Synthesis and P-glycoprotein induction activity of colupulone analogs

Article abstract of DOI:10.1039/c5ob00554j

Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol 5f displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance 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/c5ob00554j

Organic &
Biomolecular Chemistry
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Synthesis and P-glycoprotein induction activity of
colupulone analogs†‡
a,b
Cite this: DOI: 10.1039/c5ob00554j
c
d
b,d
Jaideep B. Bharate, Yazan S. Batarseh, Abubakar Wani, Sadhana Sharma,
a,b
c
d
a,b
Ram A. Vishwakarma, Amal Kaddoumi,* Ajay Kumar* and Sandip B. Bharate*
Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer’s disease (AD)
pathology. Efflux pump proteins located at the blood–brain barrier (BBB) have been reported to play an
important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter
pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic
target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is
known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In
the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp
inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were
synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for
P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human
adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 µM.
In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as
LRP1 at 1 µM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all
tested compounds, diprenylated acyl phloroglucinol 5f displayed a significant increase (29%) in Aβ trans-
port across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential
of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as
a potential anti-Alzheimer agent.
Received 19th March 2015,
Accepted 1st April 2015
DOI: 10.1039/c5ob00554j
www.rsc.org/obc
menopausal symptoms and as an alternative to hormone
Introduction
6
,7
replacement therapy.
The major constituents of hops are
8
Prenylated phloroglucinols widely occur among plants of the bitter acids (12–15% of all components). These bitter acids
Myrtaceae and Guttiferae (Clusiaceae) families, particularly in are classified as α-acids (e.g. humulone, cohumulone) and
1
–4
the genera Eucalyptus and Leptospermum and are reported to β-acids (e.g. lupulone, colupulone) (structures are shown in
5
8
possess antimicrobial properties. This class of compounds Fig. 1). The β-acid colupulone has been reported to possess
have been extensively explored synthetically due to their fasci- antibacterial and anticancer activities. In addition, it has been
2
9
nating chemical structures and potent bioactivities. The reported to stimulate expression of hepatic CYP3A enzymes
1
0
flowers of Humulus lupulus (hops) were historically used as a and pregnane-X-receptor (PXR).
preservative and flavoring agent in beer. Hops extracts are cur- PXR, a member of the nuclear receptor superfamily of pro-
rently marketed as a source of phytoestrogens to alleviate teins, modulates expression of genes involved in metabolism
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu-180001, India. E-mail: sbharate@iiim.ac.in;
Fax: +91-191-2585006; Tel: +91-191-2586333
b
Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu-180001, India
c
School of Pharmacy, The University of Louisiana at Monroe, USA.
E-mail: kaddoumi@ulm.edu
d
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal
Road, Jammu-180001, India. E-mail: ajaykmahajan@hotmail.com
†
IIIM Publication number IIIM/1764/2015.
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/
Fig. 1 Chemical structures of β-acids from hops; and general structures
c5ob00554j
of designed series for synthesis.
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and clearance of wide range of structurally diverse endogenous
and exogenous molecules. PXR is known to regulate the
expression of P-glycoprotein (P-gp), a major efflux transport
protein widely distributed in various tissues such as intestinal
epithelium, hepatic canalicular membrane, proximal tubular
of kidneys and endothelial cells of the blood–brain barrier
(BBB). Two recent independent clinical studies have reported
that Alzheimer’s disease (AD) patients have decreased clear-
ance of CNS amyloid-β (Aβ) compared to healthy volun-
1
1,12
teers.
P-gp in the clearance of Aβ,
induce P-gp have the potential to emerge as novel AD Scheme 1 Synthesis of tetraalkylated acylphloroglucinols 3a–k.
Several studies have reported the important role of
1
3–15
thus drugs that are able to
Reagents and conditions: (a) R
1 3 2 2 2
COCl, AlCl , CS , PhNO , N atm, 50 °C,
therapeutics.
1
h, 50–70%; (b) R X, 10% KOH, rt, 30 min, 55–78%.
2
The bicyclic polyprenylated phloroglucinol natural product
hyperforin is reported to activate PXR, and induce P-gp
expression, and has also been reported to show memory
enhancement in animal models.
1
6
1
7
1
8,19
Colupulone is a simpli-
fied triprenylated phloroglucinol natural product possessing
1
0
potent PXR activation ability similar to hyperforin. Previous
1
0
17
literature on colupulone and related compounds clearly
indicates the potential of this scaffold to increase Aβ clearance.
So far, no medicinal chemistry efforts have been reported on
this unique scaffold for P-gp induction activity. Therefore,
based on the known PXR activation properties of colupulone
and related compounds, and as a part of our efforts in this
20,21
area,
we initiated a medicinal chemistry program to
explore its SAR for P-gp induction and evaluate its utility in Aβ
clearance across the BBB. The modifications were planned at
both the prenyl (site A) and acyl (site B) domains as depicted
in Fig. 1. A series of tetra- and di-alkylated analogs of colu-
pulone were synthesized and evaluated for their P-gp induction
activity using in vitro rhodamine 123 efflux assays in LS-180
Scheme 2 Synthesis of dialkylated acylphloroglucinols 5a–j. Reagents
and conditions: (a) R COOH, BF -etherate, 100 °C, 2 h, 55–75%; (b) R X,
NaOMe, 60 °C, N atm, 3 h, 50–78%.
1
3
2
2
cells. Further, the effect of these compounds on Aβ transport (Scheme 2). All products were characterized by NMR, IR, MS,
across BBB was also studied.
and HR-ESIMS analysis.
Biology
All synthesized compounds were screened for P-gp induction
activity using a rhodamine 123 based efflux assay in P-gp over-
expressing human colorectal adenocarcinoma LS-180 cells. In
Results and discussion
Chemistry
Colupulone is a triprenylated acylphloroglucinol compound. a MTT assay, treatment of compounds up to 48 h was not toxic
For synthesis, we aimed to synthesize tetra- and diprenylated to LS-180 cells with IC50 > 10 µM; thus, for a P-gp induction
acylphloroglucinols. The variation in the prenyl group by repla- assay, we used 5 µM as a test concentration. LS-180 cells
cing it with geranyl or simple alkyls was also planned. These treated with 5 µM of each compound for 48 h displayed a
two series (tetra- and di-substituted) of acylphloroglucinols significant induction in P-gp activity, as displayed by the
were synthesized starting from commercially available phloro- increased efflux of rhodamine-123 (Fig. 2a and b). The exact
glucinol (1a). The first step involved in their synthesis is structure–activity relationship could not be established as
Friedel–Craft’s acylation of phloroglucinol either using AlCl3 most of the compounds displayed almost similar level of P-gp
or BF
3
-etherate. The use of AlCl
3
and nitrobenzene as a solvent induction activity (Fig. 2). However, the combination of allyl or
substituent) with an isobutanoyl or propanoyl sub-
primarily produced mono-acyl phloroglucinols 2a–c, which on prenyl (R
2
further treatment with alkyl halides in alkali at room tempera- stituent (-COR substituent) was somewhat better than others.
