Fluorous-tag assisted synthesis of bile acid-bisphosphonate conjugates: Via orthogonal click reactions: An access to potential anti-resorption bone drugs

Article abstract of DOI:10.1039/c7ob00774d

The synthesis of a small collection of novel bile acid-bisphosphonate (BA-BP) conjugates as potential drug candidates is reported. The disclosed methodology relied on the installation of azide and thiol functionalities at the head and tail positions, respectively, of the BA scaffold and its subsequent decoration by orthogonal click reactions (copper-catalyzed azide-alkyne cycloaddition, thiol-ene or thiol-yne coupling) to introduce BP units and a fluorophore. Because of the troublesome isolation of the target conjugates by standard procedures, the methodology culminated with the functionalization of the BA scaffold with a light fluorous tag to rapidly and efficiently purify intermediates and final products by fluorous solid-phase extraction.

Full text of DOI:10.1039/c7ob00774d

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Biomol. Chem., 2017, DOI: 10.1039/C7OB00774D.
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DOI: 10.1039/C7OB00774D
Journal Name
ARTICLE
Fluorous-Tag Assisted Synthesis of Bile Acid-Bisphosphonate
Conjugates via Orthogonal Click Reactions: An Access to Potential
Anti-Resorption Bone Drugs
Received 00th January 20xx,
Accepted 00th January 20xx
Chiara Massarenti,a Olga Bortolini,a Giancarlo Fantin,a Dario Cristofaro,a Daniele Ragno,a Daniela
Perrone,*a Elena Marchesi,a Gianluca Toniolob and Alessandro Massi*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
The synthesis of a small collection of novel bile acid-bisphosphonate (BA-BP) conjugates as potential drug candidates is
reported. The disclosed methodology relied on the installation of azide and thiol functionalities at the head and tail positions,
respectively, of the BA scaffold and its subsequent decoration by orthogonal click reactions (copper-catalyzed azide-alkyne
cycloaddition, thiol-ene or thiol-yne coupling) to introduce BP units and a fluorophore. Because of the troublesome isolation
of the target conjugates by standard procedures, the methodology culminated with the functionalization of the BA scaffold
with a light fluorous tag to rapidly and efficiently purify intermediates and final products by fluorous solid-phase extraction.
ibandronate and zoledronic acid through the generation of
molecular complexes with lysine-linked deoxycholic acid.6 Our
group also contributed to this area of research reporting on the
synthesis and biological evaluation of the chenodeoxycholic-
derived bisphosphonate 1
(Figure 1).7 This BA-BP conjugate was
Introduction
prepared by a stepwise synthesis strategy and exhibited high
affinity toward hydroxyapatite. Significantly, together with the
lower cytotoxicity compared to neridronate in L929 murine
Geminal bisphosphonates (BPs) are stable analogues of
pyrophosphate and represent an important class of bioactive
compounds, which are currently employed for the treatment
and prevention of several bone disorders such as bone
metastasis, myeloma, rheumatoid arthritis, osteoporosis, and
Paget’s disease.1 BPs mainly act by decreasing osteoclast
activity and inducing osteoclast apoptosis.2 Structure-activity
relationship (SAR) studies highlighted that bioactivity of BPs is
strictly dependent on the nature of substituents installed at the
geminal position of the bisphosphonic moiety, as confirmed by
the evolution of this class of drugs across three generations of
active molecules.3 Despite their successful use, bioavailability
remains a critical feature of BPs since these highly hydrophilic
derivatives are poorly absorbed from the gastrointestinal tract
after oral administration.4 A number of delivery systems have
been investigated to overcome bioavailability limitations of BPs
and improve patient compliance including liposome
encapsulation, use of nanoparticles and co-administration with
