Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2017.08.033

Forskolin C₁-isoxazole derivatives (3,5-regioisomers) (11a–e, 14, 15a–h and 15, 16a–g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell l

Full text of DOI:10.1016/j.bmcl.2017.08.033

Accepted Manuscript
Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer ac-
tivity against breast cancer cell lines
Srinivas Burra, Vani Voora, Ch. Prasad Rao, P. Vijay Kumar, Rama Krishna
Kancha, G.L. David Krupadanam
PII:
S0960-894X(17)30834-X
http://dx.doi.org/10.1016/j.bmcl.2017.08.033
BMCL 25227
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2017
21 May 2017
16 August 2017
Please cite this article as: Burra, S., Voora, V., Rao, h.P., Vijay Kumar, P., Kancha, R.K., David Krupadanam, G.L.,
Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.033
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity
against breast cancer cell lines
a
b
a
a
Srinivas Burra , Vani Voora , Prasad Rao. Ch , Vijay Kumar. P , Rama Krishna
Kancha b and David Krupadanam. G. La ∗
aNatural products lab, Dept. of. Chemistry, Osmania University, Hyderabad-500007, INDIA.
bMolecular Medicine and Therapeutics Laboratory, CPMB, Osmania University, Hyderabad-500007, INDIA.
ARTICLE INFO
ABSTRACT
Forskolin C1-isoxazole derivatives (3,5-regioisomers) ( 11a-e, 14,15a-h and 15,16a-g ) were
synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were
tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of
the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not
against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds
11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against
MCF-7 cell line with an IC50 < 1µM. The derivative 14f exhibited highest activity in both p53-
positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC50 of 0.5µM.
Article history:
Received
Revised
Accepted
Available online
Keywords:
Forskolin
Isoxazoles
Breast cancer
1,3-Dipolar cycloadditions
Regioselectivity
2009 Elsevier Ltd. All rights reserved
.
OH
O
O
OH
H
H
Breast cancer is the most common cancer in
O
O
O
S
F
O
F
H
H
OH
OH
H
H
CF3
HO
O
O
the women worldwide. Natural products such as
Docetaxel, Paclitaxel, Eribuline, Vinblastine and
Doxorubicin were used for the treatment of breast
cancer 1-4. Importantly, diterpenoids such as
Forskolin (1), Fulvestrant (2), Megestrol (3),
Formestane (4), Atamestane (5) and Exemestane
(6) were shown to have anti-cancer activity
(Figure-1), specifically against estrogen receptor-
7
Megestrol
(3)
Fulvestrant
(2)
Forskolin
(1)
O
O
O
H
H
H
H
H
H
H
H
H
O
O
O
OH
Exemestane
(6)
Atamestane
(5)
Formestane
(4)
Figure-1: Some diterpenes as breast cancer agents.
Forskolin,
a
labdane diterpene natural
product, has been isolated from the roots of an
14
Indian sub-continent plant Coleus forskohlii
.
positive breast cancer 5-13
.
Forskolin exhibits a wide range of pharmacological
properties such as anti-obesity 15, Cardiovascular 16,
asthma 17, glaucoma 18, antihypertensive, inotropic
19,20, anti-cancer activity 21,22, stimulates the
———
∗ Corresponding author. Tel.: +0-9849128145 ;
e-mail: gldkrupa@gmail.com