1
ture produced tetra-alkylated acylphloroglucinol products 3a–k Compounds of this combination demonstrated increased
in 55–78% yields (Scheme 1).
The treatment of phloroglucinol (1a) with carboxylic acids damine-123 intracellular accumulation by ≥35% when com-
in the presence of BF -etherate produced diacylphloroglucinols pared to untreated control cells due to P-gp induction.
a–c, which on treatment with alkyl halides in the presence of Available experimental data strongly suggest that impaired
NaOMe produced dialkylated acylphloroglucinols 5a–j clearance of Aβ across the BBB might largely contribute to the
activity of P-gp as confirmed by the decreased percent of rho-
3
4
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Fig. 3 Effect of compounds on in vitro Aβ42 uptake in LS-180 cells.
#
The data are expressed as mean ± SD of n = 3 experiments. Indicates
significantly different from control cells in the absence of the P-gp
inhibitor elacridar, * indicates significant difference from control cells in
the presence of elacridar, and ! indicates significantly different from
control cells in the absence of elacridar; p < 0.05. ns, not significant
from control.
results suggest a modulatory effect of the tested compounds,
not only on P-gp function, but also on other Aβ transport pro-
Fig. 2 P-gp induction activity of tetra-alkylated (a) and dialkylated (b)
acylphloroglucinols in LS-180 cells. The P-gp induction activity of com-
pounds was tested at 5 µM and was measured in terms of the % intra- teins that oppose the efflux function of P-gp. In the absence of
cellular accumulation of rhodamine 123 (Rh123)/total protein (μg) inside elacridar, the compounds 3e, 3g, 3k and 5f increased the intra-
LS-180 cells. The decrease in % intracellular accumulation (compared to
control) of Rh123 indicates induction of P-gp. Rifampicin was used as a
of an uptake transporter(s) of Aβ such as the low-density lipo-
reference P-gp inducer. On the X-axis legends, RF indicates the rifampi-
cellular accumulation of Aβ42 indicating a possible induction
protein receptor-related protein 1 (LRP1). To confirm the con-
cin. The statistical comparisons were made between control vs. com-
pounds. The p value <0.5 was considered to be significant. p Value tribution of LRP1 to the enhanced uptake of Aβ , western blot
4
2
*
<0.5, **<0.01, ***<0.001. All values are shown as average of three analysis was performed to evaluate the effect of the com-
experiments ± SD.
pounds on LRP1 protein expression in addition to P-gp follow-
ing treatment of cells with 1 μM concentrations. The results
are shown in Fig. 4. All compounds were able to induce P-gp at
formation of Aβ brain deposits and AD progression. Also, it 1 μM concentration by 1.6- to 3.6-fold compared to untreated
has been demonstrated that the efflux transporter P-gp plays a cells. On the other hand, only compounds 3k and 5f were able
substantial role in the elimination of Aβ40 and Aβ42 from the to significantly induce LRP1 by 10 and 16%, respectively, but
1
3,22–24
brain across the BBB.
report the impact of P-gp up-regulation on the intracellular compounds 3k and 5f could explain the increase in intracellu-
accumulation of Aβ42 in vitro.
lar levels of Aβ42 compared to the control (in the absence of
Accordingly, in this study we not the compounds 3e, 3g, and 5h. The increase in LRP1 by
The most potent five compounds 3e, 3g, 3k, 5f and 5h were elacridar). The increase in Aβ42 uptake by 3g and to a lesser
further investigated for their effect on Aβ uptake. To determine extent by 3e could be related to the up-regulation of another
the impact of P-gp up-regulation on the uptake of Aβ, the cellu- Aβ42 transport protein(s) other than LRP1, which could be
lar uptake of Aβ42 was evaluated following compound treat- expressed either at the luminal or abluminal side of the BBB.
ment in the presence or absence of elacridar (2.5 μM), a P-gp To test this, we conducted transport studies in a better represen-
inhibitor. Fig. 3 shows the effect of compound treatment on tative model for the BBB using mouse brain endothelial cells.
the intracellular accumulation of Aβ42 with and without elacri-
Compounds 3k and 5f were selected to evaluate their effect
dar in LS-180 cells, which were compared to vehicle treated on Aβ transport across the mouse brain endothelial bEnd3
cells (control). Inhibition studies with elacridar were per- cells as a BBB model. These two compounds were chosen as
formed to specifically elucidate the effect of the compounds representative compounds from the tetraalkylated acylphloro-
on P-gp. As anticipated, the co-treatment with elacridar signifi- glucinol (3k) and dialkylated acylphloroglucinol (5f) series.
cantly increased the intracellular levels of Aβ42 as a result of Results from the MTT assay indicated treatment of bEnd3 cells
P-gp inhibition with all compounds except 3k (p < 0.05); with the compounds 3k and 5f at 1 μM concentration for 48 h
however, in the absence of elacridar, the increased levels of were not toxic to the cells and were comparable to those
P-gp were not able to decrease Aβ42 cellular uptake to levels observed with the control treated cells. Among the two tested
lower than those with control treated cells (Fig. 3). These compounds, only compound 5f (at 1 μM for 48 h) resulted in a
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Conclusion
In conclusion, the colupulone scaffold has shown promising
P-gp induction activity in LS-180 cells. The diprenylated trike-
tone compound 5f showed increase in the Aβ efflux in LS-180
and increase in its transport across bEnd3 cells, indicating its
promise to enhance Aβ clearance from AD brains by targeting
P-gp. Thus, results presented in this work indicate promise of
this scaffold for development as an anti-Alzheimer agent.
Further studies are in progress to evaluate the compound 5f
in vivo and verify the effect on P-gp activity at the BBB and Aβ
clearance.
