Design, synthesis and P-gp induction activity of aryl phosphonate esters: Identification of tetraethyl-2-phenylethene-1,1-diyldiphosphonate as an orally bioavailable P-gp inducer

Article abstract of DOI:10.1039/c6md00300a

The clearance of amyloid-beta is mediated by the P-glycoprotein (P-gp) transporter pump located at the blood brain barrier. Therefore, the induction of P-gp has been considered as a potential therapeutic strategy for the treatment of Alzheimer's disease. The expression of P-gp is regulated through a nuclear receptor, pregnane X receptor (PXR). Thus, herein we investigated the potential of a known PXR activator, diphosphonate ester SR12813 (6a), for P-gp induction activity and further studied its structure-activity relationship. The diphosphonate ester SR12813 along with three series of analogs, viz. aryl alkylidene, aryl alkynyl, and aryl α-amino phosphonate esters, were synthesized and screened for P-gp induction activity in P-gp overexpressing adenocarcinoma LS180 cells using rhodamine 123 efflux assay. The parent compound SR12813 along with several new analogs displayed P-gp induction activity at 5 μM. Western blot analysis indicated that tetraethyl-2-phenylethene-1,1-diyldiphosphonate (6c) and tetraethyl-2-(anthracene-10-yl)ethene-1,1-diyldiphosphonate (6s) showed 7-8-fold increase in P-gp expression in LS180 cells. The diphosphonate ester 6c displayed excellent aqueous solubility, no cytochrome P450 inhibition liability and no efflux pump substrate liability. Furthermore, it exhibits an excellent oral pharmacokinetic profile in BALB/c mice with AUC_0-∞ of 2067 ng h mL^-1 and 37.6% oral bioavailability. The results presented here clearly indicate the potential of this scaffold to increase the clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent.

Full text of DOI:10.1039/c6md00300a

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DOI: 10.1039/C6MD00300A
Paper
Design, synthesis and Pꢀgp induction activity of aryl phosphonate esters:
Identification of tetraethylꢀ2ꢀphenyletheneꢀ1,1ꢀdiyldiphosphonate as an
orally bioavailable Pꢀgp inducer‡
Sudhakar Manda,a,b Abubakar Wani,b,c Sonali S. Bharate,d Ram A. Vishwakarma,a,b Ajay Kumarc,* and
5
Sandip B. Bharatea,b,*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: XXX
The clearance of amyloidꢀbeta is mediated by Pꢀglycoprotein (Pꢀgp) transporter pump located at the blood brain barrier.
Therefore, the induction of Pꢀgp has been considered as a potential therapeutic strategy for treatment of Alzheimer’s disease.
10 The expression of Pꢀgp is regulated through a nuclear receptor ꢀ pregnaneꢀXꢀreceptor (PXR). Thus, herein we investigated the
potential of a known pregnaneꢀXꢀreceptor (PXR) activator diphosphonate ester SR12813 (6a) for Pꢀgp induction activity and
further studied its structureꢀactivity relationship. A diphosphonate ester SR12813 along with a three series of analogs viz. aryl
alkylidene, aryl alkynyl, and aryl αꢀamino phosphonate esters were synthesized and screened for Pꢀgp induction activity in Pꢀgp
overexpressing adenocarcinoma LS180 cells using rhodamineꢀ123 efflux assay. The parent compound SR12813 along with
15 several new analogs displayed Pꢀgp induction activity at 5 µM. The westernꢀblot analysis indicated that tetraethylꢀ2ꢀ
phenyletheneꢀ1,1ꢀdiyldiphosphonate (6c) and tetraethylꢀ2ꢀ(anthraceneꢀ10ꢀyl)etheneꢀ1,1ꢀdiyldiphosphonate (6s) showed 7ꢀ8 fold
increase in Pꢀgp expression in LS180 cells. The diphosphonate ester 6c displayed excellent aqueous solubility, no cytochrome
P450 inhibition liability and no efflux pump substrate liability. Furthermore, it possesses excellent oral pharmacokinetic profile
in BALB/c mice with AUC0ꢀ∞ of 2067 ngꢁh/mL and 37.6% oral bioavailability. The results presented here clearly indicate the
20 potential of this scaffold to increase the clearance of brain Aβ across the BBB and thus its promise for development as potential
antiꢀAlzheimer agents.
