
0.45
0.37
0.72
>10
1.05
0.86
3.60
0.58
0.61
0.32
0.27
0.64
3-F,4-NHCSNH -
4-C(NH)3
-
-
-
-
-
a
b
a
b
NH 3-F,4-NHCONH -
NH 3-F,4-NHCSNH -
-
-
-
-
O
O
O
O
O
O
O
O
-
-
-
-
H
COOH
CH3
COOH
7c
CH Ph
COOH
2
7
d
H
CH CH CH(CH
)2
2
2
3
7
e
O
O
-
CH CH(CH B(OH)2
)2
-
4.87
4.01
2
3
2
8
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