Discovery of a marine-derived bis-indole alkaloid fascaplysin, as a new class of potent P-glycoprotein inducer and establishment of its structure-activity relationship

Article abstract of DOI:10.1016/j.ejmech.2015.10.049

The screening of IIIM natural products repository for P-gp modulatory activity in P-gp over-expressing human adenocarcinoma LS-180 cells led to the identification of 7 natural products viz. withaferin, podophyllotoxin, 3-demethylcolchicine, agnuside, rese

Full text of DOI:10.1016/j.ejmech.2015.10.049

European Journal of Medicinal Chemistry 107 (2016) 1e11
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of a marine-derived bis-indole alkaloid fascaplysin, as a new
class of potent P-glycoprotein inducer and establishment of its
structureeactivity relationship
,
, b
Sudhakar Manda a b, Sadhana Sharma c, Abubakar Wani c, Prashant Joshi a
,
Vikas Kumar b d, Santosh K. Guru c, Sonali S. Bharate d, Shashi Bhushan b
,
,
, c
Ram A. Vishwakarma a b, Ajay Kumar c **, Sandip B. Bharate a
,
,
, b, *
a Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
b Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
d Preformulation Laboratory, PKPD Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The screening of IIIM natural products repository for P-gp modulatory activity in P-gp over-expressing
human adenocarcinoma LS-180 cells led to the identification of 7 natural products viz. withaferin,
podophyllotoxin, 3-demethylcolchicine, agnuside, reserpine, seseberecine and fascaplysin as P-gp in-
ducers. Fascaplysin (6a), a marine-derived bis-indole alkaloid, was the most potent among all of them,
showing induction of P-gp with EC50 value of 25 nM. P-gp induction is one of the recently targeted
Received 14 August 2015
Received in revised form
10 October 2015
Accepted 28 October 2015
Available online 31 October 2015
strategy to increase amyloid-b clearance from Alzheimer brains. Thus, we pursued a medicinal chemistry
of fascaplysin to establish its structureeactivity relationship for P-gp induction activity. Four series of
analogs viz. substituted quaternary fascaplysin analogs, D-ring opened quaternary analogs, D-ring
Keywords:
Fascaplysin
opened non-quaternary analogs, and
b-carbolinium analogs were synthesized and screened for P-gp
b
-carboline
induction activity. Among the total of 48 analogs screened, only quaternary nitrogen containing analogs
6aeg and 10a, 10hel displayed promising P-gp induction activity; whereas non-planar non-quaternary
analogs 9aem, 13aen, 15aeh were devoid of this activity. The P-gp induction activity of best compounds
was then confirmed by western-blot analysis, which indicated that fascaplysin (6a) along with 4,5-
difluoro analog of fascaplysin 6f and D-ring opened analog 10j displayed 4e8 fold increase in P-gp
P-glycoprotein induction
Acetylcholinesterase
Quaternary analogs
Alzheimer's disease
expression in LS-180 cells at 1
tylcholinestease (AChE), an enzyme responsible for neuronal loss in Alzheimer's disease. Thus, fasca-
plysin and its analogs showing promising P-gp induction along with AChE inhibition at 1 M, with good
mM. Additionally, compounds 6a and 6f also showed inhibition of ace-
m
safety window (LS-180: IC50 > 10
m
M, hGF: 4
m
M), clearly indicates their promise for development as an
anti-Alzheimer agent.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
hallmarks viz. diffuse neuronal loss with a particular involvement
of the cholinergic system, extracellular protein deposits (amyloid-
b
Alzheimer's disease (AD) is the most common form of senile
dementia and the fourth highest cause of disability and death in the
elderly. It is characterized by the presence of three main brain
plaques) and intracellular protein deposits (neurofibrillary tangles,
NFTs) [1e3]. All current therapies are based on the cholinergic
hypothesis and demonstrate only symptomatic treatment. P-
glycoprotein (P-gp) is highly expressed on the luminal surface of
brain capillary endothelial cells and contributes to the BBB. The
recent two independent clinical studies [4,5] observed that AD
* Corresponding author. Medicinal Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu 180001, India.
** Corresponding author.
E-mail addresses: ajaykmahajan@hotmail.com (A. Kumar), sbharate@iiim.ac.in
(S.B. Bharate).
patients have decreased clearance of CNS amyloid-
healthy volunteers. The rate of A production is same as that in
healthy volunteers; whereas rate of clearance is impaired by
25e30%. High levels of A then initiates cascade of events
b compared to
b
b
http://dx.doi.org/10.1016/j.ejmech.2015.10.049
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

2
S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
culminating in neuronal damage and death manifesting as pro-
gressive dementia of the AD type. There is a direct link between P-
was screened for P-gp induction activity in P-gp overexpressing
human adenocarcinoma LS-180 cells using rhodamine 123 (Rh123)
gp and A
b
metabolism in vivo and the P-gp activity at the BBB could
cell exclusion method and rifampicin (10
mM) was used as a positive
affect risk for developing AD as well as provide a novel diagnostic
and therapeutic target [6]. Thus, it is evident that drugs that have
ability to increase levels of P-gp should increase amyloid-b clear-
ance. Large number of plant extracts and their natural products
have been reported to possess potent P-gp induction activity,
including rifampicin, St. John's Wort (Hypericum perforatum)
extract and its constituent hyperforin (1) [7e11]. Similarly, the
prenylated phloroglucinol colupulone (2) isolated from Hops
extract is a potent activator of PXR (a nuclear receptor of P-gp) [12].
control. The test concentration of 5 M was used for preliminary
m
screening. This concentration was selected in order to avoid chance
of missing any possible P-gp inducer in the first step. This screening
resulted in identification of 7 natural products as promising P-gp
inducers viz. withaferin, podophyllotoxin, 3-demethylcolchicine,
agnuside, reserpine, sesibiricin and fascaplysin. The P-gp induction
activity and structures of these 7 hits are shown in Fig. 2.
2.2. Synthesis of fascaplysin and its analogs to establish
Recently oleocanthal (3) [13] has been reported to increase Ab-
structureeactivity relationship
transport across BBB via P-gp induction. Our group also demon-
strated that P-gp inducers (colupulone analog 4 [14] and quinoline
A series of quaternary fascaplysin analogs (series 1) were syn-
thesized using our recently reported two-step strategy [23] as
depicted in Scheme 1. Tryptamine (7) was treated with 2-chloro
substituted phenyl glyoxals 8aem in presence of Pd/C in acetic
5 [15]) modulates A
ucts are potential source for identification of P-gp inducer leads for
AD drug development via amyloid- clearance strategy.
b-transport. Thus, it is clear that natural prod-
b
In continuation to our efforts in the area of discovery of P-gp
modulators from natural products [14e17], herein we report dis-
covery of fascaplysin (6a), a marine-derived bisindole alkaloid, as a
new class of P-gp inducers. The P-gp induction activity of fasca-
plysin was discovered while screening in-house natural products
repository for P-gp modulation activity.
acid which produced
b-carbolines 9aem (Schemes 1 and 2). b-
Carbolines 9aeg on heating at 220 ꢀC for 20 min resulted in ring
closure to produce fascaplysin (6a) and its analogs 6beg in 70e80%
yield (Scheme 1).
Next, we synthesized D-ring opened fascaplysin analogs (series
2) with keeping quaternary nitrogen intact using the protocol
Fascaplysin (6a) is a fused benzoyl-linked b-carbolinium alka-
shown in Scheme 2. b-Carbolines 9a and 9hem on treatment with
loid isolated from marine sponge Fascaplysinopsis Bergquist sp.
collected in the South Pacific near the Fiji Island as an unusual
antimicrobial pigment [18]. It is reported to possess cytotoxicity in
mouse leukemia L-1210 cells and cervical cancer HeLa cells [19], a
methyl iodide gave quaternary salts 10a and 10hel in 60e65% yield
(Scheme 2). D-ring opened non-quaternary analogs 9aem (series
3), prepared in Schemes 1 and 2, were also used for P-gp screening.
Further, a series of
4) of fascaplysin were synthesized starting from commercially
available tetrahydro- -carboline (11). Reaction of tetrahydro-
carboline (11) with different acyl halides 12aen in CH2Cl2 in
presence of sodium hydroxide produced N-acyl -carbolines 13aen
b-carboline based non-planar analogs (series
Cdk-4 specific inhibitory activity (IC50 0.35
mM) [20e22], and
acetylcholinesterase (AChE) inhibitory activity [23]. Herein, we
report its P-gp induction activity, and preliminary structur-
eeactivity relationship, along with its AChE inhibition activity. The
chemical structures of compounds 1e6 are shown in Fig. 1.
b
b-
b
in 63e82% yield (Scheme 3). Similarly a series of N-alkylated
carbolines 15aeh were synthesized by treatment of tetrahydro-
b
-
-
b
carboline (11) with different heterocycle linked alkyl halides 14aeh
2. Results and discussion
as depicted in Scheme 3 [24].
2.1. Screening of IIIM compound repository for P-gp induction
activity
2.3. P-gp induction activity
The in-house natural products repository (302 natural products)
All synthesized compounds from all four series (total 48
O
OH
O
O
OH
CHO
O
O
O
O
OH
HO
CHO
Hyperforin (1)
Colupulone (2)
Oleocanthal (3)
OCF3
Cl
C
N
D
E
A
B
HO
O
O
O
N
H
O
OEt
N
O
OH
O
O
4
5
Fascaplysin (6a)
Fig. 1. Structures of P-gp inducer natural products 1e3, our P-gp inducer leads 4e5, and bis-indole alkaloid fascaplysin (6a).