1
Introduction
recent years it has been investigated up to the clinical trial stage
for its potential in Alzheimer’s disease.11ꢀ13 The oliveꢀoilꢀderived
oleocanthal (2) enhances amyloidꢀbeta clearance via upregulation
of Pꢀgp and LRP1 expression.5 Recently, our group has identified
Deposition of amyloidꢀbeta (Aβ) in the brain has been considered
25 as a major hallmark for progression of the Alzheimer’s disease
(AD). Studies have demonstrated that the clearance mechanism
40
a natural product colupulone based synthetic analog 3,5ꢀ
of amyloidꢀbeta gets hampered in Alzheimer patients.1, The
2
dihydroxyꢀ4,4ꢀbis(3ꢀmethylbutꢀ2ꢀenyl)ꢀ2,6ꢀdipropionylcyclohexa
ꢀ2,5ꢀdienone (3) as an inducer of Pꢀgp and LRPꢀ1.14 Fascaplysin
(4)15 and a quinoline derivative 516 are other Pꢀgp inducer leads
discovered by us (Figure 1).
clearance of Aβ is mediated by the Pꢀgp transporter pump located
at the blood brain barrier (BBB).3ꢀ9 Thus, it is evident that drugs
30 which can induce Pꢀgp expression at BBB will have a great
potential to emerge as novel AD therapeutics. With this
hypothesis, several Pꢀgp inducers have been identified in recent
years which showed significant efficacy in Alzheimer’s disease
inꢀvivo models.5 Antiꢀtubercular drug rifampicin (1) is the widely
35 known Pꢀgp inducer which enhances Aβ clearance,10 and thus in
HO
OH
OH OH
AcO
O
O
CHO
CHO
NH
O
N
O
N
O
HO
OH
N
O
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative
O
Medicine, Canal Road, Jammu-180001, India
Rifampicin (1)
(-)-Oleocanthal (2)
OCF3
bAcademy of Scientific & Innovative Research (AcSIR), CSIR-Indian
Institute of Integrative Medicine, Canal Road, Jammu-180001, India
cCancer Pharmacology Division, CSIR-Indian Institute of Integrative
Medicine, Canal Road, Jammu-180001, India
O
O
O
Cl
N
O
dPreformulation Laboratory, PKPD Toxicology and Formulation
Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180001, India
HO
OH
O
O
N
H
N
COOEt
*E-mail: sbharate@iiim.ac.in (SBB); ajaykmahajan@hotmail.com (AK).
Fax: +91-191-2589333; Tel: +91-191-2589006
Colupulone analog 3
Fascaplysin (4)
Quinoline derivative 5
45
‡ The authors declare no competing interests.
Figure 1. Structures of known Pꢀgp inducers
Electronic supplementary information (ESI) available for experimental
details. See DOI: XXX
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The pregnane X receptor (PXR), a member of the nuclear
receptor superfamily of proteins regulates the expression of Pꢀgp,
located in various tissues such as intestinal epithelium, hepatic
canalicular membrane, proximal tubular of kidneys and
endothelial cells of the bloodꢀbrain barrier (BBB). Thus, it is
quite obvious that activators of PXR could be a potential starting
point to identify promising Pꢀgp inducer leads. A diphosphonate
Results and discussion
SR12813 is an alkylidene tetraethyl bisphosphonate ester,
containing three main structural components viz. substituted aryl
30 (bearing two tertꢀbutyl and one hydroxyl group), alkylidene
linker, and bisphosphonate ester. As this scaffold has never been
studied for Pꢀgp induction activity, here one of the objective was
also to establish preliminary structureꢀactivity relationship. For
this purpose, modifications were planned on ester portion, aryl
35 moiety as well as on the alkylidene linker, as depicted in Figure
2. Three series of phosphonate esters were synthesized as
depicted in Figure 2.
5
ester SR12813 (6a), apart from its
potent antiꢀ
hypocholesterolemic activity,17, 18 is a potent activator of PXR.19
10 In continuation to our efforts on discovery of Pꢀgp inducers as
potential antiꢀAlzheimer therapeutics,14ꢀ16 herein our objective
was to investigate the Pꢀgp induction potential, and establish
structureꢀactivity relationship of a PXR activator SR12813 (6a).
