Synthesis, screening and docking analysis of hispolon analogs as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2016.11.047

A series of 20 hispolons/dihydrohispolons were synthesized and characterized by spectral data. These compounds were subjected to in vitro antitubercular activity screening against Mycobacterium tuberculosis (H37Rv) strain. The synthesized compounds showed

Full text of DOI:10.1016/j.bmcl.2016.11.047

Accepted Manuscript
Synthesis, Screening and Docking Analysis of Hispolon analogs as Potential
Antitubercular Agents
Neduri V. Balaji, B. Hari Babu, Gottumukkala V. Subbaraju, Kurre Purna
Nagasree, Muthyala Murali Krishna Kumar
PII:
S0960-894X(16)31199-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.047
BMCL 24442
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2016
12 November 2016
17 November 2016
Please cite this article as: Balaji, N.V., Hari Babu, B., Subbaraju, G.V., Purna Nagasree, K., Krishna Kumar, M.M.,
Synthesis, Screening and Docking Analysis of Hispolon analogs as Potential Antitubercular Agents, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.047
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Synthesis, Screening and Docking Analysis of Hispolon analogs as
Potential Antitubercular Agents
Neduri V.Balajia, B.Hari Babua*, Gottumukkala V. Subbarajub, KurrePurna Nagasreec,
Muthyala Murali Krishna Kumarc*
aDepartment of Chemistry, Acharya Nagarjuna University, Guntur, AP-India
bNatsol Laboratories Private Limited, II Floor, Research & Development Building,
Ramky Commercial Hub, J. N. Pharmacity, Visakhapatnam, India
cMedicinal Chemistry Research Labs, College of Pharmaceutical Sciences, Andhra
University, Visakhapatnam – 530 003
Abstract: A series of 20 hispolons/dihydrohispolons were synthesized and characterized by
spectral data. These compounds were subjected to in vitro antitubercular activity screening
against Mycobacterium tuberculosis(H37Rv) strain. The synthesized compounds showed
varied antitubercular activity ranging from 100 - 1.6μg/mL. Among the screened compounds,
four compounds (H1, H2, H3 and H15) have shown moderate activity with MIC 25 μg/mL.
Potent activities were observed for the dihydrohispolon derivative H14 (MIC 1.6 μg/mL)
followed by H13 (6.25 μg/mL) and H17 (12.5 μg/mL), H19 (3.125μg/Ml). Docking
simulations gave good insights on the possible interactions between the tested compounds
and β-keto acyl synthase enzyme (mtbFabH). Drug-inhibitor combination studies showed no
synergism with the drugs targeting mycolic acid biosynthesis (isoniazid, ethambutol and
thiolactomycin, a specific inhibitor of KAS-B enzyme) but showed significant synergism
with other drugs including rifampicin and ciprofloxacin ascertaining the drug target for
hispolons as inhibition of mycolic acid biosynthesis, probably via mtbFabH.
Keywords: Hispolons, antitubercular, ketoacyl synthase docking analysis.
*Corresponding authors:
Dr. M.M.K.Kumar – email: drmmkau@gmail.com
Dr. B. Hari Babu – email: dr.b.haribabu@gmail.com
1

Tuberculosis is one of the oldest debilitating infectious disease, responsible for 1.5
million deaths in the year 2014.1With sustained efforts of the scientific community, effective
implementation of DOTS program and public awareness on sanitation has significantly
helped reduce mortality rate due to TB by about 47% in the last decade. But the emergence of
multi drug resistance (MDR) and Extensively drug resistance TB (XDR-TB) in the last 15
years,reinstated ‘global emergency’ status to this disease.2-4It is estimated that in 2014-15
alone, 3.3% of new TB cases and 20% of previously treated cases were found to have MDR-
TB andaround 1,90,000 people died of this. It is alarming to know that about 9.7% of the
MDR-TB patients have tested positive for XDR-TB during 2014-15. MDRTB/XDR TB
complicates the treatment protocol due to scarcity of effective drugs, drug toxicity and patient
non-compliance.1,5-7 Bedaquiline (2013) and delamanid (2014) are the only two novel drugs
approved for treatment of MDR-TB.8 Hence, there remains a pressing requisite to discover
novel, potent and safe anti-TB agents.
The diversity in structure and mechanism of activity makes natural products an indispensable
source of novel lead molecules for drug discovery.9, 10Hispolon was initially isolated as an
yellow pigment from Inonotus hispidus11and then identified in a number of traditional
medicinal mushrooms, including Phellinus linteus13 and Phellinus igniarius14,which have
been used to treat various cancer types in East Asia15.Curcumin, hispolon and cinnamic
derivatives contain a common pharmacophore (α,β-unsaturated carbonyl/ acid attached to an
aromatic ring, Figure1). Curcumin, though known for its antibacterial activity showed
insignificant antitubercular activity. While cinnamic acid analogues, in general have been
also reported to possess no intrinsic activity but potentiate the activity of other antitubercular
agents16,17.However,some cinnamic acid derivatives, have been reported to possess
significant selective antitubercular activity.18Ketoacyl synthase B is the proposed target for
anti TB activity of cinnamides, which may be the reason for their inactivity against other
microbes. The fact that hispolons were not explored so far for their antitubercular potential
encouraged us to synthesize and screen hispolon analogs for possible antitubercular activity.
2

