Synthesis, biological evaluation and QSAR studies of diarylpentanoid analogues as potential nitric oxide inhibitors

Article abstract of DOI:10.1039/c4md00541d

A series of forty-five 1,5-diphenylpenta-2,4-dien-1-one analogues were synthesized and evaluated for their nitric oxide (NO) inhibition activity in IFN-γ/LPS-activated RAW 264.7 cells. Compounds 3h, 7a, 7d and 7e exhibited comparable or significantly higher activity than the standard, curcumin (IC₅₀ = 14.69 ± 0.24 μM). Compound 7d, a 5-methylthiophenyl-bearing analogue, displayed the most promising NO-inhibitory activity with an IC₅₀ value of 10.24 ± 0.62 μM. The 2D and 3D QSAR analyses performed revealed that a para-hydroxyl group on ring B and an α,β-unsaturated ketone moiety on the linker are crucial for a remarkable anti-inflammatory activity. Based on ADMET and TOPKAT analyses, compounds 3h, 7a and 7d are predicted to be nonmutagenic and to exhibit high blood-brain barrier (BBB) penetration, which indicates that they are potentially effective drug candidates for treating central nervous system (CNS) related disorders.

Full text of DOI:10.1039/c4md00541d

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Faudzi, S. W. Leong, F. Abas, M. F. F. Mohd Aluwi, K. Rullah, K. W. Lam, S. Ahmad, C. L. Tham, K. Shaari and
N. H. Lajis, Med. Chem. Commun., 2015, DOI: 10.1039/C4MD00541D.
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DOI: 10.1039/C4MD00541D
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RSCPublishing
ARTICLE
Synthesis, biological evaluation and QSAR studies of
diarylpentanoid analogues as potential nitric oxide
inhibitors
Cite this: DOI: 10.1039/x0xx00000x
S. M. Mohd Faudzia,b, S. W. Leonga, F. Abas*a,c, M. F. F. Mohd Aluwid, K. Rullahd, K.
W. Lamd, S. Ahmade, C. L. Thamf, K. Shaaria,b, and N. H. Lajis*a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
A series of fortyꢀfive 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone analogues were synthesized and
evaluated for their nitric oxide (NO)ꢀinhibition activity in IFNꢀγ/LPSꢀactivated RAW 264.7
www.rsc.org/
cells. Compounds 3h
,
7a
,
7d and 7e exhibited comparable or significantly higher activity as
5ꢀ
compared to the standard, curcumin (IC50=14.69±0.24 µM). Compound 7d,
a
methylthiophenylꢀbearing analogue, displayed the most promising NOꢀinhibitory activity with
an IC50 value of 10.24±0.62 ꢁM. The 2D and 3D QSAR analyses performed revealed that a
para-hydroxyl group on ring B and an α,βꢀunsaturated ketone moiety on a linker are crucial for
remarkable antiꢀinflammatory activity. Based on ADMET and TOPKAT analyses, compounds
3h, 7a and 7d are predicted to be nonmutagenic and to exhibit high blood−brain barrier (BBB)
penetration, which indicates that they are potentially effective drug candidates for the treating
central nervous system (CNS) related disorders.
Diarylpropanoids (such as flavonones and chalcones),
diarylheptanones (especially curcumins and their derivatives) and
Introduction
divinylketonoids (such as zerumbone) are natural products
commonly found in Zingiberaceous plants, especially Curcuma
domestica, Alpinia rafflesiana, and Zingiber zerumbet,
respectively.8,9 These compounds have been shown to display
excellent antiꢀinflammatory activity through their actions on a
number of mediators and promoters.10 Curcumin (Fig . 1) has
been one of the most investigated diarylheptanoids due to its
strong bioactivity, but its instability under physiological
conditions, poor absorbability and fast metabolism have limited
its potential practical use.11 A number of studies have shown that
a diarylpentadienone scaffold enabled their antiꢀinflammatory
properties via NOꢀproduction suppression activity and the
inhibition on proinflammatory cytokines such as TNFꢀα and ILꢀ
6.12,13 Previously, we synthesized a series of diarylpentanoid
monocarbonyl analogues13 (Fig. 1), and they were observed to
enhance the stability in vitro and improve the pharmacokinetic
profiles, particularly those related to antiꢀinflammatory
activity.13ꢀ15
Inflammation is a complex component of an animal’s response
system in response to a variety of stimuli resulting from
biological, chemical, and physical invasions or damages.
However, prolonged exposure to a stimulus can lead to a chronic
phase. It has been reported that chronic inflammation may lead to
the development of numerous diseases, including cancer,1
chronic asthma, cardiovascular diseases, diabetes, autoimmune
diseases, atherosclerosis, allergies, rheumatoid arthritis, bowel
inflammation, psoriasis, multiple sclerosis, and accelerated
aging.2
NO is a shortꢀlived bioactive free radical synthesized from Lꢀ
arginine, NADPH and molecular oxygen by the catalytic reaction
of nitric oxide synthase (NOS), which has an important function
as an intercellular messenger in the regulation of physiological
and pathophysiological mechanism in the nervous, cardiovascular
and immunological systems.3,4 Excessive generation of NO
enhances the production of diverse inflammatory mediators and
may contribute to the immunopathology of macrophageꢀ
dependent inflammation5 and degenerative diseases, including
cancer and cardiovascular disorders. Therefore, an inhibitory
effect on NO production is a promising therapeutic target for
potential antiꢀinflammatory agents. It is wellꢀdocumented that
inducible nitric oxide synthase (iNOS) is expressed in response to
various stimuli (LPS, IFNꢀγ or TNFꢀα), which causes the
production of a vast amount of NO by macrophages during the
inflammatory process,6 while the expression of several proꢀ
inflammatory mediators, such as NO, is regulated at the
transcriptional level by nuclear factorꢀκB (NFꢀκB).7
O
O
HO
OH
H3CO
OCH3
Curcumin
O
O
O
1,5-diarylpentadien-3-one
2,4-bis-benzylidenecyclopentanone
2,5-bis-benzylidenecyclohexanone
Fig. 1
Curcumin and its diarylpentanoid analogues
This journal is © The Royal Society of Chemistry 2013
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DOI: 10.1039/C4MD00541D
R2
R5
O
membered heterocyclic analogues of an acetophenone yielded the
respective products in the range of 15 to 90%. It is noteworthy
that the 5ꢀmethylꢀthiophenylꢀcontaining analogues gave better
yields (greater than 70%).
R3
R4
R1
R1 = 4-H (Series 1)
4-CH3 (Series 2)
4-OCH3 (Series 3)
4-N(CH3)2 (Series 4)
4-Cl (Series 5)
The resulting methoxylated diarylpentadienones were then
further demethylated to their polyphenolic analogues II (Series
7), using boron tribromide in dichloromethane (see Scheme 1),
with the yields ranging from 20 to 75%. All of the purified
diarylpentadienone analogues were characterized via 1Hꢀ and
13Cꢀnuclear magnetic resonance (NMR) as well as mass
spectrometry. Based on the NMR data, the couplingꢀconstant (J)
values of the double bond of the two methylene units in the
pentadiene chain are in the range of 7ꢀ8 Hz, indicating that the
compounds are in the cis configuration.
3,5-OCH3,4-OH (Series 6)
4-OH (Series 7)
Fig. 2 Representative structure of 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀ
one
O
R2
R5
O
R2
A
O
R3
R4
R3
R4
H
CH3
a
+
B
R1
R1
I
R5
R1 = 4-H (Series 1)
4-CH3 (Series 2)
4-OCH3 (Series 3)
4-N(CH3)2 (Series 4)
4-Cl (Series 5)
R2
A
O
NO inhibition in LPS/IFNꢀγꢀinduced macrophages
b
3,5-OCH3,4-OH (Series 6)
R3
R4
B
R1
The inhibitory potency of the 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone
analogues on LPS/IFNꢀγꢀstimulated inflammation was first
investigated in RAW 264.7 macrophage cells at a 50 ꢁM
concentration. Based on the preliminary evaluation, twentyꢀfive
compounds were shown to significantly inhibit the LPS/IFNꢀγꢀ
stimulated NO production, indicating that our diarylpentadienone
scaffold may possess interesting antiꢀinflammatory property. The
IC50 values of the twentyꢀfive bioactive compounds were
determined and compared to that of the positive control,
curcumin. In addition, MTT assays were performed to ensure that
the nitric oxide inhibition in the RAW 264.7 cells was not due to
cytotoxic activity. The efficacy of the 1,5ꢀdiphenylpentaꢀ2,4ꢀ
dienꢀ1ꢀone analogues on the nitrite production in the macrophage
cells is shown in Table 1. The results of the NOꢀinhibitory
screening showed that compounds 3h, 7a, 7d and 7e displayed
potent antiꢀinflammatory activity with IC50 values of less than 20
µM. Additionally, seven other compounds (1c, 3iꢀk, 3q, and 7bꢀ
c) significantly inhibited NO production, with IC50 ranged
between 20.0ꢀ30.0 µM.