Experimental section
General
All chemicals were obtained from Sigma-Aldrich Company
1
13
and used as received. H, C and DEPT NMR spectra were
recorded on Bruker-Avance DPX FT-NMR 500 and 400 MHz
instruments. Chemical data for protons are reported in parts
per million (ppm) downfield from tetramethylsilane and are
Fig. 4 (A) Representative western blots for P-gp and LRP1 in LS-180
cells treated with the compounds at 1 μM concentration. (B) Quantitative
fold increase in P-gp and LRP1 expressions. The data are expressed as
mean ± SD of n = 3 independent experiments (*p < 0.05, ***p < 0.001).
referenced to the residual proton in the NMR solvent (CDCl
3
,
7
(
.26 ppm). Carbon nuclear magnetic resonance spectra
C NMR) were recorded at 125 MHz or 100 MHz: chemical
1
3
data for carbons are reported in parts per million (ppm,
δ scale) downfield from tetramethylsilane and are referenced
significant increase (by 29%) in 25I-Aβ40 transport across the
1
to the carbon resonance of the solvent (CDCl , 77 ppm).
3
endothelial monolayer (p < 0.05), while compound 3k showed
ESI-MS and HR-ESIMS spectra were recorded on Agilent 1100
LC-Q-TOF and HRMS-6540-UHD machines. IR spectra were
recorded on a Perkin-Elmer IR spectrophotometer. Melting
points were recorded on digital melting point apparatus.
1
25
comparable I-Aβ40 transport to that of control cells (no treat-
ment), which is inconsistent with the uptake study results in
LS-180 cells. This could be due to the effect of 3k on other
efflux or influx Aβ transport proteins expressed either at the
luminal or abluminal side. In addition, these collective find- General procedure for synthesis of monoacylated
ings demonstrate that utilization of transport studies (A→B) to phloroglucinols 2a–c
evaluate the effect of the compounds on Aβ clearance is a
better model to identify hit compounds that are able to
enhance Aβ clearance from the brain. In conclusion, these
findings provide an in vitro proof of concept of the potential of
A solution of phloroglucinol (100 mg, 1 equiv.) and anhydrous
aluminium chloride (3 equiv.) in carbon disulphide (10 ml)
under an inert atmosphere was stirred at room temperature for
20 min. Nitrobenzene (15 ml) was added and temperature of
compound 5f to enhance Aβ clearance from AD brains (Fig. 5).
the reaction mixture was allowed to increase to 50 °C. Acyl
chloride (3 equiv.) was added and reaction mixture was stirred
for 30 min. On cooling, the reaction mixture was diluted with
ethyl acetate. Water was added to the resulting mixture, which
led to the formation of a white precipitate in the aqueous
layer. The organic layer was decanted and the remaining solid
residue was washed 5–6 times with EtOAc. The combined
EtOAc layer was evaporated in vacuo using a Rotavapor and the
remaining viscous oil was purified by silica gel column chromato-
graphy using n-hexane–EtOAc as an eluent to yield mono-
acylated phloroglucinols 2a–c in 50–70% yield.
1
-Acetyl-2,4,6-trihydroxy benzene (2a). Yield: 60%; white
1
solid; H NMR (CD
C NMR (CDCl , 100 MHz): δ 205.2, 172.7, 104.9, 95.7, 33.1;
3
OD, 400 MHz): δ 5.77 (s, 2H), 2.61 (s, 3H);
1
3
3
1
25
IR (CHCl
1
3
): νmax 3369, 2953, 2853, 1619, 1430, 1404, 1365,
Fig. 5 Effect of compounds 3k and 5f on
I-Aβ transport, relative
298, 1199, 1025 cm− ; ESI-MS: m/z 169.00 [M + H] ;
1
+
to control, across bEnd3 cell-based BBB model. Asterisk * indicates
+
significant difference from control (p < 0.05).
HR-ESIMS: m/z 169.0483 calcd for C H O + H (169.0495).
8 8 4
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1
-Isobutanoyl)-2,4,6-trihydroxy benzene (2b). Yield: 65%; (CDCl
3
, 400 MHz): δ 18.42 (s, 1H), 3.77–3.70 (m, 1H), 1.38
1
13
pale yellow sticky liquid; H NMR (CD OD, 400 MHz): δ 5.72 (s, 6H), 1.29 (s, 6H), 1.12 (d, J = 4.0 Hz, 6H); C NMR (CDCl3,
3
1
3
(
(
s, 2H), 3.89–3.86 (m, 1H), 1.04 (s, 3H), 1.02 (s, 3H); C NMR 100 MHz): δ 209.9, 208.6, 199.3, 196.8, 108.1, 56.9, 52.2, 35.2,
CDCl , 100 MHz): δ 212.4, 166.7, 105.3, 96.5, 40.6, 20.4; 24.2, 23.8, 19.1; IR (CHCl ): νmax 3434, 2976, 2928, 1723, 1672,
3350, 2924, 2823, 1626, 1603, 1522, 1463, 1555, 1470, 1421, 1383, 1362, 1231, 1047 cm ; ESI-MS: m/z
3
3
−
1
IR (CHCl ): ν
3
max
1
+
+
1
384, 1228, 1160, 1096 cm− ; ESI-MS: m/z 197.00 [M + H] ; 253.10 [M + H] ; HR-ESIMS: m/z 253.1601 calcd for C14
20 4
H O +
+
+
HR-ESIMS: m/z 197.0805 calcd for C10
H
12
O
4
+ H (197.0808).
H (253.1613).
1
-(3-Methyl-butanoyl)-2,4,6-trihydroxy benzene (2c). Yield:
2,2,6,6-Tetraallyl-5-hydroxy-4-(isobutyryl)cyclohex-4-ene-1,3-
1
1
7
2
6
9
2
0%; yellow solid; H NMR (CD
3
OD, 400 MHz): δ 5.70 (s, 2H), dione (3e). Yield: 65%; pale yellow sticky liquid; H NMR
.82 (d, J = 8.0 Hz, 2H), 2.15–2.05 (m, 1H), 0.86 (d, J = 8.0 Hz, (CDCl
3
, 400 MHz): δ 18.59 (s, 1H), 5.56–5.44 (m, 4H), 5.00 (dd,
13
H); C NMR (CDCl , 100 MHz): δ 207.1, 166.0, 165.8, 105.6, J = 8.0, 16.0 Hz, 8H), 3.70–3.63 (m, 1H), 2.56–2.41 (m, 8H),
3
1
3
5.8, 53.7, 44.2, 26.8, 23.2; IR (CHCl
3 3
): νmax 3369, 2955, 2923, 1.11 (d, J = 4.0 Hz, 6H); C NMR (CDCl , 100 MHz): δ 207.5,
−
1
852, 1628, 1561, 1520, 1455, 1384, 1215, 1164 cm ; ESI-MS: 204.5, 196.1, 192.8, 131.4, 130.8, 119.0, 118.3, 110.5, 63.7, 59.5,
+
m/z 211.00 [M + H] ; HR-ESIMS: m/z 211.0962 calcd for 40.1, 38.6, 34.2, 18.1; IR (CHCl ): ν
C
3400, 3079, 2979, 2927,
3
max
+
−1
11
H
14
O
4
+ H (211.0965).