adenylyl cyclase activity and increases intracellular disubstituted isomer, isoxazole H-4 appears as a
levels of cyclic AMP 23. It has been reported that singlet in the range of δ 6.59-6.76 ppm, whereas in
the order of reactivity of the hydroxyls in forskolin the 3,4-isomer, isoxazole H-5 appears downfield in
is 1-OH > 6-OH > 9-OH. 24 Generally, Heterocyclic the range of δ 7.50-8.50 ppm 29.
compounds improve the pharmacokinetic and
pharmacodynamic properties of anti-cancer drugs
A small library of isoxazole substituted
forskolin derivates was synthesized adopting 1,3-
by enhancing lipophilicity, polarity or other dipolar cycloadditions. To synthesize isoxazole as
physicochemical features. In the present study, we the sole product at C1-OH, the double bond at C-
thus introduced novel isoxazoles at C1-OH and
tested their activity on breast cancer cell lines.
1,3-dipolar cycloaddition is one of the best
methods for the synthesis of 5-membered
14,15 of forskolin (1) was initially reduced with
10% Pd/C in MeOH resulting in the formation of
14,15-dihydroforskolin (7) (Scheme 1: Step-1).
This intermediate (7) was then propargylated at 1-
heterocyclic compounds. In this reaction, 1,3-dipole OH in the presence of K2CO3 and NaI resulting in
reacts with dipolarophiles in a symmetry allowed the formation of 1-O-propargyl-14,15-
[π4s+π2s] manner. According to Sustmanns dihydroforskolin (8) and 1-O-propargyl-6-O-acetyl-
approach 25 these cycloadditions are of three types: 7-deacetyl14,15-dihydroforskolin (9) regioisomers
HOMO-Controlled (Type-I: the interaction of the in 1:1 ratio (Scheme 1: Step-2). 1,3-dipolar
30
cycloadditions were finally performed with aryl
dipole HOMO with the dipolarophile LUMO is
greatest), HOMO-LUMO Controlled (Type-II: both nitrile oxides (in situ generated from 10a-d, h) on
frontier orbital interactions are large) and LUMO- pure 1-O-propargyl-14,15-dihydroforskolin (8)
Controlled (Type-III: the interaction of the dipole resulting in the formation of the isoxazoles: 1-O-(3-
LUMO with the dipolarophile HOMO is greatest). (substituted phenyl)isoxazole-5-yl)methyl-14,15-
Earlier studies suggested that reactions involving dihydroforskolins (11a-e) (Scheme 1: Step 3) (ex.
aryl nitrile oxide 1,3-dipoles belong to Type-II procedure 31). Similarly, (Scheme-1, step-2 and 3),
26-28
molecular orbital interactions in FMO theory
.
propargylation of forskolin (1) gave two
¯
≡N+
-O ) dipole, larger HOMO regioisomers 1-O-propargyl-forskolin (12) and 1-
In nitrile oxide (C
coefficient is on electronegative oxygen, while the O-propargyl-6-O-acetyl-7-deacetyl-forskolin (13)
32
larger LUMO coefficient is on the carbon. The (Scheme-2, step-1) . Since, these compounds (12
HOMO of the 1,3-dipole when interacts with the and 13) have both terminal alkyne and alkene
LUMO of the dipolarophile favors the formation of moieties, reaction with aryl nitrile oxides (in situ
one particular regioisomer, while the LUMO of 1,3- generated from 10a-h) adopting 1,3-dipolar
dipole when interacts with the HOMO of the cycloaddition strategy furnished two products C1-
dipolarophile favors the formation of an opposite isoxazole and C13-isoxazoline substituted C1-
regioisomer. Generally, isoxazole regioisomers are isoxazole: 14a-h & 15a-h (in 3:1 ratio from
distinguished using 1H NMR. In the 3,5-

of 3,5-disubstituted isoxazole regioisomer. Among
the protons of C-13 isoxazolines (15a-h and 17a-g),
H-5‴ appeared in the range of δ 4.89-6.17 (br. s)
and two H-4‴ protons appeared in the range of δ
3.21-5.13 (m, 2H), indicating the formation of 3,5-
disubstituted-4,5-dihydroisoxazoline exclusively.
ESI-MS of 11a molecular ion peak was observed at
m/z 622 [M+Na]+, HRMS (ESI) of molecular ion
peak of 14a was observed at m/z : 615.3289
compound 12) and 16a-g & 17a-g (in 3:1 ratio from
compound 13) (Scheme-2, step-2).
All the novel forskolin derivatives were
1
characterized from their H NMR and/or 13C NMR,
1
IR and ESI-MS/HRMS-(ESI) spectra. In the H
NMR of 11a-e, 14a-h, 15a-h, 16a-g and 17a-g, C1-
isoxazole proton appeared as singlet in the range of
δ 6.43-6.91ppm indicating a proton at 4th position
of isoxazole ring, confirming exclusive formation