O
O
O
O
H3C
CH3
HO
OH
curcumin
O
antimicrobial
O
O
H3C
CH3
No antimicrobial
but antitubercular
HO
hispolon derivative (H2)
O
R
O
H3C
antitubercular
HO
cinnamic acid esters and amides
R= ORI and NHR
Figure 1 – Structure comparison of various natural products with feruloyl moiety
R1
R2
CHO
O
O
+
R3
R5
H3C
CH3
R4
tributyl borate
n-butyl amine
R1
O
O
R1
O
O
R2
R3
Acetic anhydride
R2
R3
CH3
CH3
H1 to H9
R3 = OAc
R5
R5
R4
O
R4
H-10 to H-12
Pd/C, H2
R1
O
R1
R2
R3
O
O
Acetic anhydride
R2
R3
CH3
CH3
R5
R5
R4
R4
H-13 to H-19
H-20
3

Scheme1: Synthesis of hispolon derivatives
In our ongoing effort to identify novel lead compounds from natural products with anti-
mycobacterial activity, we have synthesized (Schme 1) a series of new (H7 -12 and H17- 20)
and known (H1- 6 and H13-16) hispolon analogs and evaluated for their anti-mycobacterial
activity. Hispolons were synthesized from respective aldehyde and acetylacetone using
methods that were reported previously.19 Hydrogenation of compounds H1, H2-5 and H6-7,
yielded H19, H13-16and H17-18 respectively. Acetylation of H2, H3, H6and H17 yielded
H10, H11,H12and H20respectively. The synthesized compounds were characterized
thoroughly by the interpretation of spectral data (1H NMR, Mass and IR).
The hispolon derivatives synthesized were screened for antitubercular activity using MABA
method20and the results were presented in Table1. DMSO was used to ensure complete
solubility of the samples.Among the compounds tested,the compounds that contain 4-
hydroxyl group, H2andH6 showed antitubercular activity (MIC) at 25μg/mL. As presented in
Figure2, presence of additional substitution adjacent to 4-hydroxyl group or when 4-
hydroxyl group is protected as alkoxy(H4)or acetate (H11) has significantly decreased
antitubercular activity. Further,hydrogenation of these derivatives resulted in
dihydrohispolons (H13 – H20), which showed an unexpected increase in antitubercular
activity.Highest potency(MIC 1.6μg/mL) was observed for the dihydrohispolon
derivative,H14.
O
O
Addition of styryl units abolishes
activity
CH3
4-hydroxy group is desired
R
Substituents on nearby carbons
or protection of hydroxyl
significantly reduces activity
reduction of double bond
potentiates activity
Figure2: SAR of hispolons for anti TB activity
4

Table1: Antitubercular activity of hispolons and their derivatives
R1
R2
R3
R4
R5
MIC against
M.
S.No
Code
tuberculosisH37Rv
(µg/mL)
Hispolons
1
2
-H
-H
-OH
-OCH3
-H
-OH
-OH
-H
-H
-H
-H
25
25
H1
H2
3
-H
-OH
-H
-H
25
H3
4
-H
-H
-OCH3
-H
-H
50
H4
5
-H
-OCH3 -OCH3
-OCH3 -OH
-OCH3 -OCH3
-H -OCH3
-H
-H
100
50
H5
6
-H
-OCH3
-H
-H
H6
7
-OCH3
-OCH3
-H
-H
100
100
50
H7
8
-H
-OCH3
-H
H8
9
-OCH3 -OCH3 -OCH3
H9
10
11
12
-H
-OCH3
-H
-OAc
-OAc
-OAc
-H
-H
-H
>100
100
>100
H10
H11
H12
-H
-H
-H
-OCH3
-OCH3
-H
Dihydrohispolons
13
14
15
16
17
18
19
20
21
22
23
-H
-H
-OCH3
-H
-OH
-OH
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
6.25
1.6
H13
H14
-H
-H
-OCH3
-H
25
H15
-H
-OCH3 -OCH3
-OCH3 -OH
-OCH3 -OCH3
-H
100
H16
-H
-OCH3
-H
12.5
>100
3.125
100
H17
-OCH3
-H
H18
-OH
-OH
-H
H19
-H
-OCH3
-OAc
-OCH3
H20
>100
3.125
3.125
CURCUMIN
Pyrazinamide
Ciprofloxacin
5