Despite of the promising Pꢀgp induction activity of two natural
15 product based Pꢀgp inducer leads 3 and 4 discovered earlier, their
oral pharmacokinetic profile was very poor (%F = < 5%). The
pharmacophoric features of SR12813 indicated that this scaffold
will have better pharmacokinetic properties. Furthermore,
literature search indicated that no medicinal chemistry efforts
20 have been made on this unique scaffold for Pꢀgp induction
activity. Thus, herein, the synthesis, Pꢀgp induction activity in
LS180 cells, structureꢀactivity relationship, physicochemical
properties/ drug discovery liabilities (aqueous solubility,
solubility in other biological media’s, cytochrome P450 inhibition
25 liabilities, Cacoꢀ2 permeability) and pharmacokinetic analysis of
aryl phosphonates is presented in this paper.
Table 1. Synthesis, Pꢀgp induction activity and cytotoxicity of alkylidene diꢀphosphonate esters 6aꢀsab
Entry
R1
Ar
% accumulation of Rh123 in
LS180 cells (±SD)cd
90 ± 6ns
% cell viability of LS180
cells at 30 ꢀMd
ꢀEt
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀCH(CH3)2
ꢀEt
ꢀCH(CH3)2
ꢀCH(CH3)2
ꢀEt
ꢀPh (4ꢀOH, 3,5ꢀdiꢀtertꢀBu)
ꢀPh (4ꢀOH, 3,5ꢀdiꢀtertꢀBu)
ꢀPh
ꢀPh
ꢀPh(2ꢀNO2)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
100
100
100
92
100
74
100
100
100
86
100
100
92
66.93 ± 3.48***
58.09 ± 4.63***
73.88 ± 3.49***
65.69 ± 5.29***
68.08 ± 11***
65.32 ± 7.56***
64.8 ± 4.96***
72.45 ± 5.26***
70 ± 4.11***
ꢀPh(3ꢀBr)
ꢀPh(4ꢀN,NꢀdiMe)
ꢀPh(3,5ꢀdiꢀOMe)
ꢀPh(3,5ꢀdiꢀOMe)
ꢀPh(4ꢀF, 5ꢀOMe)
ꢀPh(3,5,6ꢀtriꢀOMe)
ꢀPh(3,4,6ꢀtriꢀOMe)
ꢀ5ꢀnitrofuranꢀ2ꢀyl
ꢀ5ꢀnitrofuranꢀ2ꢀyl
ꢀPh(3,4ꢀdimethyleneꢀdioxy)
ꢀnaphthꢀ1ꢀyl
68.74 ± 6.23***
64.07 ± 2.89***
88.3 ± 3.32ns
67.84 ± 8.61***
41
83.27 ± 4.9ns
100
100
100
100
100
100
nd
73.66 ± 8.08***
63.71 ± 7.36***
70.48 ± 2.19***
56.6 ± 3.65***
100
6q
6r
6s
ꢀnaphthꢀ1ꢀyl
ꢀquinolinꢀ5ꢀyl
ꢀanthracenꢀ10ꢀyl
ꢀ
Control
Rifampicin
ꢀ
ꢀ
ꢀ
77.3 ± 7.5***
a Reagents and conditions: (a). Nꢀmethyl morpholine, TiCl4, CCl4, dry THF, 0 °C to room temp., overnight, 50ꢀ65% yield; b product yields are mentioned
in the experimental section; c The Pꢀgp induction activity of compounds tested at 5 µM was measured in terms of the % intracellular accumulation of
rhodamine 123 (Rh123)/total protein (ꢀg) in LS180 cells. The decrease in % intracellular accumulation (compared to control) of Rh123 indicates
induction of Pꢀgp. Rifampicin (5 ꢀM) was used as a reference Pꢀgp inducer; dThe statistical comparisons were made between control vs compounds. The
p value <0.5 was considered to be significant. P value *< 0.5, **<0.01, ***<0.001. All values are shown as average of three experiments ± SD. nd: not
determined.
2
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Figure 2. Chemical structure of SR12813 and general structures
of designed series for synthesis.
induction activity (74ꢀ77% intracellular accumulation of Rh 123)
without any toxicity (Table 3). (d) αꢀamino phosphonate esters
60 showed weak activity (Table 4). Here, only marginal decrease in
the % intracellular accumulation of Rh123 levels was observed
(82ꢀ90% intracellular accumulation of Rh 123). (e) Both monoꢀ
and bisꢀphosphonate esters showed Pꢀgp induction activity.
Similarly, alkynyl monoꢀphosphonate esters also were active.
