Diastereoselective approach to rationally design tetrahydro-β-carboline-isatin conjugates as potential SERMs against breast cancer

Article abstract of DOI:10.1039/c9ra00744j

A series of tetrahydro-β-carboline-isatin conjugates, with varying substituents as well as stereochemistry at C-1 and C-5 position of tetrahydro-β-carboline (THβC) and isatin ring, were prepared and assayed for anti-proliferative efficacy on Estrogen Responsive ER(+) (MCF-7) and ER(?ve) MDA-MB-231 cell-lines. The synthesized scaffolds displayed selective anti-proliferative efficacy against MCF-7 cell-line with the most active conjugate 8b exhibiting an IC₅₀ value of 37.42 μM, comparable to that of peganumine A, a tetrahydro-β-carboline analogue, isolated from Peganum harmala. The synthesized compound 8b was also more potent than the standard drug tamoxifen (IC₅₀ = 50 μM against MCF-7). The observed activities were further corroborated via docking studies in ER-α (PDB ID: 3ERT).

Full text of DOI:10.1039/c9ra00744j

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Diastereoselective approach to rationally design
tetrahydro-b-carboline–isatin conjugates as
potential SERMs against breast cancer†
Cite this: RSC Adv., 2019, 9, 9809
Bharvi Sharma,a Amandeep Singh,a Liang Gu,b Sourav Taru Saha,b Ashona Singh-
c
c
c
b
a
*
*
Pillay, Nosipho Cele, Parvesh Singh, Mandeep Kaur and Vipan Kumar
A series of tetrahydro-b-carboline–isatin conjugates, with varying substituents as well as stereochemistry at
C-1 and C-5 position of tetrahydro-b-carboline (THbC) and isatin ring, were prepared and assayed for anti-
proliferative efficacy on Estrogen Responsive ER(+) (MCF-7) and ER(ꢀve) MDA-MB-231 cell-lines. The
synthesized scaffolds displayed selective anti-proliferative efficacy against MCF-7 cell-line with the most
active conjugate 8b exhibiting an IC50 value of 37.42 mM, comparable to that of peganumine A,
a tetrahydro-b-carboline analogue, isolated from Peganum harmala. The synthesized compound 8b was
also more potent than the standard drug tamoxifen (IC50 ¼ 50 mM against MCF-7). The observed
activities were further corroborated via docking studies in ER-a (PDB ID: 3ERT).
Received 28th January 2019
Accepted 11th March 2019
DOI: 10.1039/c9ra00744j
rsc.li/rsc-advances
anti-proliferative activity against MDA-MB-231 cell-lines with
IC50 values in the mM range.9 The anti-proliferative potential of
Introduction
THbCs along with their low cytotoxicity, attributed to the lack of
DNA intercalating properties owing to its non-planar structure,
has recently attracted the attention of the scientic fraternity for
the development of selective anti-proliferatives.8
Breast cancer (BC) is the most prevalent neoplasm amongst
women and considered as the second largest cause of death
globally aer lung cancer. In 2018, an estimated 1 735 350 new
cases of BC were diagnosed and over 609 640 deaths were ex-
pected to occur in United States.1 The occurrence of breast
cancer in India is continually increasing over the last few
decades and has now ranked number one amongst Indian
females with an age adjusted rate as high as 25.8 and mortality
of 12.7 per 100 000 women in 2017.2 Naturally occurring tetra-
hydro-b-carboline (THbC) derivatives such as vinblastine,3
reserpine,4 manzamine5 and callophycin A;6 are indole-based
alkaloids known for their diverse pharmacological potential.
Peganumine A, an octacyclic THbC analogue, extracted from the
seeds of Peganum harmala, exhibited signicant anti-
proliferative activity against MCF-7 with an IC50 value of 38.5
mM.7 N-(Biphenyl-2-yl)tryptoline (BPT) is another example of
Isatin is another indole-based moiety with proven anti-
proliferative potential via inhibition of tyrosine kinases like
VEGFR-1, VEGFR-2 and cyclic dependent kinases (CDKs).10
Sutent®, an isatin analogue (5-uoro-3-alkenyl-2 oxindole) has
been approved by the FDA for the treatment of gastrointestinal
stromal tumors and advanced renal cell carcinoma.11 Although
market is saturated with rst line drugs and potential SERMS
sanctioned by US Food and Drug Administration (FDA) viz.
tamoxifen (TAM), raloxifene having signicant anti-BC activi-
ties, yet they are also associated with adverse side effects such as
endometrial cancer, insomnia, dizziness.12 Recent reports from
our lab have shown in vitro anti-proliferative activities of ospe-
mifene–isatins against breast cancer cell lines. The inclusion of
isatin ring among these conjugates enhanced the anti-
proliferative activities in general with the most potent conju-
gate exhibiting an IC50 of 1.56 mM against MCF-7, being ꢁ30
folds potent than tamoxifen.13 The methodology was further
extended towards evaluating the anti-proliferative efficacy of
1H-1,2,3-triazole linked nitro-imidazole–isatin and isatin–fer-
rocenyl chalcone conjugates.14,15
a
saturated tricyclic framework exhibiting better anti-
proliferative potential via inhibition of mitosis and blocking
the G0/G1 phase of the cell cycle and has low cytotoxicity
(Fig. 1).8 Structurally modied tadalal derivatives displayed
aDepartment of Chemistry, Guru Nanak Dev University, Amritsar-143005, India.
E-mail: vipan_org@yahoo.com
bSchool of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3,
Wits-2050, Johannesburg, South Africa. E-mail: mandeep.kaur@wits.ac.za
In continuation to our recent reports on the synthesis of
molecular conjugates with biological relevance and their eval-
uation,16–18 the present manuscript encompasses the design,
synthesis and anti-proliferative evaluation of 1H-1,2,3-triazole
linked THbC–isatin conjugates, as depicted in Fig. 2, against
MCF-7 and MDA-MB-231 cell-lines using MTT assay. The use of
cSchool of Chemistry and Physics, University of KwaZulu Natal, P/Bag X54001,
Westville, Durban 4000, South Africa
† Electronic supplementary information (ESI) available: 1H and 13C spectral data
of the synthesized compounds along with scanned 1H and 13C NMR spectra of
representative compounds viz. 8b, 8c, 8f, 8g, 8h, 8i, 8j and Table 4. See DOI:
10.1039/c9ra00744j
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Fig. 1 Natural and synthetic structural motifs with a tetrahydro-b-carboline core.
Pictet–Spengler reaction of L-tryptophan with 30% aqueous
solution of formalin at room temperature. In the case of
aliphatic aldehydes viz. acetaldehyde, a kinetically controlled
Pictet–Spengler reaction of L-tryptophan with 40% solution of
acetaldehyde yielded cis-isomer(1S,3S)-1-methyl-2,3,4,9-tetrahy-
dro-1H-pyrido-[3,4-b]indole-3-carboxylic acid 2b as the major
product.
With a less reactive aromatic aldehyde viz. benzaldehyde, the
reaction upon heating in acetic acid followed a thermodynami-
cally controlled pathway to afford a diastereomeric mixture
which was used in subsequent steps without purication. The
treatment of THbC derived acids 2a–c with thionyl chloride in
dry ethanol afforded the corresponding esters 3a–c. Compound
3c having the phenyl substituent at C-1 was successfully puried
using column chromatography and only the trans isomer was
used in subsequent steps because of its prevalence in naturally
occurring alkaloids viz. ajmaline,21 sarpagine22 as well as the
non-natural analogue tadalal. Base-promoted N-prop-
argylation of 3a–c in dry acetonitrile resulted in the formation of
the corresponding ‘click’ precursors 4a–c (Scheme 1). The
second precursor viz. N-alkylazidoisatins 7 was obtained via
base-promoted alkylation of C-5 substituted isatin 5 with
dibromoalkane to afford 6 with successive treatment with
sodium azide (Scheme 2).
Fig. 2 Designed tetrahydro-b-carboline–isatin conjugates.
L-tryptophan in the present series of compounds is not only
owed to its natural availability but also because of the prom-
ising anti-proliferative potential of L-tryptophan derived THbC
against MDA-MB-231 cell-line. The nature of substituents as
well as the stereochemistry at C-1 of THbC and C-5 of isatin ring
was varied so as to study their effect on the anti-proliferative
activities.
Results and discussion
Chemistry
The synthetic methodology involved the enantioselective Pictet–
Spengler cyclization of L-tryptophan with various aldehydes to
afford the corresponding tetrahydro-b-carbolines with varying
stereochemistry at C-1 position.19,20 Depending upon the reac-
tivity of different aldehydes, different reaction conditions were
employed for the synthesis of corresponding THbC derived
carboxylic acids. The preparation of 2,3,4,9-tetrahydro-1H-b-
Scheme 1 Synthesis of N-propargylated tetrahydro-b-carboline ethyl
ester. (i) 30% formaldehyde, NaOH, H2O, 8 h, reflux, or (ii) 40% acet-
aldehyde, H2SO4,, 6–8 h, rt, or (iii) benzaldehyde, acetic acid, 2 h, reflux
(iv) SOCl2, EtOH, 6 h, reflux (v) propargyl bromide, acetonitrile, rt, 6–
carboline-3-carboxylic acid 2a involved the base-promoted 8 h. (vi) CuSO4$5H2O, sodium ascorbate, EtOH, rt, 8 h.
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IC50 value i.e. the necessary concentration for inhibiting 50% of
cell viability by the synthesized compounds, has been enlisted
in Table 1.
As evident from Table 1, all the synthesized conjugates
proved to be inactive against MDA-MB-231 (ERꢀ) cell-line and
were selective towards estrogen responsive MCF-7 cell-line.
Structure activity relationship analysis showed the depen-
dence of anti-proliferative activity on the nature of substituent
at C-1 and C-5 position of THbC and isatin ring respectively
while the alkyl chain length did not seem to play any substantial
role. Among un-substituted (R ¼ H) isatin based conjugates 8a–
8i, the presence of either hydrogen or methyl substituents at C-1
position of THbC resulted in the loss of activities as evident
from 8g–8i. The induction of uoro-substituent at C-5 position
of isatin ring proved to be detrimental for anti-proliferative
activities as most of the compounds were inactive except 8r,
having a phenyl substituent at C-1, exhibiting an IC50 value of
42.11 mM. Interestingly, several compounds (8k–n) showed
a negative correlation, where an increase in drug concentration
induced a decrease in growth inhibition, possibly suggesting
that these compounds are supporting cancer cell proliferation.
However, it could also suggest that at high concentrations,
these compounds might have precipitated out of culture media
and thus lost their effectiveness.
Scheme
2 Synthesis of substituted N-alkyl-azido-isatin (i) NaH,
dibromoalkane, anhy. DMF, 60 ꢂC, 6–8 h (ii) NaN3, DMF, 60 ꢂC, 2 h.
Cu-promoted azide–alkyne cycloaddition reactions of 4a–c
with 7a–f gave the desired THbC–isatin conjugates 8a–r in good
yields as shown in Scheme 3. The compounds thus synthesized
were puried using ethylacetate : hexane (80 : 20) on alumina
as a stationary phase.
The structure to the compounds was assigned on the basis of
analytical evidences and spectral data. For example, the most
active compound 2-{1-[3-(2,3-dioxo-2,3-dihydro-indol-1-yl)-
propyl]-1H-[1,2,3]triazol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8b) displayed molecular
ion peak m/z at [M + 1]+ 513.2206 in its High Resolution Mass
Spectrum (HRMS). The prominent features in its 1H NMR
include the appearance of triplet at d 1.23 corresponding to
methyl protons, multiplet corresponding to methylene (H-4) of
THbC ring at d 3.09–3.21 and singlet because of triazole-H at
d 7.67. Further, 13C NMR conrmed the assigned structure by
the appearance of characteristic absorptions at d 172.7 repre-
senting the carbonyl carbon of THbC along with d 158.5 and
183.0 corresponding to isatin ring carbonyls.
Molecular docking studies
In order to identify novel binding cores, molecular docking of
the active ligands in the binding site of estrogen receptor-a (ER-
a) was performed to determine the crucial binding interactions
responsible for the inhibitory effect observed.24 The native
ligand in the crystal structure of ER-a (PDB ID: 3ERT) is a-
moxifene which inhibited the receptor by binding to the
predominantly hydrophobic pocket. The binding interaction
prole of the ligand–protein complex largely comprised of
hydrophobic interactions with residues Leu41, Ala45, Trp78,
Leu82, Leu86, Phe99, Leu220 and Leu231. These interactions
were accompanied by two distinct hydrogen bonds with Glu48
and Arg89, two water bridges established between residues
Thr42 and Leu86 as well as a single salt bridge with amino acid
Asp46.
Anti-proliferative activities of synthesized conjugates
The newly synthesized conjugates were screened for their anti-
proliferative activities against human breast cancer cell-lines
viz. MCF-7 and MDA-MB-231 and the results were compared
with reference drugs, plumbagin23 and peganumine A.7 Detailed
results of these studies, in terms of % age growth inhibition of
MCF-7 and MDA-MB-231 cells, have been depicted in Fig. 3. The
Compound 8b shared a similar hydrophobic interaction
network to that of the native ligand. The binding landscape of
compound 8b comprised a signicant hydrophobic contribu-
tion between residues, Leu41, Leu44, Ala45, Leu49, Trp78,
Leu82, Leu220 and Leu231 with the phenyl fused heterocyclic
rings and the hydrophobic linker (bond distance ¼ 3.93/3.77,
˚
3.99, 3.81, 3.78, 3.62/3.87, 3.95, 3.95 and 3.84 A, respectively).
A single salt bridge exists between the positively charged tert-
˚
amine and Asp46 at a distance of 5.11 A. Despite the binding
prole of compound 8b being very simple, it was identied as
the most potent inhibitor (Fig. 4).
Compounds 8d, 8e and 8r demonstrated promising activity
against the MCF-7 cell-line in comparison to peganumine A.
Compounds 8d and 8e have a near identical binding interaction
prole. The binding of these compounds to the active site of ER-
a comprised an extensive network of van der Waals interactions
Scheme 3 Synthesis of 1H-1,2,3-triazole-linked tetrahydro-b-carbo-
line–isatin conjugates 8a–r (i) CuSO4, sodium ascorbate, EtOH, 6–8 h,
rt.
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Fig. 3 Representative graph of percentage growth inhibition on MCF7 and MDA-MB-231 cells at selected concentrations of test compounds
8a–r. 40 mM plumbagin was used as a positive control. Data shows mean ꢃ standard deviation S.D. (n ¼ 3), where *p < 0.05, **p < 0.01 and ***p <
0.001 significance compared to untreated control.
between residues Leu41, Leu44, Ala45, Leu49, Trp78, Leu86, Leu41, Ala45, Trp78, Leu82, Leu86, Tyr221 and Leu231 at
Phe99, Lys224 and Leu231 and the aromatic moieties of the a bond distance of 3.70, 3.75, 3.64, 3.80/3.87, 3.52, 3.99 and 3.69
˚
ligands (bond distance ¼ 4.00/3.90//3.92/3.78, 3.97, 3.68//3.76, A, respectively. Also, the ligand interaction included two
˚
3.83, 3.83//3.54, 3.87, 3.83, 3.96/3.95//3.39 and 3.85//3.82 A; hydrogen bonds between the donor residue Asp46 and the
respectively). The protein–ligand interaction was further sup- carbonyl oxygen of the ligand ethoxy group as well as the
ported by a hydrogen bond between the oxygen of the hydrogen acceptor residue Leu220 and the aromatic nitrogen (bonꢂd
ꢂ
˚
acceptor Leu220 and the triazole nitrogens (bond distance ¼ distance ¼ 3.82 and 2.93 A; bond angle ¼ 100.07 and 166.19 ;
ꢂ
ꢂ
˚
3.00//3.54 A; bond angle ¼ 136.41 //105.19 ) as well as a salt respectively). The uorine moiety of compound 8r also formed
bridge between the anion Asp46 and the tertamine (bond a distinct electrostatic interaction with residue Arg89, (bond
ꢂ
˚
˚
distance ¼ 4.27//5.06 A).
distance ¼ 3.18 A, donor angle ¼ 141.31 , acceptor angle ¼
The inhibitory activity of compound 8r was largely driven by 109.50ꢂ). The thermodynamic and conformational stability of
the existence of the hydrophobic network between residues the complexes 8b, 8d, 8e and 8r in the binding site has been well
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Table 1 IC50 values of the test compounds in MCF-7 and MDA-MB-231 cells
MDA-MB231
MDA-MB231
Entry Compound
MCF-7 (mM) (mM)
Entry Compound
MCF-7 (mM) (mM)
8a
>100
>100
8k
>100
>100
8b
8c
37.42
>100
>100
>100
8l
>100
>100
>100
>100
8m
8d
8e
8f
45.3
50
>100
>100
>100
8n
8o
8p
>100
>100
>100
>100
>100
>100
>100
8g
>100
>100
8q
>100
>100
8h
8i
>100
>100
>100
>100
>100
>100
8r
42.11
3.5
>100
4.4
Plumbagin
Peganumine A7
Tamoxifen
38.5
50
—
75
8j
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Fig. 4 Three-dimensional illustration of docked ligands interacting in the binding site of the estrogen receptor (PDB: 3ERT).
depicted by high binding affinities with energy value of ꢀ9.6, compounds have been well demonstrated in Table 3 (see ESI†).
ꢀ8.8, ꢀ9.9 and ꢀ10.5 kcal molꢀ1 respectively (Table 2). The The synthesized conjugates 8b, 8d, 8e and 8f (TPSA/A ¼ 110.01,
˚
parameters describing the physicochemical properties viz. logP 110.01, 110.01 and 110.10 respectively) possess good potential
and topological polar surface area (TPSA) of the target to permeate cell membrane. Further, the moderate positive
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Table 2 Docking of the most active compounds in ER-a
estrogen responsive MCF-7 cell line suggestive of the fact that
they can serve as molecular templates in the expansion of cancer
chemotherapies competent of targeting ER-dependent breast
cancers. Docking results further support that the most potent
compound displays great binding affinity with ER(+) owing to
occupation of binding sites via various hydrophobic, H-bonding
and cation-p interactions with ER(+) active site.
Docking score/(kcal
Ligand identier
molꢀ1
)
RMSD i.b.
RMSD u.b.
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
ꢀ9.7
ꢀ9.6
ꢀ9.7
ꢀ8.8
ꢀ9.9
ꢀ8.7
ꢀ9.3
ꢀ9.6
ꢀ10.4
ꢀ10.2
ꢀ10.2
ꢀ9.3
ꢀ9.5
ꢀ9.6
ꢀ8.9
ꢀ9.1
ꢀ10.9
ꢀ10.5
ꢀ7.9
ꢀ6.2
0.000
0.000
0.000
1.960
0.000
1.978
2.080
2.577
0.000
0.000
0.000
1.193
2.061
1.265
2.170
4.238
0.000
0.000
0.000
1.357
0.000
0.000
0.000
2.689
0.000
2.557
2.582
4.014
0.000
0.000
0.000
1.427
2.912
1.506
2.873
6.490
0.000
0.000
0.000
2.189
Experimental section
General note
All the reactions were carried out using standard techniques.
The column chromatography was carried out using silica gel
(60–120 mesh) with ethyl acetate : hexane as eluent. Melting
points were recorded in capillaries and are uncorrected. The
spectra of 1H NMR and 13C NMR spectra were recorded on JEOL
400, BRUKER 500 and 125 MHz spectrometers and obtained as
CDCl3 solutions using tetramethylsilane (TMS) as an internal
standard. Chemical shis were reported in parts per million
(ppm) and coupling constants J indicated in hertz. Mass spec-
tral data was gathered on BrukermicrOTOF QII equipment
using ESI as the source.
8q
8r
Native ligand
Plumbagin
value of partition coefficient logP clearly denes the lipophilic
nature of the compounds 8b, 8d, 8e and ability to permeate cell
membrane effectively and efficiently.
Preparation of 2-prop-2-ynyl-2,3,4,9-tetrahydro-b-carboline-3-
carboxylic acid ethyl ester 4
To a solution of secondary amine (3 mmol) and K2CO3 (3.6
mmol) in 15 mL acetone, propargyl bromide (3.6 mmol) was
added and reaction mixture was stirred overnight at room
temperature. The reaction mixture was ltered and concen-
trated under reduced pressure to yield crude product which was
puried via column chromatography using (20 : 80) ethyl-
acetate : hexane as eluent. 4a pale yellow solid; 92%; 4b pale
yellow solid 90%; 4c light brown solid 84%.
The molecular docking of the discussed conjugates has
highlighted critical pharmacophoric features of the potential
drug candidates which directly affect their inhibitory activity
against MCF-7 cell-line. It is evident that the synthesized tetra-
hydro-b-carboline–isatin conjugates are promising anti-
proliferative agents. With further derivatization, the pharmaco-
logical efficacy of the potential drug candidates can be improved
to reveal potent inhibitors. The binding pattern of the active
compound and potential anti-proliferative agents i.e. compounds
8b, 8d, 8e and 8r, respectively; emphasises the need for the active
Procedure for the synthesis of conjugates (8a–r)
ligand to comprise hydrophobic pharmacophores. Effective To the stirred solution of N-alkylazido-isatin 7 (1 mmol) and 2-
inhibitory activity can be achieved from the presence of hydrogen prop-2-ynyl-2,3,4,9-tetrahydro-b-carboline-3-carboxylic
acid
bonds created between the protein and ligand, specically with ethyl ester 4 in a mixture of ethanol : water (85 : 15), was added
the conjugate being the hydrogen bond acceptor and protein the CuSO4$5H2O (0.055 mmol) and sodium ascorbate (0.143
donor. Further to this, the interaction of the isatin conjugate in mmol). The reaction mixture was allowed to stir at rt for 7–8 h.
the binding pocket should be facilitated with the presence of Upon completion of the reaction as monitored through TLC, the
ionised non-covalent bonds. The structural incentive by intro-
ducing a halogen at position C-5 position of the isatin appears
not critical as is observed in compound 8r.
resulting mixture was extracted with ethyl acetate (2 ꢄ 30 mL)
and water (2 ꢄ 25 mL). The organic layers were combined, dried
over anhydrous Na2SO4 and concentrated under reduced pres-
sure to yield the desired conjugates (8a–r); which were puried
via chromatography using alumina as stationary phase and
ethyl-acetate : hexane (80 : 20) as eluent mixture.
Conclusion
A series of tetrahydro-b-carboline–isatin conjugates having varied
2-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]tri-
substituents at C-5 position of isatin as well as at C-1 position of azol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
THbC ring were prepared via azide alkyne cycloaddition reac- acid ethyl ester (8a). Yield 72%; light orange solid; mp 108–
tions. The compound, 8b with an optimum combination of un- 110 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.20 (t, J ¼ 7.1 Hz, 3H,
substituted THbC and isatin ring along with a propyl-chain as –CH3); 2.93–3.10 (m, 2H, H4); 3.58–3.66 (m, 2H, H3 + H5); 3.99 (s,
spacer, proved to be most potent among the synthesized scaf- 2H, H6); 4.03–4.15 (m, 5H, H5 + H2 + H8); 4.62 (t, J ¼ 5.6 Hz, 2H,
folds exhibiting an IC50 value of 37.42 mM, comparable to that of H7); 6.53 (d, J ¼ 8 Hz, 1H, Ar–H); 6.88 (t, J ¼ 7.5 Hz, 1H, Ar–H);
peganumine A. The anti-proliferative evaluation studies of 7.02–7.10 (m, 2H, Ar–H); 7.26 (d, 1H, J ¼ 7.9 Hz, Ar–H); 7.34–
synthesized conjugates revealed them to be selective against 7.41 (m, 2H, Ar–H); 7.46 (d, J ¼ 7.4 Hz, 1H, Ar–H), 7.56 (s, 1H,
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triazole-H); 8.30 (s, 1H, –NH (exchangeable with D2O)) 13C NMR 8 Hz, 1H, Ar–H); 7.41 (d, J ¼ 7.4 Hz, 1H, Ar–H); 7.46 (t, J ¼ 7.7 Hz,
(100 MHz, CDCl3) d 14.2, 24.0, 39.6, 45.0, 47.4, 48.9, 57.6, 60.4, 1H, Ar–H); 7.52 (d, J ¼ 7.3 Hz, 1H, Ar–H); 7.76 (s, 1H, triazole-H);
106.2, 110.1, 111.2, 117.5, 117.8, 119.5, 121.2, 124.0, 124.2, 8.36 (s, 1H, –NH (exchangeable with D2O)) 13C NMR (100 MHz,
125.4, 127.1, 131.2, 132.4, 135.2, 136.1, 146.2, 158.1, 172.2, 183.0 CDCl3) d 14.3, 18.2, 24.1, 27.8, 36.5, 46.4, 47.6, 49.2, 60.4, 60.7,
HRMS calcd for C27H26N6O4 [M + 1]+ 499.2049, found 499.2021. 105.8, 110.1, 110.9, 116.5, 117.8, 119.2, 121.6, 122.7, 124.2,
2-{1-[3-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-propyl]-1H-[1,2,3] 125.9, 127.5, 131.1, 136.2, 137.7, 146.8, 150.2, 158.5, 172.8, 183.2
triazol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic HRMS calcd for C29H30N6O4 [M + 1]+ 527.2362, found 527.2329.
acid ethyl ester (8b). Yield 70%; light orange solid; mp 101–
2-{1-[4-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-butyl]-1H-[1,2,3]tri-
103 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.23 (t, J ¼ 7.1 Hz, 3H, –CH3); azol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-carboline-3-
2.32–2.39 (m, 2H, H8); 3.09–3.21 (m, 2H, H4); 3.75 (t, J ¼ 6.8 Hz, carboxylic acid ethyl ester (8f). Yield 68%; light orange solid; mp
2H, H9); 3.88–3.93 (m, 2H, H3 + H5); 4.09–4.23 (m, 5H, H5 + H2 + 130–134 C; H NMR (400 MHz, CDCl3-d1) d 1.29 (t, J ¼ 7.0 Hz,
1
ꢂ
H6); 4.40 (t, J ¼ 6.4 Hz, 2H, H7); 6.88 (d, J ¼ 7.9 Hz, 1H, Ar–H); 3H, –CH3); 1.41 (d, J ¼ 6.3 Hz, 3H, –CH3); 1.59–1.63 (m, 2H, H9);
7.03–7.12 (m, 3H, Ar–H); 7.28 (d, J ¼ 7.8 Hz, 1H, Ar–H); 7.44 (d, J 1.88–1.95 (m, 2H, H8); 2.91–2.95 (m, 1H, H4); 3.06–3.12 (m, 1H,
¼ 7.4 Hz, 1H, Ar–H); 7.53–7.59 (m, 2H, Ar–H); 7.67 (s, 1H, triazole- H4); 3.61–3.70 (m, 3H, H10 + H3); 3.98–4.02 (d, J ¼ 15.7 Hz, 1H,
H); 8.03 (s, 1H,–NH (exchangeable with D2O)) 13C NMR (100 MHz, H6); 4.06–4.12 (q, J ¼ 6.4 Hz, 1H, H5); 4.19–4.25 (m, 3H, H2 + H6);
CDCl3) d (ppm): 14.3, 23.9, 27.7, 37.5, 46.3, 47.6, 49.2, 60.2, 60.8, 4.35 (t, J ¼ 6.2 Hz, 2H, H8); 6.76 (d, J ¼ 7.8 Hz, 1H, Ar–H); 6.96–
106.1, 110.2, 110.9, 117.6, 117.9, 119.3, 121.5, 123.7, 124.2, 125.8, 7.07 (m, 3H, Ar–H); 7.28 (d, J ¼ 7.6 Hz, 1H, Ar–H); 7.38–7.44 (m,
127.1, 131.2, 136.2, 138.7, 145.8, 150.3, 158.5, 172.7, 183.0 HRMS 2H, Ar–H); 7.48 (d, J ¼ 7.08 Hz, 1H, Ar–H); 7.65 (s, 1H, triazole-H);
calcd for C28H28N6O4 [M + 1]+ 513.2206, found 513.2234.
8.81 (s, 1H, –NH (exchangeable with D2O)). 13C NMR (100 MHz,
2-{1-[4-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-butyl]-1H-[1,2,3]tri- CDCl3) d 14.3, 18.5, 24.1, 27.4, 31.6, 39.2, 44.9, 49.3, 52.4, 61.2,
azol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
61.7, 105.7, 110.1, 111.1, 117.5, 117.9, 119.3, 121.4, 123.7, 123.9,
acid ethyl ester (8c). Yield 72%; light orange solid; mp 92–94 ꢂC; 125.5, 126.8, 135.4, 136.3, 138.6, 145.1, 150.5, 158.3, 173.5, 183.3
1H NMR (400 MHz, CDCl3-d1) d (ppm): 1.20 (t, J ¼ 7.1 Hz, 3H, HRMS calcd for C30H32N6O4 [M + 1]+ 541.2519, found 541.2530.
–CH3); 1.58–1.65 (m, 2H, H9); 1.87–1.95 (m, 2H, H8); 3.05–3.17
2-{1-[3-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-propyl]-1H-[1,2,3]
(m, 2H, H4); 3.65 (m, 2H, H10); 3.79 (d, J ¼ 15.2 Hz, 1H, H5); 3.86 triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-carboline-
(t, J ¼ 5.6 Hz, 1H, H3); 4.00–4.15 (m, 5H, H5 + H2 + H6); 4.35 (t, J 3-carboxylic acid ethyl ester (8g). Yield 64%; light orange solid;
¼ 6.8 Hz, 2H, H7); 6.81 (d, J ¼ 7.9 Hz, 1H, Ar–H); 7.00–7.09 (m, mp 168–170 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.17 (t, J ¼ 7.1 Hz,
3H, Ar–H); 7.27–7.29 (m, 1H, Ar–H); 7.40 (d, J ¼ 7.5 Hz, 1H, Ar– 3H, –CH3); 2.95–3.13 (m, 2H, H4); 3.69–3.72 (m, 2H, H8); 4.00–
H); 7.48–7.54 (m, 3H, Ar–H + triazole-H); 8.5 (s, 1H, –NH 4.18 (m, 5H, H2 + H6 + H3); 4.56–4.61 (m, 2H, H7); 5.41 (s, 1H,
(exchangeable with D2O)) 13C NMR (100 MHz, CDCl3) d (ppm): H5); 6.46 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.02–7.15 (m, 5H, Ar–H);
14.3, 24.0, 27.4, 29.7, 39.3, 46.2, 49.2, 49.3, 60.3, 60.8, 105.7, 7.22–7.24 (m, 2H, Ar–H); 7.27 (s, 2H, Ar–H); 7.37–7.47 (m, 3H,
110.1, 111.1, 117.5, 117.8, 119.1, 121.3, 123.1, 124.0, 125.7, Ar–H); 7.49 (s, 1H, triazole-H); 7.53 (dd, J ¼ 6.5, 0.9 Hz, 1H, –NH
127.0, 131.4, 136.3, 138.6, 145.6, 150.5, 158.4, 172.82, 183.36 (exchangeable with D2O)); 13C NMR (100 MHz, CDCl3-d1) d 14.3,
HRMS calcd for C29H30N6O4 [M + 1]+ 527.2362, found 527.2324. 24.7, 40.8, 46.0, 47.8, 56.8, 60.5, 60.9, 106.0, 109.5, 110.9, 117.3,
2-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]tri-
azol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-carboline-3-
118.2, 119.3, 121.7, 123.6, 124.2, 125.1, 125.6, 126.9, 128.1,
128.8, 129.7, 134.7, 136.5, 138.7, 140.3, 141.9, 147.5, 150.1,
carboxylic acid ethyl ester (8d). Yield 67%; light orange solid; 158.5, 172.8, 182.5 HRMS calcd for C33H30N6O4 [M + 1]+
mp 146–148 ꢂC 1H NMR (400 MHz, CDCl3) d 1.28 (t, J ¼ 7.1 Hz, 575.2362, found 575.2341.
3H, –CH3); 1.44 (d, J ¼ 6.6 Hz, 3H, –CH3); 2.70–2.75 (m, 1H, H4);
2-{1-[3-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-propyl]-1H-[1,2,3]
2.90–2.94 (m, 1H, H4); 3.82 (q, J ¼ 6.4 Hz, 1H, H5); 3.92 (d, J ¼ triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-carboline-
15.9 Hz, 1H, H6); 4.16–4.20 (m, 5H, H6 + H2 + H3 + H8); 4.61–4.76 3-carboxylic acid ethyl ester (8h). Yield 68%; light orange solid;
(m, 3H, H7 + H8); 6.39 (d, J ¼ 7.0 Hz, 1H, Ar–H); 7.00–7.14 (m, mp 163–165 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.17 (t, J ¼ 7.1 Hz,
4H, Ar–H); 7.30 (d, J ¼ 7.6 Hz, 2H, Ar–H); 7.41 (d, J ¼ 7.4 Hz, 1H, 3H, –CH3); 2.27–2.33 (m, 2H, H8); 3.20–3.22 (m, 2H, H4); 3.70–
Ar–H); 7.53 (s, 1H, triazole-H); 8.07 (s, 1H, Ar–H) 13C NMR (100 3.75 (m, 2H, H9); 3.85 (d, J ¼ 14.8 Hz, 1H, H6); 4.00–4.07 (m, 3H,
MHz, CDCl3) d 14.3, 18.3, 24.0, 45.3, 47.4, 49.3, 49.5, 60.4, 60.8, H2 + H3); 4.14 (d, J ¼ 14.7 Hz, 1H, H6); 4.30–4.34 (m, 2H, H7);
105.2, 110.2, 111.4, 117.4, 117.5, 119.6, 121.3, 124.2, 125.2, 5.51 (s, 1H, H5); 6.85 (d, J ¼ 7.9 Hz, 1H, Ar–H); 7.02–7.16 (m, 5H,
125.4, 127.1, 130.2, 136.5, 139.7, 146.0, 151.2, 158.6, 172.9, 183.1 Ar–H); 7.26–7.29 (m, 2H, Ar–H); 7.43 (s, 1H, Ar–H); 7.46–7.58 (m,
HRMS calcd for C28H28N6O4 [M + 1]+ 513.2206, found 513.2214. 4H, Ar–H); 7.64 (s, 1H, triazole-H); 7.95 (s, 1H, –NH
2-{1-[3-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-propyl]-1H-[1,2,3] (exchangeable with D2O)) 13C NMR (100 MHz, CDCl3) d 14.2,
triazol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-carboline-
24.7, 31.5, 37.5, 46.2, 47.5, 57.1, 60.5, 61.0, 106.1, 110.2, 110.9,
3-carboxylic acid ethyl ester (8e). Yield 72%; light orange solid; 117.6, 118.2, 119.3, 121.6, 123.2, 124.1, 125.7, 127.0, 128.1,
mp 141–143 ꢂC, 1H NMR (400 MHz, CDCl3) d 1.30 (t, J ¼ 7.1 Hz, 128.8, 128.9, 134.8, 136.6, 138.7, 140.6, 142.1, 147.0, 150.3,
3H, –CH3); 1.46 (d, J ¼ 6.8 Hz, 3H, –CH3), 2.27–2.34 (m, 2H, H8); 158.5, 173.1, 183.1 HRMS calcd for C34H32N6O4 [M + 1]+
2.93–2.98 (m, 1H, H4); 3.08–3.15 (m, 1H, H4); 3.68–3.73 (m, 3H, 589.2519, found 589.2542.
H9 + H3); 4.03 (d, J ¼ 15.8 Hz, 1H, H6); 4.12 (q, J ¼ 6.4 Hz, 1H,
2-{1-[4-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-butyl]-1H-[1,2,3]
H5); 4.22–4.27 (m, 3H, H2 + H6); 4.37 (t, J ¼ 6.56 Hz, 2H, H7); 6.81 triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-carboline-
(d, J ¼ 7.9 Hz, 1H, Ar–H); 7.01–7.09 (m, 3H, Ar–H); 7.27 (d, J ¼ 3-carboxylic acid ethyl ester (8i). Yield 62%; light orange solid;
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mp 158–161 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.17 (t, J ¼ 7.1 Hz,
3H, –CH3); 1.62–1.69 (m, 2H, H9); 1.88–1.94 (m, 2H, H8); 3.13–
3.24 (m, 2H, H4); 3.67–3.71 (m, 2H, H10); 3.82 (d, J ¼ 14.7 Hz, 1H,
H6); 3.98–4.07 (m, 3H, H2 + H3); 4.12 (d, J ¼ 14.6 Hz, 1H, H6);
4.32–4.36 (m, 2H, H7); 5.49 (s, 1H, H5); 6.83 (d, J ¼ 7.8 Hz, 1H,
Ar–H); 7.04–7.17 (m, 5H, Ar–H); 7.27 (s, 2H, Ar–H); 7.34–7.36 (m,
2H, Ar–H); 7.46–7.53 (m, 3H, Ar–H); 7.55 (s, 1H, triazole-H); 7.57
(s, 1H, –NH (exchangeable with D2O)) 13C NMR (100 MHz,
CDCl3) d 14.3, 24.1, 24.6, 27.5, 39.2, 46.1, 49.3, 57.0, 60.6, 60.9,
106.2, 110.2, 111.0, 117.6, 118.2, 119.3, 121.6, 122.4, 124.0,
125.7, 126.9, 128.2, 128.8, 128.9, 129.7, 134.7, 136.5, 138.6,
142.0, 147.1, 150.5, 158.4, 173.1, 183.3 HRMS calcd for
2-{1-[2-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-ethyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8m). Yield 64%; light
brown solid; mp 118–120 ꢂC; 1H NMR (400 MHz, CDCl3–)
d (ppm): 1.30 (t, J ¼ 7.1 Hz, 3H, –CH3); 1.43 (d, J ¼ 6.6 Hz, 3H,
–CH3); 2.74–2.79 (m, 1H, H4); 2.95–2.99 (m, 1H, H4); 3.87 (q, J ¼
6.4 Hz, 1H, H5); 3.95 (d, J ¼ 15.9 Hz, 1H, H6); 4.20–4.24 (m, 5H,
H6 + H2 + H3 + H8); 4.67–4.81 (m, 3H, H7 + H8); 6.43 (d, J ¼
7.0 Hz, 1H, Ar–H); 7.06–7.17 (m, 3H, Ar–H); 7.37 (d, J ¼ 7.6 Hz,
2H, Ar–H); 7.47 (d, J ¼ 7.4 Hz, 1H, Ar–H); 7.56 (s, 1H, triazole-H);
8.04 (s, 1H, Ar–H) 13C NMR (100 MHz, CDCl3) d (ppm): 14.3,
18.5, 24.6, 45.8, 47.4, 49.4, 49.7, 60.5, 60.9, 105.1, 110.3, 110.8,
116.4, 117.7, 120.4, 122.4, 124.6, 125.1, 125.7, 127.1, 131.2,
135.4, 139.6, 145.4, 151.3, 157.6, 172.8, 182.5 HRMS calcd for
C
35H34N6O4 [M + 1]+ 603.2675, found 603.2681.
2-{1-[2-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-
C
28H27FN6O4 [M + 1]+ 531.2111, found 531.2134.
[1,2,3]triazol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid ethyl ester (8j). Yield 78%; light brown solid; mp
134–135 ꢂC; 1H NMR (500 MHz, CDCl3) d 1.24 (t, J ¼ 7.1 Hz, 3H,
–CH3); 3.00–3.14 (m, 2H, H4); 3.70–3.73 (m, 2H, H3 + H5); 4.03 (s,
2H, H6); 4.06–4.19 (m, 5H, H5 + H2 + H8); 4.65 (m, 2H, H7); 6.55–
6.58 (m, 1H, Ar–H); 7.06–7.22 (m, 4H, Ar–H); 7.29 (d, J ¼ 6.8 Hz,
1H, Ar–H); 7.43 (d, J ¼ 7.7 Hz, 1H, Ar–H); 7.61 (s, 1H, triazole-H);
8.24 (s, 1H, –NH (exchangeable with D2O)) 13C NMR (125 MHz,
CDCl3) 14.2, 24.0, 40.7, 46.0, 47.5, 49.0, 59.8, 60.7, 105.8, 110.8,
112.5, 112.7, 117.7, 117.9, 119.2, 121.4, 124.2, 124.8, 125.0,
126.9, 131.1, 136.18, 146.03, 158.2, 172.5, 181.85 HRMS calcd
for C27H25FN6O4 [M + 1]+ 517.1955, found 517.1919.
2-{1-[3-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-propyl]-
1H-[1,2,3]triazol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-
3-carboxylic acid ethyl ester (8k). Yield 74%; light brown solid;
mp 128–130 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.2 (t, J ¼ 7.1 Hz,
3H, –CH3); 2.39–2.43 (m, 2H, H8); 3.18–3.32 (m, 2H, H4); 3.96 (t,
J ¼ 6.8 Hz, 2H, H9); 3.99–4.03 (m, 2H, H3 + H5); 4.12–4.34 (m,
5H, H5 + H2 + H6); 4.45 (t, J ¼ 6.4 Hz, 2H, H7); 6.98–7.01 (m, 1H,
Ar–H); 7.05–7.18 (m, 3H, Ar–H); 7.30 (d, J ¼ 7.8 Hz, 1H, Ar–H);
7.48 (d, J ¼ 7.4 Hz, 1H, Ar–H); 7.23–7.58 (m, 1H, Ar–H); 7.71 (s,
1H, triazole-H); 8.05 (s, 1H, –NH (exchangeable with D2O)) 13C
NMR (100 MHz, CDCl3) d (ppm): 14.3, 24.0, 27.9, 37.8, 47.3, 47.8,
50.2, 60.5, 61.2, 105.8, 110.1, 110.3, 116.5, 117.5, 120.1, 121.8,
124.6, 125.4, 126.3, 127.9, 131.1, 136.7, 138.9, 145.9, 150.2,
158.7, 172.4, 182.4 HRMS calcd for C28H27FN6O4 [M + 1]+
531.2111, found 531.2132.
2-{1-[4-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-butyl]-
1H-[1,2,3]triazol-4-ylmethyl}-2,3,4,9-tetrahydro-1H-b-carboline-
3-carboxylic acid ethyl ester (8l). Yield 76%; light brown solid;
mp 121–123 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.20 (t, J ¼ 7.1 Hz,
3H, –CH3); 1.60–1.67 (m, 2H, H9); 1.90–1.98 (m, 2H, H8); 3.18–
3.33 (m, 2H, H4); 3.86 (m, 2H, H10); 3.95 (d, J ¼ 15.2 Hz, 1H, H5);
4.03 (t, J ¼ 5.6 Hz, 1H, H3); 4.10–4.33 (m, 5H, H5 + H2 + H6); 4.46
(t, 2H, J ¼ 6.8 Hz, H7); 6.83 (d, J ¼ 7.9 Hz, 1H, Ar–H); 7.06–7.15
(m, 3H, Ar–H); 7.29–7.32 (m, 1H, Ar–H); 7.46 (d, J ¼ 7.5 Hz, 1H,
Ar–H); 7.48–7.54 (m, 2H, Ar–H + triazole-H); 8.43 (s, 1H, –NH
(exchangeable with D2O)) 13C NMR (100 MHz, CDCl3) d 14.3,
24.6, 27.7, 30.1, 39.5, 47.2, 49.4, 49.8, 60.6, 60.9, 105.8, 110.3,
111.2, 117.9, 118.8, 119.4, 121.7, 123.2, 124.9, 125.6, 127.7,
131.2, 136.2, 140.1, 145.4, 150.8, 158.8, 172.8, 182.3 HRMS calcd
for C29H29FN6O4 [M + 1]+ 545.2268, found 545.2291.
2-{1-[3-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-propyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8n). Yield 65%; light
brown solid; mp 112–114 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.32
(t, J ¼ 7.1 Hz, 3H, –CH3); 1.49 (d, J ¼ 6.8 Hz, 3H, –CH3), 2.30–2.37
(m, J ¼ 15.8 Hz, 2H, H8); 2.98–3.02 (m, 1H, H4); 3.06–3.18 (m,
1H, H4); 3.70–3.77 (m, 3H, H9 + H3); 4.06 (d, J ¼ 15.8 Hz, 1H, H6);
4.15 (q, J ¼ 6.4 Hz, 1H, H5); 4.25–4.30 (m, 3H, H2 + H6); 4.38 (t, J
¼ 6.56 Hz, 2H, H7); 6.82 (d, J ¼ 7.9 Hz, 1H, Ar–H); 7.13–7.22 (m,
2H, Ar–H); 7.30 (d, J ¼ 8 Hz, 1H, Ar–H); 7.48 (d, J ¼ 7.4 Hz, 1H,
Ar–H); 7.52 (t, J ¼ 7.7 Hz, 1H, Ar–H); 7.56 (d, J ¼ 7.3 Hz, 1H, Ar–
H); 7.78 (s, 1H, triazole-H); 8.32 (s, 1H, –NH (exchangeable with
D2O)) 13C NMR (100 MHz, CDCl3) d 14.3, 18.5, 24.6, 27.4, 35.5,
46.7, 47.8, 49.3, 60.1, 60.6, 105.4, 110.3, 111.4, 116.8, 117.5,
118.2, 120.8, 122.9, 124.3, 125.8, 127.6, 131.0, 136.8, 137.8,
146.4, 150.1, 158.6, 172.7, 182.8 HRMS calcd for C29H29FN6O4
[M + 1]+ 545.2268, found 545.2282.
2-{1-[4-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-butyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-methyl-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8o). Yield 67%; light
brown solid; mp 101–103 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.31
(t, J ¼ 7.0 Hz, 3H, –CH3); 1.42 (d, J ¼ 6.3 Hz, 3H, –CH3); 1.60–1.67
(m, 2H, H9); 1.90–1.94 (m, 2H, H8); 2.93–2.98 (m, 1H, H4); 3.07–
3.15 (m, 1H, H4); 3.68–3.74 (m, 3H, H10 + H3); 4.01–4.06 (d, J ¼
15.7 Hz, 1H, H6); 4.08–4.14 (q, J ¼ 6.4 Hz, 1H, H5); 4.21–4.27 (m,
3H, H2 + H6); 4.39 (t, J ¼ 6.2 Hz, 2H, H8); 6.78 (d, J ¼ 7.8 Hz, 1H,
Ar–H); 6.98–7.11 (m, 2H, Ar–H); 7.30 (d, J ¼ 7.6 Hz, 1H, Ar–H);
7.34–7.45 (m, 2H, Ar–H); 7.49 (d, J ¼ 7.0 Hz, 1H, Ar–H); 7.68 (s,
1H, triazole-H); 8.87 (s, 1H, –NH (exchangeable with D2O)). 13
C
NMR (100 MHz, CDCl3-d1) d (ppm): 14.3, 18.7, 24.5, 27.8, 31.6,
39.2, 45.0, 49.2, 52.2, 61.2, 61.8, 105.9, 110.1, 113.1, 117.5, 118.9,
119.8, 121.4, 122.7, 123.9, 126.1, 126.8, 135.4, 136.3, 138.6,
146.0, 150.4, 158.2, 173.5, 183.0 HRMS calcd for C30H31FN6O4
[M + 1]+ 559.2424, found 559.2446.
2-{1-[2-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-ethyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8p). Yield 62%; light
brown solid; mp 198–200 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.18
(t, J ¼ 7.1 Hz, 3H, –CH3); 3.01–3.14 (m, 2H, H4); 3.70–3.75 (m,
2H, H8); 4.03–4.21 (m, 5H, H2 + H6 + H3); 4.60–4.67 (m, 2H, H7);
5.47 (s, 1H, H5); 6.48 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.05–7.21 (m, 4H,
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Ar–H); 7.24–7.29 (m, 2H, Ar–H); 7.31 (s, 2H, Ar–H); 7.38–7.49 (m, the wells was then added to each well, which was incubated with
3H, Ar–H); 7.51 (s, 1H, triazole-H); 7.54 (s, 1H, –NH cells for 16 h at 37 ꢂC. The optical density of each well was read at
(exchangeable with D2O)); 13C NMR (100 MHz, CDCl3) d 14.3, 570 nm using a microtiter plate reader (Thermo Fisher Scientic
24.8, 40.7, 46.2, 47.9, 56.7, 60.5, 61.0, 106.0, 109.4, 110.9, 112.3, Multiskan GO Microplate Reader, SkanIt™ soware).
118.2, 119.4, 121.7, 123.6, 124.2, 125.1, 125.8, 126.9, 128.2,
128.9, 129.6, 134.6, 136.5, 138.4, 140.3, 141.8, 147.5, 150.6, Statistical analysis
158.7, 172.3, 182.8 HRMS calcd for C33H29FN6O4 [M + 1]+
593.2268, found 593.2287.
The statistical analysis was carried out using Excel® and IC50
values were determined using Graph pad Prism5 soware
(Hearne Scientic Soware). The experiments were performed
2-{1-[3-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-propyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-
in duplicate and the statistical signicance was calculated using
carboline-3-carboxylic acid ethyl ester(8q). Yield 64%; light
student's t-test. A p-value of less than 0.05 was used to estimate
brown solid; mp 191–193 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.17 (t,
the signicance of the observations. A Z-factor was calculated
J ¼ 7.1 Hz, 3H, –CH3); 2.29–2.34 (m, 2H, H8); 3.23–3.25 (m, 2H,
for each 96-well plate and assays having Z-factor above >0.5 were
H4); 3.72–3.78 (m, 2H, H9); 3.89 (d, J ¼ 14.8 Hz, 1H, H6); 4.01–4.08
included in the statistical analysis.25
(m, 3H, H2 + H3); 4.20 (d, J ¼ 14.7 Hz, 1H, H6); 4.34–4.37 (m, 2H,
H7); 5.54 (s, 1H, H5); 6.87 (d, J ¼ 7.9 Hz, 1H, Ar–H); 7.03–7.18 (m,
Molecular docking
4H, Ar–H); 7.27–7.30 (m, 2H, Ar–H); 7.42 (s, 1H, Ar–H); 7.47–7.60
The X-ray crystal structure of the estrogen receptor-a (PDB ID:
(m, 4H, Ar–H); 7.68 (s, 1H, triazole-H); 7.97 (s, 1H, –NH
3ERT) was obtained from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do). Using the UCSF Chimera
(exchangeable with D2O)) 13C NMR (100 MHz, CDCl3) d 14.3, 24.8,
31.6, 37.8, 46.4, 47.4, 57.5, 60.4, 61.2, 106.4, 110.3, 110.8, 117.6,
package the ligand and receptor complexes were prepared for
118.5, 119.5, 121.6, 123.1, 124.2, 125.7, 127.1, 128.2, 128.4, 129.1,
docking.26 Chem3D Ultra was used to construct a three-
134.7, 136.9, 138.9, 140.5, 142.7, 147.1, 150.2, 158.6, 173.1, 183.2
dimensional structure of the native ligand and most active
HRMS calcd for C34H31FN6O4 [M + 1]+ 607.2424, found 607.2467.
isatin conjugates. The 3D structure of the compounds was
further optimised using the force eld, MMFF94s in the Avo-
gadro package (V 1.2.0). AutoDock Tools (V 1.5.6) was used to
establish the grid box parameters. All non-polar hydrogens were
merged, Gasteiger partial atomic charges added, and rotatable
bonds assigned. The grid box of the complex had a grid spacing
2-{1-[4-(5-Fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-butyl]-
1H-[1,2,3]triazol-4-ylmethyl}-1-phenyl-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid ethyl ester (8r). Yield 68%; light
brown solid; mp 188–190 ꢂC; 1H NMR (400 MHz, CDCl3) d 1.17
(t, J ¼ 7.1 Hz, 3H, –CH3); 1.64–1.73 (m, 2H, H9); 1.92–1.97 (m,
2H, H8); 3.19–3.27 (m, 2H, H4); 3.68–3.72 (m, 2H, H10); 3.85 (d, J
˚
of 0.375 A. The parameter of the grid points are given in Table 4
¼ 14.7 Hz, 1H, H6); 4.02–4.10 (m, 3H, H2 + H3); 4.15 (d, J ¼
(ESI†). Autodock Vina was used to perform the docking of each
14.6 Hz, 1H, H6); 4.35–4.38 (m, 2H, H7); 5.51 (s, 1H, H5); 6.89 (d,
ligand into the IGF-1 receptor in triplicate.27 The docking
systems were validated by redocking the ligand of the protein
J ¼ 7.8 Hz, 1H, Ar–H); 7.09–7.21 (m, 4H, Ar–H); 7.29 (s, 2H, Ar–
H); 7.41–7.46 (m, 2H, Ar–H); 7.48–7.52 (m, 3H, Ar–H); 7.56 (s,
crystal structures into the binding site. The Protein–Ligand
1H, triazole-H); 7.59 (s, 1H, –NH (exchangeable with D2O)) 13C
Interaction Proler (PLIP) was used to analyse the non-polar
NMR (100 MHz, CDCl3) d 14.3, 24.4, 24.7, 27.8, 39.1, 46.7, 49.2,
interactions between the protein and ligand in complex.28
57.2, 60.7, 61.2, 106.8, 110.1, 111.0, 117.6, 117.9, 119.3, 121.7,
122.3, 124.0, 125.6, 126.7, 128.2, 128.7, 128.9, 129.6, 134.2,
Conflicts of interest
136.5, 138.5, 142.1, 147.21, 150.3, 158.6, 173.1, 183.1 HRMS
calcd for C35H33FN6O4 [M + 1]+ 621.2581, found 621.2543.
The authors declare no conict of interest.
Cell culturing
Abbreviations
MCF-7 cells were cultured in DMEM media with 10% FBS and 1%
penicillin–streptomycin and MDA-MB-231 cells were cultured in
BC
Breast cancer
Tetrahydro-b-carboline
Selective estrogen receptor modulators
Estrogen receptor
50% Inhibitory concentration
Structure–activity relationship
DMEM supplemented with 5% FBS and 1% penicillin–strepto-
mycin, both incubated at 37 ꢂC and 5% carbon dioxide.22
THbC
SERMs
ER
IC50
SAR
MTT assay
Cells were seeded in 96 well plates at a density of 5000 cells per well
in triplicate in media. Aer 24 hours (h), the test compounds,
diluted in complete Dulbecco's media Eagle's medium (DMEM)
were added to each well. Cells were treated with a range of different
concentrations of test compounds (1 mM, 5 mM, 10 mM, 20 mM, 50
mM, 100 mM) for 24 h at 37 ꢂC and 5% carbon dioxide. Subse-
Acknowledgements
quently, sterile 5 mL of 5 mg mLꢀ1 MTT (Sigma-Aldrich) dissolved Financial assistance from Council of Scientic and Industrial
in PBS was added to each well and incubated with cells for 2 h. Research, New Delhi, India, under CSIR-JRF fellowship CSIR Ref
Solubilization solution (10% SDS, 10 mM HCl) of equal volume to No. 09/254(0257)/2016-EMR-I gratefully acknowledged. The
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authors also would like to thank the National Research Foun- 12 C. D. Teaux, V. Leblanc, G. B. Langer, S. Parent, E. Asselin
dation of South Africa for their support in facilitating our and G. B. Rube, Steroids, 2008, 73, 1077–1089.
research (UID: 99563). We would also like to thank the Centre 13 S. Kumar, L. Gu, G. Palma, M. Kaur, A. S. Pillay, P. Singh and
for High Performance Computing based in Cape Town for V. Kumar, New J. Chem., 2018, 42, 3703.
access to computational resources. MCF7 (The European 14 S. Kumar, L. Gu, G. Palma, M. Kaur, A. S. Pillay, P. Singh and
Collection of Authenticated Cell Cultures (ECACC), UK). MDA-
MB-231 (The American Type Culture Collection (ATCC), USA).
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15 A. Singh, S. T. Saha, S. Perumal, M. Kaur and V. Kumar, ACS
Omega, 2018, 3, 5808–5813.
16 S. Kumar, T. bains, A. S. W. Kim, C. Tam, J. Kim,
L. W. Cheng, K. M. Land, A. Debnath and V. Kumar, Front.
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