II
R1 = 4-OH (Series 7)
R5
Scheme 1 Reagents and conditions: (a) NaOH, ethanol, RT
(overnight); (b) BBr3, CH2Cl2 0 °C (1h).
Continuing the interest in understanding the biological
implications of the structural features in diarylpentanoids, we
have prepared fortyꢀfive 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone
analogues (Fig. 2). Unlike the commonly studied diarylpentanoid
and curcumin series reported earlier, in which the carbonyl is
located at the middle carbon,13,16 the ketone functionality in the
new series is located at Cꢀ1 of the pentadienone linker moiety. It
would be expected that the electronic character of the carbonyl,
which may contribute in the ligandꢀreceptor interaction, may be
somewhat influenced by its position of being directly attached to
one of the two aromatic rings. Herewith, we describe the
synthesis of all of the analogues and the evaluation of their NOꢀ
suppression activity on IFNꢀγ/LPSꢀactivated RAW 264.7 cells.
To explore the interaction and effects of the diarylpentadienone
analogues on the inflammatory mediator, we performed 2D and
3D QSAR analysis. It is hoped that further understanding may be
gained through the correlations established between the
physicochemical features, electronics and steric factors and the
biological potency.
In series 1 of diarylpentadienone analogues, the most significant
NO suppression effect is exhibited by compound 1c, which
contains a metaꢀhydroxylated phenyl ring. In contrast with
compound 1b, an orthoꢀhydroxyl bearing analogue did not
contribute towards the activity enhancement. This observation
can be best explain by the presence of chelated system in
compound 1b, in comparison to the absence such system in
compound 1c. This trend was further supported by a metaꢀ
hydroxylated on ring A as displayed by analogue 3h, with the
highest inhibition potency with the IC50 of 16.3 µM. Further
comparisons of the variation of ring A in series 3, revealed three
Results and discussion
Chemistry
paraꢀhydroxylꢀbearing compounds, 3iꢀk also showed
a
1,5ꢀDiarylpentaꢀ2,4ꢀdienꢀ1ꢀone analogues I (Series 1ꢀ6) were
synthesized via ClaisenꢀSchmidt condensation by reacting the
appropriate cinnamaldehyde and substituted acetophenone in
ethanolic sodium hydroxide for 24 h (see Scheme 1). The
presence of an electronꢀdonating group at the para position in the
acetophenones (origin of ring B) appeared to improve the yield
up to 90%. Conversely, lower yields in the range of 13 to 65%
were obtained when an electronꢀwithdrawing group was present
in the aromatic ring of the cinnamaldehyde. This trend correlates
well with the results of a previous study on the preparation of
flavanones from 2’ꢀhydroxychalcone.17 The cyclization of a
chalcone containing an electronꢀdonating group afforded a high
yield, whereas the presence of electronꢀwithdrawing and bulky
groups resulted in lower product yields. The use of fiveꢀ
remarkable antiꢀinflammation property. Based on this result, it is
interesting to note that a hydroxyl group on either metaꢀ or paraꢀ
position of ring A indicated the importance of this feature in the
enhancement of NO inhibition activity as exhibited by
compounds 3hꢀk, as opposed the activity observed in the
unsubstituted ring A such as compound 3a. This finding
correlated well with previous study,18 which suggested that the
presence of hydroxyl group at metaꢀ and/or paraꢀposition. The
presence of these functionalities might contribute to higher
electron density as well as higher hydrogen bond potential of the
phenyl moiety, which could be participate in the πꢀπ interaction
or hydrogen bonding in the active site, that is important in the
NO inhibitory activity.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

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DOI: 10.1039/C4MD00541D
Table 1. NO inhibitory activity and cytotoxicity of 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone analogues on RAW 264.
O
Ar
R1
(Ring A)
(Ring B)
Compounds
Curcumin
R1 (Ring B)
ꢀ
Ar (Ring A)
ꢀ
NO inhibition (%) ± NO inhibition IC50 Cytotoxicity
IC50
S.E.M at 50 ꢁM
99.3 ± 0.2
(ꢁM) ± S.E.M
14.7 ± 0.2
(ꢁM) ± S.E.M
>100
1a
1b
1c
1d
1e
1f
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀH
phenyl
66.8 ± 0.7
<10
103.9 ± 0.6
51.1 ± 4.8
33.1 ± 4.2
<10
44.6 ± 2.5
ND
26.1 ± 0.1
71.2 ± 5.1
ND
44.3 ± 5.4
ND
48.7 ± 5.1
>100
ND
ND
2ꢀhydroxyphenyl
3ꢀhydroxyphenyl
4ꢀmethoxyphenyl
4ꢀfluorophenyl
4ꢀchlorophenyl
ND
1g
1h
1i
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀH
4'ꢀCH3
5ꢀbromoꢀ2ꢀhydroxyphenyl
5ꢀchloroꢀ2ꢀhydroxyphenyl
3,5ꢀdichloroꢀ2ꢀhydroxyphenyl
5ꢀmethylthiophenꢀ2ꢀyl
2,5ꢀdimethylfuranꢀ3ꢀyl
phenyl
phenyl
4ꢀmethoxyphenyl
4ꢀfluorophenyl
5ꢀchloroꢀ2ꢀhydroxyphenyl
2,4ꢀdimethoxyphenyl
3,5ꢀdichloroꢀ2ꢀhydroxyphenyl
4ꢀmethylphenyl
3ꢀhydroxyꢀ4ꢀmethoxyphenyl
4ꢀhydroxyꢀ3,5ꢀdimethoxyphenyl
3ꢀchloroꢀ4ꢀhydroxyphenyl
2ꢀfluoroꢀ4ꢀhydroxyphenyl
5ꢀmethylthiophenꢀ2ꢀyl
2,5ꢀdimethylfuranꢀ3ꢀyl
2,5ꢀdimethylthiophenꢀ3ꢀyl
1Hꢀpyrrolꢀ2ꢀyl
thiophenꢀ2ꢀyl
furanꢀ2yl
phenyl
4ꢀmethoxyphenyl
2ꢀhydroxyphenyl
4ꢀflurophenyl
thiophenꢀ2ꢀyl
furanꢀ2ꢀyl
4ꢀchlorophenyl
4ꢀmethoxyphenyl
5ꢀchloroꢀ2ꢀhydroxyphenyl
phenyl
4ꢀfluorophenyl
<10
<10
ND
ND
ND
68.6 ± 2.6
59.0 ± 6.0
ND
45.8 ± 1.5
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
ND
>100
ND
ND
ND
ND
ND
19.6 ± 2.1
52.6 ± 4.3
84.6 ± 1.1
73.7 ± 7.1
77.4 ± 3.4
41.6 ± 1.7
40.0 ± 3.3
26.4 ± 1.7
<10
1j
1k
2a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
4a
4b
4c
4d
4e
4f
5a
5b
5c
6a
6b
7a
7b
7c
7d
7e
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀOCH3
4'ꢀN(CH3)2
4'ꢀN(CH3)2
4'ꢀN(CH3)2
4'ꢀN(CH3)2
4'ꢀN(CH3)2
4'ꢀN(CH3)2
4'ꢀCl
<10
42.2 ± 2.5
100.2 ± 2.3
89.4 ± 3.9
98.2 ± 0.9
102.3 ± 1.6
41.7 ± 4.6
59.7 ± 1.1
44.8 ± 5.2
95.8 ± 1.7
69.6 ± 1.1
61.5 ± 2.4
51.2 ± 4.2
54.5 ± 1.3
16.4 ± 3.1
71.5 ± 4.8
32.4 ± 2.8
56.9 ± 3.5
31.2 ± 4.0
45.4 ± 3.9
<10
ND
ND
16.3 ± 2.0
21.5 ± 2.3
23.5 ± 2.4
21.8 ± 2.0
ND
28.4 ± 1.7
58.6 ± 2.1
67.6 ± 5.0
60.3 ± 5.4
ND
>100
ND
>100
68.5 ± 5.2
76.6 ± 3.7
>100
>100
ND
>100
ND
>100
ND
80.2 ± 5.1
ND
28.7 ± 4.1
5.0 ± 3.6
>100
>100
>100
28.3 ± 2.9
45.49 ± 4.9
39.8 ± 2.3
ND
57.4 ± 1.2
47.9 ± 5.5
27.5 ± 0.1
73.6 ±7.5
77.4 ± 6.2
ND
63.8 ± 4.5
ND
115.9 ± 4.8
ND
4'ꢀCl
4'ꢀCl
3',5'ꢀOCH3ꢀ4'ꢀOH
3',5'ꢀOCH3ꢀ4'ꢀOH
4'ꢀOH
4'ꢀOH
4'ꢀOH
4'ꢀOH
4'ꢀOH
70.5 ± 2.1
ND
64.1 ± 5.0
76.7 ± 1.1
101.8 ± 1.1
82.1 ± 2.7
93.4 ± 0.1
103.1 ± 1.6
110.2 ± 1.1
42.0 ± 1.6
39.3 ± 0.1
16.4 ± 0.3
27.2 ± 1.1
27.11 ± 0.7
10.2 ± 0.6
14.8 ± 1.9
4ꢀhydroxyphenyl
2,4ꢀdihydroxyphenyl
2ꢀhydroxyꢀ4ꢀmethoxyphenyl
5ꢀmethylthiophenꢀ2ꢀyl
4ꢀfluorophenyl
ND = Not determined
The introduction of electronꢀwithdrawing group on ring A regardless
of their quantity and position does not lead to significant NO
inhibitory activity, as displayed by compounds 1eꢀi in comparison to
its unsubstituted compound 1a.