65 This indicates that there is no effect of change of two
phosphonates to one and change of double bond linker with triple
bond linker.
A
series of analogues were synthesized20 starting from
5
commercially available substituted aldehydes 10. Aldehydes 10
on treatment with diphosphonate ester 11aꢀb in dry THF in
presence of titanium tetrachloride produced alkylidene
diphosphonate esters 6aꢀs and alkylidene monophosphonate
esters 7aꢀd in 55ꢀ65% yield (Table 1 and 2). Next, a series
10 alkynyl phosphonates21 were synthesized by treatment of
substituted phenylacetylenes 12 with diethylchloro phosphate 13
using nꢀBuLi as a base. Three alkynyl phosphonates 8aꢀc were
prepared (Table 3). Further, we synthesized αꢀamino
phosphonates22 by treatment of substituted aldehydes 10 with
15 anilines 14 and diethyl phosphite 15 using silica perchloric acid
as a catalyst. Two αꢀamino phosphonate esters 9aꢀb were
prepared (Table 4). All synthesized compounds were
characterized by 1H NMR, 13C NMR and MS analysis.
In general, amongst the four series tested, series one
(alkylidene diꢀphosphonate esters) was most promising, wherein
70 the phenyl alkylidene diꢀphosphonate ethyl ester 6c and
anthracenꢀ10ꢀyl alkylidene diꢀphosphonate ethyl ester 6s showed
the lowest % of intracellular accumulation of rhodamineꢀ123
(58% and 56%, respectively), in comparison to untreated control
cells, suggesting both compounds as the most potent Pꢀgp
75 inducers. Furthermore, the Pꢀgp induction activity of these two
compounds was better than the positive control rifampicin. Both
these compounds does not showed any toxicity to LS180 cells at
30 µM. Results are shown in Table 1.
All synthesized compounds were screened for Pꢀgp
20 induction activity using rhodamine 123 based efflux assay in Pꢀ
gp expressing human colorectal adenocarcinoma LS180 cells.
Prior to screening compounds for Pꢀgp induction activity in
Table 2. Synthesis, Pꢀgp induction activity and cytotoxicity of
LS180 cells, the cytotoxic effect of all compounds was 80 alkylidene monoꢀphosphonate esters 7aꢀdab
investigated in these cells in order to select the nonꢀtoxic test
25 concentration. In MTT assay, all compounds were nonꢀtoxic to
LS180 cells with IC50 > 30 µM, except compound 6n which
showed 41% cell viability at 30 µM. Therefore, for Pꢀgp
induction assay, we selected 5 µM as a test concentration. LS180
cells treated with 5 µM of each compound, for 48 h, displayed
30 significant induction of Pꢀgp activity, as displayed by the reduced
intracellular accumulation of rhodamineꢀ123, compared to
control, as a result of increased efflux of rhodamineꢀ123 (Table
1ꢀ4).
Entry
R1 Ar
% accumulation
% cell
of Rh123 in LS180 viability of
cells (±SD)cd
LS180 cells
at 30 ꢀMd
ꢀEt ꢀPh(4ꢀCl)
7a
7b
76.38 ± 3.69***
91.31 ± 1.95ns
82
ꢀEt ꢀPh(3,5ꢀdiꢀF)
100
In alkylidene diꢀphosphonate esters series (6aꢀs), several
35 newer analogs displayed promising ability to decrease the
intracellular accumulation of Rh123 levels in LS180 cells. The
SR12813 showed moderate Pꢀgp induction activity at 5 µM.
However, several newly synthesized analogs showed promising
Pꢀgp induction activity. Particularly, the compound 6c showed
40 reduction in % accumulation of Rh123 levels up to 58% (control
= 100%; positive control rifampicin – 77%). Similarly, another
new compound 6s also showed similar level of potent Pꢀgp
induction activity (56% intracellular levels of Rs123).