S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
3
Fig. 2. P-gp induction activity (a) and structures (b) of natural product hits identified from screening of institutional natural product repository in LS-180 cells (Rifampicin was used
as a positive control; the test concentration of 10 M was used for rifampicin and 5 M for all test compounds). The statistical comparisons were made between control versus
compounds. The p value <0.5 was considered to be significant. P value *<0.5, **<0.01, ***<0.001, ns no significance.
m
m
compounds) were screened for P-gp induction activity in P-gp
overexpressing adenocarcinoma LS180 cells at 1 M. The decrease
in the % intracellular accumulation (compared to control) of Rh123
indicates induction of P-gp. Rifampicin (10 M) was used as a
reference P-gp inducer. Statistical comparisons were made be-
tween control versus compounds by using Bonferroni test. Fasca-
plysin (6a) was found to display P-gp induction activity at 1 mM, as
indicated by decreased intracellular accumulation of Rh123 levels
up to 57% in LS180 cells.
As shown in Table 1, fascaplysin (6a) and its analogs 6f and 10j
m
showed reduced % intracellular rhodamine-123 levels (57%, 49%
and 70%, respectively), in comparison to untreated control cells
(100%), indicating their effect as a P-gp inducer. The effect of these
key compounds was also checked at lower concentrations. As
shown in Table 2, these compounds showed significant decrease in
the intracellular Rh123 levels at very low concentration, 62.5 nM.
The EC50 of fascaplysin was determined and was found to be 25 nM.
The promising p-glycoprotein induction activity of these com-
m
Several fascaplysin analogs showed ability to induce p-glyco-
protein as shown in Table 1. The obtained P-gp screening results
revealed important structureeactivity relationship features. All
quaternary nitrogen containing analogs 6aeg (series 1), 10a and
10hel (series 2) showed promising P-gp induction activity. How-
ever, the D-ring opened analogs without quaternary nitrogen
pounds was further confirmed by western-blot analysis at 1 mM.
Upon exposure to compounds 6a, 6f and 10j, the P-glycoprotein
expression was found to be significantly increased (by 4e8 fold)
(Fig. 3).
All compounds were tested for in-vitro cytotoxicity in LS-180
cells using MTT assay at 10
mM and 48 h treatment time, wherein
9aem (series 3) were inactive. The tetrahydro
(non-planar, non-quaternary) 13aen and 15aeh (series 4) were
also devoid of P-gp induction activity at 1 and 5 M. Thus, it is
evident that, upon opening of the D-ring of fascaplysin while
maintaining C-ring nitrogen in quaternary form (analog 10a,
10hel), the P-gp induction activity was maintained. However, on
removal of quaternary status of the C-ring nitrogen, the P-gp in-
duction activity was completely diminished. This indicates that, the
opening of D-ring is tolerated; as far as the quaternary status of C-
ring nitrogen is maintained.
b
-carboline analogs
most of the compounds showed >50% cell viability (IC50 > 10 m
M)
(Table 1). Furthermore, the best compound fascaplysin (6a) was
also screened for cytotoxicity in normal human gingival fibroblast
hGF cells. It showed cytotoxicity in this cell line with IC50 value of
m
4
mM. These cytotoxicity results indicated that there is a good
therapeutic index of these compounds for P-gp induction.
2.4. AChE inhibition activity
Recently, fascaplysin (6a) was reported as AChE inhibitor [23].

4
S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
O
X
R2
N
D
NH2
H
C
a
R1
B
A
E
O
N
N
X
R1
H
O
H
R2
8a: R1 = R2 = H, X = Cl
7
9a: R1 = R2 = H, X = Cl
8b: R1 = H, R2 = 4-Cl, X = Cl
8c: R1 = 4-Cl, R2 = 5-F, X = Cl
8d: R1 = 3-Cl, R2 = 4-Cl, X = Cl
8e: R1 = H, R2 = 6-Cl, X = F
8f: R1 = 4-F, R2 = 5-F, X = Cl
8g: R1 = H, R2 = 5-Cl, X = Cl
9b: R1 = H, R2 = 4-Cl, X = Cl
9c: R1 = 4-Cl, R2 = 5-F, X = Cl
9d: R1 = 3-Cl, R2 = 4-Cl, X = Cl
9e: R1 = H, R2 = 6-Cl X = F,
9f: R1 = 4-F, R2 = 5-F, X = Cl
9g: R1 = H, R2 = 5-Cl, X = Cl
b
X
R1
3
2
1
4
N
C
E
A
D
B
N
H
5
R2
6
O
6a: R1 = R2 = H, X = Cl
6b: R1 = H, R2 = 4-Cl, X = Cl
6c: R1 = 4-Cl, R2 = 5-F, X = Cl
6d: R1 = 3-Cl, R2 = 4-Cl, X = Cl
6e: R1 = H, R2 = 6-Cl, X = F
6f: R1 = 4-F, R2 = 5-F, X = Cl
6g: R1 = H, R2 = 5-Cl, X = Cl
Scheme 1. Synthesis of fascaplysin and its quaternary analogs 6aeg. Reagents and conditions: (a). 10% Pd/C, AcOH, reflux, 3 h, 65e85%; (b). 220 ꢀC, 20 min, 70e80%.
I
O
NH2
N
N
D
CH3
C
C
H
a
b
A
A
CH3I
B
B
Ar
N
N
Ar
N
Ar
E
O
H
H
H
O
O
7
9a: Ar = Ph (2-Cl)
9h: Ar = Ph
9i: Ar = indole-3-yl
9j: Ar = Ph (2-Br)
9k: Ar = Ph (2-F)
9l: Ar = Ph (4-CF3)
9m: Ar = Ph (2,4-dimethoxy)
10a: Ar = Ph (2-Cl)
10h: Ar = Ph
10i: Ar = indole-3-yl
10j: Ar = Ph (2-Br)
10k: Ar = Ph (2-F)
10l: Ar = Ph (4-CF3)
8a: Ar = Ph (2-Cl)
8h: Ar = Ph
8i: Ar = indole-3-yl
8j: Ar = Ph (2-Br)
8k: Ar = Ph (2-F)
8l: Ar = Ph (4-CF3)
8m: Ar = Ph (2,4-dimethoxy)
Series 2
Series 3
Scheme 2. Synthesis of D-ring opened analogs of fascaplysin 10a, 10hel. Reagents and conditions: (a). 10% Pd/C, AcOH, reflux, 3 h, 65e85%; (b). ACN, 80 ꢀC, 12 h, 60e75%.
Based on this report, in addition to their P-gp induction activity, we
screened best identified P-gp inducers 6f and 10j for AChE inhibi-
H-bonding with the Gly-221 and H2O 737 at AChE catalytic site, as
shown in Fig. 4; and thereby it prevents the entry of substrate ACh
to the catalytic site.
tion activity at the concentration (1
activity was performed. Compound 6f showed 62% inhibition of
AChE at 1 M; whereas compound 10j showed only 7% inhibition of
mM) at which P-gp induction
m
2.5. Aqueous solubility of best compounds
AChE at this concentration.
In accordance to the recent literature precedence and the
availability of human AChE crystal structure, best compound 6f
which exhibited significant P-gp efflux function induction as well
as AChE inhibition, was further studied for its interaction with
human AChE (PDB ID: 4EY7). In previous publication [23], we have
reported that parent compound fascaplysin (6a) bounds only at the
peripheral anionic site by interacting with the Trp-286 residue and
does not interacts directly with the catalytic site residue Trp-86 (as
shown in Section S3 of supporting information). Similar to fasca-
plysin, the analog 6f also interacts only with the peripheral anionic
site residue Trp-286 (corresponding residue in human AChE) by
The solubility of most potent compounds 6a, 6f, and 10j was
determined in water, PBS, SGF, and SIF. Fascaplysin (6a) showed
high solubility in water as well as biological fluids PBS, SGF, and SIF
(>1500
(>1500
solubility in SIF (<5
m
m
g/ml). Compound 6f showed high solubility in water
g/ml), moderate solubility in PBS and SGF, however less
g/ml). Compound 10j also showed high sol-
m
ubility in water as well as biological fluids PBS, SGF, and SIF (800 mg/
ml). Solubility results are shown in Table 3.
3. Conclusion
hydrophobic pep interactions. But, in addition to the hydrophobic
interactions, it enters deeper into the catalytic pocket due to the
additional bulkier halogen groups on the E-ring, and exhibits polar
In summary, from the screening of in-house natural products
repository for P-gp induction activity, we have identified fasca-
plysin as a new class of P-gp inducer. A medicinal chemistry of

S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
5
4.2. Synthesis of substituted phenyl glyoxals 8aem
O
R
O
R2
1 R1
12a-n
Cl
R2
Cl
N
The solution of SeO2 (12.79 mmol) in 1,4-dioxane/water (10 mL,
95: 5) was heated at 60 ꢀC for 3 h. Substituted acetophenones
(12.98 mmol) were added and the reaction mixture was refluxed
for 4 h. Reaction mixture was filtered and the filtrate was concen-
trated. The formation of glyoxal was confirmed by TLC and MS. The
crude products 8aem (>85% pure) were directly used for the next
step without purification [25].
