Synthesis, biological evaluation and 3D-QSAR studies of new chalcone derivatives as inhibitors of human P-glycoprotein

Article abstract of DOI:10.1016/j.bmc.2014.02.005

P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC₅₀ = 42 nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.

Full text of DOI:10.1016/j.bmc.2014.02.005

Bioorganic & Medicinal Chemistry 22 (2014) 2311–2319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and 3D-QSAR studies of new
chalcone derivatives as inhibitors of human P-glycoprotein
Zahida Parveen a,c, , Gerda Brunhofer b, , Ishrat Jabeen b, , Thomas Erker b, Peter Chiba a,
Gerhard F. Ecker b,
⇑
a Institute of Medical Chemistry, Medical University Vienna, Waehringer Strasse 10, 1090 Vienna, Austria
b University of Vienna, Department of Medicinal Chemistry, Althanstrasse 14, 1090 Vienna, Austria
c Abdul Wali Khan University Mardan, Malakand Mardan Rd, Mardan, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an impor-
tant role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number
of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported
as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but
do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates
for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated
in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed
the highest activity (IC50 = 42 nM), which is one order of magnitude higher than the activity for an equil-
ipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors,
methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric
features influencing P-gp inhibitory activity.
Received 2 December 2013
Accepted 6 February 2014
Available online 15 February 2014
Keywords:
ABCB1
MDR
P-glycoprotein
Chalcones
GRIND
Ó 2014 Published by Elsevier Ltd.
1. Introduction
find more potent ABC transporter inhibitors, natural products often
provide interesting scaffolds and serve as lead structures, as
Human P-glycoprotein (ABCB1, P-gp) is a multispecific drug
efflux transporter which mediates resistance of cancer cells to
cytotoxic drugs.1 It belongs to the family of ATP-binding cassette
transport proteins that protect cells from the toxic effect of
compounds through active outward transfer.2 It has been demon-
strated in animal models, that even low levels of expression can
cause resistance of tumors to cytotoxic drugs. Early on, the concept
of simultaneous administration of anticancer drugs with inhibitors
of P-gp has been advocated as a concept for evading resistance.
However, clinical studies have not lived up to the high expecta-
tions, and several phase II and phase III clinical studies have been
terminated prematurely because of severe side effects related to
P-gp inhibition.
recently reviewed by Klepsch et al.4 Flavonoids, polyphenolic
compounds found to be ubiquitous in the plant kingdom, have
been shown to possess inhibitory activity towards BCRP.5 In case
of P-gp, Zhang and colleagues reported that quercetin, genistein,
and morin had inhibitory activity towards P-gp mediated dauno-
mycin transport.6 Recently, a QSAR model based on naturally
occurring flavonoids was published describing their modulating
effect on P-gp.7
For chalcones, which are biosynthetic precursors of flavonoids,
detailed structure activity relationship information with respect
to their interaction with P-gp is still missing. Chalcones used in this
study contain delocalized pi-electron systems, which restrain
structural flexibility and may be regarded as planar and linear,
which renders them suitable candidates for 3D-QSAR studies. Fur-
thermore, the chalcones reported in this study are structurally
related to a set of propafenones previously studied by our group.
However, chalcones are more rigid and are uncharged compounds,
as they lack protonable nitrogens. Another important feature asso-
ciated with these compounds is that they are photoactivable and
can be used as tool compounds to label P-gp. Finally, similar to
the structurally related flavonoids, chalcones are described to pos-
sess both P-gp and BCRP inhibitory properties. Recent studies
Nevertheless,
a
proof-of-concept study illustrated that
co-administration of the anticancer agent topotecan and elacridar,
an inhibitor of both P-gp and BCRP (ABCG2, breast cancer resis-
tance protein), significantly increased the bioavailability of the
anticancer drug and reduced inter-patient variability.3 In order to
⇑
Corresponding author. Tel.: +43 4277 55110; fax: +43 4277 9551.
E-mail address: gerhard.f.ecker@univie.ac.at (G.F. Ecker).
Z.P., G.B., and I.J. contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.02.005
0968-0896/Ó 2014 Published by Elsevier Ltd.

2312
Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
showed that basic as well as non-basic chalcone derivatives
modulate ABC transporters.8–10 These data together with those
presented herein suggest that chalcones offer a new compound
class which can be used to restore sensitivity of cells to chemother-
apeutic agents (chemo-sensitizers).
2.1.2.3. (2E,4E)-5-(2-Methoxypehnyl)-1-(30,40,50-trimethoxy-
phenyl)-2,4-pentadien-1-one (5). Yield: 1.240 g (70%) of yellow
crystals; mp: 111 °C. 1H NMR (CDCl3): d 7.72–7.53 (m, 2H), 7.52–
7.23 (m, 4H), 7.17–6.89 (m, 4H), 3.95 (s, 6H), 3.93 (s, 3H), 3.90 (s,
3H). 13C NMR (CDCl3): d 189.2, 157.6, 153.0, 145.9, 142.1, 137.3,
133.7, 130.4, 127.4, 125.0, 124.3, 120.7, 111.1, 105.8, 60.9, 56.3,
55.5. MS m/z: 354 (100%, M+), 339 (73%), 195 (28%), 140 (25%),
91 (54%). Anal. Calcd for C21H22O5ꢀ0.3H2O: C, 71.16; H, 6.26.
Found: C, 70.91; H, 6.47.
2. Experimental procedure
2.1. Chemistry
2.1.2.4. 5-(2-Methoxyphenyl)-1-(40-hydroxy-30,50-dimethoxy-
Unless otherwise stated, all chemicals were obtained from
Sigma–Aldrich or TCI Europe and were of analytical grade. Melting
points were determined on a Kofler hot stage apparatus and
are uncorrected. The 1H and 13C NMR spectra were recorded on a
Bruker Avance DPx200 (200 and 50 MHz). Chemical shifts are
reported in d units (ppm) relative to Me4Si line as internal standard
and J values are reported in Hertz. Mass spectra were obtained by a
Hewlett Packard (GC: 5890; MS: 5970) spectrometer. The purity of
the synthesized compounds was established by combustion analy-
sis with a Perkin-Elmer 2400 CHN elemental analyzer and was
within 0.4%. Solutions in organic solvents were dried over anhy-
drous sodium sulphate.
phenyl)-2,4-pentadien-1-one (6).
Yield: 0.240 g (28%) yellow
solid; mp: 44–51 °C. 1H NMR (CDCl3): d 7.78–6.87 (m, 10H), 5.99
(s, broad, 1H), 3.98 (s, 6H), 3.90 (s, 3H). 13C NMR (CDCl3): d
188.6, 157.5, 146.8 (2C), 145.4, 139.3, 137.0, 130.3, 129.9, 127.5,
127.4, 125.1, 124.2, 120.7, 111.1, 105.7(2C), 56.5 (2C), 55.5. MS
m/z: 340 (100%, M+), 309 (8%), 246 (6%), 181 (35%), 115 (63%). Anal.