[M+NH4]+, 15a was observed at : m/z 747.3491 indicating the presence of 6-acetyl group at axial
[M+H]+, 16a was observed at: m/z 615.3288 position, while 7-OCOCH3 of 11a, 14a and 15a
[M+NH4]+ and 17a was observed at : m/z 747.3495
[M+H]+.
protons showed no nOe with other protons
indicating the presence of 7-acetyl group at
equatorial position 32. For compounds 15a and 17a,
NOESY correlations for final isoxazole
products 11a, 14a, 15a, 16a and 17a (See figure-2) nOe was observed between 1) H-12(a) proton and H-
revealed that the isoxazole proton H-4ʹ showed nOe 14 2) H-15 proton and H-2″″/H-6″″, and 3) 4″″a-
with aromatic protons H-2″ / H-6″ while 4″a-OCH3
OCH3 protons and H-3″″/H-5″″ (for NOESY
protons showed nOe with H-3″/H-5″ indicating that spectra see supporting information).
they are spatially closer. For compounds 11a, 14a
and 15a, 6-OH proton showed nOe with 8-CH3, H-5
Aryl nitrile oxides which were used in above
reactions (Scheme-1 & Scheme-2) were generated
proton showed nOe with 9-OH/1-OCH2, and 9-OH in situ from substituted benzaldehyde oximes (10a-
proton showed nOe with H-12(a) indicating that h), in the presence of NaOCl as oxidant and
these protons are at the axial position. Further, 6- triethylamine as the base. 10a-h were synthesized
OCOCH3 protons of 16a and 17a showed nOe with
from substituted benzaldehydes in the presence of
H-2a / 4(a)-CH3 / 10-CH3 and H-2a respectively hydroxylamine hydrochloride and sodium acetate
H3C
CH3
CH3
13
CH3
13
O
CH3
O
11
O
15
O
15
14
H
CH
12
3H
15
14
H
CH3
CH
12
3H
O
H
CH3
CH
12
3H
O
H
14
13
H
H
11
CH3
O
11
2
CH3
OH
CH3
OH
2
10
2
10
H
10
9
H
9
H
9
7
8
1
O
8
1
7
8
1
7
H
H
CH3
CH3
H
4
H
H
6
H
5
6
3
H3C
H2C
H3C
H2C
4
4
H3C
H2C
5
3
6
5
O
OH
H
3
O
OH
H
O
OH
H
H
OH
O
O
H
H
H
H
5'
O
H
H
H
H
5'
H
H
H
O
5'
O
' O
1'
O
N
4'
1
1'
H
5' a
4'
3'
H
N
H
6' '
6' '
N
3'
3'
5' '
H
H
H
H
H
H
5' '
2' '
H
2' '
H
3' '
OCH3
4' 'a
3' '
OCH3
OCH3
4' 'a
H
(11a)
(14a)
(16a)
CH3
H3C
O
CH3
H
CH3
CH
12
O
O
11
3H
O
1' ' '
1' ' '
H
CH3
CH
12
3H
O
H
11
13
14
CH3
OH
O
H
2
13
14
O
CH3
O
10
2
H
9
N
3' ' '
10
H
H
N
3' ' '
H
9
7
8
1
H
H
8
7
1
H
H
H
H
15
4
6' ' ' '
4
H
5
6
3
H
6' ' ' '
H3C
H2C
H
15
O
OH
H
H
6
H
5
3 H3C
O
O
OH
H
H
OH
H2C
5'
H
H
H
H
H
H
CH3
2' ' ' '
H
H
H
H
5'
5' ' ' '
2' ' ' '
5' ' ' '
O
4'
O
N
3' ' ' '
1'
5' a
4'
H
OCH3
H
O
' ' ' '
OCH3
1'
3'
3
H
3'
H
N
6' '
6' '
5' '
H
H
H
5' '
OCH3
H
2' '
H
2' '
H
3' '
3' '
OCH3
4' 'a
4' 'a
(15a)
(17a)
Figure-2: NOESY Correlations of 11a, 14a, 15a, 16a and 17a