1715, 1669, 1639, 1555, 1433, 1293 cm ; ESI-MS: m/z 357.10
+
+
[
(
M + H] ; HR-ESIMS: m/z 357.2078 calcd for C22
357.2060).
-Hydroxy-4-(isobutyryl)-2,2,6,6-tetra(prop-2-ynyl)cyclohex-
28 4
H O + H
General procedure for synthesis of tetraalkylated
phloroglucinols 3a–k
5
To the solution of monoacylated phloroglucinols 2a–c 4-ene-1,3-dione (3f). Yield: 55%; pale yellow sticky liquid;
1
(
(
3
1 equiv.) in 10% aq. KOH solution, was added alkyl halides
4 equiv.) and the reaction mixture was allowed to stir for 2.89–2.77 (m, 8H), 2.05 (s, 4H), 1.22 (d, J = 4 Hz, 6H); C NMR
0 min at rt. It was then acidified with HCl and extracted with (CDCl , 100 MHz): δ 208.1, 202.3, 194.8, 190.9, 111.0, 73.2,
EtOAc. The organic layer was washed with brine solution and 72.5, 63.0, 59.3, 34.9, 31.9, 29.7, 29.6, 29.4, 26.1, 24.4, 22.7,
dried over sodium sulphate. The solvent was evaporated 19.2, 14.2; IR (CHCl ): νmax 3400, 3290, 2923, 2852, 1726, 1673,
H NMR (CDCl , 400 MHz): δ 18.46 (s, 1H), 3.84–3.78 (m, 1H),
3
1
3
3
3
−
1
+
in vacuo using a Rotavapor and the crude product was purified 1558, 1425, 1383, 1156 cm ; ESI-MS: m/z 349.00 [M + H] ;
by silica gel (# 60–120) column chromatography to get tetra- HR-ESIMS: m/z 349.1435 calcd for C H O + H (349.1434).
+
2
2
20 4
alkylated products 3a–k in 55–78% yield.
5-Hydroxy-4-isobutyryl-2,2,6,6-tetrakis(3-methylbut-2-en-1-yl)-
4
-Acetyl-2,2,6,6-tetraallyl-5-hydroxycyclohex-4-ene-1,3-dione cyclohex-4-ene-1,3-dione (3g). Yield: 68%; light yellow liquid;
1
1
(
3a). Yield: 60%; pale yellow sticky liquid; H NMR (CDCl3,
H NMR (CDCl , 400 MHz): δ 18.61 (s, 1H), 4.92–4.86 (m, 4H),
3
4
2
1
1
3
1
3
00 MHz): δ 18.31 (s, 1H), 5.58–5.46 (m, 4H), 5.05 (dd, J = 8.0, 3.66–3.59 (m, 1H), 2.65–2.59 (m, 2H), 2.54 (dd, J = 8.0, 12.0 Hz,
1
3
0.0 Hz, 8H), 2.67–2.51 (m, 8H), 2.46 (s, 3H); C NMR (CDCl
00 MHz): δ 204.5, 200.4, 195.3, 193.2, 131.4, 130.7, 119.2, Hz, 6H); C NMR (CDCl , 100 MHz): δ 208.3, 207.5, 197.8,
3
,
4H), 2.28 (d, J = 8.0 Hz, 2H), 1.60–1.50 (m, 24H), 1.16 (d, J = 8.0
1
3
3
3
18.4, 111.7, 63.6, 59.3, 40.0, 38.7, 26.5; IR (CHCl ): νmax 3421, 194.5, 136.4, 136.3, 134.6, 134.5, 119.0, 118.9, 118.4, 118.3,
080, 3011, 2981, 2926, 2854, 1715, 1668, 1640, 1555, 1433, 112.5, 66.8, 65.8, 61.1, 37.1, 35.6, 33.7, 32.0, 29.8, 29.4, 26.0,
363, 1028 cm− ; ESI-MS: m/z 329.10 [M + H] ; HR-ESIMS: m/z 25.9, 22.7, 20.4, 19.2, 17.9, 17.8, 14.2; IR (CHCl ): ν
1
+
3429,
max
3
+
29.1749 calcd for C20
H
24
O
4
+ H (329.1747).
2966, 2925, 2855, 1713, 1671, 1554, 1447, 1377, 1294,
-Acetyl-5-hydroxy-2,2,6,6-tetra(prop-2-ynyl)cyclohex-4-ene- 1096 cm− ; ESI-MS: m/z 469.20 [M + H] ; HR-ESIMS: m/z
1
+
4
1
+
1
4
2
1
,3-dione (3b). Yield: 76%; brown solid; H NMR (CDCl3, 469.3329 calcd for C H O + H (469.3312).
30 44 4
00 MHz): δ 18.06 (s, 1H), 2.83–2.70 (m, 8H), 2.58 (s, 3H),
.05–2.02 (m, 4H); C NMR (CDCl
94.2, 191.1, 112.3, 73.3, 72.7, 62.7, 59.1, 26.7, 25.9, 24.4; yellow sticky liquid; H NMR (CDCl , 400 MHz): δ 18.66
5-Hydroxy-4-(isobutyryl)-2,2,6,6-tetrakis((E)-3,7-dimethylocta-
1
3
3
, 100 MHz): δ 202.3, 200.9, 2,6-dienyl)cyclohex-4-ene-1,3-dione (3h). Yield: 72%; pale
1
3
IR (CHCl
3
): νmax 3289, 2923, 2852, 2120, 1726, 1645, 1599, (s, 1H), 5.06 (t, J = 8.0 Hz, 4H), 4.93 (t, J = 8.0 Hz, 4H),
−
1
+
1
424, 1363, 1197, 1094 cm ; ESI-MS: m/z 321.10 [M + H] ; 3.73–3.66 (m, 1H), 2.67–2.62 (m, 2H), 2.57–2.50 (m, 4H),
+
HR-ESIMS: m/z 321.1123 calcd for C H O + H (321.1121).