Calcd for C20H20O5ꢀ0.1H2O: C, 70.20; H, 5.95. Found: C, 70.13; H,
5.71.
2.1.2.5. (E)-3-(2-Methylthiophenyl)-1-(30,40,50-trimethoxyphen-
yl)-2-propen-1-one (7). Yield: 0.496 g (64%) yellow crystals;
mp: 83–86 °C. 1H NMR (CDCl3): d 8.22 (AB-system, J = 15.6 Hz,
1H), 7.71–7.59 (m, 1H), 7.48–7.27 (m, 6H), 3.95 (s, 6H), 3.94 (s, 3H),
2.50 (s, 3H). 13C NMR (CDCl3): d 189.8, 153.1 (2C), 142.3, 141.7,
139.9, 134.1, 133.3, 130.4, 127.3, 127.0, 125.5, 124.3, 106.2 (2C),
60.9, 56.3, 16.5. MS m/z: 344 (1%, M+), 297 (100%), 236 (5%), 195
(13%), 149 (88%). Anal. Calcd for C19H20O4S: C, 66.26; H, 5.85.
Found: C, 66.22; H, 5.91.
2.1.1. General synthesis procedure for compounds 1 and 2
To a suspension of 5 mmol of the corresponding benzaldehyde,
7.5 mmol (3.220 g) (1,3-dioxolan-2-yl-methyl)triphenylphospho-
nium bromide and 0.005 g 18-crown-6 in anhydrous THF and
under argon atmosphere 20.8 mmol (0,499 g) NaH were added
carefully. The reaction mixture was stirred till the reaction was
completed (monitoring by TLC). Then, the mixture was cooled to
0 °C and first water and then 10% HCl were carefully added. After
60 min stirring at room temperature, the mixture was extracted
with ethyl acetate, 10% HCl and water. The combined organic
phase was dried over Na2SO4 and the solvent was removed in
vacuo. The so-obtained crude product was purified by flash
chromatography.
2.1.2.6. 5-(2-Methylthiophenyl)-1-(30,40,50-trimethoxyphenyl)-
2,4-pentadiene-1-one (8). Yield: 0.509 g (55%) yellow crystals.
mp: 114.5–115.5 °C. 1H NMR (CDCl3): d 7.76–7.42 (m, 3 H), 7.40–
6.86 (m, 7 H), 3.95 (s, 6H), 3.94 (s, 3H), 2.48 (s, 3H). 13C NMR
(CDCl3): d 189.0, 153.1 (2C), 144.8, 142.3, 138.6, 138.3, 135.1,
133.4, 129.4, 128.4, 127.3, 126.2, 125.6, 125.2, 105.8 (2C), 60.9,
56.3 (2C), 16.5. MS: m/z 370 (M+, 65%), 355 (46%), 297 (60%), 187
(100%), 149 (90%). Anal. Calcd for C21H22O4S: C, 68.08; H, 5.99.
Found: C, 67.74; H, 6.01.
2.1.1.1. (E)-2-Methylthiocinnamaldehyde (1) and 3,4,5-Trimeth-
oxycinnamaldehyde (2).
See Ref. 11.
2.1.2. General Synthesis Procedure for Compounds 3–23
A solution of the 2.5 mmol of the appropriate acetophenone,
indanone, tetralone derivative or 1,3-diacteylbenzene and 2 mL
50% NaOH in 10 mL ethanol was stirred at room temperature for
30 min. Then, 2.5 mmol (or 5 mmol with 1,3-diacetylbenzene) of
the corresponding benzaldehyde or cinnamaldehyde derivative,
dissolved in 1 mL ethanol, were added and stirred at room temper-
ature After conversion of the starting compounds was completed
as monitored by TLC, the reaction mixture was poured into ice
water and acidified with 10% HCl to pH 6. The so-formed solid
was filtered off and the crude product was further purified by
recrystallization in ethanol.
2.1.2.7. 2-[1-(4-Dimethylaminophenyl)-methylidene]-5,6-dim-
ethoxyindan-1-one (9).
Yield: 0.457 g (57%) yellow crystals.
mp: 196–203 °C. 1H NMR (CDCl3): d 7.68–7.49 (m, 3H), 7.34 (s,
1H), 6.97 (s, 1H), 6.73 (d, J = 9.0 Hz, 2H), 3.99 (s, 3H), 3.95 (s, 3H),
3.90 (s, 2H), 3.04 (s, 6H). 13C NMR (CDCl3): d 193.3, 154.7, 150.9,
149.4, 144.3, 133.3, 132.4 (2C), 131.7, 130.6, 123.4, 111.9 (2C),
107.2, 104.9, 56.2, 56.1, 40.7 (2C), 32.4. MS: m/z 323 (M+, 100%),
280 (13%), 237 (14%), 161 (15%), 121 (20%). Anal. Calcd for
C20H21NO3: C, 74.28; H, 6.55; N, 4.33. Found: C, 74.07; H, 6.50; N,
4.25.
2.1.2.8. 2-[3-(4-Dimethylaminophenyl)-prop-2-en-yliden]-5,6-
dimethoxyindan-1-one (10). Yield: 0.422 g (48%) orange crystals.
mp: 210–213 °C. 1H NMR (CDCl3): d 7.50–7.29 (m, 4H), 7.02–6.61
(m, 5H), 3.99 (s, 3H), 3.93 (s, 3H), 3.72 (s, 2H), 3.01 (s, 6H). 13C NMR
(CDCl3): d 192.5, 154.8, 150.9, 149.4, 143.8, 142.2, 134.1, 133.3,
132.7, 128.7 (2C), 124.6, 119.9, 112.0 (2C), 107.2, 104.8, 56.2, 56.1,
40.2 (2C), 30.2. MS: m/z 349 (M+, 100%), 334 (17%), 175 (19%), 144
(25%), 117 (27%). Anal. Calcd for C22H23NO3: C, 75.62; H, 6.64; N,
4.01. Found: C, 75.35; H, 6.60; N, 3.96.
2.1.2.1. (E)-3-(2,4,6-Trimethylphenyl)-1-(20-methoxyphenyl)-2-
propen-1-one (3).
See Ref. 12.
2.1.2.2. (E)-1-(3,4-Dimethoxyphenyl)-3-(4-methoxy-1-naph-
thyl)-2-propen-1-one (4). Yield: 0.174 g (20%) yellow solid; mp:
112–116 °C. 1H NMR (CDCl3): d 8.62 (AB-system, J = 15.3, 1H),
8.38–8.21 (m, 2H), 7.89 (d, J = 8.2 Hz, 1H), 7.77–7.49 (m, 5H), 7.00–
6.84 (m, 2H), 4.05 (s, 3H), 3.99 (s, 3H), 3.97 (s, 3H). 13C NMR
(CDCl3): d 188.6, 157.6, 153.1, 149.2, 141.0, 132.8, 131.6, 127.5,
126.0, 125.6, 124.8, 123.2, 122.9, 122.6, 121.9, 110.8, 110.0, 103.7,
56.1, 56.0, 55.7. MS m/z: 348 (72%, M+), 165 (100%), 158 (40%), 139
(91%), 79 (55%). Anal. Calcd for C22H20O4ꢀH2O: C, 75.45; H, 5.81.