24
25
6.25
1.6
Streptomycin
Ethambutol
Isoniazid
26
27
28
0.8
1.6
Rifampicin
12.5
Thiolactomycin
An attempt was made to study the ligand-receptor interactions between the tested
compounds and the ketoacylsynthase enzyme (mtbFabH) by performing docking simulations
on the X-ray crystal structure of Kas-B retrieved from protein databank (1hzp.pdb). The
active site of the enzyme was identified with the help of the co-crystallized ligand.21The
condensation site of this enzyme is constituted by Asn274, Cys112 and His244 triad. VINA
was used for performing docking simulations and binding energy calculation.22 The
applicability of docking and scoring functions in VINA was validated by redocking
experiment.23, 24 Here we have performed the docking simulation of the co-crystallized ligand
(extracted from 1hzp.pdb) in the receptor pocket and compared the lowest energy docked
conformation to that of original x-ray crystallographic atomic coordinates. In our study VINA
could successfully predict the atomic coordinates of the co-crystallized ligand with an
rmsd<2.0 A°.
The ligands were evaluated by binding affinity of the compounds (∆Gb, kcal/mol), ligand
efficiency (LE, binding affinity contribution per a non-hydrogen atom)25,26, hydrogen
bonding and pose analysis. The NMR analysis of hispolons and their dihydro derivatives
clearly showed significant presence (≈ 80-90%, see the supplementary data) of ‘enol’ isomer
in polar solvent (DMSO). Hence we considered both the isomers in preparation of the
compound library. The results obtained from this experiment, including amino acids
participating in ligand-receptor interactions are represented in Table2. All the active
compounds in this series were found to have LE above 0.4. The compound ‘enol’ isomers of
H3 (MIC 25μg/mL) and it’s dihydro derivative H14(MIC 1.6μg/mL) showed the best LE
values of -0.44 and -0.46, respectively.
In the docking analysis it was found that, phenolic ring of the hispolon H3 showed strong
hydrogen bond interactions with Asn274 and His244 the critical amino acids responsible for
recognition and transfer of fatty acyl moiety onto Cys112 (Figure 3).27 Two more strong
hydrogen bond interactions were observed between the enolic hydroxyl and keto group of the
6

hispolon to the amino acids Gly209 and Asn247 respectively. The reduction of double bond
in H3 resulted in increasing lipophilicity and flexibility of the diketo’tail’ allowing it to form
much stronger hydrophobic interactions as seen in H14, which may be responsible for
appropriate alignment of the ligand and increase in its potency. Addition of second hydroxyl
group (H19) gave slightly less potent (MIC 3.12 μg/mL) derivative than the monohydroxy
derivative (H14). Further increase in substitution on benzene created steric hindrance and the
aromatic unit can no longer fit in the receptor pocket, hence resulting in reversal of the
docking pose (‘tail’ first). Methylation of the phenolic hydroxyl resulted in significant
reduction in hydrogen bond interactions with Asn274. This may be the reason for very week
bioactivity observed with H7 (MIC 100 μg/mL) and other hispolons with trimethoxyphenyl
unit.
The results obtained from the docking simulations strongly supported the need for 4-
hydroxyphenyl group and reduction of double bond for optimal bioactivity as observed in the
SAR analysis. Altogether, the interactions with the catalytic site amino acid Asn274 seems to
be the deciding factor for the bioactivity of these molecules. The need for acidic aromatic unit
attached to a lipophilic tail and docking results helped us draw parallels between mode of
inhibition observed for hispolons and platensin, a potent KAS inhibitor isolated from an
extract of Streptomyces platensis.28-30
Figure 3: Docking conformation of active compound6-(4-hydroxyphenyl)hexane-2,4-
dione (H14) in the active site of FAS-II enzyme.
7

Table3: Combined drug-inhibitor activity against M. tuberculosis H37Rv assessed by
MABA method
S.No
Drugs
Enhancement of drug activities
(x/y coefficients)
against M. tuberculosis H37rv
1
2
H3+EMB
H3+INH
H3+RIF
-
-
++ (0.062)
++ (0.062)
+ (0.25)
+ (0.125)
-
3
4
H3+CIP
5
H3+PYZ
H3+STR
H3+TLM
H14+EMB
H14+INH
H14+RIF
H14+CIP
H14+PYZ
H14+STR
H14+TLM
6
7
8
-
9
-
10
11
12
13
14
++ (0.062)
+++ (0.031)
+ (0.25)
+ (0.25)
-
Efforts were made to ascertain the drug target for hispolons using drug-inhibitor
combination studies.31 As shown in the table 3, the compound H3 and H14 showed synergism
when combined with rifampicin, ciprofloxacin, pyrazinamide and streptomycin. The test
compounds did not show any synergism with isoniazid, ethambutol and thiolactomyn which
are known to be inhibitors of mycolic acid biosynthesis. As evident from these results that
hispolons act via enzymes involved in mycolic acid biosynthesis as their target and most
probably via inhibition of fatty acid synthase II as also indicated by the computational
studies.
Anti -TB therapy usually spreads from 3 months to one year and has high tendency to
cause hepatotoxicity.32,33 Hence, it is desirable for new anti TB agent to have selective
antagonistic activity and safety towards liver. The hispolons and dihydrohispolons were
screened for antibacterial activity against gram +ve (S. aureus), gram-ve (E.coli). None of
8

them were found be active at 100μg/mL concentration. The compounds H13 and H14 were
earlier reported by us19 as non-toxic to normal human cells upto 100μM and thus making
these very good lead molecules for antitubercular activity.19a,b` In addition, hispolons were
reported to possess potential hepatoprotective activity.34 Hence our study successfully
identified potential anti TB lead molecules with double advantage. We plan to carry forward
lead optimization and mechanistic studies on these compounds.
Table 2. Docking results of hispolon analogues on Fas-II enzyme
S.No Code
Docking
score
(kcal/Mol)
LE
(kcal/Mol/
atom)
MIC against
M. tuberculosis
H37Rv (µg/mL)
Ligand-receptor
interactions
1
2
-7.0
-7.1
-0.43
-0.41
25
25
Asn247, Ala246, Asn274
H1
H2
Ala246, Asn274, Gly152,
Arg249, Arg36
3
-6.6
-0.44
25
Asn274, His244, Ala246,
H3
Asn247, Gly209, Phe213
Arg36, Ala246, Asn247
Asn247, Ala246
4
5
6
7
8
-6.8
-6.3
-6.2
-6.4
-5.3
-0.42
-0.35
-0.32
-0.32
-0.26
50
100
50
H4
H5
H6
H7
H8
Asn274, Ala246, pro210
Asn247, Ala246
100
100
Asn247, Ala246, Gly152,
Phe213, Arg249
9
-6.3
-6.6
-7.1
-6.4
-7.1
-0.31
-0.33
-0.39
-0.29
-0.42
50
Pro210, Asn274
H9
10
11
12
13
>100
100
Ala246, Gly209
H10
H11
H12
H13
Arg36, Gly152, Ala246
Ala246, Arg249, Asn247
>100
6.25
Asn274, Ala246, Asn247,
Gly152
14
-6.9
-0.46
1.6
Asn274, His244, Ala246,
Asn247,Gly209, Phe213
Asn274, Gly209, Arg36
H14
15
16
-6.5
-6.7
-0.41
-0.37
25
H15
H16
100
Ala246, Arg151, Gly152,
Gly209
17
-6.9
-0.37
12.5
Asn274, Asp246, Asn247,
Arg36
H17
18
19
-6.5
-6.7
-0.32
-0.44
>100
3.125
Ala246, Gly209, Asn247
H18
H19
Asn247, Ala246, Asn274,
Gly209, Ala306
9