2.38–2.32 (m, 2H), 2.01–1.89 (m, 16H), 1.68–1.56 (m, 36H),
2
0
16 4
1
3
4
-Acetyl-5-hydroxy-2,2,6,6-tetrakis(3-methylbut-2-enyl)cyclohex- 1.18 (s, 6H) ; C NMR (CDCl
3
, 100 MHz): δ 208.0, 207.2, 198.1,
-ene-1,3-dione (3c). Yield: 55%; yellow sticky liquid; H NMR 194.6, 139.9, 138.5, 131.5, 131.4, 124.3, 124.1, 124.0, 123.9,
CDCl , 400 MHz): δ 18.21 (s, 1H), 4.90–4.85 (m, 4H), 2.74–2.59 123.8, 118.8, 118.7, 118.1, 118.0, 117.7, 112.0, 109.9, 109.8,
1
4
(
(
3
1
3
m, 8H), 2.50 (s, 3H), 1.59–1.52 (m, 24H); C NMR (CDCl
3
,
66.8, 65.7, 61.3, 61.2, 40.1, 40.0, 36.4, 35.4, 33.8, 29.7, 29.6,
1
1
2
00 MHz): δ 207.2, 206.4, 196.7, 193.4, 135.3, 135.5, 117.9, 26.6, 25.7, 22.7, 19.3, 17.6, 16.4, 16.2, 16.1, 14.2; IR (CHCl ):
3
17.3, 116.9, 111.5, 65.7, 64.7, 60.0, 36.1, 34.5, 32.6, 30.9, 28.7,
ν
3434, 2923, 2965, 1712, 1669, 1555, 1446, 1382, 1151,
max
8.4, 24.9, 24.8, 21.7, 19.4, 18.7, 17.1, 17.0, 16.9, 16.8; 1097 cm− ; ESI-MS: m/z 741.50 [M + H] ; HR-ESIMS: m/z
1
+
+
IR (CHCl
1
4
3
): νmax 3391, 2982, 2928, 1720, 1666, 1550, 1453, 1362, 741.5829 calcd for C50
H
76
O
4
+ H (741.5816).
259, 1028 cm− ; ESI-MS: m/z 441.29 [M + H] ; HR-ESIMS: m/z
1
+
4-(3-Methylbutanoyl)-5-hydroxy-2,2,6,6-tetramethylcyclohex-
+
41.3006 calcd for C28
H
40
O
4
+ H (441.2999).
4-ene-1,3-dione (3i). Yield: 60%; pale yellow sticky liquid;
1
5
-Hydroxy-4-(isobutyryl)-2,2,6,6-tetramethylcyclohex-4-ene-
3
H NMR (CDCl , 400 MHz): δ 18.39 (s, 1H), 2.88 (d, J = 4.0 Hz,
1
1
,3-dione (3d). Yield: 78%; pale yellow sticky liquid; H NMR 2H), 2.20–2.13 (m, 1H), 1.45 (s, 6H), 1.36 (s, 6H), 0.99
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1
3
(
3 3
d, J = 4.0 Hz, 6H); C NMR (CDCl , 100 MHz): δ 209.0, 202.6, 96.6, 54.5, 26.8, 23.6; IR (CHCl ): νmax 3252, 2958, 2931, 1620,
98.5, 195.9, 108.4, 55.9, 51.3, 46.2, 30.9, 28.7, 28.3, 25.0, 23.2, 1416, 1340, 1272, 1195 cm− ; ESI-MS: m/z 295.10 [M + H] ;
1
+
1
2
1
[
(
+
2.8, 21.6; IR (CHCl
3
): νmax 3400, 2957, 2924, 2853, 1722, 1671, HR-ESIMS: m/z 295.1535 calcd for C16
H
22
O
5
+ H (295.1540).
−
1
559, 1465, 1383, 1260, 1047 cm ; ESI-MS: m/z 267.10
M + H] ; HR-ESIMS: m/z 267.1588 calcd for C H O + H
15 22 4
267.1591).
-(3-Methylbutanoyl)-2,2,6,6-tetraallyl-5-hydroxycyclohex-4-
ene-1,3-dione (3j). Yield: 66%; pale yellow sticky liquid; The sodium metal (0.15 g) was added to methanol (3 ml) to
+
+
General procedure for synthesis of dialkylated phloroglucinols
a–j
5
4
1
3
H NMR (CDCl , 400 MHz): δ 18.50 (s, 1H), 5.55–5.44 (m, 4H), form a NaOMe solution. To this solution was added the
5
2
0
2
4
2
.01 (dd, J = 8.0, 16.0 Hz, 8H), 2.79 (d, J = 8.0 Hz, 2H), methanolic solution of diacylated phloroglucinol (1 mmol) fol-
.57–2.45 (m, 4H), 2.42 (d, J = 8.0 Hz, 4H), 2.19–2.09 (m, 1H), lowed by alkyl halides (5 mmol). The solution was refluxed
1
3
.92 (d, J = 8.0 Hz, 6H); C NMR (CDCl
03.9, 196.9, 194.0, 132.4, 131.8, 120.1, 119.4, 112.7, 64.8, 60.2, poured into 4 N HCl to make pH of the reaction mixture acidic
7.3, 41.2, 39.5, 25.5, 22.7; IR (CHCl ): ν 3400, 3079, 2958, (pH 6.0). The reaction mixture was extracted with ethyl acetate
3 2
, 100 MHz): δ 205.8, under a N atmosphere for 3 h. Then, the reaction mixture was
3
max
−
1
925, 28.52, 1715, 1669, 1639, 1555, 1435, 1384, 1039 cm
;
and organic layer was washed with 4 N HCl followed by brine
+
ESI-MS: m/z 371.20 [M + H] ; HR-ESIMS: m/z 371.2230 calcd solution and finally dried over sodium sulphate. Solvent was
+
for C H O + H (371.2217).
evaporated in vacuo using a Rotavapor and the crude product
-(3-Methylbutanoyl)-5-hydroxy-2,2,6,6-tetrakis(3-methylbut- was purified by silica gel (# 60–120) column chromatography
-enyl)cyclohex-4-ene-1,3-dione (3k). Yield: 67%; brown sticky to yield products 5a–j in 50–78% yield.