14a-h
N
NH
b
a
N
H
13a-n
N
H
15a-h
N
H
11
NH
N
N
O
15a: R2
=
13h: R1 =
N
13a: R1 =
13b: R1 =
O
Cl
N
NO
2 13i: R1 =
15b: R2
=
S
4.3. General procedure for preparation of b-carbolines 9aem
N
H
O
CF3
To the solution of tryptamine (7, 6.25 mmol) and substituted
phenyl glyoxals 8aem (7.5 mmol) in glacial acetic acid (15 mL) was
added 10% Pd/C catalyst (20 mol%) and reaction mixture was
refluxed for 3 h. The reaction mixture was filtered through celite
and the filtrate was concentrated on rotary evaporator to get crude
product which on silica gel column chromatography (20% ethyl
13c: R1 =
15c: R2
=
13j: R1 =
N
O
O
NO2
N
CH3
15d: R2
15e: R2
15f: R2
15g: R2
=
N
N CH3
13k: R1 =
13l: R1 =
13d: R1 =
13e: R1 =
O
H3C
NO2
acetate/hexane) gave corresponding
yield.
b-carbolines 9aem in 65e85%
=
=
N
O
Br
S
O
Cl
S
4.3.1. 2-Chlorophenyl(9H-pyrido[3,4-b]indol-1-yl)methanone (9a)
Yellow solid; yield: 85%; m.p. 203e205 ꢀC; HPLC: >99%
13m: R1 =
N
13f: R1 =
13g: R1 =
(tR ¼ 16.71 min); 1H NMR (500 MHz CDCl3, ppm):
d 10.42 (brs, NH,
=
N
N
H3C
1H), 8.56 (d, J ¼ 4.8 Hz, 1H), 8.20e8.15 (m, 2H), 7.66e7.59 (m, 3H),
7.53e7.35 (m, 4H); 13C NMR (100 MHz, CDCl3, ppm):
d 197.57,
13n: R1 =
N
15h: R2
=
S
141.25, 138.78, 138.39, 136.80, 135.46, 132.77, 132.06, 131.88, 131.83,
131.16, 130.01, 129.90, 126.48, 121.89, 120.70, 119.16, 112.10; IR
(CHCl3): nmax 3421, 3058, 2360, 2340, 2360, 1699, 1646, 1626, 1592,
1430, 1212 cmꢂ1; ESI-MS: m/z 307.06 [MþH]þ; HR-ESIMS: m/z
307.0619 calcd for C18H12ClN2O þ Hþ (307.0632).
Series 4
Scheme 3. Synthesis of non-planar and non-quaternary analogs of fascaplysin 13aen
and 15aeh. Reagents and conditions: (a) CH2Cl2, NaOH, 0 ꢀC, 15 min then rt, 3 h,
63e82%; (b) K2CO3, CH3CN, 3 h, rt, 66e78%.
4.3.2. (2,4-Dichlorophenyl)(9H-pyrido[3,4-b]indol-1-yl)methanone
(9b)
Yellow solid; yield: 68%; m.p. 212e215 ꢀC; HPLC: >99%
fascaplysin led to the establishment of SAR of this scaffold and
identified analog 10j which showed 8-fold increase in P-gp
(tR ¼ 25.90 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.39 (s, NH,
expression in LS-180 cells, indicating its promise to enhance A
b
1H), 8.55 (d, J ¼ 4.0 Hz, 1H), 8.20e8.17 (m, 2H), 7.65e7.53 (m, 4H),
7.42e7.38 (m, 2H); 13C NMR (100 MHz, CDCl3, ppm):
d 196.42,
clearance from AD brains. Furthermore, compounds 6a, 6f and 10j
showed good solubility in water as well as simulated body fluids.
Thus, results presented in this work, indicates promise of this
scaffold for development as an anti-Alzheimer agent.
141.27, 138.82, 136.89, 136.73, 136.70, 135.20, 133.19, 132.99, 132.03,
129.62, 127.04, 126.75, 121.97, 121.16, 120.73, 119.36, 112.12; IR
(CHCl3): nmax 3433, 3368, 3061, 2923, 2851, 1899, 1653, 1625, 1555,
1493, 1463, 1429, 1373, 1282, 1259, 1245, 1149, 1118, 1095, 1064,
1050, 1013 cmꢂ1; ESI-MS: m/z 341.02 [MþH]þ; HR-ESIMS: m/z
341.0221 calcd for C18H11Cl2N2O þ Hþ (341.0242).
4. Experimental section
4.1. General
4.3.3. (2,4-Dichloro-5-fluorophenyl)(9H-pyrido[3,4-b]indol-1 yl)
methanone (9c)
All chemicals were obtained from SigmaeAldrich Company and
used as received. 1H, 13C and DEPT NMR spectra were recorded on
Brucker-Avance DPX FT-NMR 500 and 400 MHz instruments.
Chemical data for protons are reported in parts per million (ppm)
downfield from tetramethylsilane and are referenced to the resid-
ual proton in the NMR solvent (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm).
Carbon nuclear magnetic resonance spectra (13C NMR) were
recorded at 125 MHz or 100 MHz: chemical data for carbons are
Yellow solid; yield: 65%; m.p. 209e211 ꢀC; HPLC: >99%
(tR ¼ 29.26 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.39 (s, NH,
1H), 8.54 (d, J ¼ 8.0 Hz, 1H), 8.17e8.15 (m, 2H), 7.63e7.51 (m, 3H),
7.42e7.36 (m, 2H); 19F NMR (376.5 Hz, CDCl3, ppm):
to ꢂ117.03 (m, 1F); 13C NMR (100 MHz, CDCl3, ppm):
d
ꢂ116.99
d
194.92,
156.36 (1JCF ¼ 150.51 Hz), 141.24, 138.88, 136.92, 134.68, 132.09,
131.66, 129.69, 127.43, 123.73, 121.98, 121.25, 120.65, 119.58, 118.21,
117.91, 112.13; IR (CHCl3): nmax 3368, 2920, 2850, 2343, 1653, 1625,
1596, 1572, 1493, 1462, 1430, 1391, 1315, 1282, 1256, 1204, 1174,
1149, 1118, 1090, 1062, 1017 cmꢂ1; ESI-MS: m/z 359.01 [MþH]þ; HR-
ESIMS: m/z 359.0145 calcd for C18H10Cl2FN2O þ Hþ (359.0148).
reported in parts per million (ppm,
d scale) downfield from tetra-
methylsilane and are referenced to the carbon resonance of the
solvent (CDCl3, 77.16 ppm; CD3OD, 49.0). ESI-MS and HRMS spectra
were recorded on Agilent 1100 LC-Q-TOF and HRMS-6540-UHD
machines. IR spectra were recorded on PerkineElmer IR spectro-
photometer. Melting points were recorded on digital melting point
apparatus. HPLC analysis was done on Shimadzu HPLC system
(model: LC-6AD) equipped with a PDA detector (model: SPD-
4.3.4. (2,3,4-Trichlorophenyl)(9H-pyrido[3,4-b]indol-1-yl)
methanone (9d)
Yellow solid; yield: 75%; m.p. 216e218 ꢀC; HPLC: 91%
M20A) using Inertsil C8 (3.5
water (50: 50) mobile phase by isocratic elution at flow rate of 1 ml/
m
, 4.6 ꢁ 250 mm) column using ACN:
(tR ¼ 17.73 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.34 (s, NH,
1H), 8.53 (t, J ¼ 3.6 Hz,1H), 8.18 (t, J ¼ 7.2 Hz, 2H), 7.65e7.53 (m, 3H),
min.
7.41e7.38 (m, 2H); 13C NMR (100 MHz, CDCl3, ppm):
d 195.84,

6
S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
Table 1
In-vitro P-gp induction and cytotoxic activity of fascaplysin and its analogs in LS-180 cells.a
Entry
% Rh123 accumulation in LS-180 cells at 1
m
M after 48 hb,c
% viability of LS-180 cells at 10 m
Mc
Control
Rifampicin
6a
6b
6c
6d
6e
6f
6g
10a
10h
10i
10j
10k
10l
9a
9b
9c
9d
9e
100
100
nd
67.1 4.6***
57.58 4.7***
57.1 9.2***
55.5 6.9***
57.8 8.3***
59.2 6.7***
49.6 1.2***
58.7 7.8***
71.4 8.4**
75.2 9.1**
77.6 7.6**
70.1 6.7**
79.0 6.0**
78.5 5.8**
105.4 4.4ns
97.3 0.2ns
106.1 0.5ns
104.1 1.1ns
89.6 4.1***
112.4 5.3**
102.7 3.4ns
94.6 2.6ns
99.4 1.4ns
104.8 1.8ns
100.9 4.1ns
106.6 1.8ns
107.8 4.1ns
57.5 3.7
66.2 8.1
36.8 6.8
57.4 8.4
63.4 10.2
63.5 3.6
67.9 6.9
69.7 9.0
77.5 0.3
69.1 9.4
69.1 5.9
69.8 7.1
75.7 4.9
89.4 7.6
98.8 4.0
92.4 8.6
84.9 9.3
60.1 1.4
81.9 5.6
69.6 7.8
67.5 4.6
78.7 3.8
80.3 2.8
98.4 3.9
105.6 2.6
82.2 2.7
9f
9g
9h
9i
9j
9k
9l
9m
nd: not determined.
a
All series 4 compounds 13aen and 15aeh were devoid of P-gp induction activity at 5
m
M.
M; and was measured in terms of the % intracellular accumulation of rhodamine 123/total protein (
inside LS-180 cells. The decrease in % intracellular accumulation (compared to control) of Rh123 indicates induction of P-gp. Rifampicin (10 M) was used as a reference
value <0.5 was considered to be significant. value*<
b
P-gp induction activity of compounds was checked at 1
m
mg)
m
P-gp inducer. The statistical comparisons were made between control versus compounds. The
0.5,**<0.01,***<0.001, ns no significance.
p
P
c
Values are shown as average of three experiments SD.
1197, 1148, 1107, 1064 cmꢂ1; ESI-MS: m/z 374.80 [MþH]þ; HR-
ESIMS: m/z 374.9865 calcd for C18H10Cl3N2O þ Hþ (374.9853).