This observation suggested that low electron density of ring A was
disfavour in enhancing the activity. A similar trend was also clearly
demonstrated by the respective halogenated diarylpentadienone
analogues, 3c, 3d, 3f, 4d, 5a, 5c and 6b, whereby the presence of
electronꢀwithdrawing group in ring A was accompanied with
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DOI: 10.1039/C4MD00541D
reduced inhibitory activity. This finding is consistent with the Among the 4ꢀfluorophenyl containing analogues including 1e, 3c,
previous reports, which suggested that the introduction of halogen 4d, 6b and 7e, the highest inhibitory activity was displayed by
moiety in phenyl ring of chalcone19 and NSAIDs derivative U0126,20 analogue 7e. This notable result illustrate that the presence of 4’ꢀ
lowered the antiꢀinflammatory activity.
hydroxyl group on ring B, emerges to be a very important
requirement for antiꢀinflammatory activity, as compound 7e
exhibited three fold better activity than compound 4d (4’ꢀ
dimethylamino substituted ring B) and two fold better inhibition than
6b (3’,5’ꢀdimethoxyꢀ4’ꢀhydroxy substituted ring B). This trend may
be explained by the fact that hydroxyl group can act as a stronger
hydrogen bond acceptor than dimethylamino group in physiological
pH environment. In this slightly basic condition, the hydroxyl group
would form the negatively charged oxygen atom and possesses a
stronger nucleophilic character, which in turn increases its tendency
as hydrogen acceptor, in comparison to dimethylamino group. In
addition, the bulky nature of 4’ꢀdimethylamino and 3’,5’ꢀdimethoxyꢀ
4’ꢀhydroxy may also contribute to its mild activity. On the other
hand, the introduction of 4’ꢀmethoxy group in ring B (compound 3c)
did not improve the NO inhibitory activity. This must be due to its
lower affinity for hydrogen bonding as compared to hydroxyl
moiety.
In contrast to the halogenated compounds previously mentioned with
relatively poor activities, the analogues containing paraꢀhydroxylꢀ
bearing halogenated ring A such as compounds, 3j and 3k showed a
significant NO inhibition property. This observation implied that the
presence of 4ꢀhydroxyl group in ring A may have veiled the negative
impact of the electronꢀwithdrawing group.
We also investigated the role of electronꢀdonating group (methoxy
and methyl) in ring A on the antiꢀinflammatory activity in series 1.
The methoxylated compound 1d was twoꢀfold less active than the
unsubtituted ring A analogue, 1a, suggesting the high electron
density is not important in improving NO inhibition. The same
conclusion could also be drawn based on the reduced bioactivity
displayed by analogues 3b, 3e, 3g, 4b, and 5b. This was further
supported by Manna et al,21 who showed that electronꢀreleasing
group (methoxy, methyl, diꢀmethoxy) of 3ꢀcyanoꢀ2(1H) pyridones
resulted in weaker antiꢀinflammatory activity, possibly due to steric
hindrance and/or lack of hydrogen bonding capability.20
The importance of 4’ꢀhydroxyl group over 4’ꢀmethoxy and
unsubstituted ring B could be clearly seen when ring A is composed
of 5ꢀmethylthiophenꢀ2ꢀyl moiety. Compound 7d exhibited six fold
On the other hand, the replacement of phenyl group with
heterocyclic scaffold, such as thiophenyl (1j) and furanyl (1k) in
ring A resulted in mild NO inhibitory activity with IC50 values of
68.6 µM and 59.0 µM, respectively. It is worth noting that furanyl
exhibits a higher potential than thiophenyl ring due to its capability
of forming hydrogen bond via its nonꢀbonding electron pair, in
comparison to sulphur species in which hydrogen bond formation is
more susceptible towards aromatic π system, rather than with the
unshared electron pair localized on S atom.22 Further variation in NO
inhibition activity of heterocycleꢀcontaining analogues are
exemplified by compounds 3lꢀq and 4eꢀf, where the furanylꢀ
containing ring A (3q) exhibited the highest activity. This result
were in close agreement with those of Qiu et al, which stated that
thiopheneꢀbearing 4ꢀarylidene curcumin analogues demonstrated a
reduction in activity in comparison to furanylꢀbearing, indicating the
S replacement of the O atom in heterocycle made a negative impact
to its activity.23
better NO inhibition activity than compound 1j,
a ring B
unsubstituted 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone while compound 3l
displayed poor activity. These findings correlated well with the
results of previous work in which a compound possessing an α,βꢀ
unsaturated ketone with 4’ꢀhydroxylphenyl ring significantly
suppressed the TNFꢀαꢀinduced NFꢀκB activation.7 On the other
hand, a significant role of 4’ꢀmethoxy over 4’ꢀdimethylamino group
on ring B in enhancing the NO inhibition has been proven by
compounds 3p and 3q over compounds 4e and 4f, when ring A is
composed of thiophenyl and furanyl rings. This could be clarified by
the fact that the oxygen atom of the methoxy group has higher
hydrogen bond accepting character as compared to the nitrogen atom
of dimethylamino. Although the pKa of dimethylamino group (pka
~10.61) is slightly higher than methoxy group (pKa ~10.55), which
confer its higher basicity with increasing hydrogen bond acceptor
effect, the introduction of two methyl groups increases the steric
bulk around the nitrogen atom. As the result water molecules are
hindered from solvating the protonated form of dimethylamino
compounds and prevents stabilization of this ion. This, in turn,
decreases its hydrogen bond acceptor character of the respective
compounds24.
The strategy to demethylate the methoxyꢀbearing diarylpentadienone
to form hydroxylated analogues had increased the bioactivity
significantly, as displayed by analogues in series 7. The 5ꢀ
methylthiophenꢀ2ꢀyl bearing analogue, compound 7d recorded as the
most potent NO inhibitor with IC50 value of 10.7 µM. It is worth
noting that the introduction of an electronꢀwithdrawing (fluoro) and
heterocyclic groups positively affects the NO inhibitory property in
this series, in comparison to the respective analogues in series 1ꢀ6.
This change in inhibitory activity may be rationalized by the crucial
factor of the 4’ꢀhydroxyl group in ring B, which mitigated the
negative contributions of two groups mentioned above, resulting in
the alteration of their interaction with the mediators.
In summary, both rings (A and B) play a significant role in
enhancing the antiꢀinflammatory activity in the diarylpentadienꢀ1ꢀ
one compounds. It is apparent that the presence of 4’ꢀhydroxyl group
on ring B (analogues of series 7) and either a 3ꢀhydroxyl or 4ꢀ
hydroxyl groups on ring A (such as compounds 1c and 3hꢀk) are the
features that contribute to NO inhibitory activity.
Quantity structureꢀactivity relationship (QSAR) analysis
2D QSAR
Further structureꢀactivity analyses of the variation in ring B were
also performed to correlate the structural features with the NO
inhibition activity. Among the diarylpentadienone analogues having
phenyl group in ring A, all compounds composed of phenyl (1a), 4’ꢀ
methylphenyl (2a), 4’ꢀmethoxyphenyl (3a), 4’ꢀdimethylaminophenyl
(4a) and 3,5ꢀdimethoxyꢀ4ꢀhydroxyphenyl (6a) on ring B displayed
mild to poor activity (see Table 1). Based on this result, it is possible
to assume that difference in electron density on ring B did not
significantly affected the inhibitory potency of compounds.
The 2D QSAR was performed to establish a consistent relationship
between the biological activity of drugs and the effect of
physicochemical and structural parameters on the chemical
reactivity. The following equation shows the details of the optimal
QSAR model generated by the GFA studies utilizing Discovery
Studio 3.1, where r2 is the correlation coefficient against 17 training
compounds, r2 (adj) is the r2 adjusted value for the number of terms
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4MD00541D
in the model, and r2(pred) is the predictive r2 determined for the eight coefficient of variation) and the LOOꢀpredicted variance was found
test compounds. Only linear terms were used for the development of to be 61.10%; the lower Friedman L.O.F value of 0.0199 signifies
the model, and pIC50 (ꢀlog IC50) was used as the dependent variable. that the GFA model fitted the data well.