adsorption enhancing agents such as surfactants, salicylates,
and bile acids (BAs).5 Indeed, the unique features of facial
amphiphilic BAs have been successfully exploited by Park and
co-workers for improving the intestinal permeability of
fibroblast culture cells, the conjugate
1 displayed a higher
activity in inhibition of osteoclastogenesis.7 These promising
results encouraged us to set-up a modular and general synthetic
strategy to rapidly explore the chemical space around the BA
scaffold through the efficient generation of a small collection of
BA-BP conjugates eventually functionalized with additional
molecular portions such as fluorophores. Therefore, herein we
report on the design and synthesis of the multi-functional BA
scaffold I suitably equipped with azide and thiol groups at the
‘head’ and ‘tail’ positions of the BA unit, respectively, for further
elaborations via orthogonal click reactions (Figure 1). The
copper-catalyzed azide-alkyne cycloaddition (CuAAC),8 thiol-
ene coupling (TEC),9 and the thiol-yne coupling (TYC)9c,10 were
selected for the scope. On the other hand, the high potential
and versatility of the above click reactions performed
sequentially or simultaneously have been amply documented in
materials science and bioconjugation studies.11 Although not
initially planned, the post-reaction phase of BA-BP conjugates
synthesis was addressed in this study by taking advantage of the
incorporation of a light fluorous tag onto the BA moiety
(scaffold II) to facilitate the purification of click intermediates
and the target conjugates. In fact, tagged substrates can be
efficiently separated from nonfluorous-tagged side-products by
fluorous solid-phase extraction (F-SPE).12 Noteworthy, while
fluorous technology has been widely utilized for the synthesis
of biomolecules such as peptides and carbohydrates,13 its
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ARTICLE
Journal Name
application to the elaboration of BA and BP derivatives is
unprecedented to the best of our knowledge.
R
R
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DOI: 10.1039/C7OB00774D
CO2H
CO2Me
MeOH, H2SO4
100 °C, 1 h
24
O
ONa
3
P
OH
OH
HO
R'
HO
R'
HO
P
2 R = OH; R' = C7 α-OH CA
3 R = H; R' = C7 α-OH CDC
4 R = H; R' = H LC
6 R = OH; R' = C7 α-OH
7 R = H; R' = C7 α-OH
8 R = H; R' = H
ONa
O
1
5 R = H; R' = C7 β-OH UDC
9 R = H; R' = C7 β-OH
HO
OH
I2, Ph3P
Im, 1,3-dioxolane
RT, 30 min.
linker
tail
linker
tail
SH
SH
R
R
R
R
(TEC/TYC)
(TEC/TYC)
CO2Me
CO2Me
NaN3
DMF, RT, 6 h
II
I
N3
N3
R'
head
(CuAAC)
head
(CuAAC)
N3
R'
I
R'
= Fluorous Tag
(Fluorous Separations)
14 R = OH; R' = C7 α-OH
15 R = H; R' = C7 α-OH
16 R = H; R' = H
10 R = OH; R' = C7 α-OH
11 R = H; R' = C7 α-OH
12 R = H; R' = H
Fig 1 Biologically active chenodeoxycholic-derived bisphosphonate 1 (Ref. 7) and the
multi-functional bile acid scaffolds I and II designed for this study.
17 R = H; R' = C7 β-OH
13 R = H; R' = C7 β-OH
Scheme 1 Synthesis of the head-functionalized BA derivatives 14-17.
The feasibility of S-trityl deprotection was successfully tested
using the chenodeoxycholic acid compound 24, which was
selected as the model substrate for this study in virtue of the
biological activity displayed by the previously synthesized
Results and Discussion
The synthesis of the orthogonally functionalized BA scaffolds of
type
different hydrophobic nature, namely the two human primary
BAs cholic ( ) and chenodeoxycholic (CDC) acids , the
secondary human BA lithocholic LC acid and the
ursodeoxycholic (UDC) acid (Scheme 1). Introduction at the
head position (C3) of the -azide functionality was
I commenced with the selection of four bile acids with
chenodeoxycholic-derived bisphosphonate 1 7
.
Therefore,
C
2-3
treatment
of
protected
24
with
trifluoroacetic
(
)
4,
acid/triethylsilane in dichloromethane (room temperature, 15
min) afforded the target thiol derivative 27 in 93% isolated yield
(Scheme 2).