ꢀEt ꢀPh(3ꢀBr, 4ꢀ
OMe)
7c
70.49 ± 3.86***
97.7 ± 4.49ns
100
100
100
100
nd
ꢀEt ꢀPh(3ꢀBr, 4ꢀF)
7d
Control
Rifampicin
ꢀ
ꢀ
ꢀ
ꢀ
77.3 ± 7.5***
a
Reagents and conditions: (a). NꢀMethyl morpholine, TiCl4, CCl4, dry
b
THF, 0 °C to room temp., overnight, 55ꢀ65% yield; product yields are
mentioned in the experimental section; cThe Pꢀgp induction activity of
85 compounds tested at 5 µM was measured in terms of the % intracellular
accumulation of rhodamine 123 (Rh123)/ total protein (ꢀg) in LS180
cells. The decrease in % intracellular accumulation (compared to control)
of Rh123 indicates induction of Pꢀgp. Rifampicin (5 ꢀM) was used as a
reference Pꢀgp inducer; d The statistical comparisons were made between
A precise structureꢀactivity relationship was not observed;
45 however some of the key features noticed were: (a) In case of 90 control vs compounds. The p value <0.5 was considered to be significant.
P value *< 0.5, **<0.01, ***<0.001. All values are shown as average of
three experiments ± SD. nd: not determined.
alkylidene diꢀphosphonate esters, the unsubstituted aryl moiety
was preferred over substituted aryl moiety (e.g. compound 6c and
6s versus 6e, 6h, 6k, 6m, 6p; Table 1). (b) In case of alkylidene
monoꢀphosphonates, the aryl moiety substituted with one halogen
50 atom showed 76% accumulation of Rh123. Substitution of aryl
moiety with two halogen atoms, results in reduced Pꢀgp induction
activity (e.g. 7b and 7d – both contain two halogen atoms – 91
and 97% accumulation of Rh123 levels). However, when aryl
moiety containing one halogen atom was substituted with one
55 electronꢀdonating group (e.g. OMe), Pꢀgp induction activity was
increased (e.g. 7c – 70% Rh123 levels; Table 2). (c) The
alkynylphosphonate esters, in general displayed good Pꢀgp
Table 3. Synthesis, Pꢀgp induction activity and cytotoxicity of
95 alkynylphosphonate esters 8aꢀcab
Entry
Ar
%
% cell viability
of LS180 cells at
accumulation
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(a)
(b)
of Rh123 in
LS180 cells
(±SD)cd
30 ꢀMd
Figure 3. The effect of alkylidene bisꢀphosphonate esters 6c and
6s (5 ꢀM) on Pꢀgp expression in LS180 cells. (a) Westernꢀblot
35 (b) quantitation results of Pꢀgp expression.
8a
Ph
77.64 ± 4.31*
100
Next, the EC50 for Pꢀgp induction was determined for
SR12813 (6a) and best new analogs 6c and 6s along with
rifampicin (Table 5). Both newly synthesized phosphonate esters
40 6c and 6s showed better Pꢀgp induction activity (EC50 = 0.27ꢀ0.29
µM) than SR12813 (6a: EC50 = 1.25 µM). The compound 6s
possesses 4.3ꢀ4.6ꢀfold higher EC50 value than SR12813 (6a). The
EC50 of new analogs 6c and 6s was comparable to that of positive
control rifampicin.
8b
Ph (4ꢀCF3)
74.63 ± 0.64** 100
75.16 ± 2.61** 100
8c
Ph (3ꢀCF3)
Control
Rifampicin
ꢀ
ꢀ
100
100
77.3 ± 7.5***
nd
a Reagents and conditions: a). nꢀBuLi, dry THF, ꢀ78 °C, 2 h, 75ꢀ85%;
b
c
product yields are mentioned in the experimental section; The Pꢀgp
induction activity of compounds tested at 5 µM was measured in terms of
the % intracellular accumulation of rhodamine 123 (Rh123)/total protein
(ꢀg) in LS180 cells. The decrease in % intracellular accumulation
(compared to control) of Rh123 indicates induction of Pꢀgp. Rifampicin
(5 ꢀM) was used as
45 Table 5. Pꢀgp induction activity in terms of EC50 values of 6a, 6c
and 6s
5
d
a
reference Pꢀgp inducer;
The statistical
Entry
EC50 (µM)
1.250
comparisons were made between control vs compounds. The p value <0.5
was considered to be significant. P value *< 0.5, **<0.01, ***<0.001. All
SR12813 (6a)
10 values are shown as average of three experiments ± SD. nd: not
determined.
0.271
6c
0.290
6s
Rifampicin
0.226
Table 4. Synthesis, Pꢀgp induction activity and cytotoxicity of αꢀ
amino phosphonate esters 9aꢀba
After obtaining two interesting Pꢀgp inducer leads based on the
known PXR activator SR12813 scaffold, next we decided to
50 perform their preclinical characterization. Both compounds 6c
and 6s displayed excellent thermodynamic aqueous solubility in
water, PBS, SGF and SIF (S = >1.5 mg/ml) (Table 6).