The list of molecules involved and their calculated parameter values
According to the GFA equation, three dominant molecular fragments
(Fig. 3) were determined by this model. All of the molecular
fragments, A (EPFP_6: ꢀ66486510), B (EPFP_6: 838692040) and C
(EPFP_6: ꢀ653303157), have positive regression coefficients,
indicating that all three features are important for the inhibition
potency. Molecular fragment B, with a hydroxyl group at the paraꢀ
position in ring B, enhances the NOꢀinhibitory activity more than the
for the model are presented in Table 2.
Table 2. IC50, pIC50, number of rotatable bonds and MFPSA values of active
compounds.
Number of
rotatable
bond
Compound
1a
1c
1d
1j
1k
3a
3h
3i
3j
3k
3m
3o
3p
3q
4a
4b
4d
5b
6a
6b
7a
7b
7c
7d
7e
IC50
pIC50
4.351
4.583
4.148
4.164
4.23
4.339
4.787
4.669
4.63
MFPSA
0.072
0.15
0.098
0.18
0.115
0.098
0.178
0.167
0.153
0.161
0.134
0.164
0.206
0.154
0.07
0.091
0.068
0.09
0.178
0.173
0.22
0.283
0.227
0.247
0.077
fragments
A and C. This trend has been substantiated by
44.6 ± 2.5
26.1 ± 0.1
71.2 ± 5.1
68.6 ± 2.6
59.0 ± 6.0
45.8 ± 1.5
16.3 ± 2.0
21.5 ± 2.3
23.5 ± 2.4
21.8 ± 2.0
39.8 ± 2.3
57.4 ± 1.2
47.9 ± 5.5
27.5 ± 0.1
73.6 ±7.5
77.4 ± 6.2
63.8 ± 4.5
70.5 ± 2.1
42.0 ± 1.6
39.3 ± 0.1
16.4 ± 0.3
27.2 ± 1.1
27.1 ± 0.7
10.2 ± 0.6
14.8 ± 1.9
4
4
5
4
4
5
6
6
5
5
5
5
5
5
5
6
5
5
6
6
4
4
5
4
demethylated compounds 7a, 7d and 7e, which displayed stronger
activity than that of their respective methoxyꢀcontaining
diarylpentadienone (compounds 3b, 3l and 3c).
The number of rotatable bonds is also an important descriptor
influencing the activity. GFA analysis suggested that the number of
rotatable bond present in the diarylpentadienone system should never
be higher than five, above which causes NO inhibition by the
molecules.
4.662
4.34
4.241
4.32
Additionally, the higher value of MFPSA is undesirable for potency
of the compounds. The effect of this factor is demonstrated by
comparing the NO inhibitory activities of compounds 1a, 1j and 1k,
which clearly indicated that the presence of a heterocyclic ring
moiety lowered the activity. However, this MFPSA descriptor only
plays a minor role in the activity because of its relatively small
coefficient value. In contrast, fragment B of the EPFP fingerprint
plays a larger role in enhancing NO inhibition. This trend is
displayed by the most potent candidates, compound 7d.
4.561
4.134
4.111
4.195
4.152
4.377
4.406
4.784
4.565
4.567
4.99
A randomization test at a 95% confidence level was performed to
determine the significance and reliability of the generated GFA data,
and the results are shown in Table 3. The resulting random r2 value
of 0.7798 is considerably lower than the nonꢀrandom r2 value of
0.9682 from the GFA equation, suggesting that the model generated
was significant and was not obtained by chance.
4.831
4
GFA equation:
EPFPs code
Fragment fingerprint Regression
coefficient
pIC50 = 3.8276 + 0.2744 * Count<EPFP_6:ꢀ66486510> + 0.52132 *
Count<EPFP_6:838692040> 0.3257 Count<EPFP_6:ꢀ
+
*
A
OH
+ 0.2744
653303157> − 0.0035719 * Molecular_PolarSurfaceArea + 0.43089
* <5.2815−Num_RotatableBonds>
EPFP_6: ꢀ
66486510
*
*
Parameter values generated:
n (number of compounds in
analysis)=25; r2 = 0.9681; r2(adj) = 0.9535; r2(pred) = 0.9182; RMS
(root mean square) residual error = 0.0507; Friedman L.O.F (lack of
fit) = 0.0199; and q2 (cross validation correlation coefficient)=
0.6110.
*
B
*
*
*
+ 0.52132
+ 0.3257
EPFP_6:
838692040
O
*
The term EPFP_6 is a fingerprint for molecular characterization. The
use of this descriptor in model generation allows the identification of
the molecular features that favor inhibition and are nonꢀpromoting.
Num_rotatableBonds is the number of rotatable bonds, and
Molecular_FractionalPolarSurfaceArea (MFPSA) is the total surface
belonging to polar atoms and is calculated as the ratio of the polar
surface area divided by the total surface area. These types of
descriptors permit us to predict the transport properties of drugs.
C
*
EPP_6: ꢀ
653303157
*
O
*
Fig. 3 EPFPs equation descriptors
Both the fingerprint EPFP_6, and Num_rotatableBonds show
positive effects on the NOꢀinhibitory potency, whereas
molecular_FractionalPolarSurfaceArea has the opposite effect.
Based on the GFA equation, 95.35% of the variance (adjusted
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DOI: 10.1039/C4MD00541D
Table 3. Randomization test of GFA equation.
Eq. No.
Model 1
0.9681
95%
r2 from nonꢀrandom model
Confidence level
Total trials
49
49
0.7798
±0.17
Nonꢀrandom r2 < random r2
Mean value of r2 form random trials
Standard deviation of random trials
3D QSAR
Fig. 4 Structural allignment of compounds using templateꢀbasd technique
based on core of analogue 7d
The use of 3DꢀQSAR analysis employing the comparative molecular
field analysis (CoMFA) method facilitated the correlation of the
biological activity with the 3D structures of potential drug molecules
as well as the van der Waals potentials and Coulombic terms, which
represent the steric and electrostatic potential factors.
Partial least squares (PLS) analysis was used to linearly correlate the
CoMFA interaction energies and the inhibitory activity. The IC50
values were converted into pIC50 (ꢀlog IC50) and used as the
dependent variables in the 3D QSAR analysis. The steric and
electrostatic CoMFA potentials were calculated at each lattice
intersection of a regularly spaced grid of 1.0 Å. The most potent
compound in the series, compound 7d, was used as a template, and
the rest of the molecules were aligned at the common α,βꢀ
unsaturated ketone fragment using a docking method. The final,
aligned conformation of all ligands is displayed in Fig. 4.
From the total of 25 compounds involved in this analysis, 17
molecules were incorporated into the training set and eight
candidates were in the test set in order to generate the CoMFA
model (Table 2). The statistical validity of the model generated was
judged by the high values of q2 (more than 0.5) and r2 (more than
0.9). The 3DꢀQSAR model gave a high crossꢀvalidated q2 value of
0.536 and a nonꢀcrossꢀvalidated correlation coefficient r2 of 0.988,
which signified the correlation. A graph depicting the actual versus
predicted activity of the training set and the test set compounds is
shown in Fig. 5. The CoMFA contour maps developed were
displayed in Fig. 6A and Fig. 6B, denoting the steric and
electrostatic potential factors, respectively.
Fig. 5 The actual versus predicted activity of training and test sets
In Fig. 6B, the blue region represents the favorable electrostatic area
with positive charge, whereas the red region indicates the favorable
electrostatic area with negative charge.25 There is a major red region
around Cꢀ4’ of ring B in the electrostatic map, suggesting that a
substituent with a negative charge on the ring system would result in
higher potency. The presence of electronꢀdonating groups, such as
the 4’ꢀOH moiety in ring B, enhances the antiꢀinflammatory activity,
as demonstrated by compounds 3h, 7a and 7d. In contrast, the
analogues containing 4’ꢀCl, 4’ꢀN(CH3)2 and 3’,5’ꢀOCH3,4’ꢀOH in
ring B were found to be weak, as shown by compounds in series 4,
5 and 6. This finding is in agreement with the results of 2D QSAR
analysis earlier, where compounds possessing molecular fragment B
showed higher bioactivity. In the steric contour map (Fig. 6A), no
green region is observed, indicating that a bulky substituent is not
favorable to increase the NOꢀinhibition activity. Additionally, the
heavy yellow shadow on Cꢀ2,4 on ring A and Cꢀ3’,4’,5’ of ring B
implied that a less bulky functional group is preferred to enhance the
activity.
A
B
Fig. 6 (A) Steric contour maps. Green region represent favorable bulky
groups region, while yellow maps represent disfavorable bulky group area.
(B) Electrostatic potential contour maps. Blue region indicate positive charge
electrostatic favorable areas whereas, red contour electronegative groups are
favorable in enhancing activity.
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4MD00541D
toxicity caused by the drugꢀlike molecules (e.g., liver injury). All
compounds except 1c and 3h are hepatotoxins based on the
calculated results.
ADMET Analysis
ADMET analysis is a computationally designed tool for predicting
pharmacokinetic properties, including absorption, distribution,
metabolism, excretion and toxicity, for a collection of molecules
based on their molecular structure within an organism. This ADMET
prediction is crucial in drug discovery and development to improve
the efficiency of drug candidates and to reduce the cost and time by
optimizing the screening and testing only on the most promising
compounds with acceptable properties.
