Synthesis and antiproliferative activity of novel (+)- usnic acid analogues

Article abstract of DOI:10.1080/10286020.2019.1603220

Twenty one novel (+)- usnic acid-based analogues belonging to three classes such as enamines, imines, and pyrazoles were synthesized. All the synthesized compounds were characterized by their spectral data (¹H NMR, ¹³C NMR, IR, and H

Full text of DOI:10.1080/10286020.2019.1603220

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis and antiproliferative activity of novel (+)-
usnic acid analogues
Uppuluri Venkata Mallavadhani, Nagi Reddy Vanga, Kancharana
Balabhaskara Rao & Nishanth Jain
To cite this article: Uppuluri Venkata Mallavadhani, Nagi Reddy Vanga, Kancharana
Balabhaskara Rao & Nishanth Jain (2019): Synthesis and antiproliferative activity of
novel (+)- usnic acid analogues, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2019.1603220
To link to this article: https://doi.org/10.1080/10286020.2019.1603220
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1603220
Synthesis and antiproliferative activity of novel (þ)- usnic
acid analogues
Uppuluri Venkata Mallavadhania, Nagi Reddy Vangaa, Kancharana Balabhaskara
Raob and Nishanth Jainb
aCentre for Natural Products & Traditional Knowledge, CSIR-Indian Institute of Chemical Technology,
b
Hyderabad 500007, India; Applied Biology Department, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500007, India
ABSTRACT
ARTICLE HISTORY
Received 26 November 2018
Accepted 27 March 2019
Twenty one novel (þ)- usnic acid-based analogues belonging to
three classes such as enamines, imines, and pyrazoles were syn-
thesized. All the synthesized compounds were characterized by
their spectral data (1H NMR, 13C NMR, IR, and HRMS). The synthe-
sized compounds were evaluated for their antiproliferative activity
against a panel of four human cancer cell lines including HeLa
(cervix), MDA-MB-231 (breast), A549 (lung), and MiaPaca (pan-
creas) by employing SRB cell proliferation assay. Screening results
indicated that all synthesized compounds showed enhanced
activity than the parent compound. Most significantly, com-
pounds 2e and 4a showed potent antiproliferative activity against
all the cancer cell lines tested. Compounds 2e and 4a arrested
the cell cycle in G2/M phase and induced apoptosis in HeLa cells.
In view of significant antiproliferative activity, compounds 2e and
4a can be considered as lead molecules for further development.
KEYWORDS
Antiproliferative activity;
apoptosis; cell cycle arrest;
enamine; pyrazole; (þ)-
usnic acid
1. Introduction
Usnic acid is a dibenzofuran derivative, found uniquely in lichens as secondary
metabolite. It is especially abundant in lichen genera such as Alectoria, Cladonia,
Evernia, Lecanora, Ramalina, and Usnea. In addition to its broad spectrum of bio-
logical activities such as antimicrobial, antiviral, antiprotozoal, antiproliferative, anti-
inflammatory, and analgesic activities, the lichen extracts containing usnic acid have
CONTACT Uppuluri Venkata Mallavadhani
mallavadhani@iict.res.in; uvmavadani@yahoo.com
Centre for
Natural Products & Traditional Knowledge, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2019.1603220.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
U. V. MALLAVADHANI ET AL.
been used as crude drugs throughout the world. As a pure substance, it has been for-
mulated in creams, toothpaste, mouthwash, deodorants, and sunscreen products
[1–3]. High natural abundance, functional group diversity, and the broad spectrum of
biological activities of usnic acid made it as a good target for chemists and biologists
around the world. Till date various types of moieties such as enamines, chalcones,
thiazoles, sulphides, aurones, flavones, and coumarins were introduced on ring A and
ring C of usnic acid, of which usnic acid enamines showed promising biological activ-
ities [4–9]. Many research groups around the world synthesized a large variety of
usnic acid enamines with potent biological activities such as antiviral and anticancer
activities [5–7, 10–12]. Amines containing pharmacophoric moieties such as morpho-
lines, piperazines, and pyridines were selected and coupled with usnic acid to obtain
usnic acid enamines [13]. Pyrazole, a nitrogen-containing five membered heterocyclic
ring, is an important pharmacophore in medicinal chemistry. Pyrazole moiety is present
in many marketed drugs like celecoxib, antipyrine, phenylbutazone, novalgine, ramifena-
zone, apixaban, fipronil, rimonabant, and pyrazofurin. Pyrazole derivatives exhibit
diverse biological activities such as anticancer, antimicrobial, analgesic, anti-inflamma-
tory, antihypertensive, antiepileptic, and antidepressant [14]. Phenylhydrazines containing
different electron releasing and withdrawing groups were selected and coupled with
usnic acid to obtain usnic acid-based pyrazoles. Based on above facts, we have now syn-
thesized some usnic acid-based enamines and pyrazoles and evaluated for their antiproli-
ferative activity against four cancer cell lines.
2. Results and discussion
2.1. Chemistry
(þ)- Usnic acid (1) was isolated from the lichen Usnea longissima in 1% yield accord-
ing to our reported procedure [15]. The protocols adopted for the synthesis of novel
(þ)- usnic acid-based enamines and imines are presented in Scheme 1. (þ)- Usnic acid
(1 eq.) was refluxed with appropriate alkylamines containing different heterocyclic rings
(2.2 eq.) in ethanol to afford the enamines (2a-2f) in 48–56% yields and imines 3a-3f
in 37–45% yields [11, 16, 17]. Compounds 2a-2f and 3a-3f were thoroughly character-
1
ized by their spectral data (1H & 13C NMR, IR, and HRMS). In H NMR spectra of
compounds 2a-2f, a three proton singlet appeared between d 2.60 and 2.68 correspond-
ing to C-12 methyl (enamine methyl) and in 13C NMR spectra, the carbon signal
1
appeared between d 188.2 and 190.4 corresponding to C-3 carbonyl. In H NMR spec-
tra of compounds 3a-3f, a three proton singlet appeared between d 2.70 and 2.85 cor-
responding to C-14 methyl (imine methyl). The protocols adopted for the synthesis of
(þ)- usnic acid-based pyrazoles (4a-4i) are presented in Scheme 2. They were synthe-
sized by following the previously reported procedure [18]. (þ)- Usnic acid was refluxed
with appropriate phenylhydrazines and pyridine in ethanol to get the (þ)- usnic acid
pyrazoles 4a-4i in 81–92% yields. The synthesized pyrazoles were thoroughly character-
ized by their physical and spectral data and by comparing with the literature values
[18]. In 13C NMR spectra, the characteristic pyrazole ring carbon (carbon adjacent to
sp3 nitrogen) appeared between d 151.3 and 152.4.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthesis of compounds 2a-2f and 3a-3f.
Scheme 2. Synthesis of compounds 4a-4i.