15
Entry
Ar1 Ar2
%
% cell
accumulation
of Rh123 in
LS180 cells
(±SD)bc
viability of
LS180 cells
at 30 ꢀMc
Table 6. Aqueous solubility of SR12813 (6a) and its analogs 6c and 6s
Mediaa
Solubility (µg/ml)
82.56 ± 5.32ns
90.85 ± 3.21ns
100
100
100
100
nd
9a
ꢀPh ꢀPh
6a
6c
6s
9b
ꢀPh ꢀPh (4ꢀCl)
Water
PBS
SGF
SIF
200
200
200
200
>1500
>1500
>1500
40
400
Control
Rifampicin
ꢀ
ꢀ
>1500
>1500
>1500
77.3 ± 7.5***
a
b
Reagents and conditions: (a). nꢀBuLi, dry THF, ꢀ78 °C, 2 h, 80ꢀ85%;
product yields are mentioned in the experimental section; cThe Pꢀgp
induction activity of compounds tested at 5 µM was measured in terms of
the % intracellular accumulation of rhodamine 123 (Rh123)/total protein
20 (ꢀg) in LS180 cells. The decrease in % intracellular accumulation
(compared to control) of Rh123 indicates induction of Pꢀgp. Rifampicin
(5 ꢀM) was used as a reference Pꢀgp inducer; dThe statistical comparisons
were made between control vs compounds. The p value <0.5 was
considered to be significant. P value *< 0.5, **<0.01, ***<0.001. All
25 values are shown as average of three experiments ± SD. nd: not
determined.
55 aPBS: phosphate buffer saline (pH 7.4); SGF: simulated gastric
fluid (pH 1.2); SIF: simulated intestinal fluid (pH 6.8)
Next, the Cacoꢀ2 permeability and CYP liability was determined
for compound 6c. The cytochrome P450 liability was assessed on
60 four key drug metabolizing enzymes CYP3A4, CYP2D6,
CYP2C9, CYP2C19 at 10 ꢀM. Compound 6c showed 19, 20, 0,
and 0% inhibition of these enzymes at tested concentration,
indicating that this compound does not possess any CYP liability
(<50% inhibition at 10 ꢀM). In cacoꢀ2 permeability assay, the
65 efflux ratio of 0.9 indicated that it is not a substrate of efflux
pumps. The pharmacokinetic behaviour of compound 6c was then
evaluated in BALB/c mice using oral and IV dosing.23 A single
10 mg/kg oral dose and 1.0 mg/kg intravenous dose was given to
the BALB/c mice. Compound showed good plasma exposure on
70 oral administration with AUC0ꢀt and AUC0ꢀ∞ of 1968 and 2067
ngꢁh/mL, respectively. Furthermore, compound has good
elimination halfꢀlife (3.48 h). The in vivo blood clearance
following intravenous dosing was found to be 30.4 mL. minꢀ1.kgꢀ1
with a large volume of distribution 1.81 L.kgꢀ1. Compound 6c
75 showed good oral bioavailability (37.6%) (Figure 4).
The promising Pꢀgp induction activity of these compounds
was further confirmed by westernꢀblot (WB) analysis. In the WB
30 analysis, the exposure of LS180 cells with compounds 6c and 6s
at 5 ꢀM, resulted in 7.2 and 7.8ꢀfold increase in the Pꢀgp
expression in LS180 cells (Figure 3).
6s
6c
6s
6c
4
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human colon adenocarcinoma cell line; MTT, (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀ
35 yl)ꢀ2,5ꢀdiphenyltetrazolium bromide); Pꢀgp, Pꢀglycoprotein; PXR,
pregnaneꢀXꢀreceptor; Rh123, rhodamine 123; SAR, structureꢀactivity
relationship.
IV 1 mg/Kg
10000
1000
100
PO 10 mg/Kg
Notes and references
10
1. K. G. Mawuenyega, W. Sigurdson, V. Ovod, L. Munsell, T. Kasten,
40
J. C. Morris, K. E. Yrasheski and R. J. Bateman, Science 2010, 330,
1774.