TOPKAT Analysis
Safe drugs with minimal deleterious side effects still remain the
major goal of medicinal chemistry. Additional toxicology studies
must be run to assess the potential toxicities caused by drugs, as they
may lead to drugꢀcandidate attrition during preclinical and clinical
development.29 To improve the efficiency of drug discovery,
The ADMET analysis of ten selected molecules was performed on analysis is conducted using Toxicity Prediction by Komputer
six important parameters, including the aqueous solubility (AS), Assisted Technology (TOPKAT). The main goal of this analysis is
human intestinal absorption (HIA), blood−brain barrier (BBB), to determine the potential ecotoxicity, toxicity, mutagenicity, and
cytochrome P450 2D6 (CYP2D6), plasma protein binding (PPB), reproductive or developmental toxicity of the selected compounds.
and hepatotoxicity (HT), via Discovery Studio 3.1. The ADMET
Table 4. Result of ADMET predictions on selected parameters of
prediction results were compiled in Table 4.
selected compounds.
According to Lipinski,26 solubility is still the most important issue in
drug discovery because many negative impacts can be intensified by
lowerꢀsolubility drugꢀlike molecules, including relatively poor
absorption and bioavailability after oral dosing. Thus, improved
absorption and solubility are necessary to enhance the
pharmacokinetic properties in the human body.
AS
HIA
BBB
High
CYP2D6
Nonꢀ
HT
PPB
3
Good
Good
Nonꢀ
Bound
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
inhibit
Nonꢀ
hepatotoxin
Nonꢀ
hepatotoxin
Hepatotoxin
20 Low
21 Low
22 Low
23 Low
41 Good
42 Good
43 Good
45 Low
46 Low
Good
Good
Good
Good
Good
Good
Good
Good
Good
High
Bound
Bound
Bound
Bound
Bound
Bound
Bound
Bound
Bound
High
Based on the ADMET findings, compounds 1c, 7a, 7b, and 7c
displayed sufficient aqueous solubility and intestinal absorption,
indicating that they can serve as good oral drugs, in comparison to
3hꢀk, 7d, and 7e that had poor aqueous solubility.
High
Hepatotoxin
Hepatotoxin
Hepatotoxin
Hepatotoxin
Hepatotoxin
Hepatotoxin
Hepatotoxin
High
The blood−brain barrier (BBB) is normally associated with the
delivery process involved in central nervous system (CNS). The
BBB penetration implies the ability of drugꢀlike molecules to pass
through the blood−brain barrier to reach their therapeutic target in
the brain. High BBB permeation is crucial for CNSꢀactive
compounds but is undesirable for CNSꢀinactive compounds in order
to avoid CNS side effects.27 As shown in Table 4, all the ten
compounds possessed medium to very high BBB penetration,
signifying that they are effective candidates for treating CNS related
disorders.
High
Medium
Medium
High
inhibit
Nonꢀ
inhibit
Very
high
AS = aqueous solubility; HIA = human intestinal absorption; BBB = blood brain barrier;
CYP2D6 = cytochrome P450 2D6; PPB = protein plasma binding; HT = hepatotoxicity
Cytochrome P450 2D5 (CYP2D6) is an enzyme involved in the
metabolism of drugs in the liver, and its inhibition is usually caused
by the drugꢀdrug interactions. These drugꢀdrug interactions normally
occur when two drugs are orally coꢀadministered and compete for
the same metabolizing enzyme (involved in ADME), causing one
drug to be bound by isozyme, while the other drug is excluded from
metabolism.28 Notably, whenever the drug is metabolized too
quickly, the drug potency will decrease, but when the drug is
metabolized too slowly, the concentration may increase to a toxic
level. As presented, all compounds show negative results for
CYP2D6 inhibition, indicating their tolerance towards CYP2D6.
Toxicity studies (TOPKAT) follow the same procedure as used in
calculating the ADMET properties. In this study, the same ten
selected molecules were analyzed using toxicity predictors,
including the aerobic biodegradability, mutagenicity, rodent
carcinogenicity, ocular irritancy, skin irritancy and skin sensitization.
The results of TOPKAT are presented in Table 5.
Based on the toxicityꢀprediction findings, all of the selected
compounds are nonmutagenic, and nonbiodegradable, except for
compound 3h. Unfortunately, compounds 7a and 7d are predicted to
be carcinogenic. In addition, all candidates are skin sensitizers, and
compounds 3hꢀk, 7d, and 7e are ocular irritants. Compounds 7d and
7e are expected to exhibit skin irritancy.
Plasma proteins may adsorb a significant amount of drug molecules
once they have entered the bloodstream and to be distributed to their
specific therapeutic targets. Protein plasma binding (PPB) can affect
the pharmacokinetics of drug substances because the bound
molecules are temporarily shielded from metabolism. Hence, only
the unbound drugs exhibit noteworthy pharmacological effects. In
this experiment, all compounds displayed strong binding interactions
between the drug molecules and plasma protein, which suggested
that dosing issues must be addressed to achieve the desirable
therapeutic concentration for treatment. The last descriptor to be
considered is hepatotoxicity, which predicts the potential organ
In an attempt to develop new NO inhibitors as potent antiꢀ
inflammatory agents, a series of 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone
analogues were synthesized and evaluated for their biological
activities. Compound 7d is the most promising NO inhibitor from
the entire series. The combination of two computational techniques,
the 2D and 3D QSAR has helped provide the most significant
correlation of structural descriptors and steric and electrostatic
This journal is © The Royal Society of Chemistry 2012
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DOI: 10.1039/C4MD00541D
factors with the biological reactivity. In addition, ADMET and
TOPKAT analyses facilitated the understanding of the drug
efficiency and possible toxicity of each compounds. The information
obtained in this study suggested that the paraꢀhydroxyl group on
ring B and an α,βꢀunsaturated ketone moieties are important for the
NOꢀinhibition activity.
Chemical stability test
There are several drawbacks that limit the potential development of
curcumin as a therapeutic agent. Various clinical studies strongly
indicate that one such drawback is its low stability. Stability of a
compound in solution is necessary for in vivo oral dosing,
particularly in acidic, basic and physiological conditions of the
gastrointestinal (GI) tract in order to improve compounds
pharmacokinetic (PK) performance and the in vivo pharmacological
activity. Therefore, we tested the chemical stability of the most
active compound, 7d and curcumin in phosphate buffer containing
1% DMSO in different pH’s including the acidic (pH 2.4),
physiological (pH 7.0) and basic (pH 12.7) conditions, using an
absorption spectrum assay. Fig. 7 illustrates the changes in
ultraviolet absorptions of curcumin and compound 7d, respectively.
Curcumin is very stable in acidic environment, but the poor chemical
stability of curcumin could be observed from its maximal absorption
peaks gradually decreased over time at pH 7. However, the stability
of curcumin is increases at higher pH (>11.7). Conversely, the
absorption peak of the compound 7d showed no significant changes
in all different pH conditions, which indicated that our most active
compound was chemically more stable in vitro, in comparison to
curcumin.
Table 5. Result of TOPKAT analysis of selected compounds.
AB
AM
RC
OI
SI
SS
Nonꢀ
Nonꢀ
Nonꢀ
Nonꢀ
Nonꢀ
3
Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ
20 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ
21 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ
22 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ
23 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ
41 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ Nonꢀ
42 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ Nonꢀ Nonꢀ
43 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ
45 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
Nonꢀ Nonꢀ Nonꢀ
46 Degradable Mutagen Carcinogen Irritant Irritant Sensitizer
AB = aerobic biodegradability; AM = Ames mutagenicity; RC = rodent carcinogenicity;
OI = ocular irritancy; SI = skin irritancy; SS = skin sensitization
Fig. 7 Ultravioletꢀvisible absorption spectra of curcumin and 7d at different pH
.
8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4MD00541D
Cꢀ5”), 128.4 (Cꢀ3’, Cꢀ5’), 128.2 (Cꢀ2’, Cꢀ6’), 127.3 (Cꢀ2”, Cꢀ6”),
126.9 (Cꢀ3), 125.4 (Cꢀ1). EIMS, m/z: 234 (M+).
Conclusions
In an attempt to develop new NO inhibitors as potent antiꢀ
inflammatory agents, a series of 1,5ꢀdiphenylpentaꢀ2,4ꢀdienꢀ1ꢀ
one analogues were synthesized and evaluated for their
biological activities. Compound 7d is the most promising NO
inhibitor from the entire series. The combination of two
computational techniques, the 2D and 3D QSAR has helped
provide the most significant correlation of structural descriptors
and steric and electrostatic factors with the biological reactivity.
In addition, ADMET and TOPKAT analyses facilitated the
understanding of the drug efficiency and possible toxicity of
each compounds. The information obtained in this study
suggested that the paraꢀhydroxyl group on ring B and an α,βꢀ
unsaturated ketone moieties are important for the NOꢀinhibition
activity.
(3ꢀHydroxyphenyl)ꢀ5ꢀphenylpentaꢀ2,4ꢀdienꢀ1ꢀone (1c)
Yellow solid (21.2%), m.p. 115ꢀ116 °C. 1H NMR (500 MHz, CDCl3,
δ ppm): 6.36 (br. s, 1H, 5’ꢀOH), 7.03 ꢀ 7.06 (m, 2H, 3ꢀH, 4ꢀH), 7.09
(d, 1H, 1ꢀH), 7.34 ꢀ 7.39 (m, 5H, 2”ꢀH, 3”ꢀH, 4”ꢀH, 5”ꢀH, 6”ꢀH),
7.50 ꢀ 7.54 (m, 3H, 2’ꢀH, 3’ꢀH, 4’ꢀH), 7.59 (s, 1H, 6’ꢀH), 7.60 ꢀ 7.65
(m, 1H, 2ꢀH). 13C NMR (126 MHz, CDCl3, δ ppm): 190.7 (C=O),
156.4 (Cꢀ5’), 145.5 (Cꢀ2), 142.5 (Cꢀ4), 139.5 (Cꢀ1’), 136.0 (Cꢀ1”),
129.9 (Cꢀ3’), 129.3 (Cꢀ4”), 128.9 (Cꢀ3”, Cꢀ5”), 127.4 (Cꢀ2”, Cꢀ6”),
126.8 (Cꢀ3), 125.2 (Cꢀ1), 120.9 (Cꢀ2’), 120.3 (Cꢀ4’), 115.0 (Cꢀ6’).
EIMS, m/z: 250 (M+), HRESIMS m/z 249.0997 [MꢀH]ꢀ (calculated
for C17H14O2, 249.0994).
(3ꢀHydroxyꢀ4ꢀmethoxyphenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)pentaꢀ2,4ꢀ
dienꢀ1ꢀone (3h)
1
Yellow solid (54.9%), m.p. 72ꢀ73°C. H NMR (500 MHz, acetoneꢀ
d6, δ ppm): 3.83 (s, 3H, 4”ꢀOCH3), 3.93 (s, 3H, 4’ꢀOCH3), 6.91 ꢀ
7.01 (m, 3H, 3’ꢀH, 3ꢀH, 4ꢀH), 7.08 (d, J = 4.4 Hz, 2H, 3”ꢀH, 5”ꢀH),
7.31 (d, J = 14.7 Hz, 1H, 1ꢀH), 7.55 (d, J = 7.8 Hz, 3H, 6’ꢀH, 2”ꢀH,
6”ꢀH), 7.61 ꢀ 7.69 (m, 2H, 2ꢀH, 2’ꢀH), 8.73 (br. s, 1H, 5’ꢀOH). 13C
NMR (126 MHz, acetoneꢀd6, δ ppm): 187.1 (C=O), 160.5 (Cꢀ4”),
151.3 (Cꢀ4’), 147.6 (Cꢀ5’), 143.5 (Cꢀ2), 140.6 (Cꢀ4), 130.6 (Cꢀ1’),
129.1 (Cꢀ3), 128.6 (Cꢀ2”, Cꢀ6”), 125.1 (Cꢀ1), 124.1 (Cꢀ1”), 123.0
(Cꢀ2’), 114.5 (Cꢀ6’), 114.2 (Cꢀ3”, Cꢀ5”), 111.0 (Cꢀ3’), 55.3 (4”ꢀ
OCH3), 54.7 (4’ꢀOCH3). EIMS, m/z: 310 (M+), HRESIMS m/z
309.1209 [MꢀH]ꢀ (calculated for C19H18O4, 309.1205).
Experimental
Materials and instrumentations
All chemicals and reagents were purchased from Merck, Sigmaꢀ
Aldrich and Thermo Fischer Scientific. All solvents were dried and
distilled prior to use. The solvents used in the spectroscopic
measurements were of spectroscopic grade. The reaction mixtures
were extracted with organic solvents, dried over anhydrous
magnesium sulfate and then concentrated in vacuo. To monitor the
progress of a reaction, thinꢀlayer chromatography was performed on
aluminum TLC sheets of silica gel 60 F254 (layer thickness 0.2 mm)
from Merck. Column chromatography purification was performed on
silica gel 60 (70ꢀ230 mesh ASTM, Merck). Mass spectra were
measured on a GCMSꢀQP5050A (Shimadzu, Kyoto, Japan) Mass
Spectrometer. Highꢀresolution MS (HRMS) was determined using
Acquity Ultraꢀperformance Liquid Chromatography (UPLC)ꢀPDA
system coupled to Synapt HighꢀDefinition Mass Spectrometry
(HDMS) quadrapoleꢀorthogonal acceleration timeꢀofꢀflight (oaTOF)
detector (Waters Corporation, USA) and equipped with an ESI
source. Nuclear magnetic resonance spectra were recorded using a
Varian 500 MHz NMR Spectrometer (Varian Inc., Palo Alto, CA),
and the melting points were determined using a FisherꢀJohns melting
point apparatus.
(4ꢀHydroxyꢀ3,5ꢀdimethoxyphenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)pentaꢀ
2,4ꢀdienꢀ1ꢀone (3i)
Yellowish orange solid (15.4%), m.p. 90ꢀ91°C. 1H NMR (500 MHz,
acetoneꢀd6, δ ppm): 3.97 (s, 3H, 4”ꢀOCH3), 4.05 (s, 6H, 3’ꢀ, 5’ꢀ
OCH3), 7.10 (d, J = 8.6 Hz, 2H, 3”ꢀH, 5”ꢀH), 7.18 ꢀ 7.23 (m, 2H, 1ꢀ
H, 4ꢀH), 7.42 ꢀ 7.50 (m, 1H, 3ꢀH), 7.53 (s, 2H, 2’ꢀH, 6’ꢀH), 7.63 ꢀ
7.71 (m, 3H, 2ꢀH, 2”ꢀH, 6”ꢀH), 8.25 (br. s, 1H, 4’ꢀOH). 13C NMR
(126 MHz, acetoneꢀd6, δ ppm): 187.2 (C=O), 160.6 (Cꢀ4”), 147.7
(Cꢀ3’, Cꢀ5’), 143.7 (Cꢀ2), 141.0 (Cꢀ4), 140.7 (Cꢀ4’), 129.2 (Cꢀ2”, Cꢀ
6”), 129.1 (Cꢀ1’), 128.7 (Cꢀ1”), 125.1 (Cꢀ1), 124.2 (Cꢀ3), 114.3 (Cꢀ
3”, Cꢀ5”), 106.2 (Cꢀ2’, Cꢀ6’), 55.8 (3’, 5’ꢀOCH3), 54.8 (4”ꢀOCH3).
EIMS, m/z: 340 (M+), HRESIMS m/z 339.1314 [MꢀH]ꢀ (calculated
for C20H20O5, 339.1311).
(3ꢀChloroꢀ4ꢀhydroxyphenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)pentaꢀ2,4ꢀ
dienꢀ1ꢀone (3j)
Yellow solid (34.7%), m.p. 143ꢀ144°C. 1H NMR (500 MHz, CDCl3,
δ ppm): 3.85 (s, 3H, 4”ꢀOCH3), 6.11 (br. s, 1H, 4’ꢀOH), 6.90 ꢀ 6.93
(m, 3H, 4ꢀH, 3”ꢀH, 5”ꢀH), 6.98 ꢀ 7.02 (m, 2H, 1ꢀH, 3ꢀH), 7.11 (d, J =
8.6 Hz, 1H, 3’ꢀH), 7.45 (d, J = 8.3 Hz, 2H, 2”ꢀH, 6”ꢀH), 7.59 ꢀ 7.64
(m, 1H, 2ꢀH), 7.86 (d, J = 8.6 Hz, 1H, 2’ꢀH), 8.04 (s, 1H, 6’ꢀH). 13C
NMR (126 MHz, CDCl3, δ ppm): 183.1 (C=O), 159.1 (Cꢀ4’), 156.4
(Cꢀ4”), 139.7 (Cꢀ2), 136.7 (Cꢀ4), 129.2 (Cꢀ2”, Cꢀ6”), 127.0 (Cꢀ1’, Cꢀ
1”), 126.1 (Cꢀ3), 124.9 (Cꢀ6’), 124.5 (Cꢀ1), 120.9 (Cꢀ5’), 119.9 (Cꢀ
3’), 111.9 (Cꢀ2’), 110.0 (Cꢀ3”, Cꢀ5”), 50.8 (4’ꢀOCH3). EIMS, m/z:
314.5 (M+), HRESIMS m/z 313.0712 [MꢀH]ꢀ (calculated for
C18H15ClO3, 313.0710).
Chemistry
General procedure for the synthesis of I (Series 1ꢀ6). Equimolar
quantities of the appropriate cinnamaldehyde (2 mmol) and
substituted acetophenone (2 mmol) were dissolved in 5 ml ethanol.