5
3
a
accomplished through a double nucleophilic displacement with
retention of configuration following our previously disclosed
procedure with some improvements.14 The synthetic sequence
is detailed in the Experimental Section for the hitherto
unreported lithocholic series. Accordingly, the BAs 2-5 were
initially converted into the corresponding methyl esters 6-9 by
R
14 R = OH; R' = C7 α-OH CA
15 R = H; R' = C7 α-OH CDC
16 R = H; R' = H LC
CO2H
0.5 M LiOH
MeOH, 50 °C, on
17 R = H; R' = C7 β-OH UDC
N3
R'
18 R = OH; R' = C7 α-OH
19 R = H; R' = C7 α-OH
20 R = H; R' = H
the classical Fisher esterification method, then the 3b-iodides
10-13 were obtained by treatment with I2 and Ph3P in the
presence of imidazole and 1,3-dioxolane. The second
nucleophilic substitution was finally performed with sodium
21 R = H; R' = C7 β-OH
ClCO2Et, Et3N
then TrSCH2CH2NH2 22
THF, 0 °C to RT
azide in DMF at room temperature to give the 3a-azide
derivatives 14-17 in satisfactory overall yields (44-63%, Scheme
O
O
R
2
2
1).
HN
HN
TFA, Et3SiH
DCM, RT
2
2
The synthesis of the di-functional BA scaffolds of type
I was
SH
STr
completed with the introduction at the tail position (C24) of a
trityl-protected thiol group through a cysteamine linker
(Scheme 2). Hence, the methyl esters 14-17 were hydrolyzed
with 0.5 M LiOH to the corresponding free acids 18-21
(quantitative yields), which in turn were converted into the
amides 23-26 by coupling with S-trityl cysteamine 22 through
the mixed anhydride method in the presence of ethyl
chloroformate (45-83%). The derivatives 23-26 constituted a set
of BA scaffolds suitably functionalized for subsequent CuAAC
and TEC/TYC orthogonal click reactions upon removal of the S-
trityl protecting group. This step was planned to be performed
just before the TEC/TYC to avoid undesired sulphur oxidation
side-reactions.
N3
OH
N3
R'
27
23 R = OH; R' = C7 α-OH
24 R = H; R' = C7 α-OH
25 R = H; R' = H
26 R = H; R' = C7 β-OH
Scheme
2 Synthesis of the S-protected BA derivatives 23-26 and of the
orthogonally functionalized chenodeoxycholic scaffold 27
.
The clickable BP reagents 29,31-32 utilized in the present
investigation were prepared as reported in the literature with
some modifications (Scheme 3). Accordingly, the tetraethyl but-
3-ene-1,1-diyl-bisphosphonate 2915 was synthesized by
treatment of the commercially available methylene
bisphosphonate 28 with KOtBu and allyl iodide in dry toluene at
0 °C; although the formation of the double alkylation product
2 | J. Name., 2012, 00, 1-3
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ARTICLE
could not be avoided by this strategy, the reduction of reaction of the substrates, the TEC was next attempted using a slight
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DOI: 10.1039/C7OB00774D
time and temperature allowed to recover the target mono-allyl excess (1.5 equiv.) of the alkene 29. While these conditions
derivative 29 in acceptable 40% yield after column guaranteed the full consumption of thiol 27
,
the
chromatography.
The
tetraethyl
ethene-1,1-diyl- chromatographic separation of the target conjugate 34 (>95%
bisphosphonate 3116 was obtained in 80% yield by a two-step NMR yield) from the allyl BP 29 was troublesome and
sequence involving the initial formation of the BP intermediate impracticable; in fact, the lengthy elution from the column even
30 (paraformaldehyde, Et2NH, MeOH, 80 °C) followed by with high polar mobile phases (this is typical of BP-containing
elimination under acidic conditions in the presence of molecular products) resulted in the collection of 34 contaminated by 29
.