1
0
2
4
6
8
10
2. D. M. E. van Assema, M. Lubberink, M. Bauer, W. M. van der Flier,
R. C. Schuit, A. D. Windhorst, E. F. I. Comans, N. J. Hoetjes, N.
Tolboom, O. Langer, M. Müller, P. Scheltens, A. A. Lammertsma
and B. N. M. van Berckel, Brain, 2012, 125, 181ꢀ189.
3. F. C. Lam, R. Liu, P. Lu, A. B. Shapiro, J. M. Renoir, F. J. Sharom
and P. B. Reiner, J. Neurochem., 2001, 76, 1121ꢀ1128.
4. H. Qosa, A. H. Abuznait, R. A. Hill and A. Kaddoumi, J. Alzheimers
Dis., 2012, 31, 151ꢀ165.
Time (h)
Parameters
Dose (mg/kg)
t1/2,β (h)
AUC0ꢀt (ngꢁh/mL)
AUC0ꢀ∞ (ngꢁh/mL)
Cmax (ng/mL)
C0 (ng/mL)
CL (mL/min/Kg)
Vd (L/Kg)
IV
1
PO
10
3.48
1968
2067
870
ꢀ
ꢀ
ꢀ
45
0.69
543
549
811
1153
30.4
1.81
1.10
4.0
ꢀ
50 5. A. H. Abuznait, H. Qosa, B. Busnena, K. A. El Sayed and A.
Kaddoumi, ACS Chem. Neurosci., 2013, 19, 973ꢀ982.
Vdss (L/Kg)
Tlast (h)
tmax (h)
ꢀ
ꢀ
6. A. H. Abuznait, C. Cain, D. Ingram, D. Burk and A. Kaddoumi, J.
Pharm. Pharmacol., 2011, 63, 1111–1118.
0.50
37.6
7. J. R. Cirrito, R. Deane, A. M. Fagan, M. L. Spinner, M. Parsadanian,
%F
ꢀ
55
60
65
M. B. Finn, H. Jiang, J. L. Prior, A. Sagare, K. R. Bales, S. M. Paul,
B. V. Zlokovic, D. PiwnicaꢀWorms and D. M. Holtzman, J. Clin.
Invest., 2005, 115, 3285ꢀ3290.
Figure 4. Pharmacokinetic analysis of compound 6c in BALB/c
mice. The plasma versus plasma concentration profile of
compound in BALB/c mice is shown [t1/2,ß: terminal half life;
AUC0ꢀt: the area under the plasma concentrationꢀtime curve from
0 to last measurable time point; AUC0ꢀ∞: area under the plasma
concentrationꢀtime curve from time zero to infinity; Cmax
maximum observed plasma concentration; C0: extrapolated
concentration at zero time point; CL: clearance; Vd: volume of
8. S. Vogelgesang, I. Cascorbi, E. Schroeder, J. Pahnke, H. K. Kroemer,
W. Siegmund, C. KunertꢀKeil, L. C. Walker and R. W. Warzok,
Pharmacogenetics, 2002, 12, 535ꢀ541.
5
:
9. D. Kuhnke, G. Jedlitschky, M. Grube, M. Krohn, M. Jucker, I.
Mosyagin, I. Cascorbi, L. C. Walker, H. K. Kroemer, R. W. Warzok
and S. Vogelgesang, Brain Pathol., 2007, 17, 347ꢀ353.
10. H. Qosa, A. H. Abuznait, R. A. Hill and A. Kaddoumi, J. Alzh. Dis.,
2012, 151ꢀ165.
10 distribution; Vdss: volume of distribution at steady state; Tlast: time
at which last concentration was found; F: bioavailability].
Conclusion
11. D. W. Molloy, T. I. Standish, Q. Zhou, G. Guyatt and D. S. GROUP,
Int. J. Geriatr. Psychiatry, 2013, 28, 463ꢀ470.
In conclusion, the alkylidene phosphonate scaffold has shown
promising Pꢀgp induction activity in LS180 cells. The alkylidene
15 bisphosphonate 6c strongly induced the expression of Pꢀgp which
plays major role in enhancing Aβ efflux across the BBB.
Furthermore, this compound 6c possessed excellent aqueous
solubility, no CYP and efflux pump liability; and has excellent
pharmacokinetic properties with oral bioavailability of 37%.
20 Thus, the results presented herein, demonstrate the potential of
this scaffold for investigation as amyloidꢀβ clearing agent and
thereby as a potential antiꢀAlzheimer treatment.