Found: C, 75.42; H, 5.90.
2.1.2.9. 6-Methoxy-2-[1-naphthalen-2-yl-methylidene]-3,4-dih-
ydro-2H-naphthalen-1-one (11).
See Ref. 13.
2.1.2.10. 6,7-Dimethoxy-2-(3-(3,4,5-trimethoxyphenyl)-2-pro-
penylidene)-1-tetralone (12). Yield: 0.282 g (27%) yellow solid.

Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
2313
mp: 89–94 °C. 1H NMR (CDCl3): d 7.62 (s, 1H), 7.51 (AB-system,
J = 10.4 Hz, 1H) 7.17–6.87 (m, 2H), 6.82–6.64 (m, 3H), 3.96 (s, 3H),
3.95 (s, 3H), 3.88 (s, 6H), 3.16–2.88 (m, 4H). 13C NMR (CDCl3): d
186.1, 153.4, 153.3, 148.2, 140.4, 138.3, 135.1, 134.1, 132.4, 126.9,
122.9, 109.9, 109.5, 104.2 (2C), 61.0, 56.2 (2C), 56.0 (2C), 28.5, 26.3.
MS: m/z 410 (M+, 100%), 395 (37%), 379 (40%), 231 (35%), 175
(41%). Anal. Calcd for C24H26O6: C, 70.23; H, 6.38. Found: C, 70.01;
H, 6.39.
105.8, 59.7, 55.7. MS: m/z 342 (M+, 100%), 327 (29%), 299 (24%),
195 (47%), 103 (26%). Anal. Calcd for C19H18O6: C, 66.66; H, 5.30.
Found: C, 66.52; H, 5.28.
2.1.2.17. 4-[(E)-3-Oxo-3-ferrocenyl-1-propenyl]benzoic acid
(19). Yield: 0.405 g (45%) red crystals. mp: >350 °C. 1H NMR
(DMSO-d6): d 13.12 (s, broad, 1H), 8.30–7.82 (m, 4H), 7.67 (AB-
system, J = 15.6 Hz, 1H), 7.53 (AB-system, J = 15.6 Hz, 1H), 5.07 (s,
2H), 4.68 (s, 2H), 4.22 (s, 5H). 13C NMR (DMSO-d6): d 192.1, 158.4,
139.3, 138.6 (2C), 131.9, 128.9, 126.0 (2C), 80.7, 73.2 (2C), 70.0
(5C), 69.9 (2C). MS: m/z 360 (M+, 100%), 165 (76%), 121 (33%), 102
(15%), 56 (28%). Anal. Calcd for C20H16O3Fe: C, 66.69; H, 4.48.
Found: C, 66.43; H, 4.23.
2.1.2.11. 6-Methoxy-2-(3-(3,4,5-trimethoxyphenyl)-2-propeny-
lidene)-1-tetralone (13). Yield: 0.399 g (42%) yellow crystals.
mp: 152–156 °C. 1H NMR (CDCl3): d 8.10 (d, J = 8.7 Hz, 1H), 7.51
(AB-system, J = 10.2 Hz, 1H), 7.15–6.84 (m, 3H), 6.78–6.68 (m, 3H),
3.92 (s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 3.00 (s, 4H). 13C NMR (CDCl3):
d 186.2, 163.4, 153.4, 145.8, 140.5, 135.2, 134.3, 132.4, 130.6,
127.3, 122.9, 113.2, 112.4, 104.2 (2C), 61.0, 56.2 (2C), 55.4, 29.2,
26.1. MS: m/z 380 (M+, 100%), 365 (46%), 349 (47%), 161 (69%), 115
(33%). Anal. Calcd for C23H24O5: C, 72.61; H, 6.36. Found: C, 72.36;
H, 6.31.
2.1.2.18. 1-(3,4-Dimethoxyphenyl)-3-[3-(3,4-dimethoxyphenyl)-
3-oxo-1-propenyl]phenyl)-2-propen-1-one (20). Yield: 0.161 g
(14%) pale yellow solid. mp: 121–125 °C. 1H NMR (CDCl3): d 7.95–
7.79 (m, 3H), 7.76–7.57 (m, 8H), 7.56–7.44 (m, 1H), 6.95 (d,
J = 8.3 Hz, 2H), 3.99 (s, 12H). 13C NMR (CDCl3): d 188.2 (2C), 153.4
(2C), 149.3 (2C), 143.0 (2C), 135.8 (2C), 131.1 (2C), 129.8 (2C),
129.5, 128.3, 123.1 (2C), 122.5 (2C), 110.7 (2C), 110.0 (2C), 56.1
(2C), 56.1 (2C). MS: m/z 458 (M+, 54%), 293 (51%), 165 (100%), 137
(17%), 77 (26%). Anal. Calcd for C28H26O6ꢀ0.5H2O: C, 71.93; H, 5.82.
Found: C, 71.82; H, 6.05.
2.1.2.12. 6-Methoxy-2-(3-(3,4,5-trimethoxyphenyl)-2-propeny-
lidene)-1-indanone (14). Yield: 0.445 g (49%) yellow crystals.
mp: 182–186 °C. 1H NMR (CDCl3): d 7.51–7.29 (m, 3H), 7.19 (dd,
J = 8.3 Hz, J = 2.5 Hz, 1H), 7.02–6.82 (m, 2H), 6.74 (s, 2H), 3.93 (s,
6H), 3.89 (s, 3H), 3.86 (s, 3H), 3.84–3.78 (m, 2H). 13C NMR (CDCl3):
d 193.5, 159.5, 153.4, 141.9, 141.6, 140.5, 136.7, 133.2, 131.9,
126.9, 123.7, 123.7, 105.6, 104.4 (2C), 61.0, 56.2 (2C), 55.6, 29.8.
MS: m/z 366 (M+, 100%), 351 (44%), 335 (46%), 165 (24%), 82 (24%).
Anal. Calcd for C22H22O5: C, 72.12; H, 6.05. Found: C, 71.85; H, 6.04.
2.1.2.19. 3-{3-[3-Oxo-3-(3,4,5-trimethoxyphenyl)-1-propenyl]
phenyl}-1-(3,4,5-trimethoxyphenyl)-2-propen-1-one (21).