2.2. Biological evaluation
2.2.1. In vitro cytotoxicity
The synthesized compounds 2a-2f, 3a-3f (enamines), and 4a-4i (pyrazoles) were eval-
uated for in vitro cytotoxicity against a panel of four human cancer cell lines such as
HeLa (cervix), MDA-MB-231 (breast), A549 (lung), and MIAPaCa (pancreas) by employ-
ing sulforhodamine B (SRB) cell proliferation assay [19]. Nocodazole was used as a posi-
tive control. The results are summarized in Table 1. The compounds were treated in

4
U. V. MALLAVADHANI ET AL.
Table 1. IC50 values of synthesized compounds.
IC50 (lM)
Compound
HeLa
MDA MB-231
A549
MiaPaca
1
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
4b
4c
88.20 0.31
22.71 0.77
19.37 0.91
30.51 0.28
36.24 0.46
4.12 0.31
40.43 0.48
28.56 0.91
49.43 0.34
46.10 0.43
50.14 0.32
33.31 0.41
35.27 0.87
6.19 0.76
83.21 0.57
41.28 0.97
40.9 0.21
79.62 0.15
25.61 0.29
27.37 0.43
26.73 0.46
30.56 0.81
3.90 0.17
29.73 0.98
42.64 0.67
70.67 0.54
52.64 0.32
45.31 0.46
29.34 0.41
39.11 0.34
5.90 0.03
61.71 0.41
43.42 0.29
51.41 0.29
68.43 0.56
34.63 0.46
39.48 0.96
38.81 0.22
36.21 0.31
0.91 0.28
65.76 0.82
26.64 0.69
24.59 0.98
27.52 0.81
24.31 0.27
5.99 0.27
33.13 0.78
49.98 0.92
86.31 0.28
41.10 0.22
39.17 0.23
35.0 0.74
34.18 0.29
6.85 0.94
75.31 0.5
58.12 0.55
62.64 0.62
41.34 0.64
40.19 0.27
30.27 0.82
40.31 0.24
40.2 0.51
0.97 0.84
61.49 0.52
19.11 0.35
40.73 0.46
21.64 0.78
30.62 0.45
4.93 0.51
26.53 0.18
39.51 0.61
69.49 0.99
60.53 0.96
40.4 0.36
16.2 0.38
29.94 0.68
7.39 0.47
58.21 0.28
66.91 0.25
55.91 0.26
50.21 0.33
24.52 0.28
36.34 0.51
37.46 0.88
30.34 0.59
0.95 0.26
4d
4e
4f
4g
4h
4i
43.64 0.59
25.28 0.95
28.34 0.53
35.34 0.61
43.14 0.42
0.89 0.99
Nocodazole
5-doses according to the NCI-60 protocol. Subsequently, after 48 h of treatment, the
absorbance of the SRB was monitored and based on it IC50 values were determined by
interpolation after plotting percent growth versus compounds concentrations. In addition,
these results also indicate that the compounds bind a common target that this expressed
in all the cell lines. Most of the synthesized compounds showed better antiproliferative
activity than the parent compound. Among (þ)- usnic acid enamines (2a-2f and 3a-3f),
compound with imidazole substituent (2e) showed potent antiproliferative activity against
all tested cell lines with IC50 values: 4.12 0.31 lM (HeLa), 3.90 0.17 lM (MDA-MB-
231), 5.99 0.27 lM (A549), and 4.93 0.51 lM (MIAPaCa). Compounds 2a, 2b, 2c, 2d,
and 2f showed moderate IC50 values (19.37 0.91 ꢀ 40.73 0.46 lM) against all the tested
cancer cell lines. From the close analysis of IC50 values (Table 1), it is evident that com-
pounds with monosubstitution (2a-2f) showed better cytotoxicity than that of com-
pounds with disubstitution (3a-3b) against all the tested cell cancer lines.
Among (þ)- usnic acid pyrazoles (4a-4i), compound 4a showed potent antiproli-
ferative activity against all the tested cell lines with IC50 values: 6.19 0.76 lM
(HeLa), 5.90 0.03 lM (MDA-MB-231), 6.85 0.94 lM (A549), and 7.39 0.47 lM
(MIAPaCa). Compounds with methoxyphenyl and nitrophenyl groups (4f, 4g, 4h,
and 4i) showed moderate antiproliferative activity against all tested cell lines with
IC50 values 24.52 0.28–43.14 0.42 lM. Compounds with chlorophenyl and bromo-
phenyl substituents (4b, 4c, 4d, and 4e) exhibited enhanced antiproliferative activity
(IC50: 40.9 0.21–83.21 0.57 lM) when compared to the parent compound against
most of the tested cell lines. From the close analysis of IC50 values, it is evident that
compounds with methoxyphenyl and nitrophenyl substituents (4f, 4g, 4h, and 4i)
exhibited better antiproliferative activity than the compounds with chlorophenyl and
bromophenyl substituents (4b, 4c, 4d, and 4e).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 1. Anti-mitotic effect of 2e and 4a by FACS analysis: Induction of cell cycle G2/M arrest by
compounds. HeLa cells were harvested after treatment at 10 lM for 24 h. DMSO treated cells were
served as vehicle control, respectively. The percentage of cells in each phase of cell cycle was
quantified by flow cytometry.
2.2.2. Cell cycle analysis
The screening results revealed that compounds 2e and 4a showed significant antiprolifer-
ative activity against human cervical cancer cell line HeLa with IC50 values
4.12 0.31 lM and 6.19 0.76 lM, respectively. To understand whether this cell growth
inhibition of human cervical cancer cells was due to cell cycle arrest, we performed a cell
cycle analysis study [20]. Compounds 2e and 4a were used at 10 lM concentrations for
24 h. Subsequently, the cells were harvested and fixed with ice cold 70% ethanol.
The fixed cells were later stained with propidium-iodide and the cell cycle profile was
monitored. Notably, 2e (52.3%) and 4a (66.7%) treated cells showed an accumulation in
G2/M phase (Figure 1). Based on these results it can be concluded that 2e and 4a arrest
cells in G2/M phase and possibly this arrest contributes to their anti-cancer effects.
2.2.3. Immunofluorescence of tubulin
HeLa cells were arrested in G2/M phase and showed altered morphology, wherein
these cells rounded up with prominent disappearance of cytoplasmic staining, as
compared to untreated cells. Since the compounds arrested in G2/M phase, we per-
formed immunofluorescence of tubulin protein to understand the phenotype elicited
by the compounds [19]. Interestingly, 2e and 4a treated cells showed typical G2/M

6
U. V. MALLAVADHANI ET AL.
Figure 2. Effect of 2e and 4a on microtubules and nuclear condensation. HeLa cells were inde-
pendently treated with 2e and 4a at 10 lM concentration for 24 h. Following the termination of
experiment, cells were fixed, and stained for tubulin. DAPI was used as counter stain. The merged
images of cells stained for tubulin and DAPI are represented. Paclitaxel was used as posi-
tive control.
arrest phenotype. Moreover, most of the compound treated cells displayed in the field
have a centered nuclear staining as evidenced by 4’,6-diamidino-2-phenylindole
(DAPI). Thus, our results further corroborate with flow cytometry analysis that 2e
and 4a indeed arrest cells in G2/M phase of cell cycle (Figure 2).