Table 2
The percentage intracellular Rh123 levels in LS-180 cells after treatment with
compounds 6a, 6f and 10j at nanomolar concentrations.
Compound, nM
% intracellular Rh123 levels in LS-180 cellsa
4.3.5. (2-Chloro-6-fluorophenyl)(9H-pyrido[3,4-b]indol-1-yl)
methanone (9e)
6a
6f
10j
Yellow solid; yield: 76%; m.p. 218e220 ꢀC; HPLC: 91%
0
100
100
100
(tR ¼ 10.64 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.31 (s, NH,
62.5
125
250
500
42.89 1.61
39.28 5.41
43.71 4.01
34.54 0.21
35.54 0.77
33.79 3.73
35.29 3.50
30.62 2.85
80.02 7.03
41.78 0.91
42.34 7.23
33.65 2.89
1H), 8.47 (d, J ¼ 4.0 Hz, 1H), 8.11 (t, J ¼ 4.0 Hz, 2H), 7.58e7.56 (m,
2H), 7.36e7.25 (m, 3H), 7.09e7.05 (m, 1H); 19F NMR (376.5 Hz,
CDCl3, ppm):
CDCl3, ppm):
d
d
ꢂ111.98 to ꢂ112.02 (m, 1F); 13C NMR (100 MHz,
192.38, 157.70 (1JCF ¼ 248.5 Hz), 139.23, 137.23,
a
Values are shown as average of three experiments SD.
134.48, 133.31, 129.93, 129.31, 127.60, 125.81, 125.64, 123.52, 119.99,
119.18, 118.70, 117.73, 112.29 (2JCF ¼ 21.37 Hz) 110.14; IR (CHCl3):
nmax 3412, 3053, 2923, 2852, 1889, 1783, 1654, 1627, 1602, 1567,
1495, 1463, 1449, 1432, 1376,1317, 1284, 1254, 1242, 1174, 1148, 1118,
1093, 1064, 1019 cmꢂ1; ESI-MS: m/z 325 [MþH]þ; HR-ESIMS: m/z
325.0540 calcd for C18H11ClFN2O þ Hþ (325.0538).
141.22, 138.95, 138.71, 136.89, 135.76, 134.77, 132.67, 132.07, 129.71,
128.21, 127.38, 122.01, 121.25, 120.67, 119.58, 112.13; IR (CHCl3): nmax
3467, 3431, 3094, 2925, 2853, 2350, 2071, 1930, 1915, 1656, 1637,
1625, 1573, 1492, 1464, 1429, 1361, 1325, 1314, 1282, 1254, 1214,
Fig. 3. The effect of fascaplysin 6a and its analogs 6f, 10j on P-gp expression in LS-180 cells at 1 mM. (a) Western-blot (b) quantitation results of P-gp expression.

S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
7
ppm): d 195.56, 141.04, 138.07, 137.59, 137.34, 136.29, 132.42, 131.68,
131.25, 129.30, 128.07, 121.83, 120.85, 120.78, 118.54, 112.03; IR
(CHCl3): nmax 3430, 3378, 3055, 2922, 2850, 1899, 1640, 1619, 1596,
1574, 1492, 1464, 1426, 1373, 1317, 1247, 1212, 1149, 1118, 1087, 1060,
1013 cmꢂ1; ESI-MS: m/z 273.10 [MþH]þ; HR-ESIMS: m/z 273.1015
calcd for C18H13N2O þ Hþ (273.1022).
4.3.9. (1H-indol-3yl)(9H-pyrido[3,4-b]indol-1-yl)methanone (9i)
Yellow solid; yield: 65%; m.p. 220e222 ꢀC; HPLC: 91%
(tR ¼ 16.83 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.75 (s, NH,
1H), 9.45 (d, J ¼ 4.0 Hz, 1H), 8.71 (s, NH, 1H), 8.70 (s, 1H), 8.57 (d,
J ¼ 8.0 Hz, 1H), 8.18e8.14 (m, 2H), 7.60 (d, J ¼ 4.0 Hz, 2H), 7.48 (d,
J ¼ 8.0 Hz, 1H), 7.39e7.35 (m, 3H); 13C NMR (100 MHz, CDCl3 ppm):
d
187.33,141.56,138.43,137.87,137.81,136.97,135.92,135.00,130.72,
128.64, 127.18, 122.88, 122.04, 121.65, 120.02, 119.78, 117.93, 114.21,
112.98, 112.31; IR (CHCl3): nmax 3452, 3413, 3248, 3148, 3055, 2926,
2853, 2564, 2351, 2069, 1892, 1851, 1778, 1633, 1597, 1554, 1507,
1490,1467,1442,1423,1385,1352,1317,1249,1216,1193,1135,1052,
1013 cmꢂ1; ESI-MS: m/z 312 [MþH]þ; HR-ESIMS: m/z 312.1132
calcd for C20H14N3O þ Hþ (312.1131).
Fig. 4. Interaction of compound 6f with the active site of AChE.
Table 3
Solubility of selected compounds 6a, 6f and 10j.
Entry
Solubility (
m
g/ml)a,b
PBS
4.3.10. (2-Bromophenyl)(9H-pyrido[3,4-b]indol-1-yl)methanone
(9j)
Water
SGF
SIF
6a
6f
10j
>1500
>1500
800
>1500
80
800
>1500
80
800
>1500
<5
800
Yellow solid; yield: 65%; m.p. 204e206 ꢀC; HPLC: >99%
(tR ¼ 14.97 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.48 (s, NH,
1H), 8.57 (d, J ¼ 4.0 Hz, 1H), 8.18 (t, J ¼ 4.0 Hz, 2H), 7.70e7.56 (m,
a
Thermodynamic experimental solubility.
4H), 7.49e7.26 (m, 3H); 13C NMR (100 MHz, CDCl3, ppm):
d 198.28,
b
PBS, phosphate buffer saline; SGF, simulated gastric fluid; SIF, simulated intes-
141.25, 140.33, 138.85, 136.93, 135.13, 133.17, 131.94, 131.32, 129.85,
129.57, 126.95, 121.98, 121.08, 120.71, 120.12, 119.28, 112.15; IR
(CHCl3): nmax 3424, 3057, 2922, 2851, 2611, 1900, 1715, 1653, 1624,
1591, 1564, 1493, 1462, 1429, 1378, 1316, 1283, 1263, 1248, 1213,
1161, 1149, 1094, 1043, 1012 cmꢂ1; ESI-MS: m/z 351.01 [MþH]þ; HR-
ESIMS: m/z 351.0124 calcd for C18H12BrN2O þ Hþ (351.0127).
tinal fluid.
4.3.6. (2-Chloro-4,5-difluorophenyl)(9H-pyrido[3,4-b]indol-1 yl)
methanone (9f)
Yellow solid; yield: 74%; m.p. 236e238 ꢀC; HPLC: 90%
(tR ¼ 14.70 min); 1H NMR (400 MHz, DMSO-d6, ppm):
d 12.28 (s,
NH, 1H), 8.50e8.46 (m, 2H), 8.34 (d, J ¼ 6.4 Hz, 1H), 7.94e7.84 (m,
4.3.11. (2-Fluorophenyl)(9H-pyrido[3,4-b]indol-1-yl)methanone
(9k)
3H), 7.64 (t, J ¼ 6.0 Hz, 1H), 7.35 (t, J ¼ 6.0 Hz, 1H); 19F NMR
(376.5 Hz, DMSO-d6, ppm):
d
ꢂ133.60 to ꢂ133.71 (m, 1F), ꢂ139.60
Yellow solid; yield: 70%; m.p. 198e200 ꢀC; HPLC: 90%
to ꢂ139.70 (m, 1F); 13C NMR (100 MHz, DMSO-d6, ppm):
d 193.70,
(tR ¼ 14.72 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.44 (s, NH,
149.91 (1JCF ¼ 233.41 Hz), 148.10 (1JCF ¼ 247.74 Hz), 141.93, 137.94,
136.20, 135.27, 134.74, 131.39, 129.27, 125.93, 121.97, 120.58, 120.01,
119.86, 118.87, 118.57, 113.10; IR (KBr): nmax 3423, 3059, 2922, 2850,
1900, 1794, 1646, 1628, 1592, 1565, 1463, 1452, 1430, 1378, 1339,
1317, 1286, 1248, 1208, 1062 cmꢂ1; ESI-MS: m/z 343 [MþH]þ; HR-
ESIMS: m/z 343.0438 calcd for C18H10ClF2N2O þ Hþ (343.0444).
1H), 8.57 (d, J ¼ 4.0 Hz, 1H), 8.16 (t, J ¼ 4.0 Hz, 2H), 7.79e7.76 (m,
1H), 7.62e7.53 (m, 3H), 7.37e7.19 (m, 3H); 19F NMR (376.5 Hz,
CDCl3, ppm):
CDCl3, ppm):
d
d
ꢂ111.34 to ꢂ111.40 (m, 1F); 13C NMR (100 MHz,
195.58, 160.50 (1JCF ¼ 252.77 Hz), 141.15, 138.70,
136.72, 135.74, 133.02, 131.19, 131.17, 129.45, 123.89, 121.92, 120.98,
120.74, 119.16, 116.33, 112.09; IR (CHCl3): nmax 3398, 3051, 2923,
2852, 1649, 1629, 1612, 1596, 1579, 1566, 1494, 1463, 1448, 1428,
1378, 1338, 1282, 1264, 1246, 1147, 1118, 1102, 1085, 1061,
1020 cmꢂ1; ESI-MS: m/z 291.09 [MþH]þ; HR-ESIMS: m/z 291.0926
calcd for C18H12FN2O þ Hþ (291.0928).