were heated at reflux in ethanol for 1hour to afford only 10 derivatives displayed more than 2-fold anti-
oximes (10a-h) with excellent yields (80-96%) 29.
cancer activity against BT-474 cell line, of which 8
Forskolin was previously shown to have
5
activity against breast cancer cell lines
.
derivatives displayed more than 10-fold activity
Furthermore, the anti-proliferative activity of (Table 1). Notably,
9
forskolin derivatives
forskolin was shown to be mediated by the tumor displayed very high anti-cancer activity at sub
suppressor protein p53 6. Thus, we tested the anti-
micro-molar concentrations (IC50 < 1 µM) against
Table-1: Anti cancer activity of forskolin derivatives
Anti-cancer activity (IC50 in µM)
BT-474 Compound
Compound
Forskolin
MCF-7
MCF-7
BT-474
63.3+5.7
>100
52.6+4.6
79.6+18.7
15f
10+0
>100
0.5+0
*
>100
*
8
11a
14g
15g
0.5+0
2.1+0.1
9.6+2
0.5+0
>100
29.3+10.6
7+2
0.5+0
5+1
11b
11c
11d
11e
-
14h
15h
13
35.3+10
30+0
>100
>100
>100
>100
28.3+12.3
>100
>100
1.6+0.2
2.6+1.1
9.1+0.2
57.3+9.4
79.3+14.4
12+2.6
0.5+0
16a
17a
16b
17b
16c
17c
16d
17d
16e
17e
16f
17f
16g
17g
-
>100
-
>100
12
0.5+0
4+1
14a
15a
14b
15b
14c
15c
14d
15d
14e
15e
14f
3+0.8
1.1+0.4
0.5+0
4.3+1.1
>100
4.4+0.3
*
4+1
*
>100
>100
55.3+9.2
>100
*
*
32+7.5
59+14.7
84.6+15.5
3.3+0.5
0.5+0
>100
>100
>100
1.3+0.5
>100
*
5.6+1.1
*
>100
*
*
75+22.9
7+2.6
5.6+2
0.5+0
>100
0.5+0
*
*
* = formed in very less amounts
cancer activity of forskolin derivatives on both p53- the p53-positive MCF-7 cell line. Of these, the
positive MCF-7 and p53-negative BT-474 cell lines compound 14f (1-O-(3-(4-benzyloxyphenyl-
33. The parental forskolin displayed cellular IC50 of isoxazole-5-yl)methyl-forskolin) with an acetyl
63.3+5.7 µM against MCF-7 but lacked any anti-
cancer activity against the BT-474 cell line (IC50
group at 7th position exhibited the highest activity
>
in both the MCF-7 and BT-474 cell lines with an
100 µM) (Table 1). Of the 32 derivatives tested, 24 IC50 of 0.5µM (Table 1). On the contrary, the
compounds showed more than 2-fold higher anti- compound 16f (1-O-(3-(4-benzyloxyphenyl-
cancer activity than the parental forskolin against isoxazole-5-yl)methyl-6-O-acetyl-7deacetyl-
MCF-7 cell line, of which 18 derivatives showed forskolin) with an acetyl group at 6th position
more than 10-fold activity (Table 1). However, lacked anti-cancer activity with cellular IC50 values