Yield:
0.519 g (40%) white solid. mp: 152–154 °C. 1H NMR (CDCl3): d 77.94–
7.80 (m, 3H), 7.72 (dd, J = 7.6 Hz, J = 1.3 Hz, 2H), 7.61–7.47 (m, 3H),
7.30 (s, 3H), 3.97 (s, 12H), 3.95 (s, 6H). 13C NMR (CDCl3): d 188.9 (2C),
153.2 (4C), 143.6 (2C), 135.7 (4C), 133.2 (2C), 129.8 (2C), 129.6, 128.7,
122.6 (2C), 106.1 (4C), 61.0 (2C), 56.4 (4C). MS: m/z 518 (M+, 100%),
323 (36%), 195 (95%), 152 (24%), 136 (23%). Anal. Calcd for C30H30O8: C,
69.49; H, 5.83. Found: C, 69.19; H, 5.85.
2.1.2.13. 6,7-Dimethoxy-2-(3-(3,4,5-trimethoxyphenyl)-2-pro-
penylidene)-1-indanone (15). Yield: 0.709 g (72%) yellow
crystals. mp: 193–195 °C. 1H NMR (CDCl3): d 7.39–7.29 (m, 2H),
6.99–6.87 (m, 3H), 6.73 (s, 2H), 3.99 (s, 3H), 3.93 (s, 9H), 3.89 (s,
3H), 3.78 (s, 2H). 13C NMR (CDCl3): d 182.4, 155.2, 153.4, 149.5,
144.0, 141.1, 136.5, 132.4, 132.1, 131.7, 123.8, 107.2, 104.9, 104.3
(2C), 61.0, 56.2, 56.2 (2C), 56.1, 30.1. MS: m/z 396 (M+, 100%), 381
(36%), 365 (52%), 175 (22%), 165 (25%). Anal. Calcd for C23H24O6: C,
69.68; H, 6.10. Found: C, 69.44; H, 6.05.
2.1.2.20. (E)-3-(3,4,5-Trimethoxyphenyl)-1-{3-[(E)-3-(3,4,5-tri-
methoxyphenyl)-2-propenoyl]phenyl}-2-propen-1-one (22). Yield:
0.052 g (4%) pale yellow solid. mp: 145–148 °C. 1H NMR (CDCl3): d
8.63 (s, 1H), 8.23 (dd, J = 7.7 Hz, J = 1.6 Hz, 2H), 7.78 (AB-system,
J = 15.5 Hz, 2H), 7.67 (t, J = 7.7 Hz, 2H), 7.46 (AB-system,
J = 15.5 Hz, 2H), 6.90 (s, 4H), 3.94 (s, 12H), 3.92 (s, 6H). 13C NMR
(CDCl3): d 189.8 (2C), 153.5 (4C), 146.0 (2C), 138.7 (2C), 132.3 (2C),
130.0 (2C), 129.0, 128.2, 120.8 (2C), 105.8 (4C), 61.0 (2C), 56.1 (4C).
MS: m/z 518 (M+, 100%), 487 (38%), 221 (33%), 206 (20%), 193
(15%). Anal. Calcd for C30H30O8ꢀ0.1H2O: C, 69.25; H, 5.85. Found:
C, 69.06; H, 5.92.
2.1.2.14. 6,7-Dimethoxy-2-(3-(4-hydroxy-3-methoxyphenyl)-2-
propenylidene)-1-indanone (16). Yield: 0.559 g (64%) yellow
crystals. mp: 114–120 °C. 1H NMR (CDCl3): d 7.41–7.28 (m, 2H),
7.19–7.05 (m, 1H), 7.03–6.69 (m, 5H), 5.87 (s, 1H), 3.99 (s, 3H), 3.96
(s, 3H), 3.94 (s, 3H), 3.76 (s, 2H). 13C NMR (CDCl3): d 192.5, 155.1,
149.5, 146.9, 146.7, 144.0, 141.4, 135.6, 132.4, 132.4, 129.1, 122.2,
121.3, 114.8, 109.1, 107.2, 104.9, 56.2, 56.1, 55.9, 30.1. MS: m/z 352
(M+, 100%), 337 (34%), 335 (25%), 176 (16%), 101 (21%). Anal. Calcd
for C21H20O5ꢀ0.2H2O: C, 70.85; H, 5.78. Found: C, 70.67; H, 5.71.
2.1.2.21. 5-(3,4,5-Trimethoxyphenyl)-1-{3-[5-(3,4,5-trimethoxy-
phenyl)-2,4-pentadienoyl]phenyl}-2,4-pentadien-1-one (23). Yield:
0.271 g (19%) yellow solid. mp: 142–145 °C. 1H NMR (CDCl3): d
8.57 (s, 1H), 8.18 (dd, J = 7.8 Hz, J = 1.5 Hz, 2H), 7.78–7.54 (m, 3H),
7.17 (AB-system, J = 14.8 Hz, 2H), 7.06–6.91 (m, 4H), 6.75 (s, 4H),
3.92 (s, 12H), 3.89 (s, 6H). 13C NMR (CDCl3): d 189.5 (2C), 153.4
(4C), 145.4 (2C), 142.5 (2C), 139.5 (2C), 138.5 (2C), 132.2 (2C),
131.6 (2C), 129.0, 128.1, 126.3 (2C), 124.6 (2C), 104.5 (4C), 61.0
(2C), 56.1 (4C). MS: m/z 570 (M+, 4%), 218 (100%), 204 (19%), 188
(55%), 117 (27%). Anal. Calcd for C34H34O8ꢀ0.3H2O: C, 70.89; H,
6.08. Found: C, 70.80; H, 6.00.
2.1.2.15. 5-Methoxy-2-(3-(2-methoxyphenyl)-2-propenylidene)-
1-indanone (17). Yield: 0.490 g (64%) orange crystals. mp: 151–
154 °C. 1H NMR (CDCl3): d 7.80 (d, J = 8.5 Hz, 1H), 7.56 (dd,
J = 7.8 Hz, J = 1.5 Hz, 1H), 7.44–7.25 (m, 3H), 7.15–6.85 (m, 5H),
3.89 (s, 6H), 3.78 (s, 2H). 13C NMR (CDCl3): d 192.2, 164.9, 157.4,
151.8, 136.5, 135.9, 133.1, 131.8, 130.2, 127.3, 125.8, 125.4, 124.9,
120.7, 115.0, 111.1, 109.7, 55.6, 55.5, 30.5. MS: m/z 306 (M+, 100%),
199 (37%), 145 (30%), 115 (26%), 101 (22%). Anal. Calcd for
C20H18O3: C, 78.41; H, 5.29. Found: C, 78.14; H, 5.98.
2.1.2.22.
Methyl-2-hydroxy-3-[3-(2-methoxyphenyl)-prop-2-
2.1.2.16. 4-[(E)-3-Oxo-3-(30,40,50-trimethoxyphenyl)-1-propenyl]
benzoic acid (18). Yield: 0.445 g (52%) yellow crystals. mp:
223 °C. 1H NMR (DMSO-d6): d 13.1 (s, 1H), 8.08 (AB-system
J = 15.7 Hz, 1H), 8.01 (s, 4H), 7.78 (AB-system J = 15.7 Hz, 1H),
7.45 (s, 2H), 3.90 (s, 6H), 3.77 (s, 3H). 13C NMR (DMSO-d6): d 187.3,
166.4, 152.5, 142.0, 141.7, 138.3, 132.2, 131 6, 129.2, 128.5, 123.5,
enyliden]cyclohexene-1-carboxylate (24). The compound was
synthesized according to the procedure described previously:14 to
a solution of 10 mmol (1.702 g, 1.6 mL) ethyl 2-cyclohexanone
carboxylate and 10 mmol (1.522 g, 1.5 mL) diazabicycloundecene
(DBU) in 20 mL dry methanol 10 mmol (1.622 g) 2-methoxycin-
namicaldehyde, dissolved in 10 mL dry methanol, added. The

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Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
reaction mixture stirred under argon atmosphere at room tem-
perature for 40 hours. Then, it was cooled to 0 °C, the obtained
precipitate was filtered off and recrystallized in ethanol. Yield:
0.599 g (20%) yellow crystals. mp: 125–128 °C. 1H NMR (CDCl3): d
12.22 (s, 1H), 7.52 (dd, J = 7.7 Hz, J = 1.5 Hz, 1H), 7.36–7.09 (m, 4H),
7.05–6.81 (m, 2H), 3.87 (s, 3H), 3.79 (s, 3H), 2.70–2.52 (m, 2H),
2.50–2.35 (m, 2H), 1.88–1.67 (m, 2H). 13C NMR (CDCl3): d 173.2,
165.1, 157.1, 132.1, 130.4, 130.0, 129.2, 126.9, 126.2, 124.7, 120.7,
111.0, 99.6, 55.5, 51.6, 25.7, 23.2, 22.1. MS: m/z 300 (M+, 57%), 268
(100%), 209 (13%), 134 (60%), 91 (47%). Anal. Calcd for C18H20O4: C,
71.98; H, 6.71. Found: C, 71.96; H, 6.72.
called GRIND approach aims to extract the information enclosed
in the molecular interaction fields (MIFs) and compress it into
new types of variables whose values are independent of the spatial
position of the molecule studied. Most relevant regions are ex-
tracted from the MIF by an optimization algorithm that uses the
intensity of the field at a node and the mutual node-node distances
between the chosen nodes as a scoring function. At each point, the
interaction energy (Exyz) is calculated as a sum of Lennard–Jones
energy (Elj), Hydrogen bond (Ehb) and Electrostatic (Eal)
interactions.
X
X
X
Exyz ¼
Elj þ
Eel þ
Ehb
2.2. Biological evaluation of chalcones
Default values for Grid Step (0.5 Å) and probes (DRY represent-
ing Hydrophobic interaction, O (Carbonyl Oxygen) representing
hydrogen bond acceptor groups, N1 (Amide Nitrogen) representing
H-bond donor groups and TIP representing a shape descriptor)
were used for computation of the MIF. MIF discretization was per-
formed by the AMANDA algorithm using default values for probe
cutoff (DRY = ꢁ0.5, O = ꢁ2.6, N1 = ꢁ4.2, TIP = ꢁ0.74).17 Nodes with
an energy value below this cutoff were discarded. Large Auto and
Cross Correlation (CLACC) algorithm was used for encoding the
prefiltered nodes into GRIND thus producing most consistent vari-
ables as compared to MACC.18
2.2.1. Cell lines
The resistant CCRF vcr1000 cell line was maintained in RPMI
1640 medium containing 10% fetal calf serum (FCS) and 1000 ng/
mL vincristine. The selecting agent was washed out 1 week before
the experiments. This cell line was selected due to its distinct P-gp
expression.
2.2.2. Inhibition of daunorubicin efflux
IC50 values for daunorubicin efflux inhibition were determined
as reported.15 Briefly, cells were sedimented, the supernatant was
removed by aspiration, and the cells were resuspended at a density
of 1 ꢀ 106/mL in RPMI 1640 medium containing daunorubicin (Sigma
QSAR. Molecular structures were built with the builder function
of MOE19 version 2010 and energy minimised. Partial charges were
assigned by MMFF94 force field. 2D molecular descriptors, includ-
ing atom and bond counts, connectivity indices, partial charge
descriptors, pharmacophore feature descriptors and general phys-
icochemical descriptors were calculated by using software MOE
version 2010. PLS analysis was performed with the MOE QSAR
model tool and the predictive ability of the models was determined
by leave one out cross validation (LOO).
Chemical Co., St. Louis, MO) at a final concentration of 3 lmol/L. Cell
suspensions were incubated at 37 °C for 30 min. Tubes were chilled
on ice and centrifuged at 500g in an Eppendorf 5403 centrifuge
(Eppendorf, Hamburg, Germany). Supernatants were removed,
and the cell pellet was resuspended in medium pre-warmed to
37 °C containing either no inhibitor or compounds at various con-
centrations ranging from 20 to 200 lM, depending on the solubility
and expected potency of the inhibitor. Eight concentrations (serial
1:3 dilution) were evaluated for each inhibitor. After 60, 120, 180
and 240 s, aliquots of the incubation mixture were transferred to
tubes containing an equal volume of ice-cold stop solution (RPMI
O
O
(a)
(i)
H
H
1: R4=2-SCH3
R4
R4
2: R4=3,4,5-(OCH3)3
medium containing GPV31 at a final concentration of 5 lmol/L).
1-2
Zero time points were determined by immediately pipetting dau-
norubicin-preloaded cells into ice cold stop solution. Samples
drawn at the respective time points were kept in an ice water bath
and measured within 1 h on a Becton Dickinson FACSCalibur flow
cytometer (Becton Dickinson, Vienna, Austria). Viable cells were se-
lected by setting appropriate gates for forward and side scatter. The
excitation and emission wavelengths were 482 nm and 558 nm,
respectively. Five thousand gated events were accumulated for
the determination of mean fluorescence values.
(b)
(c)
O
O
O
+
(ii)
R1
H
n
n
R3
R1
R4
R4
R3
R2
R2
3-17
O
O
O
(iii)
H
Ar
+
Ar
CH3
OMe
OH
18-19
O
O
2.2.3. Mathematical model for determination of inhibitor IC50
values
Initial efflux rates were calculated from the time dependent lin-
ear decrease in mean flourescence through the use of linear regres-
sion analysis. IC50 values were determined from dose response
curves of inhibitor concentration versus initial efflux rates. Data
3: R1=2-OCH3, R2=R3=H, R4=2,4,6-(CH3)3, n=0
4: R1=3,4-(OCH3)2, R2=R3=H, R4=2,3-(-CH=CH-CH=CH-), 4-OCH3, n=0
5: R1=3,4,5-(OCH3)3, R2=R3=H, R4=2-OCH3, n=1
6: R1=3,5-(OCH3)2, 4-OH, R2=R3=H, R4=2-OCH3, n=1
7: R1=3,4,5-(OCH3)3, R2=R3=H, R4=2-SCH3, n=0
8: R1=3,4,5-(OCH3)3, R2=R3=H, R4=2-SCH3, n=1
points were fitted according to the equation V ¼ V0 ꢁ ðV0 ꢁ Vinf
Þ
9: R1=3,4-(OCH3)2, R2-R3=-CH2-, R4=4-N(CH3)2, n=0
10: R1=3,4-(OCH3)2, R2-R3=CH2, R4=4-N(CH3)2, n=1
11: R1=4-OCH3, R2-R3=-CH2-CH2-, R4=3,4-(-CH=CH-CH=CH-), n=0
12: R1=3,4-(OCH3)2, R2-R3=-CH2-CH2-, R4=3,4,5-(OCH3)3, n=1
13: R1=4-OCH3, R2-R3=-CH2-CH2-, R4=3,4,5-(OCH3)3, n=1
14: R1=3-OCH3, R2-R3=-CH2-, R4=3,4,5-(OCH3)3, n=1
15: R1=3,4-(OCH3)2, R2-R3=-CH2-, R4=3,4,5-(OCH3)3, n=1
16: R1=3,4-(OCH3)2, R2-R3=-CH2-, R4=3-OCH3, 4-OH, n=1
17: R1=4-OCH3, R2-R3=-CH2-, R4=2-OCH3, n=1
n
n
ꢀ½Iꢂ ꢃ ðICn50 þ ½Iꢂ Þ where V is the velocity of transport, V0 is the
transport velocity in the absence of inhibitor, Vinf is the efflux
velocity at infinite concentration of inhibitor (which is equal to
simple diffusion), [I] is the inhibitor concentration, IC50 is the
50% inhibitory concentration (50% occupancy value) and n is the
Hill coefficient.
2.3. Computational Models
Scheme 1. Synthesis of (a) compounds 1–2, (b) compounds 3–17 and (c)
compounds 18 and 19. Reagents and conditions: (i) (1) (1,3-dioxolan-2-yl-
methyl)triphenylphosphonium bromide, NaH, 18-crown-6, THF, room temperature
(2) 10% HCl, room temperature; (ii) 50% NaOH, EtOH, room temperature; (iii) (1)
50% NaOH, EtOH, room temperature, (2) 10% HCl, room temperature.
2.3.1. GRID-Independent molecular descriptor analysis
Molecular discovery software Pentacle version 1.06 was used
for computing alignment-independent 3D-descriptors.16 This so

Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
2315
(a)
(b)
O
O
O
O
O
+
H3C
H
H
R1
R1
R1
20: R=3,4-(OCH3)2
21: R1=3,4,5-(OCH3)3
20, 21
O
O
O
O
O
OMe
OMe
OMe
OMe
OMe
(ii)
n
n
H
CH3
CH3
+
OMe
OMe
2
22-23
OMe
OMe
22: n=0
23: n=1
(c)
OMe
OH
O
O
OMe
O
O
CH3
(iii)
O
H
CH3
+
Scheme 2. Synthesis of (a) compounds 20–21, (b) compounds 22–23 and (c) compound 24. Reagents and conditions: (i), (ii) 50% NaOH, EtOH, room temperature; (iii) DBU,
MeOH, argon, room temperature.
Table 1
Biological activity (
3. Results and discussion
lM), pIC50 values, hydrophobic surface area (Vsa_hyd) and the
number of rotatable bonds (b_rotN) for compounds 3–24
3.1. Chemistry
Codes
IC50
(l
M) SDa
pIC50
Vsa_hyd
b_rotN
Compounds 3–24 were synthesized as outlined in Schemes 1
and 2. All compounds are based on the chalcone scaffold, 1,3-di-
phenyl-2-propen-1-one, which was used as core structure. Substit-
uents on both aromatic rings are mostly methoxy groups, which
represent a pharmacophoric substructure for P-gp interaction.20
But also derivatives bearing a combination of methoxy together
with thiomethyl and tertiary amino groups showed high P-gp
inhibitory activity. Further chemical variations concerned the
length and type of the spacer linking the two phenyl rings
(vinylogous and ‘bridged’ chalcones). The substances were
obtained by base-catalyzed reaction of a corresponding acetophe-
none derivative with an appropriate cinnam- or benzaldehyde
(Claisen–Schmidt condensation). Dimerising the core structure
resulted in compounds 20–23, which were received by the same
procedure. Compound 19 is an example of a bioisosteric replace-
ment of the phenyl part of compound 18 against a ferrocene fea-
ture which is a strategy often used in medicinal chemistry.21,22 In
compound 24, one aromatic ring was exchanged by a cyclohexenol
moiety. This compound was obtained by a DBU-methanol-pro-
moted reaction of ethyl 2-cyclohexanone carboxylate and 2-meth-
oxy cinnamaldehyde.14
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8.51 1.50
2.75 0.10
1.38 0.59
12.0 0.70
0.65 0.15
0.87 0.03
0.13 0.07
0.04 0.01
3.55 0.33
0.14 0.04
1.74 0.38
1.15 0.03
2.63 2.25
2.33 0.34
3.89 0.40
141.30 5.60
107.20 8.40
0.14 0.06
0.12 0.07
0.14 0.02
0.06 0.01
20.4 0.20
5.07
5.56
5.86
4.92
6.19
6.06
6.87
7.37
5.45
6.84
5.76
5.94
5.58
5.63
5.41
3.85
3.97
6.86
6.92
6.86
7.24
4.69
269.85
315.08
338.97
303.31
318.56
350.18
298.03
329.65
262.88
371.69
343.39
327.58
355.89
291.92
270.96
265.44
192.15
406.62
463.23
463.24
526.47
252.94
4.00
6.00
8.00
7.00
7.00
8.00
4.00
5.00
2.00
7.00
6.00
6.00
7.00
5.00
4.00
7.00
4.00
10.00
12.00
12.00
14.00
5.00
a
Each IC50 determination was performed with eight concentrations, and each
assay point was determined in triplicate.
3.2. Structure activity relationship
the central benzene ring, and differ in the number of rotatable
bonds. Whereas almost no difference in biological activity exists
The whole data set covers a range of more than four orders of
magnitude difference in biological activity, with indanone deriva-
tive 10 possessing highest biological activity (IC50: 0.04 lM). Com-
between compounds 20–22, compound 23 (IC50: 0.06 lM), which
possess two additional number of rotatable bonds, is about one or-
der of magnitude more active than derivatives 20–22 (IC50; 0.14:
pounds 9 and 10, which both contain an aniline nitrogen atom and
only differ in one rotatable bond, showed a difference of 2-fold in
their biological activity. A similar trend has been observed for com-
pounds 20–23, that are among the most active chalcone deriva-
tives. They contain a similar substitution pattern on both sides of
0.12: 0.14 lM). We also synthesised indanones 14–17 and chal-
cone derivatives 3–8 and 11–13, containing different numbers
and pattern of methoxy groups. Almost no difference in biological
activity of 15 and 16 has been identified. Overall, a minor differ-
ence (ꢄfactor of 1–2) exists in the indanones 14–17. A similar

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Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
trend has been observed for chalcone derivatives, which indicates
that a different number and pattern of methoxy groups on the
indanone and chalcone scaffold and at the benzene ring on the
opposite side of the molecules is not making any significant differ-
ence for biological activity. Compounds 18 and 19 are acidic ana-
logs and comprise the least active compounds (IC50: 141.30:
107.20) (Table 1).
3.3. 2D-QSAR
In order to identify the most relevant physicochemical features
important for high P-gp inhibitory activity of chalcone analogs,
multiple linear regression analysis was performed. Use of MOE’s
Figure 3. Plot of observed vs. predicted MDR activity (expressed as log(1/IC50
)
values) using the GRIND model.
contingency23 analysis tool for identification of the most important
descriptors revealed the following equation (Eq. (1)).
Log ð1=IC50Þ ¼ 0:02 ðvsa hydÞ ꢁ 0:36 ðb rotNÞ þ 0:93
n ¼ 22; R2 ¼ 0:79; q2ðLOOÞ ¼ 0:71; RMSE ¼ 0:51:
ð1Þ
Figure 5 shows a plot of observed vs biological activity pre-
dicted by QSAR Eq. 1. An excellent QSAR model was obtained with
all predicted values within one order of magnitude from the mea-
sured ones. Descriptors contributing most to the variance in the
biological activity comprised vsa_hyd and b_rotN (Eq. 1). This indi-
cates that within this data set the number of rotatable bonds and
the hydrophobic surface area are the most important structural
attributes for high biological activity. This is in line with previous
findings by Wang and colleagues, who showed that hydrophobic
distribution within the molecules along with molecular weight,
number of rotatable bonds and energy of highest occupied orbital
(Ehomo) are important descriptors for P-gp inhibitory potency.24
However, in our case the QSAR equation reveals a negative contri-
bution of the number of rotatable bonds, which points towards an
unfavorable entropic contribution.
Figure 1. Plot of observed vs. predicted MDR-modulating activity of compounds
3–24, predicted values were obtained by leave-one-out cross validation.
Extensive QSAR studies on a large set of propafenone analogs
revealed the importance of hydrogen bond acceptors and their
strength, the distance between aromatic moieties and H-bond
acceptors as well as the influence of global physicochemical
parameters, such as lipophilicity and molar refractivity.25–27 How-
ever, for the present data set of chalcone derivatives we identified
only a poor correlation (r2 = 0.18) between clogP and biological
activity (Fig. 1). This indicates that the variance in the biological
activity of chalcone derivatives is mainly driven by the concrete
pattern of hydrophobicity distribution within the molecules, as
represented by vsa_hyd, rather than by their ability to penetrate
in the membrane bilayer. This is also in line with the findings of
Pleban et al.28 on the importance of the distribution of hydropho-
bicity within P-gp inhibitors. Later on this was further confirmed
by König et al., by using hydrophobic moments as QSAR descrip-
tors.29 Finally, this is additionally supported by the recent X-ray
structure of mouse P-gp, which shows a large inner cavity exhibit-
ing several hydrophobic patches for space directed hydrophobic
interactions.30
Figure 2. PCA score plot showing that the whole data set is divided into three
groups; the upper right part contains flexible and large compounds, the lower
portion contains less flexible and small compounds (mostly indanones and
tetralones); the left part of the diagram contains all compounds having a hydrogen
bond donor group.

Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
2317
Figure 4. PLS Coefficient correlograms showing the descriptors which are directly (positive values) or inversely (negative values) correlated to the biological activity. The
activity particularly increases with the increase in (DRY-DRY), (DRY-TIP) and (TIP-TIP) descriptor values.
Figure 5. (a) DRY-DRY hot spots (yellow color) which represents two hydrophobic regions 16.00–16.40 Å apart, present in most active compounds. (b) Shows steric hot spots
(green color) which makes three important boundaries (A, B and C) for most potent inhibitors of ABCB1 where A–B: 10.00–10.40 Å and A–C: 22.00–22.40 Å. (c) Represent the
distance range of a hydrophobic substructure (yellow hot spot) from molecular extreme (A) (17.60–18.00 Å) (green hot spot). (d) Shows a carboxylic acid group (N1: blue hot
spot) present very close (1.60–2.00 Å) to one of the molecular boundary (encircle), having negative effect to biological activity, while (e) represent the same pharmacophoric
features at longer distance (20.80–21.20 Å) showing positive contribution towards biological activity. (f) N1–N1 hot spots (blue color) representing two H-bond acceptors at a
distance of 9.20–9.60 Å which is favorable for biological activity of most of the compounds.

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Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
3.4. 3D-QSAR
also evidence for the importance of a distinct three dimensional
shape of inhibitors of P-gp.34
Due to its rigid scaffold, chalcones represent versatile tools for
3D-QSAR studies. In recent years especially GRID-independent
descriptors (GRIND) gained a lot of attraction in the field. GRIND
descriptors are based on molecular interaction field (MIF) calcula-
tions and are alignment-independent, thus allowing the analysis of
structurally diverse data series,31 23 3D conformations of the mol-
ecules in the data set were obtained from their 2D co-ordinates by
using program CORINA32 GRIND descriptors were derived by com-
puting molecular interaction fields (MIF) and by identifying the re-
gions with maximum field intensity at relative distances by using
the AMANDA algorithm implemented in software Pentacle version
1.06.33 The Consistently Large Auto and Cross Correlation (CLACC)
algorithm was used for encoding the prefiltered nodes into GRIND.
The values obtained from the analysis were represented directly in
correlograms plots, where the product of node–node energies ver-
sus distance separating the nodes is reported.
Also our model elucidates the importance of an optimal shape
of the ligands and identifies three important steric hot spots
(‘edges’) A, B and C. In the molecules where hot spot (A) is
10.00–10.40 Å apart from (B), this represents two edges related
to two methoxy substitutions at positions 6 and 7 of indanone
and tetralone derivatives (Fig. 5b). However, in compounds
20–23, which contain 3,4,5-tri methoxy groups, it represent the
distance between the 3- and 4-annelated methoxy groups. The
3rd steric hot spot (C) represents the substituted benzene ring on
the opposite side of the indanone and tetralone scaffold, which is
at a distance of 22.00–22.40 Å from edge (A) in most of the active
(IC50 <1 lM) compounds. This further confirms the importance of
distinct 3D shape requirements for inhibitors of P-gp.
It seems that out of three identified steric hot spots, (A) repre-
sent the most favorable one as it serve as an anchor to measure
the distances to a hydrophobic feature (Fig. 5c). Analyzing the most
First, the data set was examined using principal component
analysis (PCA). The first two components explained 43% of the var-
iance in the GRIND descriptors and separate the data mainly on ba-
sis of their pharmacophoric pattern (Fig. 2). Thus, compounds 6, 16,
24 in the upper left cluster share a similar pharmacophore having
one hydrogen band donor (OH). Compounds 20, 21, 22, 23 which
contain two carbonyl groups and show higher flexibility, are lo-
cated in the upper right hand side of the plot. Interestingly, acid
18, which exhibits pharmacophoric features from both clusters
(two C@O, one OH), is located almost in the middle of these two
clusters. Compounds with negative PC values are relatively small
(mostly indanones and tetralones) and contain one carbonyl group,
1–5 methoxy groups, but no hydrogen bond donor.
In order to analyze the underlying important pharmacophoric
patterns, PLS multivariate data analysis correlating the biological
activity with the complete set of GRIND variables (790) was carried
out using the AMANDA algorithm.31 The PLS analysis resulted in a
two-latent variable model with an r2 = 0.85. However, the cross-
validation of the model yielded q2 LOO values of 0.26, which is
quite unsatisfying. Thus, to reduce the high number of variables,
a variable selection was applied by using FFD factorial selection
implemented in Pentacle.33 The resulting number of active vari-
ables decreased from 523 to 422 which improved the quality of
the model (r2 = 0.98, q2LOO = 0.66). Figure 3 shows the plot of
the experimental versus predicted biological activities. Analysis
of the PLS coefficients profile of the 3rd Latent Variable (LV) of
the PLS model illustrates the identification of key descriptors for
high biological activity (Fig. 4). Activity increases strongly with
high value of the descriptors DRY-DRY, TIP-TIP, Dry-TIP, and N1-
TIP (Table 2).
active compounds (IC50 <1 lM) reveals the presence of a hydro-
phobic region around the substituted benzene ring at the opposite
side at a distance of 17.60–18.00 Å from edge (A). Optimal shape
and hydrophobicity was also identified in other studies as major
physicochemical parameters responsible for high affinity of flavo-
noid derivatives.35–37 Furthermore, Cianchetta and co-workers
identified the same features at a distance of 20.5 Å apart from each
other in selected substrates of P-gp.38
In order to further explore the hydrogen bonding related prop-
erties, a distance matrix of hydrogen bond acceptors from all three
steric hot spots as well as their mutual distances were computed
by GRIND descriptors. In some of the compounds edge (A) again
represents an anchor point for corresponding distance calculations.
Interestingly, two different distance ranges having opposite behav-
ior have been identified. First, compounds with low activity values
show a hydrogen bond acceptor at a distance of 1.60–2.00 Å from a
steric hot spot (Fig. 5d). In contrast, both features far apart (20.80–
21.20 Å) from each other is seen in the most active compounds
(IC50 <1 lM) (Fig. 5e). This indicates that potent P-gp inhibitors
show an elongated structure and have a hydrogen bond acceptor
far from the edges of the molecules.
The number and pattern of H-bond acceptor groups is a subject
of various publications. Seelig defined two patterns of H-bond
acceptors and proposed that P-gp ligands may contain two or more
H-bond acceptors which are separated either 2.5 0.3 Å and/or
4.6 0.6 Å apart from each other.39,40 Interestingly, no consistency
has been observed in the distance profile between different pairs of
H-bond acceptors in chalcone derivatives. A distance of 9.20–
9.60 Å has been identified between two carbonyl groups of 20–
23 and between one carbonyl group and a region between the
6- and 7-methoxy group of the highly active indanone derivative
The GRIND model indicates the presence of two hydrophobic
moieties, which are localized at two of the three steric hot spots
10 (IC50: 0.04
lM) (Fig. 5f). However, this distance is not consis-
identified, (Fig. 5a and b) in the most active (IC50 <1
lM) P-gp
tently present in all compounds having IC50 values <1 l
M. Interest-
inhibitors. Most of the QSAR studies in the past two decades
pointed towards the importance of hydrophobic substructures for
high P-gp inhibitory potency.25–27 Furthermore, by using a MIF-
based pharmacophore model, Broccatelli et al recently provided
ingly, a similar distance (8.80–9.20 Å) has been found to be
important for activity of conformationally rigid benzopyr-
ano[3,4b][1,4]oxazine-type inhibitors of P-gp.41 In another study,
performed on flavonoid derivatives, a distance of 8.00 Å between
Table 2
GRID-independent descriptors that are highly correlated to biological activity of chalcone derivatives 3–24
Variable
Distance
Correlogram
Comment
40
16.00–16.40 Å
22.00–22.40 Å
17.60–18.00 Å
1.60–2.00 Å
20.80–21.20 Å
18.80–19.20 Å
DRY-DRY
TIP-TIP
DRY-TIP
N1-TIP
N1-TIP
DRY-N1
Represents two large hydrophobic groups, remains highly consistent throughout the length of the correlogram
Distance between two steric hot spots of the molecule
Distance of a hydrophobic group to one particular steric hot spot
Distance between a hydrogen bond acceptor and a steric hot spot , showing negative contribution to biological activity
Distance between a hydrogen bond acceptor and a steric hot spot, showing beneficial contribution to potency
Distance between one of the two hydrophobic moieties to a hydrogen bond acceptor
292
518
715
763
442

Z. Parveen et al. / Bioorg. Med. Chem. 22 (2014) 2311–2319
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4. Conclusion
With the current study we present a series of novel chalcone
derivatives which in part show P-gp inhibitory activity in the nano-
molar range. Based on a set of 22 compounds covering different
modifications of the chalcone scaffold, two predictive QSAR models
were established in order to elucidate the molecular features
responsible for high biological activity. 2D-QSAR analysis revealed
the importance of the hydrophobic surface area and the number of
rotatable bonds. Interestingly, in contrast to several other com-
pound classes, there was only a poor correlation between overall
lipophilicity of the compounds and their P-gp inhibitory activity.
This indicates that for chalcones hydrophobic areas directly con-
tribute to ligand binding. This is further exemplified by the GRIND
analysis, which identified three hydrophobic hot spots in the mol-
ecules. Furthermore, distinct distances between these hydrophobic
features and H-bond acceptors have been exemplified. Remark-
ably, these compounds do not contain a basic nitrogen atom. Fur-
thermore, they exhibit a quite rigid and planar structure, which
renders them quite unique in the chemical space of P-gp inhibitors.
Thus, chalcones represent an interesting new class of P-gp ligands
which will deserve further investigation.
Acknowledgements
We acknowledge Austrian Science Foundation, grants F3502
and F3509 for financial support and Higher Education Commission
Pakistan for giving scholarship to Z.P. and I.J. to complete this
study.
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