4.3.7. (2,5-Dichlorophenyl)(9H-pyrido[3,4-b]indol-1-yl)methanone
(9g)
Yellow solid; yield: 80%; m.p. 210e212 ꢀC; HPLC: 92%
(tR ¼ 12.97 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.32 (s, NH,
1H), 8.56 (d, J ¼ 4.0 Hz, 1H), 8.20e8.18 (dd, J ¼ 4.0, 8.0 Hz, 2H),
7.65e7.56 (m, 3H), 7.43e7.38 (m, 3H); 13C NMR (100 MHz, CDCl3,
4.3.12. (4-Trifluoromethylphenyl)(9H-pyrido[3,4-b]indol-1-yl)
methanone (9l)
ppm):
d 196.08, 141.21, 139.70, 138.97, 136.85, 134.95, 132.55, 132.0,
131.68, 131.14, 131.10, 130.15, 129.72, 129.64, 121.99, 121.19, 119.47,
112.12; IR (CHCl3): nmax 3416, 3069, 3086, 3054, 3040, 2955, 2924,
2853, 2614, 2094, 1941, 1914, 1898, 1784, 1651, 1625, 1595, 1509,
1493, 1453, 1430, 1393, 1315, 1283, 1270, 1256, 1246, 1210, 1149,
1121, 1099, 1063, 1010 cmꢂ1; ESI-MS: m/z 341.06 [MþH]þ; HR-
ESIMS: m/z 341.0206 calcd for C18H11Cl2N2O þ Hþ (341.0242).
Yellow solid; yield: 75%; m.p. 201e203 ꢀC; HPLC: >99%
(tR ¼ 36.47 min); 1H NMR (400 MHz, CDCl3, ppm):
d 8.50 (d,
J ¼ 4.0 Hz, 1H), 8.37e8.26 (m, 4H), 7.86 (d, J ¼ 8.0 Hz, 2H), 7.75 (d,
J ¼ 8.0 Hz, 1H), 7.61 (t, J ¼ 8.0 Hz, 1H), 7.35 (t, J ¼ 8.0 Hz, 1H); 19F
NMR (376.5 Hz, CDCl3, ppm):
CDCl3, ppm): d 194.57, 141.08, 140.56, 138.24, 137.38, 135.52, 133.60,
d
ꢂ63.03 (s, 3F); 13C NMR (100 MHz,
133.34, 131.95, 130.38, 129.54, 124.99, 124.96, 122.77, 121.92, 121.04,
120.75, 119.04, 112.08; IR (CHCl3): nmax 3393, 3066, 2922, 2852,
2675, 1904, 1696, 1649, 1626, 1598, 1578, 1514, 1494, 1428, 1404,
1328, 1317, 1249, 1217, 1167, 1067 cmꢂ1; ESI-MS: m/z 341.09
[MþH]þ; HR-ESIMS: m/z 341.0894 calcd for C19H12F3N2O þ Hþ
(341.0896).
4.3.8. Phenyl (9H-pyrido[3,4-b]indol-1-yl)methanone (9h)
Yellow solid; yield 80%; m.p. 180e182 ꢀC; HPLC: >99%
(tR ¼ 17.11 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.45 (s, NH,
1H), 8.63e8.61 (m, 1H), 8.32 (d, J ¼ 8.0 Hz, 2H), 8.20e8.17 (m, 2H),
7.63e7.52 (m, 5H), 7.37e7.35 (m, 1H); 13C NMR (100 MHz, CDCl3,

8
S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
4.3.13. (2,4-Dimethoxyphenyl)(9H-pyrido[3,4-b]indol-1yl)
methanone (9m)
121.88, 120.81, 114.83; IR (KBr): nmax 3453, 2924, 1637, 1508,
1070 cmꢂ1; ESI-MS: m/z 339 [MꢂCl]þ; HR-ESIMS: m/z 339.0093
calcd for C18H9Cl2N2Oþ (339.0086).
Yellow solid; yield: 66%; m.p. 233e235 ꢀC; HPLC: 93%
(tR ¼ 7.26 min); 1H NMR (400 MHz, CDCl3, ppm):
d 10.40 (s, NH,1H),
8.54 (d, J ¼ 4.0 Hz, 1H), 8.18e8.11 (m, 2H), 7.69e7.60 (m, 3H),
4.4.5. 6-Chloro fascaplysin (6e)
7.36e7.26 (m, 1H), 6.63e6.59 (m, 2H); 3.89 (s, 3H), 3.78 (s, 3H); 13
NMR (100 MHz, CDCl3, ppm): 196.86, 163.44, 160.30, 141.11,
C
Brick red solid; yield: 74%; m.p. 231e233 ꢀC; HPLC: 93%
d
(tR ¼ 5.39 min); 1H NMR (400 MHz, CD3OD, ppm):
d 9.27 (d,
138.27, 137.05, 136.72, 133.01, 31.47, 129.15, 121.79, 120.85, 120.63,
118.28,111.98,101.48, 99.26, 55.82, 55.52; IR (KBr): nmax 3368, 3056,
30,003, 2925, 2850, 1731, 1644, 1623, 1577, 1503, 1493, 1463, 1427,
1315, 1282, 1160, 1131, 1087, 1062, 1030 cmꢂ1; ESI-MS: m/z 333
[MþH]þ; HR-ESIMS: m/z 333.1232 calcd for C20H17N2O3 þ Hþ
(333.1233).
J ¼ 6.0 Hz, 1H), 8.85 (d, J ¼ 6.0 Hz, 1H), 8.36 (d, J ¼ 8.0 Hz, 1H), 8.19
(d, J ¼ 8.0 Hz, 1H), 7.82e7.74 (m, 2H), 7.71e7.69 (m, 1H), 7.60 (d,
J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 7.6 Hz, 1H); 13C NMR (100 MHz, CD3OD,
ppm): d 180.46, 149.74, 149.0, 138.71, 136.06, 135.04, 133.99, 130.21,
127.48, 126.92, 125.27, 124.71, 124.43, 123.39, 122.47, 121.10, 115.20,
114.70; IR (KBr): nmax 3460, 2925, 1637, 1510, 1020 cmꢂ1; ESI-MS:
m/z 305.0 [MꢂF]þ; HR-ESIMS: m/z 305.0480 calcd for
4.4. General procedure for synthesis of fascaplysin analogs 6aeg
C
18H10ClN2Oþ (305.0476).
The b
-carbolines 9aeg (0.163 mmol) were heated at 220 ꢀC for
15 min. The reaction was cooled and the product was recrystallized
from CH2Cl2/diethyl ether producing corresponding quaternary
fascaplysin analogs 6aeg in 70e80% yield.
4.4.6. 4,5-Difluoro fascaplysin (6f)
Brick red solid; yield: 75%; m.p. 240e242 ꢀC; HPLC: 92%
(tR ¼ 5.68 min); 1H NMR (400 MHz, CD3OD, ppm):
d 9.18 (d,
J ¼ 6.4 Hz, 1H), 8.82 (d, J ¼ 6.4 Hz, 1H), 8.54 (d, J ¼ 6.4 Hz, 1H), 8.33
(d, J ¼ 8.0 Hz, 1H), 7.83 (d, J ¼ 6.8 Hz, 1H), 7.76e7.73 (m, 1H),
7.66e7.64 (m, 1H), 7.40e7.35 (m,1H); 19F NMR (376.5 Hz, DMSO-d6,
4.4.1. Fascaplysin (6a)
Brick red solid; yield: 80%; m.p. 230e232 ꢀC; HPLC: 96%
(tR ¼ 9.96 min); 1H NMR (500 MHz, CD3OD, ppm):
d
9.40 (d,
ppm):
d
ꢂ134.60 to ꢂ134.71 (m, 1F), ꢂ140.60 to ꢂ140.70 (m, 1F);
13C NMR (100 MHz, CD3OD, ppm):
d
181.1, 161.21 (1JCF ¼ 255.05 Hz),
J ¼ 5.6 Hz, 1H), 8.98 (d, J ¼ 5.8 Hz, 1H), 8.48 (d, J ¼ 7.9 Hz, 1H), 8.35
(d, J ¼ 8.0 Hz,1H), 8.08 (d, J ¼ 7.3 Hz, 1H), 7.96 (t, J ¼ 7.7 Hz, 1H), 7.89
(t, J ¼ 7.7 Hz, 1H), 7.79 (d, J ¼ 8.3 Hz, 1H), 7.73 (t, J ¼ 7.4 Hz, 1H), 7.54
149.22, 144.05 (2JCF ¼ 213.54 Hz), 136.43, 133.33, 131.02, 128.27,
128.02, 125.60, 125.49, 124.91, 124.16, 121.44, 120.01, 18.87, 114.91,
114.71; IR (KBr): nmax 3467, 2825, 1638, 1509, 1087 cmꢂ1; ESI-MS:
m/z 307 [MꢂCl]þ; HR-ESIMS: m/z 307.0679 calcd for C18H9F2N2Oþ
(307.0677).
(t, J ¼ 7.4 Hz, 1H); 13C NMR (100 MHz, CD3OD, ppm):
d 183.25,
148.89, 148.75, 142.92, 138.32, 135.91, 133.12, 132.75, 127.59, 126.85,
125.57, 125.23, 124.66, 123.62, 121.26, 121.19, 116.49, 114.68; IR
(CHCl3): nmax 3368, 2923, 2359, 1621, 1506, 1046 cmꢂ1; ESI-MS: m/z
271.09 [MꢂCl]þ; HR-ESIMS: m/z 271.0843 calcd for C18H11N2Oþ
(271.0866).