2
3
30 4
4
2
1
liquid; H NMR (CDCl , 400 MHz): δ 18.48 (s, 1H), 4.88 (t, J =
2,6-Diacetyl-3,5-dihydroxy-4,4-dimethylcyclohexa-2,5-dienone
3
1
8
2
1
1
1
1
1
.0 Hz, 4H), 2.80 (d, J = 8.0 Hz, 2H), 2.66–2.61 (m, 2H), (5a). Yield: 77%; pale yellow sticky liquid; H NMR (CDCl
.55–2.48 (m, 4H), 2.33–2.25 (m, 2H), 2.22–2.15 (s, 1H), 400 MHz): δ 19.22 (s, 1H), 18.81 (s, 1H), 2.65 (s, 3H), 2.54
.61–1.53 (m, 24H), 1.97 (J = 4.0 Hz, 6H); C NMR (CDCl3, (s, 3H), 1.38 (s, 6H); C NMR (CDCl , 100 MHz): δ 202.2,
00 MHz): δ 207.6, 203.7, 197.4, 136.1, 134.6, 118.9, 118.3, 201.3, 200.7, 194.9, 187.5, 106.3, 105.5, 52.1, 29.2, 27.7, 24.7,
13.6, 65.5, 61.0, 47.7, 36.8, 33.9, 31.9, 26.0, 25.8, 25.7, 22.7, 24.3; IR (CHCl
9.7, 17.9, 17.8, 14.0; IR (CHCl ): νmax 3434, 2926, 2871, 1740, 1453, 1362, 1259, 1028 cm ; ESI-MS: m/z 238.90 [M + H] ;
3
,
1
3
13
3
3
): νmax 3391, 2982, 2928, 1720, 1666, 1550,
−
1
+
3
−
1
+
14 5
656, 1545, 1451, 1384, 1212, 1164 cm ; ESI-MS: m/z 483.20 HR-ESIMS: m/z 239.0915 calcd for C12H O + H (239.0914).
+
+
[M + H] ; HR-ESIMS: m/z 483.3464 calcd for C31
H
46
O
4
+ H
2,6-Diacetyl-4,4-diallyl-3,5-dihydroxycyclohexa-2,5-dienone
(5b). Yield: 70%; pale yellow sticky liquid; H NMR (CDCl3,
1
(483.3469).
4
00 MHz): δ 19.29 (s, 1H), 18.95 (s, 1H), 5.50–5.42 (m, 2H),
.02–4.92 (m, 4H), 2.77–2.68 (m, 6H), 2.61 (t, J = 8.0 Hz, 4H);
General procedure for synthesis of diacyl phloroglucinols 4a–c
5
1
3
A solution of phloroglucinol (1 equiv.) and acids (4 equiv.) in
C NMR (CDCl , 100 MHz): δ 201.2, 200.1, 198.6, 196.9, 190.9,
3
BF
3
-etherate were refluxed at 100 °C for 2.5 h. The reaction 185.7, 130.3, 129.5, 128.8, 118.1, 117.6, 117.0, 106.5, 57.9, 53.1,
): νmax 3435, 3080, 3006, 2927, 2854, 1660,
mixture was cooled to room temperature, and then into 40.9, 27.5; IR (CHCl
crushed ice and extracted with ethyl acetate (100 ml × 3). The 1642, 1550, 1456, 1363, 1028 cm ; ESI-MS: m/z 291.00
combined organic layers were evaporated in vacuo using a [M + H] ; HR-ESIMS: m/z 291.1223 calcd for C16
3
−
1
+
+
H
18
O
5
+ H
Rotavapor and the crude product was purified by silica gel (291.1227).
(
#
100–200) column chromatography to yield diacylated
phloroglucinols 4a–c in 55–75% yields.
,3-Diacetyl-2,4,6-trihydroxy benzene (4a). Yield: 60%; white
2,6-Diacetyl-3,5-dihydroxy-4,4-bis(3-methylbut-2-enyl)cyclo-
hexa-2,5-dienone (5c). Yield: 76%; pale yellow sticky liquid;
1
1
H NMR (CDCl
3
, 400 MHz): δ 19.23 (s, 1H), 18.85 (s, 1H), 4.71
1
solid; H NMR (CD OD, 400 MHz): δ 5.83 (s, 1H), 2.61 (s, 6H); (t, J = 8.0 Hz, 2H), 2.63 (s, 6H), 2.54 (s, 4H), 1.49 (s, 12H);
3
1
3
13
C NMR (CDCl
3
, 100 MHz): δ 205.2, 172.4, 104.9, 95.7, 33.1,
3
C NMR (CDCl , 100 MHz): δ 206.4, 200.2, 195.9, 193.8, 135.2,
3
1
2
1.0; IR (CHCl ): νmax 3391, 2923, 1620, 1429, 1366, 1291, 133.7, 117.9, 117.2, 112.6, 64.6, 59.8, 59.4, 36.9, 35.6, 32.8,
3
197, 1025 cm− ; ESI-MS: m/z 210.90 [M + H] ; HR-ESIMS: m/z 28.6, 26.4, 24.9, 24.8, 16.8; IR (CHCl ): ν
1
+
3400, 2969, 2925,
max
2856, 1659, 1550, 1452, 1384, 1364, 1200, 1083 cm ; ESI-MS:
3
+
−1
11.0603 calcd for C10
H
10
O
5
+ H (211.0601).
+
1
,3-Dipropanoyl-2,4,6-trihydroxy benzene (4b). Yield: 55%; m/z 347.10 [M + H] ; HR-ESIMS: m/z 347.1844 calcd for
1
+
pale yellow solid; H NMR (CD OD, 400 MHz): δ 5.76 (s, 1H), C H O + H (347.1853).
3
20 26 5
1
3
3
1
ν
.09–3.04 (m, 4H), 1.13 (t, J = 8 Hz, 6H); C NMR (CDCl
00 MHz): δ 208.2, 172.1, 104.6, 95.8, 38.4, 8.8; IR (CHCl
3
,
3,5-Dihydroxy-4,4-dimethyl-2,6-dipropionylcyclohexa-2,5-
1
3
): dienone (5d). Yield: 72%; pale yellow sticky liquid; H NMR
3264, 2939, 1622, 1588, 1420, 1399, 1274, 1191, (CDCl , 400 MHz): δ 19.47 (s, 1H), 18.98 (s, 1H), 3.16–3.13
max
3
034 cm− ; ESI-MS: m/z 239.00 [M + H] ; HR-ESIMS: m/z (m, 2H), 3.03–2.97 (m, 2H), 1.42 (s, 6H), 1.19–1.13 (m, 6H);
1
+
1
2
+
13
39.0916 calcd for C12
H
14
O
5
+ H (239.0914).
3
C NMR (CDCl , 100 MHz): δ 209.0, 204.8, 199.6, 197.3, 187.0,
1
,3-Di-(3-methyl-butanoyl)-2,4,6-trihydroxy benzene (4c). Yield: 107.8, 55.3, 50.7, 33.7, 32.1, 28.7, 23.4, 22.8, 8.0; IR (CHCl3):
1
7
(
1
5%; pale yellow solid; H NMR (CD
s, 1H), 2.94 (d, J = 4.0 Hz, 4H), 2.23 (s, 2H), 0.98 (d, J = 4.0 Hz, 1381, 1339, 1225 cm ; ESI-MS: m/z 267.00 [M + H] ;
2H); C NMR (CDCl , 100 MHz): δ 208.1, 173.4, 170.3, 105.7, HR-ESIMS: m/z 267.1220 calcd for C H O + H (267.1227).
3 14 18 5
3
OD, 400 MHz): δ 5.80
νmax 3331, 2981, 2939, 2877, 2856, 1721, 1670, 1563, 1471,
−
1
+
1
3
+
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4
,4-Diallyl-3,5-dihydroxy-2,6-dipropionylcyclohexa-2,5-dienone
2,6-Bis(3-methylbutanoyl)-3,5-dihydroxy-4,4-bis((E)-3,7-di-
1
(
5e). Yield: 66%; brown sticky liquid; H NMR (CDCl3, methylocta-2,6-dienyl)cyclohexa-2,5-dienone (5j). Yield: 69%;
00 MHz): δ 19.55 (s, 1H), 19.05 (s, 1H), 5.50–5.46 (m, 2H), pale yellow sticky liquid; H NMR (CDCl , 400 MHz): δ 19.56
3
1
4
5
2
.04–4.94 (m, 4H), 3.18 (d, J = 8.0 Hz, 2H), 3.08 (d, J = 4.0 Hz, (s, 1H), 19.05 (s, 1H), 5.33 (t, J = 8.0 Hz, 2H), 5.08 (t, J = 8.0 Hz,
H), 2.80–2.75 (m, 2H), 2.66–2.60 (m, 2H), 1.20–1.16 (m, 6H); 2H), 3.92 (d, J = 4.0 Hz, 4H), 2.99–2.92 (m, 1H), 2.78–2.62
C NMR (CDCl
31.7, 131.0, 119.7, 119.1, 107.9, 59.9, 43.2, 42.5, 35.1, 33.6, (m, 6H); C NMR (CDCl
9.7, 8.9, 8.5; IR (CHCl ): ν 3400, 2980, 2926, 1682, 1657, 193.8, 188.6, 140.4, 139.4, 131.6, 123.8, 120.5, 117.4, 109.9,
1
3
3
, 100 MHz): δ 206.2, 204.9, 198.2, 192.7, 188.0, (m, 1H), 2.10–1.82 (m, 12H), 1.74–1.51 (m, 24H), 0.98–0.94
1
3
1
2
1
[
(
3
, 100 MHz): δ 204.8, 203.0, 200.1,
3
max
−
1
555, 1451, 1380, 1260, 1186, 1063 cm ; ESI-MS: m/z 319.10 68.7, 57.8, 49.9, 48.2, 39.8, 39.6, 39.1, 26.7, 26.37, 25.7, 25.6,
+
+
M + H] ; HR-ESIMS: m/z 319.1538 calcd for C18
319.1540).
54 5
,5-Dihydroxy-4,4-bis(3-methylbut-2-enyl)-2,6-dipropionylcy- 567.30 [M + H] ; HR-ESIMS: m/z 567.4048 calcd for C36H O +
H
22
O
5
+ H
25.5, 22.7, 22.6, 22.4, 17.7, 16.4; IR (CHCl
3
): νmax 3368, 2962,
−
1
2926, 1656, 1545, 1451, 1383, 1329, 1087 cm ; ESI-MS: m/z
+
3
+
clohexa-2,5-dienone (5f). Yield: 50%; pale yellow sticky liquid; H (567.4044).
H NMR (CDCl , 400 MHz): δ 19.48 (s, 1H), 18.91 (s, 1H), 4.73
t, J = 8.0 Hz, 2H), 3.14–3.10 (m, 4H), 2.71–2.58 (m, 4H), 1.51
3
s, 12H), 1.21–1.10 (m, 6H); C NMR (CDCl , 100 MHz): Human colorectal adenocarcinoma LS-180 cells were pur-
1
3
Cell culture and treatments
(
(
1
3
δ 205.9, 205.0, 203.4, 198.4, 192.5, 191.8, 187.2, 134.6, chased from ECACC, England. These cells were grown in MEM
1
1
1
16.7, 111.6, 107.0, 59.3, 37.0, 36.3, 34.0, 33.8, 32.5, 28.7, 24.8, growth medium. The media for cell line was supplemented
6.8, 7.9, 7.7; IR (CHCl ): νmax 3400, 2973, 2925, 1656, 1551, with 1% MEM non-essential amino acids along with 10% FCS,
379, 1337, 1259, 1212, 1085 cm ; ESI-MS: m/z 375.10 100 U penicillin G and 100 µg per ml of streptomycin. Cells
3
−
1
+
+
[M + H] ; HR-ESIMS: m/z 375.2166 calcd for C22
H
30
O
5
+ H
were grown in 5% CO
compounds were dissolved in DMSO for treatment of LS-180
,1′-(4,6-Dihydroxy-5,5-dimethyl-2-oxocyclohexa-3,6-diene- cells, while the untreated control cultures received only the
,3-diyl)bis(3-methylbutan-1-one) (5g). Yield: 78%; pale yellow vehicle (DMSO < 0.2%).
2
at 37 °C with 95% humidity. All the test
(375.2166).
1
1
1
sticky liquid; H NMR (CDCl
3
, 400 MHz): δ 19.44 (s, 1H),
9.02 (s, 1H), 2.98 (d, J = 8.0 Hz, 2H), 2.86 (d, J = 8.0 Hz,
H), 2.22–2.11 (m, 2H), 1.41 (s, 6H), 0.98–0.95 (m, 12H); All synthesized compounds were screened for their ability to
P-gp-induction assay in LS-180 cells
1
2
1
3
20
C NMR (CDCl
3
, 100 MHz): δ 203.6, 201.8, 200.4, 193.8, 186.9, induce P-gp using the rhodamine 123 cell exclusion method.