2.2.4. Immunoblot assays
Thus, based on our results that 2e and 4a show a G2/M arrest associated with an
altered phenotype, we further investigated for the markers of G2/M by immunoblot
analysis [19]. Cyclin-B1/Cdk1 is master regulator of mitosis, thus, we elucidated for
the protein levels of Cyclin-B1 in 2e and 4a treated cells. In addition, nocodazole and
paclitaxel were used as positive controls. Notably, 2e and 4a showed a robust induc-
tion of Cyclin-B1 protein similar to controls. Dimethyl sulfoxide (DMSO) treated
cells did not exhibit any induction Cyclin-B1 protein (Figure 3). Further, we analyzed
whether 2e or 4a target endogenous tubulin. As the results so far support the sugges-
tion, to investigate this, we treated HeLa cells with 2e or 4a for 24 h and subsequently
analyzed for tubulin protein in soluble and insoluble fractions. 2e and 4a markedly
increased the soluble fraction to tubulin similar to nocodazole, a microtubule

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 3. 2e and 4a induce cell cycle arrest. A. HeLa cells were treated with 10 lM concentration
of compound (2e, 4a) for 24 h, nocodazole (Noc) and paclitaxel were used as controls. Cyclin-B1
and Tubulin were detected by specific antibodies. B. Distribution of tubulin analyzed by immuno-
bloting in 2e and 4a treated HeLa cells: The cells were treated with 10 lM of 2e, 4a,
Combretastatin and Paclitaxel for 24 h. Subsequently, the cells were permeabilized with Hepes-buf-
fer containing triton-x-100 and analyzed for Tubulin protein by Western blotting analysis.
depolymerizing agent. In contrast, paclitaxel increased the polymerized or insoluble
faction of tubulin in HeLa cells (Figure 3). Hence, it can be concluded that 2e and 4a
are microtubule depolymerizing agents which exert their cytotoxicity by potently
arresting cells in mitosis followed by apoptosis.
3. Conclusion
In total 21 (þ)- usnic acid-based enamines, imines, and pyrazoles were synthesized in
very good yields and evaluated for their antiproliferative activity against four cancer cell
lines. All the synthesized compounds exhibited enhanced antiproliferative activity when
compared to the parent compound. Especially, compounds 2e and 4a showed potent
antiproliferative activity against all tested cell lines with IC50 values
3.90 0.17–7.39 0.47 lM. Compounds 2e and 4a arrested the cell cycle in G2/M phase
in HeLa cells. It can be concluded that 2e and 4a are microtubule depolymerizing agents
which exert their cytotoxicity by potently arresting cells in mitosis followed by apoptosis.
These two molecules can be considered as lead compounds for further development.
4. Experimental
4.1. Chemistry
All the solvents (RANKEM, India) were distilled before use. All reagents were pur-
chased from commercial sources (Sigma-Aldrich, Alfa Aesar, India) and used as

8
U. V. MALLAVADHANI ET AL.
received. All reactions were monitored by silica gel 60 F254 glass TLC plates (Merck,
India). Column chromatography was performed on ACME grade 60–120 mesh silica
gel. Melting points were determined on a Bu€chi melting point apparatus and are
uncorrected. IR spectra were recorded on Nicollet 740 FTIR spectrophotometer in
1
KBr disks. H and 13C NMR spectra were recorded on Bruker 300 MHz and Varian
500 MHz spectrometers using TMS as internal standard. Chemical shifts were
reported as d values in parts per million (ppm) and coupling constants (J) were
reported in Hertz (Hz). The following abbreviations were used for NMR signals:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, dd ¼ doublet of doublet,
brs ¼ broad singlet, Ar-H ¼ aryl protons. Mass spectra were obtained on Agilent ESI-
QTOF instrument.
4.1.1. Isolation of (þ)- usnic acid
(þ)- Usnic acid was isolated from the lichen Usnea longissima in 1% yield according
to our reported procedure [15].
4.1.2. General procedure for the synthesis of compounds 2a-2f and 3a-3f
To a solution of 1 (2.5 g, 0.726 mmol) in ethanol (18 ml) was added appropriate alkyl-
amine (1.597 mmol) and the resulting mixture was refluxed for 4–12 h under stirring
[11, 16]. The reaction mixture was then concentrated under reduced pressure and the
resulting residue was chromatographed over a column of silica gel to afford pure
compounds 2a-2f and 3a-3f.
4.1.2.1. (R,E)-6-Acetyl-2-[1-(morpholinoamino)ethylidene]-7,9-dihydroxy-8,9b-dime-
thyldibenzofuran-1,3(2H,9bH)-dione (2a). Pale yellow solid (0.174 g, 56%); mp:
93–96 ꢁC; IR (KBr) ꢀmax: 2650–3428, 1698, 1626, 1557, 1458, 1369, 1280, 1187 cmꢂ1
;
1H NMR (300 MHz, CDCl3þDMSO-d6): d 1.71 (3H, s, H-10), 2.07 (3H, s, H-15),
2.68 (6H, s, H-12, H-14), 2.92 (4H, t, J ¼ 4.7 Hz, H-2’, H-6’), 3.85 (4H, t, J ¼ 4.7 Hz,
H-3’, H-5’), 5.80 (1H, s, H-4), 11.90 (1H, s, 9-OH), 13.36 (1H, s, 7-OH), 13.97
(1H,brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 6.8 (C-15), 16.5 (C-12), 30.5
(C-14), 31.2 (C-10), 55.0 (C-2’, C-6’), 56.4 (C-9b), 65.2 (C-3’, C-5’),100.0 (C-6), 100.6
(C-4), 101.7 (C-2), 104.3 (C-9a), 107.0 (C-8), 155.1 (C-5a), 157.5 (C-9), 162.6 (C-7),
173.4 (C-11), 173.6 (C-4a), 189.2 (C-3), 197.5 (C-1), 199.9 (C-13); HRESIMS: m/z
429.1651 [M þ H]þ (cacld for C22H25N2O7, 429.1656).
4.1.2.2. (R,E)-6-Acetyl-2-[1-(2-morpholinoethylamino)ethylidene]-7,9-dihydroxy-8,9b-
dimethyldibenzofuran-1,3(2H,9bH)-dione (2b). Pale yellow solid (0.172 g, 52%); mp:
83-85 ꢁC; IR (KBr) ꢀmax: 2656–3449, 1699, 1624, 1555, 1460, 1370, 1286, 1188 cmꢂ1
;
1H NMR (300 MHz, CDCl3): d 1.71 (3H, s, H-10), 2.10 (3H, s, H-15), 2.55 (4H, t,
J ¼ 4.5 Hz, H-2’, H-6’), 2.66–2.73 (8H, m, H-12, H-14, H-17), 3.55–3.58 (2H, m, H-
16), 3.78 (4H, t, J ¼ 4.5 Hz, H-3’, H-5’), 5.79 (1H, s, H-4), 12.05 (1H, brs, 9-OH),
13.37 (1H, s, 7-OH); 13C NMR (75 MHz, DMSO-d6): d 7.5 (C-15), 18.6 (C-12), 31.0
(C-14), 31.8 (C-10), 40.4 (C-16), 52.7 (C-2’, C-6’), 55.2 (C-17), 56.1 (C-9b), 66.2 (C-
3’, C-5’), 100.7 (C-6), 101.6 (C-4), 102.3 (C-2), 105.1 (C-9a), 106.2 (C-8), 155.7 (C-

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
5a), 157.6 (C-9), 162.5 (C-7), 172.6 (C-11), 174.3 (C-4a), 188.5 (C-3), 197.0 (C-1),
200.8 (C-13); HRESIMS: m/z 457.1961 [M þ H]þ (cacld for C24H29N2O7, 457.1969).
4.1.2.3. (R,E)-6-Acetyl-2-[1-(2-(piperazin-1-yl)ethylamino)ethylidene]-7,9-dihydroxy-
8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (2c). Pale yellow solid (0.161 g, 49%);
mp: 103-105 ꢁC; IR (KBr) ꢀmax: 2660–3427, 1700, 1623, 1555, 1460, 1370, 1287,
1
1188 cmꢂ1; H NMR (500 MHz, CDCl3): d 1.70 (3H, s, H-10), 2.10 (3H, s, H-15),
2.57 (4H, t, J ¼ 4.5 Hz, H-2’, H-6’), 2.65–2.71 (8H, m, H-12, H-14, H-17), 3.00 (4H, t,
J ¼ 4.4 Hz, H-3’, H-5’), 3.56–3.60 (2H, m, H-16), 5.79 (1H, s, H-4), 13.35 (1H, brs, 7-
OH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 6.5 (C-15), 17.1 (C-12), 30.1 (C-14),
30.9 (C-10), 40.7 (C-16), 45.5 (C-3’, C-5’), 54.2 (C-2’, C-6’), 55.0 (C-17), 55.7 (C-9b),
100.1 (C-6), 101.0 (C-4), 101.4 (C-2), 104.1 (C-9a), 106.2 (C-8), 154.8 (C-5a), 157.2
(C-9), 162.1 (C-7), 172.4 (C-11), 173.7 (C-4a), 188.6 (C-3), 197.4 (C-1), 199.5 (C-13);
HRESIMS: m/z 456.2112 [M þ H]þ (cacld for C24H30N3O6, 456.2129).
4.1.2.4. (R,E)-6-Acetyl-2-[1-(4-methylpiperazin-1-ylamino)ethylidene]-7,9-dihydroxy-
8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (2d). Pale yellow solid (0.169 g, 53%);
mp: 78-80 ꢁC; IR (KBr) ꢀmax: 2642–3422, 1699, 1629, 1553, 1457, 1365, 1286,
1
1188 cmꢂ1; H NMR (500 MHz, CDCl3): d 1.70 (3H, s, H-10), 2.09 (3H, s, H-15),
2.33 (3H, S, H-7’), 2.56–2.61 (4H, m, H-3’, H-5’), 2.67 (3H, s, H-12), 2.78 (3H, s, H-
14), 2.93 (4H, t, J ¼ 4.4 Hz, H-2’, H-6’), 5.70 (1H, s, H-4), 11.96 (1H, brs, 9-OH),
13.35 (1H, s, 7-OH), 14.09 (1H, brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d
6.2 (C-15), 15.8 (C-12), 29.8 (C-14), 30.5 (C-10), 44.2 (C-7’), 52.3 (C-3’, C-5’), 53.7
(C-2’, C-6’), 55.5 (C-9b), 99.2 (C-6), 99.7 (C-4), 101.0 (C-2), 103.6 (C-9a), 105.9 (C-
8), 154.4 (C-5a), 156.7 (C-9), 161.8 (C-7), 172.4 (C-11), 172.8 (C-4a), 188.2 (C-3),
196.6 (C-1), 199.2 (C-13); HRESIMS: m/z 442.1958 [M þ H]þ (cacld for
C23H28N3O6, 442.1972).
4.1.2.5. (R,E)-6-Acetyl-2-[1-(3-(1H-imidazol-1-yl)propylamino)ethylidene]-7,9-dihy-
droxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (2e). Pale yellow solid (0.157 g,
48%); mp: 80-82 ꢁC; IR (KBr) ꢀmax: 2650–3444, 1701, 1626, 1555, 1467, 1368, 1283,
1
1186 cmꢂ1; H NMR (300 MHz, CDCl3þDMSO-d6): d 1.71 (3H, s, H-10), 2.06 (3H, s,
H-15), 2.22–2.28 (2H, m, H-17), 2.59 (3H, s, H-12), 2.68 (3H, s, H-14), 3.44–3.50
(2H, m, H-16), 4.17 (2H, t, J ¼ 6.6 Hz, H-18), 5.79 (1H, s, H-4), 7.01 (1H, S, H-4’),
7.07 (1H, S, H-5’), 7.55 (1H, S, H-2’), 11.90 (1H, s, 9-OH), 13.36 (1H, s, 7-OH),
13.51 (1H, brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 6.9 (C-15), 17.6 (C-
12), 29.6 (C-17), 30.6 (C-14), 31.4 (C-10), 39.9 (C-16), 43.2 (C-18), 56.5 (C-9b), 100.6
(C-6), 101.7 (C-4), 101.8 (C-2), 104.4 (C-9a), 107.1 (C-8), 118.1 (C-5’), 129.3 (C-4’),
136.5 (C-2’), 155.2 (C-5a), 157.6 (C-9), 162.7 (C-7), 173.4 (C-11), 174.7 (C-4a), 189.5
(C-3), 197.6 (C-1), 200.1 (C-13); HRESIMS: m/z 452.1798 [M þ H]þ (cacld for
C24H26N3O6, 452.1816).
4.1.2.6.
dimethyldibenzofuran-1,3(2H,9bH)-dione (2f). Pale yellow solid (0.170 g, 55%); mp:
75-77 ꢁC; IR (KBr) ꢀmax: 2650–3420, 1701, 1628, 1554, 1460, 1363, 1286, 1188 cmꢂ1
(R,E)-6-Acetyl-2-[1-(piperidin-1-ylamino)ethylidene]-7,9-dihydroxy-8,9b-
;

10
U. V. MALLAVADHANI ET AL.
1H NMR (500 MHz, CDCl3): d 1.50 (2H, brs, H-4’), 1.71–1.77 (7H, m, H-10, H-3’,
H-5’), 2.09 (3H, s, H-15), 2.67 (3H, s, H-12), 2.76–2.87 (7H, m, H-14, H-2’, H-6’),
5.78 (1H, s, H-4), 12.04 (1H, brs, 9-OH), 13.35 (1H, s, 7-OH), 13.98 (1H, brs, NH);
13C NMR (125 MHz, CDCl3þDMSO-d6): d 7.4 (C-15), 17.2 (C-12), 22.9 (C-4’), 24.9
(C-3’, C-5’), 31.2 (C-14), 31.9 (C-10), 56.7 (C-9b, C-2’, C-6’), 100.4 (C-6), 101.2 (C-
4), 102.2 (C-2), 105.0 (C-9a), 107.9 (C-8), 155.8 (C-5a), 158.2 (C-9), 163.4 (C-7),
173.7 (C-11), 174.3 (C-4a), 190.4 (C-3), 198.1 (C-1), 200.5 (C-13); HRESIMS: m/z
427.1869 [M þ H]þ (cacld for C23H27N2O6, 427.1863).
4.1.2.7. (R,E)-2-[1-(Morpholinoamino)ethylidene]-6-[(E)-1-(morpholinoimino)ethyl]-
7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (3a). Pale yellow solid
(0.145 g, 39%); mp: 100-102 ꢁC; IR (KBr) ꢀmax: 2650–3430, 1694, 1623, 1554, 1457,
1
1366, 1268, 1188 cmꢂ1; H NMR (500 MHz, CDCl3): d 1.69 (3H, s, H-10), 2.13 (3H,
s, H-15), 2.59 (3H, s, H-12), 2.79 (3H, s, H-14), 2.86 - 2.91 (8H, m, H-2’, H-6’, H-2’’,
H-6’’), 3.84 - 3.88 (8H, m, H-3’, H-5’, H-3’’, H-5’’), 5.73 (1H, s, H-4), 11.40 (1H, brs,
9-OH), 14.14 (1H, brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 7.3 (C-15),
16.5 (C-12), 17.3 (C-14), 31.3 (C-10), 54.8 (C-2’, C-6’), 55.1 (C-2’’, C-6’’), 56.5 (C-
9b), 65.2 (C-3’, C-5’), 65.3 (C-3’’, C-5’’), 98.5 (C-6), 100.1 (C-4), 100.9 (C-2), 103.6
(C-9a), 107.0 (C-8), 153.3 (C-5a), 153.5 (C-9), 160.3 (C-7), 167.2 (C-13) 173.4 (C-11),
174.2 (C-4a), 189.5 (C-3), 198.0 (C-1); HRESIMS: m/z 513.2330 [M þ H]þ (cacld for
C26H33N4O7, 513.2343).
4.1.2.8. (R,E)-2-[1-(2-Morpholinoethylamino)ethylidene]-6-[(E)-1-(2-morpholino-eth-
ylamino)ethyl]-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione
(3b).
Pale yellow solid (0.181 g, 44%); mp: 80-83 ꢁC; IR (KBr) ꢀmax: 2664–3450, 1695, 1623,
1
1553, 1461, 1367, 1286, 1188 cmꢂ1; H NMR (300 MHz, CDCl3): d 1.70 (3H, s, H-10),
2.11 (3H, s, H-15), 2.60 (8H, t, J ¼ 4.6 Hz, H-2’, H-6’, H-2’’, H-6’’), 2.68–2.74 (10H,
m, H-12, H-14, H-17, H-19), 3.52–3.67 (4H, m, H-16, H-18), 3.85 (8H, t, J ¼ 4.6 Hz,
H-3’, H-5’, H-3’’, H-5’’), 5.80 (1H, s, H-4), 11.75 (1H, brs, 9-OH), 13.47 (1H, s, 7-
OH); 13C NMR (75 MHz, DMSO-d6): d 7.3 (C-15), 18.6 (C-12), 19.1 (C-14), 32.0 (C-
10), 40.9 (C-16), 43.3 (C-18), 52.8 (C-2’, C-6’), 52.9 (C-2’’, C-6’’), 55.3 (C-17), 56.3
(C-19), 57.2 (C-9b), 66.2 (C-3’, C-5’), 66.3 (C-3’’, C-5’’), 99.0 (C-6), 101.5 (C-4),
103.1 (C-2), 105.1 (C-9a), 107.0 (C-8), 155.8 (C-5a), 157.7 (C-9), 170.3 (C-7), 170.6
(C-13) 173.2 (C-11), 174.5 (C-4a), 188.6 (C-3), 197.5 (C-1); HRESIMS: m/z 569.2953
[M þ H]þ (cacld for C30H41N4O7, 569.2969).
4.1.2.9. (R,E)-2-[1-(2-(Piperazin-1-yl)ethylamino)ethylidene]-6-[(E)-1-(2-(piperazin-1-
yl)ethylamino)ethyl]-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione
(3c). Pale yellow solid (0.185 g, 45%); mp: 92–95 ꢁC; IR (KBr) ꢀmax: 2650–3430, 1697,
1
1623, 1554, 1458, 1369, 1287, 1188 cmꢂ1; H NMR (500 MHz, CDCl3): d 1.73 (3H, s,
H-10), 2.07 (3H, s, H-15), 2.50 - 2.62 (8H, m, H-2’, H-6’, H-2’’, H-6’’), 2.65–2.85
(10H, m, H-12, H-14, H-17, H-19), 2.93 (8H, t, J ¼ 4.4 Hz, H-3’, H-5’, H-3’’, H-5’’),
3.55–3.70 (4H, m, H-16, H-18), 5.80 (1H, s, H-4), 12.20 (1H, brs, 9-OH), 13.22 (1H,
brs, 7-OH), 13.37 (1H, brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 6.7 (C-
15), 17.2 (C-12), 17.6 (C-14), 31.0 (C-10), 40.9 (C-16), 43.2 (C-18), 45.7 (C-3’, C-5’),

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
45.9 (C-3’’, C-5’’), 53.8 (C-2’, C-6’), 54.0 (C-2’’, C-6’’), 55.0 (C-17), 55.9 (C-19), 56.3
(C-9b), 98.6 (C-6), 100.5 (C-4), 102.3 (C-2), 104.2 (C-9a), 107.0 (C-8), 154.6 (C-5a),
157.4 (C-9), 169.5 (C-7), 170.5 (C-13) 172.7 (C-11), 173.7 (C-4a), 188.4 (C-3), 197.9
(C-1); HRESIMS: m/z 567.3288 [M þ H]þ (cacld for C30H43N6O5, 567.3289).
4.1.2.10. (R,E)-2-[1-(4-Methylpiperazin-1-ylamino)ethylidene]-6-[(E)-1-(4-methylpipera-
zin-1-ylimino)ethyl]-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (3d).
Pale yellow solid (0.167 g, 43%); mp: 87–90 ꢁC; IR (KBr) ꢀmax: 2720–3426, 1695, 1624,
1
1553, 1457, 1365, 1280, 1187 cmꢂ1; H NMR (300 MHz, CDCl3þDMSO-d6): d 1.68
(3H, s, H-10), 2.10 (3H, s, H-15), 2.33 (6H, S, H-7’, H-7’’), 2.58 - 2.63 (11H, m, H-
12, H-3’, H-5’, H-3’’, H-5’’), 2.78 (3H, s, H-14), 2.89 - 2.95 (8H, m, H-2’, H-6’, H-2’’,
H-6’’), 5.71 (1H, s, H-4), 11.43 (1H, s, 9-OH), 13.94 (1H, brs, 7-OH), 14.87 (1H, brs,
NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 6.7 (C-15), 15.7 (C-12), 16.6 (C-14),
30.5 (C-10), 44.2 (C-7’), 44.3 (C-7’’), 52.3 (C-3’, C-5’), 52.5 (C-3’’, C-5’’), 53.5 (C-2’,
C-6’), 53.7 (C-2’’, C-6’’), 55.6 (C-9b), 97.7 (C-6), 99.2 (C-4), 100.1 (C-2), 102.7 (C-
9a), 105.8 (C-8), 152.4 (C-5a), 152.7 (C-9), 159.6 (C-7), 165.9 (C-13) 172.5 (C-11),
173.1 (C-4a), 188.4 (C-3), 196.9 (C-1); HRESIMS: m/z 539.2968 [M þ H]þ (cacld for
C28H39N6O5, 539.2976).
4.1.2.11. (R,E)-2-[1-(3-(1H-Imidazol-1-yl)propylamino)ethylidene]-6-[(E)-1-(3-(1H-imi-
dazol-1-yl)propylamino)ethyl]-7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-
dione (3e). Pale yellow solid (0.178 g, 44%); mp: 88–91 ꢁC; IR (KBr) ꢀmax
:
2644–3452, 1698, 1624, 1553, 1467, 1365, 1284, 1186 cmꢂ1 1H NMR (300 MHz,
;
CDCl3þDMSO-d6): d 1.72 (3H, s, H-10), 2.06 (3H, s, H-15), 2.24–2.29 (4H, m, H-
17, H-20), 2.60 (3H, s, H-12), 2.69 (3H, s, H-14), 3.42–3.50 (4H, m, H-16, H-19),
4.22–4.36 (4H, m, H-18, H-21), 5.80 (1H, s, H-4), 7.02 (2H, S, H-4’, H-4’’), 7.08
(2H, S, H-5’, H-5’’), 7.55 (2H, S, H-2’, H-2’’), 12.00 (1H, s, 9-OH), 13.38 (1H, s, 7-
OH), 13.52 (1H, brs, NH); 13C NMR (75 MHz, CDCl3þDMSO-d6): d 7.4 (C-15),
17.6 (C-12), 18.6 (C-14), 29.8 (C-17), 30.8 (C-20), 31.6 (C-10), 40.5 (C-16), 43.0 (C-
19), 43.6 (C-18), 44.2 (C-21), 57.0 (C-9b), 99.1 (C-6), 101.8 (C-4), 101.9 (C-2),
103.5 (C-9a), 108.1 (C-8), 118.1 (C-5’), 118.3 (C-5’’), 129.4 (C-4’), 129.5 (C-4’’),
136.4 (C-2’), 136.6 (C-2’’), 154.5 (C-5a), 155.2 (C-9), 170.0 (C-7), 170.6 (C-13)
173.9 (C-11), 174.9 (C-4a), 189.8 (C-3), 198.5 (C-1); HRESIMS: m/z 559.2653
[M þ H]þ (cacld for C30H35N6O5, 559.2663).
4.1.2.12. (R,E)-2-[1-(Piperidin-1-ylamino)ethylidene]-6-[(E)-1-(piperidin-1-ylimino)ethyl]-
7,9-dihydroxy-8,9b-dimethyldibenzofuran-1,3(2H,9bH)-dione (3f). Pale yellow solid
(0.136 g, 37%); mp: 72–74 ꢁC; IR (KBr) ꢀmax: 2655–3439, 1697, 1624, 1555, 1459,
1
1365, 1280, 1187 cmꢂ1; H NMR (500 MHz, CDCl3): d 1.48–1.53 (4H, m, H-4’, H-
4’’), 1.72–1.79 (11H, m, H-10, H-3’, H-5’, H-3’’, H-5’’), 2.11 (3H, s, H-15), 2.67 (3H,
s, H-12), 2.85–2.95 (11H, m, H-14, H-2’, H-6’, H-2’’, H-6’’), 5.71 (1H, s, H-4), 11.95
(1H, brs, 9-OH), 13.36 (1H, S, 7-OH), 13.92 (1H, brs, NH); 13C NMR (125 MHz,
CDCl3þDMSO-d6): d 7.3 (C-15), 17.1 (C-12), 18.2 (C-14), 22.8 (C-4’), 22.9 (C-4’’),
24.8 (C-3’, C-5’), 24.9 (C-3’’, C-5’’), 31.8 (C-10), 56.6 (C-9b, C-2’, C-6’), 56.7 (C-2’’,
C-6’’), 99.0 (C-6), 100.4 (C-4), 101.2 (C-2), 104.1 (C-9a), 108.0 (C-8), 153.6 (C-5a),

12
U. V. MALLAVADHANI ET AL.
153.8 (C-9), 161.3 (C-7), 168.4 (C-13) 173.9 (C-11), 174.4 (C-4a), 190.0 (C-3), 198.6
(C-1); HRESIMS: m/z 509.2749 [M þ H]þ (cacld for C28H37N4O5, 509.2758).
4.1.3. General procedure for the synthesis of compounds 4a-4i
The (þ)- usnic acid-based pyrazole hybrids (4a-4i) were synthesized by following the
previously reported procedure [18,21]. (þ)- Usnic acid (1 eq) was refluxed with
appropriate phenyl hydrazine (1 eq) and pyridine (1 eq) in ethanol for 12 h. After
completion, the reaction mixture was concentrated under reduced pressure and chro-
matographed over silica gel column to get the pure compounds 4a-4i in
81–92% yields.
4.1.3.1. (R)-8-Acetyl-5,7-dihydroxy-3,4a,6-trimethyl-1-phenyl-1H-benzofuro[3,2-f]inda-
zol-4(4aH)-one (4a). Pale yellow solid, yield: 92%, mp: 196–198 ꢁC; IR (KBr) ꢀmax
:
3083, 2984, 2926, 1675, 1626, 1511, 1477, 1364, 1288, 1170, 1046, 760 cmꢂ1; H NMR
(300 MHz, CDCl3): d 1.81 (3H, s), 2.11 (3H, s), 2.59 (3H, s), 2.64 (3H, s), 6.24 (1H,
s), 7.44-7.58 (5H, m), 11.12 (1H, s), 13.31 (1H, s); 13C NMR (125 MHz, CDCl3): d
7.4, 13.2, 30.4, 31.1, 60.3, 89.2, 101.4, 103.9, 108.2, 110.6, 123.9, 128.7, 129.6, 137.8,
148.1, 151.4, 156.2, 157.6, 163.5, 172.9, 196.1, 200.3; HRESIMS: m/z 417.1442
[M þ H]þ (cacld for C24H21N2O5, 417.1445).
1
4.1.3.2. (R)-8-Acetyl-1-(3-chlorophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4b). Pale yellow solid, yield: 81%, mp: 101–103 ꢁC; IR (KBr)
1
ꢀmax: 3082, 2984, 2927, 1675, 1625, 1475, 1370, 1289, 1178, 1046, 777 cmꢂ1; H NMR
(300 MHz, CDCl3): d 1.81 (3H, s), 2.10 (3H, s), 2.58 (3H, s), 2.65 (3H, s), 6.22 (1H,
s), 7.44-7.51 (3H, m), 7.62-7.63 (1H, m), 11.02 (1H, s), 13.32 (1H, s); 13C NMR
(125 MHz, CDCl3): d 7.4, 13.1, 30.4, 31.1, 60.4, 89.2, 101.4, 103.9, 108.2, 110.7, 121.9,
124.2, 128.7, 130.4, 135.5, 138.6, 148.1, 151.6, 156.2, 157.6, 163.5, 173.3, 196.1, 200.3;
HRESIMS: m/z 451.1057 [M þ H]þ (cacld for C24H20N2O5Cl, 451.1055).
4.1.3.3. (R)-8-Acetyl-1-(4-chlorophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4c). Pale yellow solid, yield: 88%, mp: 109–110 ꢁC; IR (KBr)
1
ꢀmax: 3083, 2985, 2926, 1675, 1625, 1498, 1370, 1289, 1178, 1046, 830 cmꢂ1; H NMR
(500 MHz, CDCl3): d 1.81 (3H, s), 2.11 (3H, s), 2.57 (3H, s), 2.64 (3H, s), 6.21 (1H,
s), 7.53 (4H, brs), 11.04 (1H, s), 13.31 (1H, s); 13C NMR (100 MHz, CDCl3): d 7.4,
13.2, 30.4, 31.1, 60.3, 88.9, 101.4, 103.8, 108.3, 110.7, 125.0, 129.8, 134.5, 136.3, 148.2,
151.6, 156.1, 157.5, 163.5, 173.2, 196.1, 200.3; HRESIMS: m/z 451.1042 [M þ H]þ
(cacld for C24H20N2O5Cl, 451.1055).
4.1.3.4. (R)-8-Acetyl-1-(3-bromophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4d). Pale yellow solid, yield: 82%, mp: 122–124 ꢁC; IR (KBr)
1
ꢀmax: 3082, 2985, 2926, 1675, 1624, 1473, 1362, 1289, 1178, 1046, 756 cmꢂ1; H NMR
(300 MHz, CDCl3): d 1.81 (3H, s), 2.11 (3H, s), 2.57 (3H, s), 2.66 (3H, s), 6.24 (1H,
s), 7.41-7.63 (3H, m), 7.75–7.80 (1H, m), 11.02 (1H, s), 13.33 (1H, s); 13C NMR
(100 MHz, CDCl3): d 7.4, 13.2, 30.4, 31.2, 60.4, 88.9, 101.5, 103.8, 108.3, 110.8, 122.3,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
123.1, 126.9, 130.8, 131.7, 138.9, 148.3, 151.7, 156.1, 157.5, 163.5, 173.4, 196.1, 200.3;
HRESIMS: m/z 495.0536 [M þ H]þ (cacld for C24H20N2O5Br, 495.0550).
4.1.3.5. (R)-8-Acetyl-1-(4-bromophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4e). Yellow solid, yield: 85%, mp: 130–132 ꢁC; IR (KBr) ꢀmax
:
3082, 2985, 2927, 1675, 1624, 1507, 1367, 1289, 1046, 826 cmꢂ1; H NMR (500 MHz,
CDCl3): d 1.80 (3H, s), 2.10 (3H, s), 2.57 (3H, s), 2.64 (3H, s), 6.21 (1H, s), 7.47 (2H,
d, J ¼ 6.8 Hz), 7.69 (2H, d, J ¼ 6.8 Hz), 11.03 (1H, s), 13.31 (1H, s); 13C NMR
(100 MHz, CDCl3): d 7.4, 13.2, 30.4, 31.1, 60.4, 88.9, 101.5, 103.8, 108.3, 110.8, 122.4,
125.2, 132.8, 136.8, 148.2, 151.7, 156.1, 157.5, 163.5, 173.3, 196.1, 200.3; HRESIMS:
m/z 495.0541 [M þ H]þ (cacld for C24H20N2O5Br, 495.0550).
1
4.1.3.6. (R)-8-Acetyl-1-(3-methoxyphenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-ben-
zofuro[3,2-f]indazol-4(4aH)-one (4f). Pale yellow solid, yield: 87%, mp: 157–159 ꢁC;
1
IR (KBr) ꢀmax: 3083, 2985, 2926, 1675, 1624, 1473, 1365, 1289, 1046, 778 cmꢂ1; H
NMR (500 MHz, CDCl3): d 1.81 (3H, s), 2.11 (3H, s), 2.58 (3H, s), 2.64 (3H, s), 3.89
(3H, s), 6.27 (1H, s), 6.99-7.02 (1H, m), 7.12-7.14 (2H, m), 7.43-7.46 (1H, m), 11.10
(1H, s), 13.32 (1H, s); 13C NMR (100 MHz, CDCl3): d 7.4, 13.2, 30.4, 31.1, 55.6, 60.3,
89.3, 101.5, 103.9, 108.2, 109.7, 110.6, 114.4, 115.9, 130.3, 138.8, 148.2, 151.3, 156.2,
157.6, 160.5, 163.5, 172.9, 196.1, 200.3; HRESIMS: m/z 447.1540 [M þ H]þ (cacld for
C25H23N2O6, 447.1550).
4.1.3.7. (R)-8-Acetyl-1-(4-methoxyphenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzo-
furo[3,2-f]indazol-4(4aH)-one (4g). Pale yellow solid, yield: 91%, mp: 168–171 ꢁC; IR
1
(KBr) ꢀmax: 3083, 2980, 2928, 1676, 1624, 1520, 1478, 1365, 1289, 1047, 829 cmꢂ1; H
NMR (500 MHz, CDCl3): d 1.80 (3H, s), 2.10 (3H, s), 2.57 (3H, s), 2.63 (3H, s), 3.88
(3H, s), 6.17 (1H, s), 7.05 (2H, d, J ¼ 8.8 Hz), 7.48 (2H, d, J ¼ 8.8 Hz), 11.16 (1H, s),
13.32 (1H, s); 13C NMR (100 MHz, CDCl3): d 7.4, 13.2, 30.4, 31.1, 55.6, 60.2, 89.1,
101.4, 103.9, 108.1, 110.3, 114.7, 125.4, 130.8, 148.1, 151.1, 156.2, 157.6, 159.7, 163.4,
172.6, 196.0, 200.3; HRESIMS: m/z 447.1528 [M þ H]þ (cacld for
C25H23N2O6, 447.1550).
4.1.3.8. (R)-8-Acetyl-1-(3-nitrophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4h). Yellow solid, yield: 83%, mp: 116–118 ꢁC; IR (KBr) ꢀmax
:
3083, 2980, 2928, 1675, 1624, 1473, 1367, 1289, 1044, 769 cmꢂ1; H NMR (400 MHz,
CDCl3): d 1.84 (3H, s), 2.11 (3H, s), 2.59 (3H, s), 2.65 (3H, s), 6.28 (1H, s), 7.78 (1H,
t, J ¼ 8.19 Hz), 7.98–8.02 (1H, m), 8.81–8.85 (1H, m), 8.49 (1H, t, J ¼ 2.07 Hz), 10.92
(1H, s), 13.31 (1H, s); 13C NMR (75 MHz, CDCl3): d 7.4, 13.1, 30.4, 31.2, 60.6, 88.5,
101.5, 103.7, 108.4, 111.1, 118.4, 122.9, 129.2, 130.7, 138.9, 148.5, 148.7, 152.2, 156.1,
157.3, 163.5, 174.1, 196.1, 200.3; HRESIMS: m/z 462.1289 [M þ H]þ (cacld for
C24H20N3O7, 462.1295).