12. B. Yulug, L. Hanoglu, E. Kilic and W. R. Schabitz, Brain Res. Bull.,
2014, 107, 37ꢀ42.
70 13. M. B. Loeb, D. W. Molloy, M. Smieja, T. Standish, C. H. Goldsmith,
J. Mahony, S. Smith, M. Borrie, E. Decoteau, W. Davidson, A.
McDougall, J. Gnarpe, M. O'DONNell and M. Chernesky, J. Am.
Geriatr. Soc., 2004, 52, 381ꢀ387.
14. J. B. Bharate, Y. S. Batarseh, A. Wani, S. Sharma, R. A.
75
Vishwakarma, A. Kaddoumi, A. Kumar and S. B. Bharate, Organic
& biomolecular chemistry, 2015, 13, 5488ꢀ5496.
15. S. Manda, S. Sharma, A. Wani, P. Joshi, V. Kumar, S. K. Guru, S. S.
Bharate, S. Bhushan, R. A. Vishwakarma, A. Kumar and S. B.
Bharate, Eur. J. Med. Chem. , 2016, 107, 1–11.
Acknowledgements
AK is thankful to CSIR, New Delhi for the award of senior
25 research associateship. VK thanks UGC for fellowship. S.S.B.
thanks ICMR for research associateship. This research was
supported by a grant from the CSIR 12th FYP project (BSCꢀ
0205).
80 16. J. B. Bharate, A. Wani, S. Sharma, S. I. Reja, M. Kumar, R. A.
Vishwakarma, A. Kumar and S. B. Bharate, Org. Biomol. Chem.,
2014, 12, 6267ꢀ6277.
17. T. A. Berkhout, H. M. Simon, D. D. Patel, C. Bentzen, E. Niesor, B.
Jackson and K. E. Suckling, J. Biol. Chem., 1996, 271, 14376ꢀ14382.
85 18. G. B. W. Ryan E. Watkins, Linda B. Moore, Jon L. Collins, Millard
H. Lambert, Shawn P. Williams, Timothy M. Willson, Steven A.
Kliewer, Matthew R. Redinbo, Science, 2001, 292, 2329ꢀ2333.
30 Abbreviations
Aβ, amyloidꢀbeta; AD, Alzheimer’s disease; BBB, bloodꢀbrain barrier;
bEnd3, endothelial polyoma middle T antigen transformed cells of
cerebral cortex of the brain; DMSO, dimethyl sulfoxide; LS180, intestinal
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19. S. A. Jones, L. B. Moore, J. L. Shenk, G. B. Wisely, G. A. Hamilton,
D. D. McKee, N. C. O. Tomkinson, E. L. LeCluyse, M. H. Lambert,
T. M. Willson, S. A. Kliewer and J. T. Moore, Mol. Endocrinol.,
2000, 14, 27ꢀ39.
5
20. Z.ꢀY. Xue, Q.ꢀH. Li, H.ꢀY. Tao and C.ꢀJ. Wang, J. Am. Chem. Soc.,
2011, 133, 11757ꢀ11765.
21. D. Lecerclé, M. Sawicki and F. Taran, Org. Lett., 2006, 8, 4283ꢀ
4285.
22. A. K. Bhattacharya and K. C. Rana, Tetrahedron Lett., 2008, 49,
2598ꢀ2601.
10
23. The pharmacokinetic study was carried out at Jubilant Biosys Limited
Bangalore (India) on a commercial basis. These experiments were
approved by the Jubilant Biosys Institutional Animal Ethics
Committee, Bangalore, India (IAEC/JDC/2012/27) and were in
accordance with the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Ministry of
Social Justice and Environment, Government of India.
15
20
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GRAPHICAL ABSTRACT
We report P-gp induction activity of a PXR activator SR12813 and its structure-activity
relationship. SR12813 analogs displaying promising P-gp induction have been identified and
studied for aqueous solubility, CYP/ efflux liabilities, and pharmacokinetic analysis.
R1O
OR1
P
O
OR1
6c: Ar = -Ph
6s: Ar = -anthracen-10-yl
OR1
P
P
OR1
OR1
Ar
O
7
O
Ar
OEt
6
O P OEt
Alkylidene phosphonate esters
OEt
6s
6c
P
O
OEt
O
Ar
P
OR1
OR1
8
Alkynylphosphonate esters
OH
SR12813 (6a)
Ar
NH
OR1
Ar
P
OR1
α-Amino phosphonate esters
O
9