The resulting mixture was stirred for 5 min, and then 1 ml of 6M
NaOH was added dropwise over three minutes. After the addition
was completed, the reaction mixture was stirred overnight at room
temperature. The adduct solution was poured into crushed ice and
neutralized with diluted HCl. The solution was extracted by ethyl
acetate (3 x 10 ml). The organic layer was dried over anhydrous
MgSO4 and evaporated under reduced pressure. Finally, the crude
products were purified by column chromatography.30 The purity of
all compounds were >95% based on HPLC analysis.
(2ꢀFluoroꢀ4ꢀhydroxyphenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)pentaꢀ2,4ꢀ
dienꢀ1ꢀone (3k)
Orange solid (51.1%), m.p. 150ꢀ151°C. 1H NMR (500 MHz,
acetoneꢀd6, δ ppm): 3.84 (s, 3H, 4”ꢀOCH3), 6.68 (dd, J = 13.0, 2.0
Hz, 1H, 5’ꢀH), 6.80 (dd, J = 8.6, 2.0 Hz, 1H, 3’ꢀH), 6.97 (d, J = 8.6
Hz, 3H, 3”ꢀH, 5”ꢀH), 7.00 (d, J = 2.7 Hz, 1H, 1ꢀH), 7.05 ꢀ 7.14 (m,
2H, 3ꢀH, 4ꢀH), 7.38 (br. s, 1H, 4’ꢀOH), 7.51 (dd, J = 14.8, 8.2 Hz,
1H, 2ꢀH), 7.57 (d, J = 8.6 Hz, 2H, 2”ꢀH, 6”ꢀH) 7.72 ꢀ 7.80 (t, 1H, 2’ꢀ
H). 13C NMR (126 MHz, acetoneꢀd6, δ ppm): 185.8 (C=O), 162.8
(Cꢀ4’), 161.6 (Cꢀ6’), 160.5 (Cꢀ4”), 143.8 (Cꢀ2), 141.1 (Cꢀ4), 128.9
(Cꢀ2’), 128.6 (Cꢀ2”, Cꢀ6”), 127.6 (Cꢀ1”), 127.6 (Cꢀ3), 124.7 (Cꢀ1),
118.4 (Cꢀ1’), 114.0 (Cꢀ3”, Cꢀ5”), 111.9 (Cꢀ3’), 102.8 (Cꢀ5’), 54.6
1,5ꢀDiphenylpentaꢀ2,4ꢀdienꢀ1ꢀone (1a)
Yellow solid (47.8%), m.p. 102ꢀ103 °C (lit,31 102ꢀ103 °C). 1H NMR
(500 MHz, CDCl3, δ ppm): 7.02 ꢀ 7.07 (m, 2H, 3ꢀH, 4ꢀH), 7.10 –
7.13 (d, 1H, 1ꢀH), 7.32 ꢀ 7.43 (m, 3H, 3”ꢀH, 4”ꢀH, 5”ꢀH), 7.48 ꢀ 7.55
(m, 4H, 3’ꢀH, 5’ꢀH, 2”ꢀH, 6”ꢀH), 7.58 (d, J = 7.3 Hz, 1H, 4’ꢀH), 7.59
ꢀ 7.65 (m, 1H, 2ꢀH), 7.99 (d, J = 7.02 Hz, 2H, 2’ꢀH, 6’ꢀH). 13C NMR
(126 MHz, CDCl3, δ ppm): 190.5 (C=O), 144.9 (Cꢀ2), 141.9 (Cꢀ4),
138.2 (Cꢀ1’), 136.1 (Cꢀ1”), 133.1 (Cꢀ4’), 128.9 (Cꢀ4”), 128.5 (Cꢀ3”,
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C4MD00541D
(4”ꢀOCH3). EIMS, m/z: 298 (M+), HRESIMS m/z 297.1007 [MꢀH]ꢀ 270 (M+), HRESIMS m/z 269.0717 [MꢀH]ꢀ (calculated for
(calculated for C18H15FO3, 297.1005).
C16H14O2S, 269.0715).
(4ꢀFluorophenyl)ꢀ5ꢀ(4ꢀhydroxyphenyl)pentaꢀ2,4ꢀdienꢀ1ꢀone (7e)
Yellow solid (21.9%), m.p. 155ꢀ156°C. 1H NMR (500 MHz, acetone
d6, δ ppm): 6.89 (d, J = 8.6 Hz, 2H, 3”ꢀH, 5”ꢀH), 7.03 ꢀ 7.14 (m, 2H,
1ꢀH, 3ꢀH), 7.22 ꢀ 7.36 (m, 3H, 4ꢀH, 3’ꢀH, 5’ꢀH), 7.49 (d, J = 8.3 Hz,
2H, 2”ꢀH, 6”ꢀH), 7.54 ꢀ 7.66 (m, 1H, 2ꢀH), 8.13 (dd, J = 17.6, 8.4
Hz, 2H, 2’ꢀH, 6’ꢀH), 8.82 (br. s, 1H, 4”ꢀOH). 13C NMR (126 MHz,
acetoneꢀd6, δ ppm): 189.0 (C=O), 166.6 (Cꢀ4’), 160.9 (Cꢀ4”), 145.8
(Cꢀ2), 142.3 (Cꢀ4), 131.1 (Cꢀ1’), 131.0 (Cꢀ1”), 129.1 (Cꢀ2’, Cꢀ6’),
128.7 (Cꢀ2”, Cꢀ6”), 124.9 (Cꢀ3), 123.9 (Cꢀ1), 115.7 (Cꢀ3’, Cꢀ5’),
114.6 (Cꢀ3”, Cꢀ5”). EIMS, m/z: 268 (M+), HRESIMS m/z 267.0902
[MꢀH]ꢀ (calculated for C17H13FO2, 267.0900).
General procedure for the synthesis of II (Series 7). The
methoxylated diarylpentanoids (I, 0.5 mmole), was dissolved in 10
ml of dichloromethane. To it 1.5 ml of boron tribromide was added
slowly and stirred for 1h and continued for overnight at room
temperature. The reaction solution was then poured into crushed ice,
extracted with ethyl acetate and dried over anhydrous MgSO4. The
organic layer was concentrated under vacuum and the resulting
products were purified using column chromatography under
appropriate solvent system.32
1,5ꢀBis(4ꢀhydroxyphenyl)pentaꢀ2,4ꢀdienꢀ1ꢀone. (7a)
Yellow solid (30.7%), m.p. 173ꢀ174 ̊
C. 1H NMR (500 MHz,
Biology
acetoneꢀd6, δ ppm): 6.88 (d, J = 8.3 Hz, 2H, 3”ꢀH, 5”ꢀH), 6.96 (d, J
= 8.6 Hz, 2H, 3’ꢀH, 5’ꢀH), 7.02 ꢀ 7.08 (m, 2H, 3ꢀH, 4ꢀH), 7.26 (d, J =
15.0 Hz, 1H, 1ꢀH), 7.48 (d, J = 8.6 Hz, 2H, 2”ꢀH, 6”ꢀH), 7.50 ꢀ 7.58
(m, 1H, 2ꢀH), 7.98 (d, J = 8.6 Hz, 2H, 2’ꢀH, 6’ꢀH), 8.87 (br. s, 1H,
4”ꢀOH), 9.32 (br. s, 1H, 4’ꢀOH). 13C NMR (126 MHz, acetoneꢀd6, δ
ppm): 187.2 (C=O), 161.7 (Cꢀ4’), 158.7 (Cꢀ4”), 143.9 (Cꢀ2), 141.2
(Cꢀ4), 130.6 (Cꢀ2’, Cꢀ6’), 130.4 (Cꢀ1’), 128.9 (Cꢀ2”, Cꢀ6”), 128.2
(Cꢀ1”), 124.5 (Cꢀ3), 123.8 (Cꢀ1), 115.8 (Cꢀ3’, Cꢀ5’), 115.3 (Cꢀ3”, Cꢀ
5”). EIMS, m/z: 266 (M+), HRESIMS m/z 265.0947 [MꢀH]ꢀ
(calculated for C17H14O3, 265.0943).
Cell culture. The RAW 264.7 murine macrophages cells obtained
from American Type Culture Collection (ATCC, Rockville, MD)
were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
containing 10% FBS and 1% penicillin/streptomycin in a 95% air
and 5% CO2 atmosphere at 37 °C.13
Nitrite determination (Griess Assay). The RAW 264.7 cells at 90ꢀ
95% confluency were detached and seeded (50000 cells/well) into a
96ꢀwell culture plate with 50 ꢁL of DMEM and incubated for 24 h.
The cells were then stimulated in 5 mg/mL of LPS (Escherichia coli,
serotype 0111:B4) and 1 ng/ml of interferonꢀgamma (IFNꢀγ) in the
presence or absence of test compounds for 17 hours. The nitrite
production was then determined by a Griess assay. Briefly, 50 ꢁl of
(2,4ꢀDihydroxyphenyl)ꢀ5ꢀ(4ꢀhydroxyphenyl)pentaꢀ2,4ꢀdienꢀ1ꢀ
one (7b)
Orange solid (22.0%), m.p. 134ꢀ135°C. 1H NMR (500 MHz,
acetoneꢀd6, δ ppm): 6.37 (s, 1H, 5’ꢀH), 6.48 (d, J = 8.8 Hz, 1H, 3’ꢀ
H), 6.90 (d, J = 6.8 Hz, 2H, 3”ꢀH, 5”ꢀH), 7.04 ꢀ 7.20 (m, 2H, 1ꢀH, 3ꢀ
H), 7.36 (d, J = 14.7 Hz, 1H, 4ꢀH), 7.51 (d, J = 6.8 Hz, 2H, 2”ꢀH, 6”ꢀ
H), 7.61 ꢀ 7.73 (m, 1H, 2ꢀH), 7.95 (d, J = 8.3 Hz, 1H, 2’ꢀH), 8.95
(br. s, 1H, 4”ꢀOH), 9.66 (br. s, 1H, 4’ꢀOH), 13.63 (br. s, 1H, 6’ꢀOH).
13C NMR (126 MHz, acetoneꢀd6, δ ppm): 187.1 (C=O), 161.6 (Cꢀ4’,
Cꢀ6’), 158.5 (Cꢀ4”), 143.7 (Cꢀ2), 141.0 (Cꢀ4), 130.4 (Cꢀ2’), 130.1
(Cꢀ2”, Cꢀ6”), 128.7 (Cꢀ1”), 127.9 (Cꢀ3), 124.2 (Cꢀ3”, Cꢀ5”), 123.6
(Cꢀ1), 115.6 (Cꢀ1’), 115.0 (Cꢀ3’, Cꢀ5’). EIMS, m/z: 282 (M+),
HRESIMS m/z 281.0895 [MꢀH]ꢀ (calculated for C17H14O4,
281.0892).
Griess
Reagent
(1%
sulfanilamide
and
0.1%
Nꢀ(1ꢀ
naphthyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid)
was added to 50 ꢁL of cell culture supernatant. The optical density
was measured at 550 nm with a microplate reader after 5 min of
incubation at room temperature.13
Cell cytotoxicity determination (MTT assay). To determine that
the observed nitric oxide inhibition was not falsely positive due to
cytotoxic effects, cytotoxicity assay was also performed following
the culture. After removal of the supernatant, 100 µL DMEM and 20
µL of 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
(MTT, 5 mg/ml) were added to all of the wells. The plate was then
incubated in 5 % CO2 at 37°C for 4 h. The supernatants in all wells
were discarded, and the formazan crystals formed were dissolved in
dimethyl sulfoxide (DMSO) and further incubated for 15 min at
room temperature. The absorbance was measured at 570 nm using a
microplate reader.13
(2ꢀHydroxyꢀ4ꢀmethoxyphenyl)ꢀ5ꢀ(4ꢀhydroxyphenyl)pentaꢀ2,4ꢀ
dienꢀ1ꢀone (7c)
Brown solid (67.7%), m.p. 192ꢀ193°C. 1H NMR (500 MHz, acetoneꢀ
d6, δ ppm): 3.90 (s, 3H, 4’ꢀOCH3), 6.48 (s, 1H, 5’ꢀH), 6.54 (d, J =
8.8 Hz, 1H, 3’ꢀH), 6.91 (d, J = 8.3 Hz, 2H, 3”ꢀH, 5”ꢀH), 7.06 ꢀ 7.21
(m, 2H, 1ꢀH, 3ꢀH), 7.38 (d, J = 14.7 Hz, 1H, 4ꢀH), 7.52 (d, J = 8.3
Hz, 2H, 2”ꢀH, 6”ꢀH), 7.65 ꢀ 7.74 (m, 1H, 2ꢀH), 8.00 (d, J = 8.8 Hz,
1H, 2’ꢀH), 8.91 (br. s, 1H, 4”ꢀOH), 13.69 (br. s, 1H, 6’ꢀOH). 13C
NMR (126 MHz, acetoneꢀd6, δ ppm): 187.1 (C=O), 166.5 (Cꢀ4’, Cꢀ
6’), 164.7 (Cꢀ4”), 144.9 (Cꢀ2), 142.2 (Cꢀ4), 131.8 (Cꢀ2’), 129.0 (Cꢀ
2”, Cꢀ6”), 127.8 (Cꢀ1”), 124.0 (Cꢀ3), 122.2 (Cꢀ1), 115.7 (Cꢀ3”, Cꢀ
5”), 115.4 (Cꢀ1’), 107.8 (Cꢀ3’), 102.7 (Cꢀ5’), 55.3 (4’ꢀOCH3). EIMS,
m/z: 296 (M+), HRESIMS m/z 295.1053 [MꢀH]ꢀ (calculated for
C18H16O4, 295.1049).
Computational analysis
2DꢀQSAR. The QSAR model generated by the genetic function
approximation (GFA) technique is used to establish the important
features that contribute to the biological activity of a molecule, based
on the experimental pIC50 (ꢀlog IC50). The descriptors used include
the fingerprint EPFP_6, molecular polar surface area and number of
rotatable bonds.
5ꢀ(4ꢀHydroxyphenyl)ꢀ1ꢀ(5ꢀmethylthiophenꢀ2ꢀyl)pentaꢀ2,4ꢀdienꢀ
1ꢀone (7d)
Dark green solid (52.9%), m.p. 135ꢀ137°C. 1H NMR (500 MHz,
acetoneꢀd6, δ ppm): 2.88 (s, 3 H, 4’ꢀCH3), 6.89 (d, J = 8.3 Hz, 2H,
3”ꢀH, 5”ꢀH), 6.94 (d, J = 3.2 Hz, 1H, 3’ꢀH), 6.98 ꢀ 7.05 (m, 1H, 1ꢀ
H), 7.06 ꢀ 7.15 (m, 2H, 3ꢀH, 4ꢀH), 7.44 ꢀ 7.51 (m, 2H, 2”ꢀH, 6”ꢀH),
7.51ꢀ7.56 (m, 1H, 2ꢀH), 7.77 (d, J = 3.7 Hz, 1H, 2’ꢀH), 8.78 (br. s,
1H, 4”ꢀOH). 13C NMR (126 MHz, acetoneꢀd6, δ ppm): 181.2 (C=O),
160.5 (Cꢀ4”), 149.6 (Cꢀ1’), 144.0 (Cꢀ2), 143.7 (Cꢀ4), 141.6 (Cꢀ4’),
132.0 (Cꢀ2’), 129.0 (Cꢀ1”), 128.8 (Cꢀ2”, Cꢀ6”), 126.9 (Cꢀ3), 124.7
(Cꢀ3’) , 123.8 (Cꢀ1), 114.3 (Cꢀ3”, Cꢀ5”), 16.1 (4’ꢀCH3). EIMS, m/z:
3DꢀQSAR. Partial least squares (PLS) analysis was used to linearly
correlate the CoMFA interaction energies and the inhibitory activity.
The IC50 values were converted into pIC50 (ꢀlog IC50) and used as
dependent variables in this 3D QSAR analysis. The steric and
electrostatic CoMFA potentials were calculated at each lattice
intersection of a regularly spaced grid of 1.0 Å. The most potent
compound in the series, compound 7d, was used as a template, and
the rest of the molecules were aligned using a docking method.
10 | J. Name., 2012, 00, 1-3
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Journal Name
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View ArticAle ROnTliInCeLE
DOI: 10.1039/C4MD00541D
Then, the training set (17 compounds) and test set (8 compounds)
were generated after the minimization of ligands through a
CHARMm force field.
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ADMET and TOPKAT analysis. The tenꢀmost active compounds
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barrier (BBB), cytochrome P450 2D6 (CYP2D6), plasma protein
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(TOPKAT) analysis was run based on the aerobic biodegradability,
mutagenicity, rodent carcinogenicity, ocular irritancy, skin irritancy
and descriptors, and skin sensitization descriptors.
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compound 7d were prepared and diluted by phosphate buffer (pH of
2.4, 7.0, and 12.7), containing 1% dimethyl sulfoxide (DMSO), to a
final concentration of 20 ꢁM. The ultraviolet absorption spectra were
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This study was financially supported by Universiti Putra Malaysia
under Research University Grant Scheme (RUGS) (9301700). The
first author also gratefully acknowledges support by the Ministry of
Education Malaysia for funding her study under SLAB scheme.
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a Laboratory of Natural Products, Institute of Bioscience, Universiti Putra
Malaysia, 43400, Serdang, Selangor, Malaysia.
bDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia,
43400, Serdang, Selangor, Malaysia.
cDepartment of Food Science, Faculty of Food Science and Technology,
Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.
dDrug and Herbal Research Centre Faculty of Pharmacy, Universiti
Kebangsaan Malaysia, Jalan Raja Muda Abd. Aziz, 50300, Kuala
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eDepartment of Biochemistry, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang,
Selangor, Malaysia.
fDepartment of Biomedical Science, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.
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†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
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