1
4.1.3.9. (R)-8-Acetyl-1-(4-nitrophenyl)-5,7-dihydroxy-3,4a,6-trimethyl-1H-benzofuro[3,2-
f]indazol-4(4aH)-one (4i). Pale yellow solid, yield: 81%, mp: 125–127 ꢁC; IR (KBr)
1
ꢀmax: 3083, 2980, 2928, 1675, 1626, 1513, 1473, 1344, 1284, 1044, 847 cmꢂ1; H NMR

14
U. V. MALLAVADHANI ET AL.
(300 MHz, CDCl3): d 1.83 (3H, s), 2.10 (3H, s), 2.60 (3H, s), 2.65 (3H, s), 6.32 (1H,
s), 7.83 (2H, d, J ¼ 9.06 Hz), 8.45 (2H, d, J ¼ 9.06 Hz), 10.89 (1H, s), 13.31 (1H, s);
13C NMR (125 MHz, CDCl3): d 7.4, 13.2, 30.3, 31.1, 60.6, 88.8, 101.5, 103.6, 108.5,
111.3, 123.7, 125.2, 142.8, 146.7, 148.7, 152.4, 156.1, 157.3, 163.6, 174.1, 196.1, 200.2;
HRESIMS: m/z 462.1288 [M þ H]þ (cacld for C24H20N3O7, 462.1295).
4.2. Biology
4.2.1. Cytotoxicity assay
All cell lines (HeLa, MDA-MB-231, A549, and MiaPaca) used in this study were pur-
chased from the American Type Culture Collection (ATCC), United States. The syn-
thesized test compounds were evaluated for their in vitro anti proliferative activity in
these four different human cancer cell lines. A protocol of 48 h continuous drug
exposure was used, and a SRB cell proliferation assay was used to estimate cell viabil-
ity or growth. All the cell lines were grown in Dulbecco’s modified Eagle’s medium
(containing 10% Fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at
37 ꢁC). Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and
seeded in 96-well plates in 100 lL aliquots at plating densities depending on the dou-
bling time of individual cell lines. The microliter plates were incubated at 37 ꢁC, 5%
CO2, 95% air, and 100% relative humidity for 24 h prior to addition of experimental
drugs and were incubated for 48 h with different doses (0.01, 0.1, 1, 10, 100mM) of
synthesized compounds. After 48 h of incubation at 37 ꢁC, cell monolayers were fixed
by the addition of 10% (wt/vol) cold trichloroacetic acid and incubated at 4 ꢁC for 1 h
and were then stained with 0.057% SRB dissolved in 1% acetic acid for 30 min at
room temperature. Unbound SRB was washed with 1% acetic acid. The protein–-
bound dye was dissolved in 10 mM Tris base solution for OD determination at
510 nm using a microplate reader (Enspire, Perkin Elmer) [19].
4.2.2. Cell cycle analysis
HeLa cells in 60 mm dishes were incubated for 24 h in the presence or absence of test
compounds 2e and 4a at 10 mM concentrations. Cells were harvested with Trypsin-
EDTA, fixed with ice-cold 70% ethanol at 4 ꢁC for 30 min, ethanol was removed by
centrifugation and cells were stained with 1 ml of DNA staining solution [0.2 mg of
propidium iodide (PI), and 2 mg RNase A] for 30 min as described earlier. The DNA
contents of 20,000 events were measured by flow cytometer (BD FACSCanto II).
Histograms were analyzed using FCS express 4 plus [22].
4.2.3. Immunohistochemistry of tubulin and analysis of nuclear morphology
HeLa cells were seeded on glass cover slip, incubated for 24 h in the presence or
absence of test compounds 2e and 4a at a concentration of 10 lM. Cells grown on
cover slips were fixed in 3.5% formaldehyde in phosphate-buffered saline (PBS) pH
7.4 for 10 minutes at room temperature. Cells were pre-mobilized for 6 minutes in
PBS containing 0.5% Triton X-100 (Sigma) and 0.05% Tween-20 (Sigma). The pre-
mobilized cells were blocked with 2% Bovine serum albumin (BSA) (Sigma) in PBS
for 1 h. Later, the cells were incubated with primary antibody for tubulin from

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
15
(Sigma) at (1:200) diluted in blocking solution for 4 h at room temperature.
Subsequently the antibodies were removed and the cells were washed thrice with PBS.
Cells were then incubated with FITC labeled anti-mouse secondary antibody (1:500)
for 1 h at room temperature. Cells were washed thrice with PBS and mounted in
medium containing DAPI. Images were captured using the Olympus confocal micro-
scope FLOW VIEW FV 1000 series and analyzed with FV10ASW 1.7 series soft-
ware [19].
4.2.4. Western blot analysis of soluble versus polymerized tubulin
Cells were seeded in 12-well plates at 1 ꢃ 105 cells per well in complete growth
medium. Following treatment of cells with compounds 2e and 4a for duration of
24 h, cells were washed with PBS and subsequently soluble and insoluble tubulin frac-
tions were collected. To collect the soluble tubulin fractions, cells were permeablized
with 200 ml of pre-warmed lysis buffer [80 mM Pipes-KOH (pH 6.8), 1 mM MgCl2,
1 mM EGTA, 0.2% Triton X-100, 10% glycerol, 0.1% protease inhibitor cocktail
(Sigma-Aldrich)] and incubated for 3 min at 30 ꢁC. Lysis buffer was gently removed,
and mixed with 100 ml of 3 ꢃ Laemi’s sample buffer (180 mM Tris-Cl pH 6.8, 6%
Sodium dodecyl sulphate (SDS), 15% glycerol, 7.5% b-mercaptoethanol and 0.01%
bromophenol blue). Samples were immediately heated to 95 ꢁC for 3 min. To collect
the insoluble tubulin fraction, 300 ml of 1 ꢃ Laemi’s sample buffer was added to the
remaining cells in each well, and the samples were heated to 95 ꢁC for 3 min. Equal
volumes of samples were run on an SDS-10% polyacrylamide gel and were transferred
to a nitrocellulose membrane employing semidry transfer at 50 mA for 1 h. Blots were
probed with mouse anti-human a-tubulin diluted 1:2000 ml (Sigma) and stained with
rabbit anti-mouse secondary antibody coupled with horseradish peroxidase, diluted
1:5000 ml (Sigma). Bands were visualized using an enhanced Chemiluminescence
protocol (Pierce) and radiographic film (Kodak) [19].
Acknowledgments
We are thankful to Director, CSIR-IICT for support and encouragement.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was financially supported by UGC, Govt. of India, provided Senior Research
Fellowship to VNR.
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