4.4.7. 5-Chloro fascaplysin (6g)
Brick red solid; yield: 80%; m.p. 236e238 ꢀC; HPLC: 97%
(tR ¼ 4.80 min); 1H NMR (400 MHz, CD3OD, ppm):
d 9.27 (d,
4.4.2. 4-Chloro fascaplysin (6b)
J ¼ 5.6 Hz, 1H), 8.88 (d, J ¼ 5.6 Hz, 1H), 8.44e8.39 (m, 2H), 7.94 (d,
Brick red solid; yield: 75%; m.p. 234e236 ꢀC; HPLC: 97%
J ¼ 8.0 Hz, 1H), 7.83e7.67 (m, 3H), 7.45 (t, J ¼ 7.2 Hz, 1H); 13C NMR
(tR ¼ 5.05 min); 1H NMR (400 MHz, CD3OD, ppm):
d 9.27 (d,
(100 MHz, CD3OD, ppm):
d 181.93, 149.65, 149.04, 144.24, 143.23,
J ¼ 6.4 Hz, 1H), 8.88 (d, J ¼ 6.0 Hz, 1H), 8.45e8.39 (m, 2H), 7.94 (d,
J ¼ 8.0 Hz, 1H), 7.84e7.80 (m, 1H), 7.74e7.68 (m, 2H),7.46 (t,
136.12, 132.67, 127.78, 127.70, 125.26, 124.68, 124.16, 121.20, 120.97,
117.55,114.64; IR (KBr): nmax 3468, 2964,1637,1417,1020 cmꢂ1; ESI-
MS: m/z 305 [MꢂCl]þ; HR-ESIMS: m/z 305.0478 calcd for
J ¼ 6.8 Hz, 1H); 13C NMR (100 MHz, CD3OD, ppm):
d 182.0, 149.73,
C
18H10ClN2Oþ (305.0476).
149.08, 143.25, 136.18, 133.02, 132.99, 132.74, 127.90, 127.77, 125.37,
124.74, 124.24, 121.26, 121.09, 117.68, 11,470; IR (KBr): nmax 3468,
2925, 1637, 1418, 1021 cmꢂ1; ESI-MS: m/z 305 [MꢂCl]þ; HR-ESIMS:
m/z 305.0475 calcd for C18H10ClN2Oþ (305.0476).
4.5. General procedure for synthesis of b-carbolinium analogs 10a
and 10hel
4.4.3. 4-Chloro-5-fluoro fascaplysin (6c)
The mixture of
b-carbolines 9a and 9hem (0.1 g, 1 mmol),
Brick red solid; yield: 70%; m.p. 218e220 ꢀC; HPLC: 95%
methyl iodide (1.5 mmol) and 5 mL ACN was heated for 12 h at
80 ꢀC in sealed tube. The mixture was allowed to cool at 25 ꢀC. The
obtained products were recrystallized with dichloromethane to get
corresponding D-ring opened
10hel in 60e75% yield.
(tR ¼ 6.68 min); 1H NMR (400 MHz, CD3OD, ppm):
d 9.25 (d,
J ¼ 4.0 Hz, 1H), 8.88 (d, J ¼ 6.0 Hz, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 8.39
(d, J ¼ 7.6 Hz,1H), 7.90 (d, J ¼ 6.8 Hz,1H), 7.83e7.70 (m, 2H), 77.45 (t,
b-carbolinium analogs 10a and
J ¼ 7.6 Hz, 1H); 19F NMR (376.5 Hz, CD3OD, ppm):
d
ꢂ111.29
to ꢂ111.32 (m, 1F); 13C NMR (100 MHz, CD3OD, ppm):
d
181.24,
161.03 (1JCF ¼ 255.29 Hz), 149.04, 143.87 (2JCF ¼ 213.79 Hz), 136.25,
136.02, 133.16, 130.85, 128.10, 128.02, 127.84, 125.43, 124.84, 123.98,
121.26, 119.84, 114.73, 114.54; IR (KBr): nmax 3459, 2925, 1619,
4.5.1. 1-(2-Chloro-benzoyl)-2-methyl beta-carbolinium iodide
(10a)
1469,1048 cmꢂ1
;
ESI-MS: m/z 323 [MꢂCl]þ; HR-ESIMS: m/z
Yellow solid; yield: 60%; m.p. 220e222 ꢀC; HPLC: 97%
323.0379 calcd for C18H9ClFN2Oþ (323.0382).
(tR ¼ 31.23 min); 1H NMR (400 MHz, CD3OD, ppm):
d 8.74 (d,
J ¼ 6.4 Hz, 1H), 8.57 (d, J ¼ 6.4 Hz, 1H), 8.38 (d, J ¼ 8.0 Hz, 1H), 7.98
(d, J ¼ 8.0 Hz, 1H), 7.76e7.69 (m, 2H), 7.61e7.53 (m, 3H), 7.43 (t,
J ¼ 7.6 Hz, 1H), 4.24 (s, 3H); 13C NMR (100 MHz, CD3OD, ppm):
4.4.4. 3,4-Dichloro fascaplysin (6d)
Brick red solid; yield: 72%; m.p. 243e245 ꢀC; HPLC: 98%
(tR ¼ 7.90 min); 1H NMR (400 MHz, CD3OD, ppm):
d
10.04 (s, 1H),
d 167.73,145.98,136.76,134.44,134.23,133.94,133.53,132.28,132.0,
8.86 (d, J ¼ 8.0 Hz, 1H), 8.41 (d, J ¼ 8.0 Hz, 1H), 7.95e7.81 (m, 3H),
131.69,128.02,124.35,123.22,120.89,118.71,113.92, 54.85; IR (KBr):
nmax 3459, 2925, 1630, 1586, 1226, 1042 cmꢂ1; ESI-MS: m/z 321
[MꢂI]þ; HR-ESIMS: m/z 321.0787 calcd for C19H14ClN2Oþ
(321.0789).
7.74e7.72 (m, 1H), 7.46 (t, J ¼ 8.0 Hz, 1H); 13C NMR (100 MHz,
CD3OD, ppm):
d 181.38, 149.64, 145.78, 145.17, 144.76, 144.30,
136.73, 134.28, 130.90, 127.06, 125.67, 125.51, 124.97, 123.92, 123.67,

S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
9
4.5.2. 1-Benzoyl-2-methyl beta-carbolinium iodide (10h)
4.6. General procedure for synthesis N-acyl
N-alkylated -carbolines 15aeh
b-carbolines 13aen and
Yield solid; yield: 68%; m.p. 233e235 ꢀC; HPLC: 99%
b
(tR ¼ 26.34 min); 1H NMR (400 MHz, CD3OD, ppm):
d 8.74 (d,
J ¼ 6.4 Hz, 1H), 8.61 (d, J ¼ 6.4 Hz, 1H), 8.40 (d, J ¼ 8.0 Hz, 1H),
7.92e7.90 (m, 2H), 7.77e7.71 (m, 2H), 7.59e7.54 (m, 3H), 7.42 (t,
J ¼ 7.2 Hz, 1H), 4.25 (s, 3H), 3.11 (s, 3H); 13C NMR (100 MHz, CD3OD,
The synthesis of N-acyl b-carbolines 13aen and N-alkylated b-
carbolines 15aeh and their spectral data has been described in our
earlier paper [24].
ppm):
d 188.49, 146.78, 137.93, 136.77, 136.1, 135.74, 134.89, 134.32,
131.79, 131.14, 124.65, 123.74, 120.80, 119.41, 114.01, 46.61; IR (KBr):
nmax 3467, 2985, 2063, 1671, 1593, 1201, 1019 cmꢂ1; ESI-MS: m/z
287 [MꢂI]þ; HR-ESIMS: m/z 287.1177 calcd for C19H15N2Oþ
(287.1178).
4.7. Cell culture and treatments
Human colorectal adenocarcinoma LS-180 cells were purchased
from ECACC, England. Normal Human gingival fibroblast (hGF) cell
line was a gift from Dr. Anil Ballapure, CDRI, Lucknow, India. Cells
were grown in MEM growth medium mixed in the ratio of 1: 1. The
media for the cell lines were supplemented with 1% MEM non-
essential amino acids along with 10% FCS, 100 U penicillin G and
4.5.3. 1-(3-Indoloyl)-2-methyl beta-carbolinium iodide (10i)
Yellow solid; yield: 62%; m.p. 240e242 ꢀC; HPLC: >99%
(tR ¼ 18.30 min); 1H NMR (400 MHz, DMSO-d6, ppm):
d 8.96 (d,
100 m
g/ml of streptomycin. Cells were grown in 5% CO2 at 37 ꢀC
with 95% humidity. All the test compounds were dissolved in DMSO
for treatment of LS-180 cells, while the untreated control cultures
received only the vehicle (DMSO < 0.2%).
J ¼ 6.4 Hz, 1H), 8.87 (d, J ¼ 8.0 Hz, 1H), 8.58 (d, J ¼ 8.0 Hz, 1H), 8.42
(s, 1H), 8.17 (s, 1H), 7.80 (t, J ¼ 8.0 Hz, 1H), 7.66e7.63 (m, 2H),
7.52e7.43 (m. 3H), 4.37 (s, 3H); 13C NMR (100 MHz, DMSO-d6,
ppm):
d 178.33, 144.68, 140.86, 137.62, 136.34, 136.30, 135.03,
134.47, 133.04, 132.37, 125.20, 124.57, 123.63, 123.49, 121.84, 121.53,
119.19, 117.84, 115.92, 113.05, 40.02; IR (KBr): nmax 3467, 3419, 2930,
2855, 2427, 1634, 1618,1584,1457,1248, 882 cmꢂ1; ESI-MS: m/z 326
[MꢂI]þ; HR-ESIMS: m/z 326.1287 calcd for C21H16N3Oþ (326.1287).
4.8. P-gp induction assay
All synthesized compounds were screened for their ability to
induce P-gp using the rhodamine 123 (Rh123) cell exclusion
method. In this method, the P-gp function was evaluated in terms
of rhodamine 123 (Rh123) accumulation and efflux [26]. Briefly, the
protocol used is as follows: colorectal LS-180 cells were seeded at a
density of 2 ꢁ 104 per well of a 96-well plate and were allowed to
grow for the next 24 h. Cells were further incubated with the test
compounds to a final concentration of 1
tive control) to a final concentration of 10
48 h. The final concentration of DMSO was kept at 0.1%. The drugs
were removed and cells were incubated with HANKS buffer for
40 min before further incubation with HANKS buffer (containing 10
4.5.4. 1-(2-Bromo-benzoyl)-2-methyl beta-carbolinium iodide (10j)
Yellow solid; yield: 70%; m.p. 242e244 ꢀC; HPLC: 99%
(tR ¼ 36.33 min); 1H NMR (400 MHz, CD3OD, ppm):
d 8.75 (d,
J ¼ 6.4 Hz, 1H), 8.58 (d, J ¼ 6.4 Hz, 1H), 8.39 (d, J ¼ 8.0 Hz, 1H), 7.95
(d, J ¼ 4.8 Hz, 1H), 7.76e7.73 (m, 2H), 7.62e7.59 (m, 3H), 7.43 (t,
J ¼ 7.6 Hz, 1H), 4.21 (s, 3H); 13C NMR (100 MHz, CD3OD, ppm):
m
M and rifampicin (posi-
mM in complete media for
d
188.49, 146.78, 137.93, 136.77, 136.61, 136.35, 136.10, 135.74,
134.89, 134.32, 131.79, 131.14, 124.65, 123.74, 120.80, 119.41, 114.01,
46.61; IR (KBr): nmax 3467, 2926, 2057, 1628, 1581, 1226, 1034 cmꢂ1
;
ESI-MS: m/z 365 [MꢂI]þ; HR-ESIMS: m/z 365.0284 calcd for
19H14BrN2Oþ (365.0284).
m
M of Rh123 as a Pgp substrate) for 90 min. At the end of Rh123
treatment cells were washed four times with cold PBS followed by
cell lysis for 1 h using 200 l of lysis buffer (0.1% Triton X-100 and
0.2 N NaOH). A total of 100 l of lysate was used for reading fluo-
C
m
4.5.5. 1-(2-Fluoro-benzoyl)-2-methyl beta-carbolinium iodide
(10k)
m
rescence of Rh123 at 485/529 nm. Samples were normalized by
dividing fluorescence of each sample with total protein present in
the lysate.
Yellow solid; yield: 65%; m.p. 234e236 ꢀC; HPLC: >99%
(tR ¼ 29.61 min); 1H NMR (400 MHz, CD3OD, ppm):
d 8.77 (d,
J ¼ 6.4 Hz, 1H), 8.60 (d, J ¼ 6.4 Hz, 1H), 8.40 (d, J ¼ 8.0 Hz, 1H),
7.97e7.95 (m, 1H), 7.78e7.76 (m, 2H), 7.64e7.62 (m, 3H), 7.46 (t,
J ¼ 7.2 Hz, 1H), 4.23 (s, 3H); 19F NMR (376.5 Hz, DMSO-d6, ppm):
4.9. Cell viability assay
d
d
ꢂ124.25 to ꢂ124.32 (m, 1F); 13C NMR (100 MHz, CD3OD, ppm):
The cell proliferation assay was performed in human colorectal
adenocarcinoma LS-180 cells and normal human gingival fibroblast
(hGF) cells. Cells (1 ꢁ 104) were seeded into each well of the 96-well
184.66, 164.57, 163.13, 151.62, 146.79, 142.06, 140.48, 140.40,
136.16, 134.43, 133.28, 127.23, 124.60, 120.82, 119.43, 118.73, 118.56,
114.06, 46.46; IR (KBr): nmax 3759, 3453, 2956, 2924, 2073, 1747,
1673,1605,1632,1520,1482,1452,1333,1316,1272,1163,1150,1116,
1019 cmꢂ1; ESI-MS: m/z 305 [MꢂI]þ; HR-ESIMS: m/z 305.1083 calcd
for C19H14FN2Oþ (305.1084).
microplate for 24 h. Cells were treated with 10 mM of each com-
pound for 24 h. The MTT dye was then added to each well 4 h prior
to the termination of experiment. Formazan crystals were dissolved
in DMSO before recording absorbance at 570 nm. Cell viability of
the untreated control sample was considered to be 100%, while
viability of test samples was calculated using the following
formula:
4.5.6. 1-(4-Trifluromethyl-benzoyl)-2-methylbeta-carbolinium
iodide (10l)
Yellow solid; yield: 75%; m.p. 236e238 ꢀC; HPLC: >99%
(tR ¼ 14.72 min); 1H NMR (400 MHz, CD3OD, ppm):
d 8.78 (d,
ODðtestÞ
% Cell viability ¼
ꢁ 100
J ¼ 6.4 Hz, 1H), 8.65 (d, J ¼ 6.4 Hz, 1H), 8.41 (d, J ¼ 8.0 Hz, 1H), 8.11
(d, J ¼ 8.0 Hz, 2H), 7.89 (d, J ¼ 4.2 Hz, 2H), 7.76e7.72 (m, 1H), 7.55 (d,
J ¼ 8.4 Hz, 1H), 7.45e7.41 (m, 1H), 7.28e7.23 (m, 1H), 4.27 (s, 3H);
ODðcontrolÞ
19F NMR (376.5 Hz, DMSO-d6, ppm):
d
ꢂ61.89 (s, 3F); 13C NMR
(100 MHz, CD3OD, ppm):
d
167.19, 145.99, 136.77, 134.98, 134.46,
4.10. In vitro AChE inhibition assay
134.36, 133.54, 131.19, 126.64, 126.61, 124.34, 123.23, 120.89, 118.71,
113.92, 48.51; IR (KBr): nmax 3453, 2956, 2073, 1632, 1520,
1163 cmꢂ1; ESI-MS: m/z 355 [MꢂI]þ; HR-ESIMS: m/z 355.1042 calcd
for C20H14F3N2Oþ (355.1052).
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel, Type V-
S, 827 U/mg solid; 1256 U/mg of protein), 5,5'-dithiobis-(2-
nitrobenzoic acid) (Ellman's reagent, DTNB), acetylthiocholine

10
S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
iodide (ATChI) and donepezil were purchased from SigmaeAldrich.
sciencedirect.com.
Compounds were dissolved in water to make 10 mM stock solution.
Desired concentrations in the range of 1e100 mM were prepared by
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.049.
further dilutions with PBS. All assays were carried out in 96-well
plate in 0.1 M NaH2PO4/Na2HPO4 buffer, pH 7.2, using Biotage
Microplate Reader. AChE stock solution (1 mg/mL in 0.1 M sodium
References
phosphate buffer, pH 7.2) was diluted to produce 5
solution for enzyme inhibition studies. Each well of 96-well plate
consisted of total 200 L assay medium, comprising 16 L of
enzyme solution, 4 L of vehicle (PBS 7.2, control) or test com-
pound, 154 L of phosphate buffer (pH 7.2), 6 L of 10 mM DTNB
(final concentration of DTNB, 0.3 mM) and 20 l of 10 mM ATChI
mg/ml working
[1] Y.J. Wang, H.D. Zhou, X.F. Zhou, Clearance of amyloid-beta in Alzheimer's
disease: progress, problems and perspectives, Drug Discov. Today 11 (2006)
931e938.
[2] Alzheimers Association, Alzheimer's disease facts and figures, Alzheimers
Dement. 8 (2012) 131e168, 11, (2015), 332e384.
[3] P. Tiraboschi, L.A. Hansen, L.J. Thal, J. Corey-Bloom, The importance of neuritic
plaques and tangles to the development and evolution of AD, Neurology 62
(2004) 1984e1989.
m
m
m
m
m
m
(final concentration of ATChI, 1 mM). Initially, test compounds
(along with a parallel PBS 7.2 as a control) were pre-incubated at
37 ꢀC with the enzyme for 30 min followed by the addition of DTNB,
ATChI and PBS (pH 7.2) to make up the volume. The activity was
determined by measuring the increase in absorbance at 412 nm
after different time intervals at 37 ꢀC. Percent inhibition values
were calculated from the slopes of absorbance readings versus
different time intervals for control and test compounds. Each
concentration was assayed in triplicate [27].
[4] K.G. Mawuenyega, W. Sigurdson, V. Ovod, L. Munsell, T. Kasten, J.C. Morris,
K.E. Yrasheski, R.J. Bateman, Decreased clearance of CNS
heimer's disease, Science 330 (2010) 1774.
b-amyloid in Alz-
[5] D.M.E.v. Assema, M. Lubberink, M. Bauer, W.M.v.d. Flier, R.C. Schuit,
A.D. Windhorst, E.F.I. Comans, N.J. Hoetjes, N. Tolboom, O. Langer, M. Muller,
P. Scheltens, A.A. Lammertsma, B.N.M.v. Berckel, Bloodebrain barrier P-
glycoprotein function in Alzheimer's disease, Brain 135 (2012) 181e189.
[6] F.C. Lam, R. Liu, P. Lu, A.B. Shapiro, J.M. Renoir, F.J. Sharom, P.B. Reiner, b-
Amyloid efflux mediated by P-glycoprotein, J. Neurochem. 76 (2001)
1121e1128.
[7] M.D. Perloff, E. Stormer, L.L. von Moltke, D.J. Greenblatt, Rapid assessment of
P-glycoprotein inhibition and induction in vitro, Pharm. Res. 20 (2003)
1177e1183.
4.11. Determination of aqueous solubility by 96-well plate-based
assay
[8] R. Xie, L.H. Tan, E.C. Polasek, C. Hong, M. Teillol-Foo, T. Gordi, A. Sharma,
D.J. Nickens, T. Arakawa, D.W. Knuth, E.J. Antal, CYP3A and P-glycoprotein
activity induction with St. John's Wort in healthy volunteers from 6 ethnic
populations, J. Clin. Pharmacol. 45 (2005) 352e356.
[9] R. Tian, N. Koyabu, S. Morimoto, Y. Shoyama, H. Ohtani, Y. Sawada, Functional
induction and de-induction of P-glycoprotein by St. John's Wort and its in-
gredients in a human colon adenocarcinoma cell line, Drug Metab. Dispos. 33
(2005) 547e554.
[10] U.I. Schwarz, H. Hanso, R. Oertel, S. Miehlke, E. Kuhlisch, H. Glaeser, M. Hitzl,
G.K. Dresser, R.B. Kim, W. Kirch, Induction of intestinal P-glycoprotein by St
John's Wort reduces the oral bioavailability of talinolol, Clin. Pharmacol. Ther.
81 (2007) 669e678.
This was determined in water, PBS, SGF, and SIF as described
earlier [28].
4.12. Molecular modeling of the fascaplysin analogs with human
AChE
Docking studies were performed using Glide module of Schro-
dinger Software implemented in the Maestro modeling package
(Schrodinger, Inc., LLC, NewYork, USA) using default settings. The
E2020 (Donepezil) bound to Human AChE (PDB: 4EY7; resolution,
2.35 A0) was used in docking studies. Briefly, human AChE was
imported from protein data bank [29] and prepared using Protein
preparation wizard for H-bond optimization, heterogeneous state
generation, protonation and overall minimization. Grid file of
docking was constructed considering the bound ligand donepezil
as centroid of the ligand and binding site of 20 A0 radius box. All
ligands were sketched in maestro, prepared using Ligprep and
docked by Glide molecular docking software in XP mode.
[11] M.D. Perloff, L.L. von Moltke, E. Stormer, R.I. Shader, D.J. Greenblatt, Saint
John's Wort: an in vitro analysis of P-glycoprotein induction due to extended
exposure, Br. J. Pharmacol. 134 (2001) 1601e1608.
[12] D.G. Teotico, J.J. Bischof, L. Peng, S.A. Kliewer, M.R. Redinbo, Structural basis of
human pregnane X receptor activation by the hops constituent colupulone,
Mol. Pharmacol. 74 (2008) 1512e1520.
[13] A.H. Abuznait, H. Qosa, B.A. Busnena, K.A. El Sayed, A. Kaddoumi, Olive-oil-
derived oleocanthal enhances
b-amyloid clearance as a potential neuro-
protective mechanism against Alzheimer's disease: in vitro and in vivo
studies, ACS Chem. Neurosci. 4 (2013) 973e982.
[14] J.B. Bharate, Y.S. Batarseh, A. Wani, S. Sharma, R.A. Vishwakarma,
A. Kaddoumi, A. Kumar, S.B. Bharate, Synthesis and P-glycoprotein induction
activity of colupulone analogs, Org. Biomol. Chem. 13 (2015) 5488e5496.
[15] J.B. Bharate, A. Wani, S. Sharma, S.I. Reja, M. Kumar, R.A. Vishwakarma,
A. Kumar, S.B. Bharate, Synthesis, and the antioxidant, neuroprotective and P-
glycoprotein induction activity of 4-arylquinoline-2-carboxylates, Org. Bio-
mol. Chem. 12 (2014) 6267e6277.
[16] P. Joshi, S. Singh, A. Wani, S. Sharma, S.K. Jain, B. Singh, B.D. Gupta, N.K. Satti,
S. Koul, I.A. Khan, A. Kumar, S.B. Bharate, R.A. Vishwakarma, Osthol and cur-
cumin as inhibitors of human Pgp and multidrug efflux pumps of Staphylo-
coccus aureus: reversing the resistance against frontline antibacterial drugs,
Med. Chem. Commun. 5 (2014) 1540e1547.
[17] R. Mudududdla, S.K. Guru, A. Wani, S. Sharma, P. Joshi, R.A. Vishwakarma,
A. Kumar, S. Bhushan, S.B. Bharate, 3-(Benzo[d][1,3]dioxol-5-ylamino)-N-(4-
fluorophenyl)thiophene-2-carboxamide overcomes cancer chemoresistance
via inhibition of angiogenesis and P-glycoprotein efflux pump activity, Org.
Biomol. Chem. 13 (2015) 4296e4309.
4.13. Statistical analysis
Data are expressed as mean SD of three independent experi-
ments unless otherwise indicated. The comparisons were made
between control and treated groups or the entire intra-group using
the Bonferroni test through the Instat-2 software. p values *<0.05
were considered significant. p value *<0.05, **<0.01, ***<0.001.
[18] D.M. Roll, C.M. Ireland, Fascaplysin, an unusual antimicrobial pigment from
the marine sponge Fascaplysinopsis sp. J. Org. Chem. 53 (1988) 3276e3278.
[19] L. Nathaniel, S.L. Segraves, T.A. Johnson, S.A. Said, X. Fu, F.J. Schmitz,
H. Pietraszkiewicz, F.A. Valeriote, C. Phillip, Structures and cytotoxicities of
fascaplysin and related alkaloids from two marine phyla-fascaplysinopsis
sponges and didemnum tunicates, Tetrahedron Lett. 44 (2003) 3471e3475.
[20] R. Soni, L. Muller, P. Furet, J. Schoepfer, C. Stephan, S. Zumstein-Mecker,
H. Fretz, B. Chaudhuri, Inhibition of cyclin-dependent kinase 4 (Cdk4) by
fascaplysin, a marine natural product, Biochem. Biophys. Res. Commun. 275
(2000) 877e884.
Acknowledgments
SM and PJ are thankful to the CSIR for award of a Senior Research
Fellowship. VK thanks UGC for the award of research fellowship.
SSB is thankful to ICMR for Research Associateship. Authors thank
analytical department, IIIM for NMR, MS and IR analysis of syn-
thesized compounds. This work was supported by CSIR 12th FYP
grant BSC-0205.
[21] N.L. Segraves, S.J. Robinson, D. Garcia, S.A. Said, X. Fu, F.J. Schmitz,
H. Pietraszkiewicz, F.A. Valeriote, P. Crews, Comparison of fascaplysin and
related alkaloids: a study of structures, cytotoxicities, and sources, J. Nat. Prod.
67 (2004) 783e792.
Appendix A. Supplementary data
[22] S.B. Bharate, S. Manda, N. Mupparapu, N. Battini, R.A. Vishwakarma, Chemistry
and biology of fascaplysin, a potent marine-derived Cdk-4 inhibitor, Mini-Rev.
Med. Chem. 12 (2012) 650e664.
Supporting information available. Spectral data scans. This
material is available free of charge via the internet at http://

S. Manda et al. / European Journal of Medicinal Chemistry 107 (2016) 1e11
11
[23] S.B. Bharate, S. Manda, P. Joshi, B. Singh, R.A. Vishwakarma, Total synthesis
and anti-cholinesterase activity of marine derived bis-indole alkaloid fasca-
plysin, Med. Chem. Commun. 3 (2012) 1098e1103.
[24] S. Manda, S.I. Khan, S.K. Jain, S. Mohammed, B.L. Tekwani, I.A. Khan,
R.A. Vishwakarma, S.B. Bharate, Synthesis, antileishmanial and anti-
representative P-gp inducers, Biol. Pharm. Bull. 4 (2006) 779e784.
[27] S.B. Bharate, L. Guo, T.E. Reeves, D.M. Cerasoli, C.M. Thompson, Bisquaternary
pyridinium oximes: comparison of in vitro reactivation potency of com-
pounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-
inhibited electric eel and recombinant human acetylcholinesterase, Bioorg.
Med. Chem. 18 (2010) 787e794.
trypanosomal activities of N-substituted tetrahydro-b-carbolines, Bioorg.
Med. Chem. Lett. 24 (2014) 3247e3250.
[28] S.S. Bharate, R.A. Vishwakarma, Thermodynamic equilibrium solubility mea-
surements in simulated fluids by 96-well plate method in early drug dis-
covery, Bioorg. Med. Chem. Lett. 25 (2015) 1561e1567.
[29] C. Jonah, J.R. Michael, B. Fiana, S.C. Michael, N.G. Ebony, L. James, C.F. Matthew,
J.H. Jude, Structures of human acetylcholinesterase in complex with phar-
macologically important ligands, J. Med. Chem. 55 (2012) 10282e10286.
[25] B. Qin, X. Liu, J. Shi, K. Zheng, H. Zhao, X. Feng, Enantioselective cyanosilylation
of r,r-dialkoxy ketones catalyzed by proline-derived in-situ-prepared N-oxide
as bifunctional organocatalyst, J. Org. Chem. 72 (2007) 2374e2378.
[26] M. Kageyama, K. Fukushima, T. Togawa, K. Fujimoto, M. Taki, A. Nishimura,
Y. Ito, N. Sugioka, N. Shibata, K. Takada, Relationship between excretion
clearance of rhodamine 123 and P-glycoprotein (P-gp) expression induced by