20
-6.3
-0.28
100
Ala246, Gly157, Asn247
H20
In conclusion, a total of 20 hispolon analogues were synthesized and screened for
antitubercular activity against M.tuberculosis H37Rv. Among the tested compounds (4-
hydroxy-phenyl)hexane-2,4-dione (H14) showed highest potency with an MIC 1.6μg/mL.
Docking simulations on the enzyme mtbFas-II, clearly showed correlation between ligand
efficiency and ligand-receptor binding interactions to their observed bioactivity, indicating it
as a possible molecular target. Drug-inhibitor combination studies ascertained the protein
target for hispolons is proteins involved in mycolic acid biosynthesis, most probably
mtbFabH. The unprecedented anti TB activity in hispolons, which are known for their
hepato-protective activity, is very promising.
Acknowledgements
The authors thank M/s Natsol Laboratories, Visakhapatnam management for allowing to
conduct synthetic experiments.
References and notes
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De, P.; Veau, D.; Florence, B. B.; Chassaing, S.; Baltas, M. Cinnamic Derivatives in
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19.
a) Balaji, N. V.; Ramani, M. V.; Viana, A. G.; Sanglard, L. P.; White, J.; Vanisree
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Synthesis of (E)-6-(2,4,6-trimethoxy phenyl)hex-5-ene-2,4-dione ( H8 ):A mixture
of tributyl borate ( 35.20 g , 3.0 equiv.) and acetylacetone ( 25.54 g, 5.0 equiv.) was
taken into a round bottom flask equipped with a mechanical stirrer and the reaction
mass was stirred at 600C for 30 min. To the complex formed, was added 2,4,6-
trimethoxybenzaldehyde (10 g, 1.0 equiv.).To the above reaction mixture n-butyl
amine (3.731 g, 1.0 equiv.) was added drop wise and stirred for 90 min at 650C. The
reaction mass cooled to 550C, acidified with 1 N HCl and stirred for 1 h. The product
was extracted with ethyl acetate (2x500 mL) and the ethyl acetate extract was
concentrated under vacuo. The residue was adsorbed over silica gel (100-200 mesh)
and purified by column chromatography (25% ethyl acetate in hexane) to get the
compound H8 as an yellow solid ; Yield: 36.1% (5.1 g); mp 130-1350C; IR (KBr)
1
: 2932, 1595, 1469, 1326, 1031, 823 cm-1. H NMR (400 MHz, CDCl3) δ ppm
max
ν
15.69 (s, 1–OH), 8.02 (d, 1H, J=16 Hz), 6.85 (d,1H, J=16 Hz), 6.12 (s, 2H), 5.61 (s,
1H), 3.87 (s, 6H), 3.84 (s, 3H), 2.12 (s, 3H).HPLC purity- 99%.
Synthesis of 2-methoxy-4-(3,5-dioxohexyl)phenyl acetate (H10 ):Acetic anhydride
(2 g, 1.0 equiv.) and pyridine (1.07 g, 1.49 equiv.) were added to stirred solution of 6-
11

(4-Hydroxy-3-methoxy-phenyl)-hex-5-ene-2,4-dione (2 g, 1.0 equiv.) in CH2Cl2 (20
mL) and stirred at RT for 3h. After completion of the reaction (monitor by TLC), It
was poured into ice cold water and stirred well for 1h. The product was extracted
with CH2Cl2 (2x25 mL) and the organic layer was dried with anhydrous Na2SO4 and
concentrated under vacuum. The crude material was recrystallized from hexane and
CH2Cl2 to get the compound H10 as a pale yellow solid. Yield 72% (1.8 g); mp 91.4-
94.2 0C; IR (KBr) max: 2949 ,1756, 1638, 1610, 1514, 1375, 1250, 1032, 975 cm-1. 1H
ν
NMR (400 MHz, CDCl3) δ ppm 15.31 (s, 1-OH), 7.54 (d, 1H, J=16 Hz), 7.13 (m,
1H), 7.11 (d, 1H, J=16 Hz), 7.05 (s, 1H), 6.40 (d, 1H), 5.65 (s, 1H), 3.87 (s, 3H), 2.32
(s, 3H), 2.17 (s, 3H). HPLC purity-98 %
Synthesis of 6-(4-hydroxy-3,5-dimethoxy phenyl)hexane-2,4-dione (H17): To a
solution of (E)-6-(4-hydroxy-3,5-dimethoxy phenyl)hex-5-ene-2,4-dione (10 g, 1.0
equiv.) in 100 mL of ethyl acetate was added 10 percent palladium/carbon (150 mg)
under nitrogen. After the vessel was purged with hydrogen, the reaction mixture was
stirred under 4 atm ( 2x4 ) of hydrogen at room temperature for 2 h. After the vessel
was purged with nitrogen, the reaction mixture was filtered to remove 10 percent
palladium/carbon. The filtrate was concentrated in vacuo, and the crude was purified
by column chromatography [ethyl acetate/ hexane (30-70%)] to obtain the title
compound H17as a colorless oil; Yield:85% (8.5 g); IR (KBr) max: 3880, 3192 ,
ν
1
3006, 2962, 2582, 2435, 1949, 1609, 1518, 1030, 968 cm-1. H NMR (400 MHz,
CDCl3 ) δ ppm 15.48 (s, 1-OH), 6.40 (d, 2H), 5.47 (s, 1H), 5.43 (s, 1H), 3.86 (s, 1H)
2.88 (m, 2H), 2.58 (m, 2H), 2.04 (s, 3H). HPLC Purity- 99%.
20.
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