sieves (p-TsOH, Toluene, 4 Å MS, 110 °C). The alkyne- Isolation of pure 34 was made possible using an excess (1.5
functionalized bisphosphonate 32 was synthesized as described equiv.) of thiol 27 in the TEC since the disulphide by-product
by selective addition of sodium acetylide to 31 (THF, -15 °C) but (MS-analysis) could be easily separated in virtue of its lower
recovered in lower yield (40%) compared to that reported in the polarity. Nevertheless, the isolated yield of 34 (>95% NMR yield)
literature.16e,17 The ethylidene bisphosphonate 31 was a could not exceed the value of 64% likely because of a partial
suitable precursor for the synthesis of the thiol-functionalized irreversible adsorption of this polar compound on silica gel. A
BP 33 as well. Indeed, this clickable reagent was easily prepared similar result was detected using alumina as stationary phase.
by Michael addition of S-trityl cysteamine 22 to 31 (DCM, room Subsequently, the conversion of the tetraethyl bisphosphonate
temperature) followed by standard trityl group deprotection 34 into the corresponding bisphosphonic acid 35 was
(95% overall yield, Scheme 3).
investigated using the McKenna conditions.18 This deprotection
reaction was optimized in DCM-MeOH using an excess of
trimethylsilyl bromide (10 equiv.) in the presence of 2,6-lutidine
(13 equiv.). It is important to stress that the absence of the base
resulted in the formation of a complex reaction mixture, which
contained by-products arising from the partial hydrolysis of the
amide bond of the cysteine linker, as confirmed by MS analysis.
Isolation of the bisphosphonic acid 35 (53% yield) was finally
accomplished by size exclusion chromatography employing
Sephadex LH-20 as stationary phase.
O
P
O
P
EtO
EtO
OEt
OEt
29
KOtBu, CH2CHCH2I
Toluene, 0 °C
O
P
O
O
P
O
P
Et2NH, (CH2O)n
EtO
EtO
OEt
OEt
EtO
EtO
OEt
OEt
P
MeOH, 80 °C
28
OMe
O
30
2
HN
2
p-TsOH
Toluene, 4 Å MS, 110 °C
SH
N3
OH
27
O
P
O
O
P
O
P
EtO
EtO
OEt
NaCCH
EtO
EtO
OEt
OEt
P
OEt
THF, -15 °C
29 (0.75 equiv.)
DMPA (10 mol%)
DMF, hν 365 nm, 1 h
32
31
1. TrSCH2CH2NH2 22
DCM, RT
2. TFA, Et3SiH
DCM, RT
O
2
HN
2
O
P
O
EtO
EtO
OEt
P
S
OEt
3
N
RO
RO
OR
OR
N3
OH
H
P
P
SH
33
O
O
34 R = Et
35 R = H
Scheme 3 Synthesis of clickable reagents 29, 31-33.
2,6-lutidine (13 equiv.)
TMSBr (10 equiv.)
DCM-MeOH, 0 °C to RT
Next, having in hands the orthogonally functionalized BA
derivatives 24, 27 and the set of clickable reagents 29 31-33
,
,
Scheme 4 Tail functionalization of the BA scaffold 27 by TEC and tetraethyl BP 34
deprotection.
we investigated the efficiency of the planned click chemistry
approach towards the generation of a small collection of BA-BP
conjugates. The tail functionalization of the BA scaffold was The head functionalization of the S-protected BA scaffold 24
initially considered by coupling 27 with the allyl-substituted BP was performed by adopting the standard procedure for the
29 under standard photoinduced TEC conditions (Scheme 4). regioselective CuAAC using the alkyne-functionalized BP 32
Thus, an equimolar mixture of 27 and 29 in DMF was irradiated (Scheme 5). After some experimentation seeking efficiency and
with
a UV-lamp (lmax 365 nm) using 2,2-dimethoxy-2- purification simplicity, optimized reaction conditions consisted
phenylacetophenone (DMPA, 10 mol %) as a radical initiator. in dissolving a slight excess of 32 (1.5 equiv.) and 24 in a mixture
Since 1H and 13P NMR analysis showed only a partial conversion of Toluene/DMF (10:1) and then adding at room temperature
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