1
2
1
05.5, 104.5, 51.3, 48.2, 46.5, 25.2, 24.8, 24.4, 23.7, 23.2, 21.7, In this method, the P-gp function was evaluated in terms of
2
5
3
1.6; IR (CHCl ): νmax 2959, 2930, 1667, 1544, 1469, 1448, intracellular rhodamine 123 accumulation. Briefly, the proto-
−
1
385, 1367, 1217, 1165, 1096 cm ; ESI-MS: m/z 323.10 col used was as follows: colorectal LS-180 cells were seeded at
+
+
4
[M + H] ; HR-ESIMS: m/z 323.1856 calcd for C H O + H
a density of 2 × 10 per well of a 96 well plate and were allowed
1
8
26 5
(323.1853).
to grow for next 24 h. Cells were further incubated with the
2
,6-Bis(3-methylbutanoyl)-4,4-diallyl-3,5-dihydroxycyclohexa- test compounds, and were diluted to a final concentration of
2
,5-dienone (5h). Yield: 73%; pale yellow sticky liquid; 5 µM and rifampicin (positive control) was added to final
1
H NMR (CDCl
.52–5.46 (m, 2H), 5.04–4.94 (m, 4H), 3.01 (d, J = 8 Hz, 2H), The final concentration of DMSO was kept at 0.1%. The test
.95 (d, J = 4.0 Hz, 2H), 2.79–2.74 (m, 2H), 2.65–2.60 (m, 2H), compounds were removed and cells were incubated with
3
, 400 MHz): δ 19.58 (s, 1H), 19.09 (s, 1H), concentrations of 5 and 10 µM in complete media for 48 h.
5
2
2
1
1
4
1
1
3
.23–2.16 (m, 2H), 1.00–0.98 (m, 12H); C NMR (CDCl
00 MHz): δ 203.7, 201.3, 196.8, 190.7, 186.1, 129.6, 128.9, HANKS buffer (containing 10 µM of Rh123 as a P-gp sub-
17.4, 116.9, 111.0, 106.5, 57.9, 53.0, 47.5, 47.1, 46.1, 41.0, strate) for 90 minutes. At the end of Rh123 treatment the
0.3, 27.6, 23.8, 20.1; IR (CHCl
3
,
HANKS buffer for 40 minutes before further incubation with
3
): νmax 2959, 2929, 2872, 1659, cells were washed four times with cold PBS followed by cell
−
1
643, 1546, 1454, 1386, 1367, 1299, 1265, 1086 cm ; ESI-MS: lysis for 1 h using 200 µl of lysis buffer (0.1% Triton X 100
+
m/z 375.10 [M + H] ; HR-ESIMS: m/z 375.2175 calcd for and 0.2 N NaOH). A total of 100 µl of the lysate was used for
+
C
22
H
30
O
5
+ H (375.2166).
,6-Bis(3-methylbutanoyl)-3,5-dihydroxy-4,4-bis(3-methylbut- normalized by dividing the fluorescence of each sample with
-enyl)cyclohexa-2,5-dienone (5i). Yield: 66%; pale yellow the total protein present in the lysate.
reading fluorescence of Rh123 at 485/529 nm. Samples were
2
2
1
sticky liquid; H NMR (CDCl
3
, 400 MHz): δ 19.55 (s, 1H), 19.01
s, 1H), 4.77 (t, J = 8.0 Hz, 2H), 3.04–2.94 (m, 4H), 2.71–2.65
m, 4H), 2.23–2.11 (m, 2H), 1.55 (s, 12H), 1.00–0.97 (m, 12H); The cell proliferation assay was evaluated in human colorectal
Cell viability assay
(
(
1
3
4
3
C NMR (CDCl , 100 MHz): δ 205.5, 203.5, 199.0, 192.9, 188.3, adenocarcinoma LS-180 cells. Cells (1 × 10 ) were seeded into
1
2
2
31.8, 119.1, 108.7, 60.2, 55.2, 49.8, 49.3, 48.3, 43.3, 42.5, 29.8, each well of a 96-well microplate for 24 h. The cells were
6.0, 25.9, 25.7, 22.8, 22.7, 22.6; IR (CHCl ): νmax 3434, 2926, treated with 10 µM of each compound for 48 h. MTT dye was
3
−
1
871, 1740, 1656, 1545, 1451, 1384, 1212, 1164 cm ; ESI-MS: then added to each well 4 h prior to the termination of experi-
+
m/z 431.10 [M + H] ; HR-ESIMS: m/z 431.2795 calcd for ment. Formazan crystals were dissolved in DMSO before
C H O + H (431.2792).
+
measuring absorbance at 570 nm. The cell viability of the
2
6
38 5
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untreated control sample was considered to be 100%, while
viability of the test samples was calculated using the following
formula:
Abbreviations
Aβ
Amyloid-beta
AD
BBB
Alzheimer’s disease
Blood–brain barrier
ODðtestÞ
%
cell viability ¼
ꢀ 100
ODðcontrolÞ
bEnd3 Endothelialpolyoma middle T antigen transformed
cells of cerebral cortex of the brain
In addition, the MTT assay was performed in the mouse
brain endothelial bEnd3 cells following treatment of the com- LS-180 Intestinal human colon adenocarcinoma cell line
pounds with 1 μM of compounds 3k and 5f for 48 h.
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
P-gp
PXR
P-glycoprotein
Pregnane-X-receptor
Aβ42 uptake studies in LS-180 cells
Selected compounds were tested for their ability to induce
Rh123 Rhodamine 123
SAR
2
6
P-gp using Aβ42 as a substrate. In this method, P-gp function
was evaluated in terms of Aβ accumulation. Briefly, the proto-
col used is as follows: colorectal LS-180 cells were seeded at
Structure–activity relationship
5
0% confluency in a 48-well plate. Cells were further incubated
Acknowledgements
with the test compounds, at a final concentration of 1 µM in
complete media for 48 h. Test compounds were removed, and
cells were washed twice with fresh media. Cells were incubated
with media containing 100 nM Aβ42 with and without 2.5 μM
elacridar (a P-gp inhibitor) for 30 min. At the end of Aβ treat-
ment the cells were washed three times with cold PBS followed
by cell lysis for 30 min using 100 µl of RIPA buffer. A total
of 70 µl of lysate was used for intracellular levels of Aβ
JBB is thankful to CSIR Innovation Center project (ITR-001) for
project fellowship. AK (CSIR) is thankful to CSIR, New Delhi
for the award of senior research associateship. This research
th
was supported by a grant from the CSIR 12 FYP project
(
BSC-0205).
42
7
2
using the ELISA method as described by us previously.
Total protein concentration in lysate was used for sample
normalization.
Notes and references
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I-Aβ40 transport studies across bEnd3 cells as a BBB model
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