greater than that of the parental forskolin (Table 1). and 17b exhibited significant anti-cancer activity in
Additionally, compounds 11a, 14a, 14f, 14h, 15a p53-positive MCF-7 cell line, while compounds 14f
and 17f displayed anti-cancer activity in both cell and 17f showed anti-tumor activity in p53-negative
lines with cellular IC50 < 5 µM (Table 1). Further BT-474 cell line.
analysis of these four compounds 11a, 14a, 14f and
14h showed significant cytotoxicity than that of the
parental forskolin (Figure 3) 34.
Acknowledgements
B.S acknowledges thankful financial support from
the UGC-New Delhi, India in the form of UGC-
SRF. The authors thank the Head, Department of
Chemistry, Osmania University, Hyderabad, India
for providing laboratory facilities and CFRD, O.U,
Hyderabad, India for providing spectral data.
RKK acknowledges the support from UGC-FRP,
Government of India for the Start-up grant [No.
F.4-5(136-FRP)/2014 (BSR)] and DST-SERB for
the Extra-Mural Research Grant [No. EMR /2014/
000904].
Supplementary data
Supplementary data provided with this article as a
separate file.
In conclusion, we describe the synthesis of
3,5-disubstituted isoxazole derivatives of forskolin
(11a-e, 14a-h, 15a-h, 16a-g and 17a-g) with good
yields in a regioselective manner by Nitrile Oxide
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1H, 9-OH), 5.52 (d, J = 4.2Hz, 1H, H-7), 4.67
(d, J =13.0Hz, 1H, OCH2), 4.48–4.40 (m, 2H,
OCH2, H-6), 4.36 (br.s, 1H, H-1), 3.86 (s, 3H,
OCH3), 3.27 (d, J =15.5Hz, 1H, H-12e), 2.28-
2.22 (m, 1H,H-12a), 2.14 (s, 3H, COCH3),
2.17– 2.12 (m, 1H, H-2a), 2.10 – 2.03 (m, 1H,
H-5), 1.83 (s, 1H, 6-OH), 1.75 – 1.63 (m, 3H,
H-14, H-2e), 1.60 (s, 3H, 8-CH3), 1.50 (s, 3H,
10-CH3), 1.53 – 1.41 (m, 1H, H-3a), 1.26 (s,
3H, 4-CH3(e)), 1.20 (s, 3H,13-CH3), 1.17–
1.11(m,1H, H-3e), 1.02 (s, 3H, 4-CH3(a)), 0.89
(dd, J = 8.8, 6.1Hz, 3H, H-15). 13C NMR
(100.6 MHz, CDCl3) δ 209.50 (CO), 169.57
ʹ), 162.04 (C
30. (a) Huisgen R, Angew Chem Int Ed Engl.
1963; 2: 633. (b) Huisgen R, J Org
Chem.1968; 33: 2291. (c) Bast K, Christl M,
Huisgen R, Mack W and Sustmann R, Chem
Ber. 106, in press.
31. General method for the Synthesis of 1-O-(3-
substitutedphenyl)isoxazole-5-yl)-methyl-
14,15-dihydroforskolins (11a-e). To a stirred
solution of propargylated forskolins (8, 12
and 13) (0.22mmol) in 5ml dichloromethane,
add triethylamine (0.22mmol) and 2ml of
aqueous NaOCl (9-12%) at 00C, add
corresponding benzaldehyde oximes (10a-h)
(0.22mmol) slowly in a portion of 30min time
in each case. Reaction was stirred at room
temperature for 6-8hr. After completion of
the reaction, the mixture was dissolved in
20ml of water and extract with DCM.
Separate the organic layer, dried over
anhydrous Na2SO4, concentrated under
reduced pressure, purified by column
chromatography (EtOAc/Hexane), 60-120
mesh silica gel to afford pure compounds
(11a-e, 14a-h and 15a-h (~3:1 ratio), 16a-g
and 17a-g (~3:1 ratio)) as solids. 1-(3-(4-
Methoxyphenyl)isoxazole-5-yl)methoxy)-
(OCO), 167.90 (C-3
-4″), 161.08
ʹ), 128.25
(C-5
(C-2″,6″), 121.26 (C-1″),114.34
ʹ), 84.25 (C
(C-3″, 5″), 101.21 (C-4
-13), 82.41
(C-8), 81.21 (C-1), 76.91 (C-9), 76.25 (C-7),
69.90 (C-6), 61.54 (OCH2), 55.35 (4ʹa-
OCH3), 49.52 (C-12), 44.00 (C-5), 43.78(C-
10), 38.55(C-3), 36.43(C-4), 34.18 (C-14),
32.78(4e-CH3), 29.46(8-CH3), 24.04 (13-
CH3), 23.82 (4a-CH3), 21.11(C-2), 20.70
(OCOCH3), 20.17 (10-CH3), 7.85 (C-15).
ESI-MS: m/z 622 [M+Na] +.
32. Koteswara Reddy M, Santosh Kumar K,
Sreenivas P, David Krupadanam GL,
Janardhan Reddy K., Tetrahedron Lett. 2011;
52: 6537–6540.
14,15-dihydroforskolin (11a). Off white solid,
m.p-120-1220C, yield : 85%; IR (KBr) (cm-
1): 3498(OH), 3334(OH), 2926(CH), 2854
(CH), 1731(OCO), 1708(CO), 1608 (C=N);
1H NMR (400 MHz, CDCl3) δ 7.73 (d, J =
8.8 Hz, 2H, H-2″, 6″), 6.97 (d, J = 8.8Hz, 2H,
3″, 5″), 6.45 (s,1H, H-4ʹ (isoxazole)), 6.44 (s,
33. Cell viability assay: Breast cancer (MCF7
and BT474) cell lines were used to test the
anti-cancer activity. 2x103 cells per well were
cultured in triplicates in a 96-well plate in the
presence of novel compounds. Untreated cells
were taken as the control. 48 hours following
the treatment with indicated compounds,

alamar blue was added and cell viability was
analyzed by assaying samples at 570 nm
wavelength. Percentage inhibition of cell
viability upon treatment with drugs when
compared to that of the untreated cells was
calculated and IC50 values were derived and
shown as mean + SD in Table-1.
34. Microscopy: MCF7 cells were plated in 6-
well plates and were treated with indicated
drugs. Untreated well was taken as control.
48 hours after drug treatment, cells were
observed under microscope for morphological
changes and photographs were taken and
shown in Figure-3.

Graphical Abstract
Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer
activity against breast cancer cell lines
Srinivas Burra a, Vani Voora b, Prasad Rao. Ch a, Vijay Kumar. P a, Rama Krishna
Kancha b and David Krupadanam. G. La ∗
aNatural products lab, Dept. of. Chemistry, Osmania University, Hyderabad-500007, INDIA.
bMolecular Medicine and Therapeutics Laboratory, CPMB, Osmania University, Hyderabad-500007, INDIA.
R3
N
O
O
14
15
O
1
O
OH
9
O
O
O
O
OH
R2
O
OH
OH
7
6
O
R1
R1 = H, R2 = COCH3, 14,15-ethyl;
(11a-e)
1
2
Forskolin
(14a-h)
(16a-g)
R = H, R = COCH3, 14,15-ethylene:
(1)
1
2
R = COCH3, R =H, 14,15-ethylene: