Diverse synthesis of natural product inspired fused and spiro-heterocyclic scaffolds via ring distortion and ring construction strategies

Article abstract of DOI:10.1039/c5nj01858g

Several natural product inspired fused and spiro-heterocyclic scaffolds were prepared by ring distortion and ring construction strategies and evaluated for anti-breast cancer activity. A facile domino Pictet-Spengler lactamization (PSL) afforded nine natural product inspired indolo[2,3-a]quinolizidine and indolo[8,7-b]indolizidine scaffolds which are converted to seven other scaffolds by functional group transformation, ring distortion and ring construction strategies. In vitro screenings of this library of sixteen scaffolds with six distinct architectures against MCF7 cell lines afforded two compounds (10 and 21) with modest activity. Principal component analysis of this library against databases of FDA approved drugs, commercial compounds and FDA approved breast cancer compounds indicated an eclectic mix of structures among the molecules.

Full text of DOI:10.1039/c5nj01858g

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DOI: 10.1039/C5NJ01858G
Diverse synthesis of natural product inspired fused
and spiroꢀheterocyclic scaffolds via ring distortion
and ring construction strategy
Chandramohan Bathula,1, § Poonam Dangi,2, § Santanu Hati,1 Rahul Agarwal,2 Parthapratim
Munshi,1 Shailja Singh2, Subhabrata Sen 1, *and Ashutosh Singh.
1 Department of Chemistry, Shiv Nadar University, Post Office Shiv Nadar University, Gautam
Buddha Nagar, Uttar Pradesh 201314, India
2 Department of Life Science, Shiv Nadar University, Post Office Shiv Nadar University,
Gautam Buddha Nagar, Uttar Pradesh 201314, India
§ Equal contributor
2,3,5,6,11,11bꢀhexahydroꢀ1Hꢀindolizino[8,7ꢀb]indole, 1,2,3,6,7,12bꢀhexahydroindolo[2,3ꢀ
a]quinolizinꢀ4(12H)ꢀone, PictetꢀSpengler Lactamization, MCF7.
1

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ABSTRACT. Several natural product inspired fused and spiroꢀheterocyclic scaffolds were
prepared by ring distortion and ring construction strategies and evaluated for antiꢀbreast cancer
activity. A facile domino PictetꢀSpengler lactamization (PSL) afforded nine natural product
inspired indolo[2,3ꢀa]quinolizidine and indolo[8, 7ꢀb]indolizidine scaffolds which are converted
to seven other scaffolds by functional group transformation, ring distortion and ring construction
strategies. In vitro screenings of this library of sixteen scaffolds with six distinct architectures
against MCF7 cell lines afforded two compounds (10 and 21) with modest activity. Principal
component analysis of this library against databases of FDA approved drugs, commercial
compounds and FDA approved breast cancer compounds indicated an eclectic mix of structures
among the molecules.
Introduction
Conventional genetic approaches have been used to examine biological systems by generating
random mutations which were then screened in search of a precise cellular phenotype.1a and g
Analogous to the genetic approach, large random collections of small molecules can be screened
to identify bioactive compounds which can elucidate the roles of specific proteins in many
biological pathways. The crux of this “chemical genetic” approach is the design and synthesis of
scaffold libraries with diverse architectures that span large tracts of biologically relevant
chemical space.1 The criticality of diverse architectures stems from the fact that small molecules
interact superficially with biological macromolecules like protein, DNA and RNA.2ꢀ3
By virtue of binding to both their biosynthetic enzymes and their targets, natural products
necessarily reside in biologically relevant chemical space4 and in that sense natural product
families are libraries of preꢀvalidated, functionally diverse structures known to bear three
2

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dimensional features that are desirable to make molecules active towards diverse biological
targets.5 They are rich in sp3 carbon content (i.e. three dimensional surface) and stereogenicity,
which has been correlated with better clinical success of drug candidates.6 However, the paucity
of effective synthetic and scaleꢀup strategies in accessing the natural products opened avenues
for generating scaffolds inspired from natural products as suitable alternatives.7 Hence, it is not
surprising that the recent years have observed a stark rise in the development of synthetic
strategies to access such scaffolds that could be used therapeutically to: (a) investigate and
develop small molecules with better efficacy on known targets and (b) interrogate novel mode of
action on previously unexploited targets.8
The indolo[2,3ꢀa]quinolizidine, indolo[8, 7ꢀb]indolizidine and 3,3´ꢀpyrrolidinylspirooxoindole
ring systems are of great interest since they elucidate the characteristic structural motifs of large
family alkaloids with conspicuous and diverse biological activities including: (a) Hirsutine 1,
isolated from Uncaria rhychnophylia that inhibits neurotoxicity mediated by inflammation and
microglial activation; (b) spirooxoindole MIꢀ219 2, that binds to MDM2 with Ki = 5nM, with an
oral bioavailability of 65% in rats and much better exposure in pharmacokinetics (PK) studies in
rats; (c) (ꢀ)ꢀVincatine 3, a member of Aspidosperma alkaloids, a complex natural product that is
biologically active and encrusted with chiral centers and functional moieties which makes the
system extremely challenging for synthesis; and (d) 4 containing the indolo[8, 7ꢀb]indolizidino
framework that exhibited high binding affinity and selectivity of CCK1 receptors and also acted
as βꢀturn mimics (Figure 1).9ꢀ13 Despite the synthetic challenge, individual syntheses of these
natural products encompassed various chiral auxiliary mediated as well as catalytic
enantioselective strategies.14
3

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Figure 1. The natural products that influenced our scaffolds;
Here we describe a modular synthetic strategy involving domino PictetꢀSpengler lactamization
of βꢀaryl amines with γꢀ and δꢀoxoesters to generate a library of indolo[2,3ꢀa]quinolizidine and
indolo[8, 7ꢀb]indolizidine scaffolds (Scheme 1). Functional group transformation, ring distortion
via oxidative ring contraction (to afford the 3, 3´ꢀpyrrolidinyl oxoindoles) and ring construction
via directed enolateꢀaddition/substitution followed by a ringꢀclosure onto the builtꢀin scaffold
electrophile, afforded carbocycles and spirocycles (Scheme 1). Cellular evaluation yielded 2
candidates that inhibit the proliferation of MCF7 cells moderately when compared with the
reference drug etoposide. These compounds exhibited no cytotoxicity against the healthy COSꢀ7
cells at similar concentration as that for MCFꢀ7 cells. The diversity of the library was assessed
via a principal component analysis (PCA) against FDA approved drugs, breast cancer drugs and
a commercial compound database.
4

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Retrosynthetic analysis
O
R
N
3,3´-pyrrolidinyl
spirooxoindole
scaffolds
R1
R2
N
O
n= 0 and 1
O
R1
N
H
n= 0 and 1
N
-
,
13 14 15a/b
R1
-
10 12
Ring
distortion
Functional
group
R
transformation
NH2
R
R
Domino
Pictet-Spengler
Lactamization
O
O
N
H
O
Ring
construction
N
N
R1
-arylamines
NH
N
N
R1 =H, Me, CF3
H
H
n= 0 and 1
O
O
21
indolo[2,3-a]quinolizidine
and indolo[8, 7-b]indolizidine
scaffolds
R1
OMe
H2N
n= 0 and 1
- ,
5 9 16
and -oxoesters
Ring
construction
Ring
construction
R
N
O
O
NH
N
R
H
22
O
O
N
NH
N
H
Ph
23
Scheme 1. The retrosynthetic analysis involving ring distortion, functional group transformation
and ring construction strategy
Results and discussion
Design and synthesis of various heterocyclic scaffolds
Tryptamine and methylꢀ5ꢀoxopentanoate were chosen as the reaction partners to optimize the
domino PictetꢀSpengler lactamization reaction.15aꢀc The synthesis of such indolo[2,3ꢀ
5

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a]quinolizidine and indolo[8, 7ꢀb]indolizidine scaffolds were previously synthesized by Allin et
al. via ring transformation reactions of bicyclic lactams.15dꢀf All the reactions were executed in
presence of 3 Å molecular sieves (MS). Initially the reactions were carried out in toluene under
reflux in presence of various acid catalysts (~10 mol %) to produce 5 (Table 1, entry 1ꢀ5). Under
these conditions, trifluoroacetic acid (TFA) produced the best result (82% yield of 5) (entry 5,
Table 1). In a bid to further improve the efficiency of the reaction by lowering the catalyst
concentration,
Table 1. Optimization of Domino PictetꢀSpengler lactamization
Temperature
(T°C)
Yield
(%)a
Entry
Solvent
Conditions
1
2
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
Reflux
Reflux
Acetic Acid (10 mol%)
Polyphosphoric acid (10 mol%)
pꢀtoluenesulfonic acid (10 mol%)
Methane sulfonic acid (10 mol%)
Trifluoroacetic acid (10 mol%)
Trifluoroacetic acid (1 mol%)
TiCl4 (1 mol%)
42
38
69
41
82
25
ꢀb
3
Reflux
4
Reflux
5
Reflux
6
Reflux
7
ꢀ5→20°C
ꢀ5→20°C
ꢀ5→20°C
ꢀ5→20°C
ꢀ5→20°C
ꢀ5→20°C
8
BF3ꢀEt2O (1 mol%)
ꢀb
9
Sc(OTf)3 (1 mol%)
ꢀb
10
11
12
Yb(OTf)3 (1 mol%)
ꢀb
TiCl4 (1 mol%)/ 1eq. 2,6ꢀlutidine)
BF3ꢀEt2O (1 mol%/ 1eq. 2,6ꢀlutidine)
56
59
6

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13
CH3CN
ꢀ5→20°C
ꢀ5→20°C
Sc(OTf)3 (1 mol%)/ 1eq. 2,6ꢀlutidine
72
Yb(OTf)3 (1 mol%)/ 1eq. 2,6ꢀ
lutidine
14
CH3CN
90
a Isolated yields after column chromatography. b Isolated unreacted tryptamine
1 mol % of TFA (entry 6) proved disappointing, generating 5 in ~25% yield. Consequently the
next set of procedures were applied using diverse Lewis acid catalysts viz. titanium chloride
(TiCl4), boron trifluorideꢀdiethyl ether (BF3.OEt2), scandium triflate (Sc(OTf)3) and ytterbium
triflate (Yb(OTf)3) at much milder reaction conditions than before (15→20°C) in acetonitrile
(CH3CN) (entry 7ꢀ10). Unfortunately none of these conditions could generate the desired
product, but careful scrutiny of the literature prompted us to use one equivalent of 2, 6ꢀlutidine as
base in all the above reactions along with the catalysts (entry 11ꢀ14). To our utmost delight this
subtle change of conditions generated the desired product 5 in 56ꢀ90% yield in all the reactions,
with the best with Yb(OTf)3 (entry 14).16
Applying the optimized procedure, several other scaffolds viz. 6ꢀ9 and 16 were synthesized with
appropriate starting combos. 7 was synthesized from Dꢀtryptophan methyl ester and methyl
levulinate. 8 and 9 were synthesized from Lꢀtryptophan methyl ester with methyl levulinate and
methylꢀ5ꢀoxopentanoate respectively (Figure 2). Tryptamine when reacted with methyl
levulinate and methyl 5,5,5ꢀtrifluoroꢀ4ꢀoxopentanoate afforded scaffolds 6 and 16 respectively
(Scheme 2). The average yields ranged from 49ꢀ84%. For the chiral scaffolds the diastereomeric
ratio ranged from 60:40 to 88:12.
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Figure 2. Synthesis of fused scaffolds
Compounds 5, 7ꢀ8 were further derivatized via Nꢀalkylation of the indole nitrogen and reduction
of the ester functionality. Compound 10 was obtained in ~45% yield by methylating 5 at the
indole nitrogen with methyl iodide (MeI) and dichloromethane (DCM) under reflux in presence
of potassium hydroxide (KOH) (Scheme 2).
MeI, KOH,
Dichloromethane
40oC
O
O
N
N
a)
Yield 45%
N
N
H
5
10
OH
O
CO2Me
O
N
N
b)
N
H
N
H
DIBAL-H
THF, -78oC
11 (Yield 66%)
CO2Me
N
OH
7
O
O
N
c)
N
N
H
H
8
12 (Yield 55%)
Scheme 2. Functionalization of scaffolds 5, 7 and 8
The ester moieties in compounds 7 and 8 were reduced with diisobutyl aluminum hydride
(DIBALꢀH) in THF at ꢀ78°C to their corresponding alcohols 11 and 12. The major diastereomers
of 11 and 12 were isolated by flash chromatography and their relative configurations were
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determined by single crystal Xꢀray analysis (Figure 2a and b). This in turn also confirmed the
relative configuration of the major diastereomers of 7 and 8 respectively.
Next, the ring distortion strategy was utilized by transforming the fused scaffolds to their spiro
analogs via oxidative ring contraction. Observed first by Irikawa et al. in 1989 the reaction is
believed to initiate via electrophilic bromination from NBS in presence of water, acetic acid and
tetrahydrofuran as solvent, resulting in a bromoꢀhydroxy intermediate which undergoes ring
Scheme 3. Synthesis of spiro scaffolds
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contraction to provide the spiralꢀmotifs (Scheme 3).17a We optimized the protocol further by
using a variety of electrophilic brominating agents scouted from the literature viz. DBDMH (1,3ꢀ
dibromoꢀ5,5ꢀdimethylhydantoin),
TBPCO
(2,4,4,6ꢀtetrabromoꢀ3ꢀnꢀpentadecylꢀ2,5ꢀ
cyclohexadienone) and DPTBE (1,2ꢀdipyridiniumditribromideꢀethane).17bꢀd Starting from 5,
with DBDMH, we could improve the yields of the desired product 13 from 25→63% (Scheme
3). Hence 14 and racemic diastereomers 15a/b were synthesized similarly from 7 and 6 (Scheme
3). The structure of 15a and b were confirmed by the single crystal Xꢀray of 15a (Figure 3c).
Figure 3. XꢀRay crystal structures of (a) 11; (b) 12; (c) 15a. ORTEP view along with the atom
labels are shown at 50% probability level. Hꢀatoms are omitted for clarity.
In a bid to diversify the 3D binding surface of racemic, 16 the next endeavor focused on
constructing heterocyclic rings viz. indole, pyrrolidinone and pyrroloisoquinolinone at the carbon
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adjacent to the amide carbonyl of 16 (Scheme 4). The diversification strategy involved
methylation of indole nitrogen followed by acylation of 16 with ethyl chloroformate to afford 17.
The crude 17 underwent: (a) SNAr reaction with 2, 4ꢀdinitrofluorobenzene in presence of NaH, at
0°C in THF to generate 18; (b) Michael addition with nitrostyrene to provide 19; and finally (c)
alkylation with oꢀnitrobenzyl bromide to generate 20. The crude intermediates 18ꢀ20 were
hydrogenated in presence of 10% PdꢀC in hydrogen at atmospheric pressure that facilitated the
synthesis of the final compounds 21ꢀ23 (which were purified by column chromatography)
(Scheme 4).
Scheme 4. Diversification of 16 via ring construction strategy
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Evaluation of antiꢀbreast cancer activity
One of the purposes of this investigation was to develop methodology to generate compounds
that have the potential to be biologically active. We accept the fact that it is an unrealistic
expectation to generate potent molecules via synthetic efforts unbiased towards any particular
biological target. However, we hoped that our scaffolds may provide hits that could be used for
further elaboration into drug leads. Therefore we focused our attention on in vitro screening of
our scaffold library against the proliferation of MCFꢀ7 (Michigan Cancer Foundationꢀ7), a breast
cancer cell line employing the MTT, [3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyi)ꢀ2, 5ꢀdiphenyltetrazolium
bromide] assay, according to the method developed by Mosmann.18 All the stock solutions of the
test compounds were prepared in DMSO. MCFꢀ7 cells were seeded in a 96 well microtitre plate
and were allowed to adhere and stretch overnight. The grown cells were then treated with 50 ꢀM
compounds in 200 ꢀL overall volume. After 24 hours of compound treatment, 10 µl of MTT
(5mg/mL) was added to each well and subsequently the plate was incubated at 37°C in the dark
for about 4 h. The reduction of MTT (rate of color developed) was quantified by measuring the
absorbance at 570 nm in a spectrophotometer (Model: Spectra MAX Plus; Molecular Devices;
supported by SOFTmax PROꢀ3.0). The assay was also carried out with etoposide, a wellꢀknown
antiꢀbreast cancer compound as positive control. Treatment with DMSO alone was taken as
negative control. All the experiments were done in triplicate. It was gratifying to see that two
scaffolds (10 and 21) showed 27ꢀ31% inhibition of cell proliferation of the MCFꢀ7 cells
compared to 42% by Etoposide (Table 2). Screening the most active compounds 10 and 21 in a
48h screen against the proliferation of MCF7 cells exhibited inhibition in the range of 35ꢀ40%
(refer supplementary material). Cytotoxicity studies of 10 and 21 on nonꢀcarcinoma COSꢀ7 cell
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line via MTT assay at the same concentration used for screening i.e. 50 µM indicated no toxicity.
(refer supplementary material).
Table 2. In vitro screening of the scaffolds
% inhibitiona
Entry Compound Code
1
ꢀ
control
2
42.12
1.92
etoposide
3
5
10
13
7
4
27.61
10.84
ꢀ16.11
ꢀ9.80
ꢀ8.25
4.92
5
6
7
8
8
14
11
6
9
10
12.44
ꢀ3.45
ꢀ4.86
ꢀ4.48
7.31
11
15a
15b
9
12
13
14
16
12
21
22
23
15
ꢀ3.16
33.45
12.07
ꢀ23.90
16
17
18
a. Experiments ran in triplicates
To computationally evaluate the structural diversity of our library, we executed principal
component analysis (PCA) study of a set of various shape based and charge based molecular
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surface descriptors for our library and three reference compound collections from one thousand
five hundred and fifty FDA approved drugs, seven hundred and forty six Chembridge Screen
compounds and twenty four FDAꢀapproved breast cancer drugs. PCA utlizes definite medicinal
chemistry descriptors to determine the overall chemical space. All the descriptors calculation and
analysis was done in Molecular Operating environment software.
In Figure 4, the first two PCA components i.e PCA1 & PCA2 were plotted on X and Y axis.
In this plot it was very clear that chemical space shared by FDA approved drugs and Chembridge
Screen Compounds were very much similar. The well represented chemical space in the plot was
covered by FDA approved compounds, Chembridge Screen Compounds, twenty three FDA
approved breast cancer drugs and six (14ꢀ15a/b, 21ꢀ23) of our library compounds. The fact that
scaffold 21 tracts the chemical space similar to the breast cancer drugs, further corroborates our
screening results (Table 2). Compounds 5ꢀ10 and 12 were lying in the underrepresented area
where except few of FDA approved and Chembridge screening compounds only one of the
breast cancer drugs were present. This indicated a diverse scaffold library, where two distinct
regions in the chemical space were occupied by several scaffolds.
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Figure 4. Sixteen library compounds (black), one thousand five hundred and fifty five FDA
approved drugs (blue), seven hundred and forty six Chembridge Screening compounds (orange)
and FDA approved breast cancer drugs were used for the PCA analyses
Conclusion
Here in we have demonstrated a diverse synthesis of a library of scaffolds (5ꢀ16 and 21ꢀ23) using
domino Pictet Spengler Lactamization (PSL) reaction, followed by various ring distortion,
construction and functional group transformations (Scheme 5). During this we have developed a
novel condition based on catalytic (1 mol %) ytterbium triflate for the domino Pictet Spengler
lactamization. We also revisited an oxidative ring contraction protocol to generate new spiro
scaffolds and improved the protocol by doubling the yield via commercially available
brominating reagent DBDMH. Beginning from simple starting materials like tryptamine and
D/Lꢀtryptophan, this synthetic strategy with steps/scaffold ratio of 1 is extremely efficient in
building the scaffold library. In vitro screening against the proliferation of MCFꢀ7 cells identified
two scaffolds with moderate activity that could be refined into better molecules through structure
activity relationship studies (SAR). Finally diversity assessment of this library through PCA
analysis indicated an eclectic mix of scaffolds that also tracts new area of chemical space.
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Scheme 5. The complete library of scaffolds (5ꢀ16 and 21ꢀ23) developed from simple starting
materials viz. Tryptamine and D/LꢀTryptophan.
EXPERIMENTAL
Chemistry
General Methods. Unless otherwise noted, all reactions were carried out in flameꢀdried
glassware under a static nitrogen atmosphere in anhydrous solvent. All reagents were obtained
1
from commercially available sources and used without purification. H and 13C spectra were
recorded using Varian 300/400 and JEOL JNMꢀECX500 MHz, using CDCl3 and DMSOꢀD6 as a
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solvents. The chemical shifts are reported in parts per million (ppm) relative to CDCl3 (δ 0.00
ppm for proton NMR and δ 77.0 ppm for carbon NMR) and DMSOꢀD6 (δ 2.50 ppm for proton
NMR and δ 39.50 ppm for carbon NMR). Coupling constants are reported in hertz (Hz). The
following abbreviations are used to designate the multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. HRMS spectra acquired via Reverseꢀphase 6540 UHD
AccurateꢀMass QꢀTof LCMS, Agilent Technologies (Milford, MA). It is to be noted that the
several crystallization experiments with various solvents and at different conditions always
resulted in poor quality crystals of compound 15a. As a result the refined structure of 15a as
reported here is with relatively high Rꢀfactor. However, it certainly confirms the correct
geometry and proper conformation of the molecule.
General Procedure for the synthesis of indolo[2,3ꢀa]quinolizidine and indolo[8, 7ꢀ
b]indolizidine scaffolds (5ꢀ9 and 16)
Appropriate βꢀaryl amines (0.5 mmol, 1 equiv) and δ or γꢀoxo esters (0.5 mmol, 1 equiv) were
dissolved in CH3CN (22 mL) and activated 3 Å molecular sieves were added to the resulting
solution. Next 2,6ꢀlutidine (0.07 ml, 0.5 mmol) and Yb(OTf)3 (5 mg, 0.005 mmol) were added
and the resulting mixture was then stirred under argon at an ambient temperature of 15ꢀ20°C for
10ꢀ16h. Once thin layer chromatography (TLC) confirms the complete consumption of the
starting materials, the reaction was cooled to room temperature (rt), quenched with water and
was extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium
sulphate (Na2SO4) and was evaporated to obtain the desired crude compounds as yellow to
colorless solids. The crude solids were purified by flash column chromatography (FCC) using
ethyl acetateꢀhexane as the mobile phase, to provide the final compounds as solid or semi solid,
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1
which were characterized by HꢀNMR, 13CꢀNMR and QTofꢀHRMS. Single crystal Xꢀray
structures were obtained for few of them.
1, 2, 3, 6, 7, 12bꢀhexahydroindolo[2,3ꢀa]quinolizinꢀ4(12H)ꢀone (5). Yield: 97 mg, 81%;
colorless solid. 1H NMR (400 MHz, DMSOꢀD6):
δ 10.91 (s, 1H), 7.40 and 7.32 (2 X d, J = 8 Hz,
2H), 7.06 and 6.97 (2 X t, J = 7.2 Hz, 2H), 4.91 and 4.78 (2 X m, 2H), 2.78 (m, 1H), 2.65 (m,
13
3H), 2.28 (m, 2H), 1.79 (m, 2H), 1.63 (m, 1H). C NMR (125 MHz, DMSOꢀD6) 168.6, 137.5,
125.8, 121.2, 118.9, 117.9, 109.4, 108.4, 53.8, 31.8, 31.2, 29.3, 21.2, 18.9. QꢀTof LCMS (MS
ESI): [M + H]+ calcd for C15H16N2O 241.1335, found 241.1376.
11bꢀmethylꢀ5,6,11,11bꢀtetrahydroꢀ1Hꢀindolizino[8,7ꢀb]indolꢀ3(2H)ꢀone (6). Yield: 59 mg,
1
49%; colorless solid. H NMR (300 MHz, DMSOꢀD6):
δ 11.01 (s, 1H), 7.39 and 7.28 (d, J = 8
Hz, 2H), 7.05 and 6.95 (t, J = 7.2 Hz, 2H), 4.21 (m, 1H), 3.01 (m, 1H), 2.78 (m, 1H), 2.61 (m,
13
2H), 2.25 (m, 2H), 2.02 (t, J = 8 Hz, 1H), 1.48 (s, 3H). C NMR (125 MHz, DMSOꢀD6) 169.2,
136.3, 133.2, 126.1, 121.3, 118.7, 117.9, 111.2, 104.5, 52.3, 51.9, 49.3, 31.7, 28.9, 22.4, 19.3,
14.1. QꢀTof LCMS(MS ESI): [M + H]+calcd for C15H16N2O 241.1335, found 241.1346.
(5S,11bR)ꢀmethyl 11bꢀmethylꢀ3ꢀoxoꢀ2,3,5,6,11,11bꢀhexahydroꢀ1Hꢀindolizino[8,7ꢀb]indoleꢀ5ꢀ
1
carboxylate (7). Yield: 96 mg, 65%; yellow solid. H NMR (500 MHz, DMSOꢀD6):
δ 11.10 (s,
1H), 7.42 (d, J = 7.55 Hz, 1H), 7.31 (d, J = 8.25 Hz, 1H), 7.07 and 6.99 (t, J = 7.6 Hz, 2H), 5.27
(d, J = 6.9 Hz, 1H), 3.59 (s, 3H), 2.78 (m, 2H), 2.51 (m, 1H), 2.35 (m, 3H), 2.01 (m, 1H), 1.48 (s,
3H). 13C NMR (125 MHz, DMSOꢀD6) 172.9, 171.5, 137.8, 136.1, 126.1, 121.2, 118.7, 118.2,
111.2, 102.5, 58.9, 52.1, 47.8, 34.5, 29.9, 25.4, 21.4. QꢀTof LCMS(MS ESI): [M + H]+calcd for
C17H18N2O3299.1390, found 299.1395.
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(5R,11bS)ꢀmethyl 11bꢀmethylꢀ3ꢀoxoꢀ2,3,5,6,11,11bꢀhexahydroꢀ1Hꢀindolizino[8,7ꢀb]indoleꢀ5ꢀ
1
carboxylate (8). Yield: 83 mg, 51%; lemon yellowsolid. H NMR (500 MHz, DMSOꢀD6):
δ
11.11 (s, 1H), 7.42 and 7.30 (d, J = 8.25 Hz, 2H), 7.07 and 6.99 (t, J = 7.6 Hz, 2H), 4.34 (m,1H),
3.67 (s, 3H), 2.98 and 2.82 (m, 2H), 2.63 (m, 1H), 2.46 (m, 3H), 2.19 and 2.08 (m, 1H), 1.65 (s,
3H). 13C NMR (125 MHz, DMSOꢀD6)176.1, 170.5, 138.2, 136.1, 126.3, 121.3, 118.7, 118.0,
111.2, 105.8, 61.4, 51.7, 51.0, 31.6, 29.0, 26.8, 22.0. QꢀTof LCMS (MS ESI): [M + H]+ calcd for
C17H18N2O3 299.1390, found 299.1395.
methyl 4ꢀoxoꢀ1,2,3,4,6,7,12,12bꢀoctahydroindolo[2,3ꢀa]quinolizineꢀ6ꢀcarboxylate (9). Yield:
73 mg, 49%; grey semi solid. 1H NMR (500 MHz, DMSOꢀD6):
δ 10.99 (s, 1H), 7.43 (d, J = 7.55
Hz, 1H), 7.31 (d, J = 8.25 Hz, 1H) 7.07 and 6.98 ( t, J1 = 7.55 Hz and J2 = 8.25, 2H), 5.91(d, J =
6.15 Hz, 1H), 4.85 (d, J = 10.3 Hz, 1H), 3.56 (s, 3H), 3.30 (m, 1H), 2.91 (m, 1H), 2.61 (m, 1H),
2.51 (m, 1H), 2.42 (m, 1H), 1.91 (m, 2H), 1.58 (m, 1H).13C NMR (125 MHz, DMSOꢀD6) 171.3,
169.2, 136.3, 133.2, 126.1, 121.3, 118.7, 117.9, 111.2, 104.5, 55.0, 52.4, 51.9, 49.3, 31.7, 28.9,
22.4, 19.3. QꢀTof LCMS(MS ESI): [M + H]+calcd for C17H18N2O3 (299.1397), found 299.1387.
11bꢀ(trifluoromethyl)ꢀ5,6,11,11bꢀtetrahydroꢀ1Hꢀindolizino[8,7ꢀb]indolꢀ3(2H)ꢀone (16). Yield:
1
125 mg, 85%; pale yellow solid. H NMR (400 MHz, DMSOꢀD6):
δ 11.3 (s, 1Η), 7.48 (d, J =
7.68 Hz, 1H), 7.39 (d, J = 8.04 Hz, 1H), 7.16 (t, J = 6.92 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 4.38
13
(m, 1H), 3.32 (m, 1H), 2.85 (m, 1H), 2.67 (m, 3H), 2.46 (m, 1H), 2.29 (m, 1H). C NMR (75
MHz, DMSOꢀD6) 173.5, 136.7130.6, 127.7, 127.5, 125.5, 124.8, 122.4, 122.0, 119.1, 118.6,
111.7, 109.2, 63.4, 63.1, 62.9, 62.6, 62.1, 35.7, 29.7, 26.9, 19.9. LCMS (MS ESI): [M + H]+
calcd for C15H13F3N2O 295.1050, found 294.9500.
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12ꢀmethylꢀ1,2,3,6,7,12bꢀhexahydroindolo[2,3ꢀa]quinolizinꢀ4(12H)ꢀone (10). Compound 5
(120 mg, 0.5 mmol) was dissolved in dichloromethane (10 mL) and was treated with potassium
hydroxide (38 mg, 0.7 mmol) and methyl iodide (210 mg, 1.5 mmol) and the resulting solution
was heated at 40°C for 6h. Once TLC confirms the complete consumption of the starting
material the reaction was cooled to rt, diluted with water (10 mL) and extracted with ethyl
acetate (2 X 10 mL). The organic layer was washed with brine, dried over Na2SO4 and was
evaporated to generate the crude compound. It was purified by flash chromatography to provide
1
the desired compound. Yield: 57 mg, 45%; white solid. H NMR (500 MHz, DMSOꢀD6):
δ 7.42
(m, 2H), 7.39 (m, 1H), 7.04 (m, 1H), 4.93 (m, 2H), 3.72 (s, 3H), 2.61 (m, 4H), 2.27 (m, 2H),
13
1.83 (m, 1H), 1.51 (m, 1H). C NMR (125 MHz, DMSOꢀD6) 168.6, 137.5, 135.6, 125.8, 121.2,
118.9, 117.9, 109.4, 108.4, 53.8, 31.8, 31.2, 29.3, 21.2, 19.0. QꢀTof LCMS (MS ESI): [M + H]+
calcd for C16H18N2O 255.1492, found 255.1478.
General Procedure for the synthesis of hydroxymethyl indolizino indolones 11 and 12.
Appropriate indolizino indolone esters 7 and 8 (0.5 mmol, 1equiv) were dissolved in THF (10
mL) and cooled to ꢀ78°C and were treated with diisobutyl aluminum hydride (0.5 mmol, 1
equiv). The reactions were then slowly warmed to rt and stirred for 8ꢀ12h. Once thin layer
chromatography (TLC) confirmed the complete consumption of the starting materials, the
reactions were quenched with water and was extracted with ethyl acetate. The organic layers
were separated, dried over anhydrous sodium sulphate (Na2SO4) and evaporated to obtain the
desired crude compounds as white to light yellow solids. The crude solids were purified by flash
chromatography using ethyl acetateꢀhexane as the mobile phase, to provide the final compounds
1
13
as solid or semi solid, which were characterized by HꢀNMR, CꢀNMR and QꢀTof LCMS (MS
ESI). Single crystal Xꢀray structures were obtained for them.
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(5S, 11bR)ꢀ5ꢀ(hydroxymethyl)ꢀ11bꢀmethylꢀ5,6,11,11bꢀtetrahydroꢀ1Hꢀindolizino[8,7ꢀb]indolꢀ
3(2H)ꢀone (11). Yield: 89 mg, 66%; light yellow solid. 1H NMR (500 MHz, DMSOꢀD6):
δ
11.01
(s, 1H), 7.39 (d, J = 7.55 Hz, 1H), 7.36 (d, J = 8.25 Hz, 1H), 4.98 (t, J = 4.2 Hz, 1H), 4.09 (m,
2H), 3.59 (m, 1H), 2.76 (m, 2H), 2.58 (m, 1H), 2.21 (m, 3H), 1.59 (s, 3H). 13C NMR (125 MHz,
DMSOꢀD6) 174.9, 138.6, 136.1, 126.3, 121.0, 118.6, 117.9, 111.1, 105.7, 62.1, 61.7, 54.8, 32.2,
30.5, 25.3, 24.0. QꢀTof LCMS (MS ESI): [M + H]+ calcd for C16H18N2O2 271.1441, found
271.1453.
(5R, 11bS)ꢀ5ꢀ(hydroxymethyl)ꢀ11bꢀmethylꢀ5,6,11,11bꢀtetrahydroꢀ1Hꢀindolizino[8,7ꢀb]indolꢀ
3(2H)ꢀone (12). Yield: 65 mg, 55%; white solid. 1H NMR (500 MHz, DMSOꢀD6):
δ
11.1 (s, 1Η), 7.39 (d, J = 7.64 Hz, 1H), 7.31 (d, J = 8 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 6.98 (t,
J = 7.32 Hz, 1H), 4.92 (m, 1H), 4.54 (m, 1H), 3.31 (m, 1H), 2.92 (d, J = 8.28 Hz, 1H), 2.69 (m,
13
2H), 2.26 (m, 2H), 1.90 (m, 1H). C NMR (125 MHz, DMSOꢀD6) 172.8, 137.5, 136.2, 126.7,
121.0, 118.5, 118.1, 111.1, 102.2, 61.7, 58.4, 48.6, 35.1, 30.3, 26.8, 21.3. QꢀTof LCMS (MS
ESI): [M + H]+ calcd for C16H18N2O2 271.1441, found 271.1454.
General Procedure for the synthesis of 3, 3´ꢀpyrrolidinylspirooxoindole (13ꢀ15a/b).
To a solution of indolo[2,3ꢀa]quinolizidine and indolo[8, 7ꢀb]indolizine scaffolds (0.5 mmol, 1
equiv) in H2O: acetic acid : THF (1:1:1) at 0°C was added DBDMH (1.2 equiv) in portions. The
reaction mixture was stirred at same temperature and after TLC indicates complete consumption
of starting material, saturated sodium bicarbonate (NaHCO3) solution was added to the reaction
mixture and then extracted with EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure to provide the crude material, which
was purified by flash chromatography with EtOAcꢀHexane (4:1) as eluent.
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6',7',8',8a'ꢀtetrahydroꢀ2'Hꢀspiro[indolineꢀ3,1'ꢀindolizine]ꢀ2,5'(3'H)ꢀdione (13). Yield: 81 mg,
63%; colorless solid. 1H NMR (500 MHz, DMSOꢀD6):
δ
10.67 (s, 1H), 7.39 (t, J = 8.25 Hz,
1H), 7.09 (t, J = 7.55 Hz, 1H), 6.91 (d, J = 7.68 Hz, 1H), 6.78 (d, J = 7.4 Hz, 1H), 3.76 (m, 2H),
3.63 (m, 1H), 2.22 (m, 2H), 2.06 (m, 1H), 1.93 (m, 1H), 0.70 (m, 1H). 13C NMR (125 MHz,
DMSOꢀD6). 176.6, 168.0, 141.4, 130.1, 128.2, 123.3, 121.9, 109.9, 64.3, 56.5, 32.6, 31.2, 30.8,
29.0, 23.4, 22.1, 19.8, 14.0. QꢀTof LCMS (MS ESI): [M + H]+ calcd for C15H16N2O2 257.1288,
found 257.1288.
(3'S,
7a'R)ꢀmethylꢀ7a'ꢀmethylꢀ2,5'ꢀdioxoꢀ2',3',5',6',7',7a'ꢀhexahydrospiro[indolineꢀ3,1'ꢀ
1
pyrrolizine]ꢀ3'ꢀcarboxylate (14). Yield: 113 mg, 72%; grey solid. H NMR (500 MHz, DMSOꢀ
D6):
δ 7.39 (d, J = 6.9 Hz, 1H), 7.23 (t, J = 7.55 Hz, 1H), 6.98 (t, J = 7.55 Hz, 1H), 6.84 (d, J =
7.55 Hz, 1H), 4.54 (t, J = 8.95 Hz, 1H), 4.13 (t, J = 5.5 Hz, 2H), 3.70 (s, 3H), 2.75 (m, 1H), 2.67
13
(m, 2H), 2.16 (m, 1H), 1.78 (m, 1H), 1.67 (m, 2H), 1.47 (s, 3H), 1.36 (m, 1H). C NMR (125
MHz, DMSOꢀD6) 179.6, 175.8, 171.9, 167.0, 142.6, 131.7, 131.6, 128.7, 126.3, 125.9, 121.4,
109.6, 71.7, 67.4, 58.3, 54.0, 52.2, 29.8, 28.4, 23.2, 22.4, 13.9, 10.8. QꢀTof LCMS (MS ESI): [M
+ H]+ calcd for C17H18N2O4 315.1339, found 315.1353.
7a'ꢀmethylꢀ2',3',7',7a'ꢀtetrahydrospiro[indolineꢀ3,1'ꢀpyrrolizine]ꢀ2,5'(6'H)ꢀdione (+/ꢀ15a) and
1
(+/ꢀ15b). Yield: 94 mg, 74% (combined yield); yellow solid. (+/ꢀ) 15a: H NMR (400 MHz,
DMSOꢀD6): δ 10.41 (s, 1H), 7.35 (d, J = 7.55 Hz, 1H), 7.21 (t, J = 8.25 Hz, 1H), 7.01 (t, J = 7.55
Hz, 1H), 6.81 (d, J = 7.55 Hz, 1H), 3.65 (m, 1H), 3.17 (m, 1H), 2.61 (m, 2H), 2.25 (m, 1H), 2.18
13
(m, 1H), 1.79 (m, 1H), 1.59 (m, 1H), 1.39 (s, 3H). C NMR (75 MHz, DMSOꢀD6) 180.1, 174.8,
142.7, 128.4, 127.6, 125.6, 121.2, 109.4, 71.5, 57.4, 34.6, 33.0, 28.0, 25.6. QꢀTof LCMS (MS
ESI): MS ESI): [M + H]+ calcd for C15H16N2O2 257.1285, found 257.1290.
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(+/ꢀ) 15b. 1H NMR (400 MHz, DMSOꢀD6):
δ 10.45 (s, 1H), 7.21 (t, J = 8.25 Hz, 1H), 6.95 (t, J
= 7.55 Hz, 1H), 6.83 (d, J = 7.55 Hz, 1H), 6.65 (d, J = 7.55 Hz, 1H), 3.65 (m, 1H), 3.35 (m, 1H),
13
2.71 (m, 2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.59 (m, 1H), 1.39 (s, 3H), 1.28 (m, 1H). C NMR
(75 MHz, DMSOꢀD6) 176.5, 173.4, 141.4, 132.3, 128.2, 122.8, 121.7, 109.5, 72.7, 57.1, 35.2,
33.1, 27.4, 23.9. QꢀTof LCMS (MS ESI): [M + H]+ calcd for C15H16N2O2 257.1285, found
257.1299.
Ethyl
11ꢀmethylꢀ3ꢀoxoꢀ11bꢀ(trifluoromethyl)ꢀ2,3,5,6,11,11bꢀhexahydroꢀ1Hꢀindolizino[8,7ꢀ
b]indoleꢀ2ꢀcarboxylate (17). Compound 16 (200 mg, 0.68 mmol) was dissolved in
dichloromethane (10 mL) and was treated with 10% KOH, methyl iodide (142 mg, 1 mmol) and
catalytic amount of tetrabutyl ammonium bromide (TBAB) and the resulting mixture was stirred
at rt overnight. Once TLC indicated complete consumption of starting material, the reaction was
quenched with water, the organic layer was separated and washed with brine. It was then dried
over magnesium sulphate and evaporated to generate crude solid, which was taken to the next
step without further purification.
The crude compound from the previous step was dissolved in THF (10 mL), cooled to ꢀ78°C
and was treated with nꢀBuLi (1M in THF) (0.65 mL, 0.65 mmol). The resulting mixture was
stirred for 0.5h and was treated with ethyl chloroformate (71 mg, 0.65 mmol). The reaction was
warmed to ꢀ40°C and when LCMS indicated formation of 70% of desired compound (if the
reaction is continued longer, then the diacylated product starts to form which makes purification
difficult) the reaction was quenched with brine and was extracted with ethyl acetate. The organic
layer was further washed with brine and was dried over magnesium sulphate. The solvent was
evaporated and the crude was purified by 20% EtOAcꢀhexane as eluent to generate the desired
compound 17 as 1:1 epimeric mixture. Yield: 210 mg, 81% (combined yield); colorless liquid.
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1H NMR (400 MHz, DMSOꢀD6): ):
δ 7.55ꢀ7.51 (t, J = 16 Hz, 2H), 7.30ꢀ7.26 (t, J = 15.2 Hz,
1H), 7.14ꢀ7.10 (t, J = 15.2 Hz, 1H), 4.40ꢀ4.37 (m, 1H), 4.17ꢀ4.10 (m, 2H), 3.78 (s, 3H), 3.46ꢀ
3.42 (m, 1H), 3.00ꢀ2.95 (m, 1H), 2.82ꢀ2.80 (m, 1H), 2.79ꢀ2.78 (m, 1H), 1.26ꢀ1.20 (m, 2H), 1.19
(m, 3H). 13C NMR (75 MHz, DMSOꢀD6) 173.1, 172.3, 137.56, 134.4, 128.0, 127.8, 127.1,
126.8, 126.6, 125.6, 125.2, 122.8,122.5, 119.4, 119.1, 118.5, 111.3, 111.1, 109.8, 109.7, 58.7,
57.8, 51.5, 48.1, 44.0, 42.7, 35.7, 31.7, 31.3, 29.0, 28.7, 19.5, 19.3. LCMS (MS ESI): [M + H]+
calcd for C19H19F3N2O3 381.1421, found 381.9700.
6ꢀaminoꢀ11'ꢀmethylꢀ11b'ꢀ(trifluoromethyl)ꢀ1',5',6',11b'ꢀtetrahydrospiro[indolineꢀ3,2'ꢀ
indolizino[8,7ꢀb]indole]ꢀ2,3'(11'H)ꢀdione (21). Compound 17 (100 mg, 0.26 mmol) was
dissolved in THF (10mL), cooled to ꢀ78°C and was treated with LiHMDS in 1M solution in THF
(0.3 mL, 0.30 mmol). The resulting mixture was stirred for 0.5h and was treated with 2,4ꢀ
dinitrofluorobenzene (48.36 mg, 0.26 mmol). The reaction was warmed to rt and when LCMS
indicated complete conversion to the desired intermediate the reaction was quenched with brine
and was extracted with ethyl acetate. The organic layer was further washed with brine and was
dried over Na2SO4. The solvent was evaporated and the crude was subjected to hydrogenation.
To a solution of the above crude material in EtOAc (10 mL) was added Pd/C (50% moist)
followed by ammonium formate and stirring was continued at ambient temperature. Once TLC
indicates completion of the reaction, the reaction mixture was filtered over celite, washed the
celite with EtOAc (2x20mL). The filtrate collected was dried over anhydrous Na2SO4 and
concentrated under reduced pressure to afford the crude, which was purified by 20% EtOAcꢀ
hexane as eluent to generate the desired compound 21. Yield: 39 mg, 34%; light yellow semiꢀ
solid. 1H NMR (400 MHz, DMSOꢀd6) δ 10.75 (s, 1H), 7.60ꢀ 7.54 (dd, J1 = 7.32 Hz, J2 = 8.08 Hz,
2H), 7.32ꢀ7.30 (t, J1 = 7.6Hz, J2 = 15.2 Hz, 1H), 7.17ꢀ 7.13 (t, J1 = 7.48 Hz, J2 = 15.28 Hz, 1H),
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6.10 (s, 1H), 5.75ꢀ5.73 (d, J = 8.2 Hz, 1H), 5.50ꢀ 5.48 (dd, J = 8.2 Hz, 1H), 5.18 (s, 2H), 4.34ꢀ
13
4.30 (m, 1H), 3.78 (s, 3H); C NMR (100 MHz, DMSOꢀd6) 175.5, 171.6, 162.9, 149.9, 143.3,
138.1, 127.7, 125.2, 123.2, 122.2, 119.7, 118.9, 117.7, 111.6, 110.27, 107.0, 96.0, 63.4, 63.1,
56.5, 36.9, 34.9, 31.7, 29.0, 19.8, 18.0. LCMS (MS ESI): [M + H]+ calcd for C23H19F3N4O2
441.1533, found 441.1819.
11ꢀmethylꢀ4'ꢀphenylꢀ11bꢀ(trifluoromethyl)ꢀ1,5,6,11bꢀtetrahydrospiro[indolizino[8,7ꢀb]indoleꢀ
2,3'ꢀpyrrolidine]ꢀ2',3(11H)ꢀdione (22). Compound 17 (100 mg, 0.26 mmol) was dissolved in
THF (10mL), cooled to ꢀ0°C and was treated with NaH (60% dispersion in mineral oil) (15 mg,
0.3 mmol). The resulting mixture was stirred for 0.5h and was treated with 2ꢀnitrostyrene (40
mg, 0.26 mmol). The reaction was warmed to rt and when LCMS indicated complete conversion
to the desired compound 19, the reaction was quenched with brine and was extracted with ethyl
acetate. The organic layer was further washed with brine and was dried over magnesium
sulphate. The solvent was evaporated and the crude was taken subjected to hydrogenation.
To a crude ethyl acetate (EtOAc) (10 mL) solution of the above intermediate, was added Pd/C
(10% w/w) followed by ammonium formate and stirring was continued at ambient temperature.
Once TLC indicates completion of the reaction, the reaction mixture was filtered over celite,
washed the celite with EtOAc (2x20mL). The filtrate collected was dried over anhydrous Na2SO4
and concentrated under reduced pressure to afford the crude, which was purified by 20% EtOAcꢀ
hexane as eluent to generate the desired compound 22. Yield: 51 mg, 43%; white solid. 1H NMR
(400 MHz, DMSOꢀd6) δ 8.48ꢀ8.44 (d, J = 14.8 Hz, 1H), 7.55ꢀ7.50 (m, 1H), 7.48ꢀ7.19 (m, 2H),
7.12ꢀ7.05 (m, 1H), 6.87ꢀ6.85 (d, J = 7.64 Hz, 1H), 6.77ꢀ6.73 (t, J = 15 Hz, 1H), 6.63ꢀ6.52 (m,
2H), 6.35ꢀ6.32 (t, J = 15.24 Hz, 1H), 4.02ꢀ3.97 (m, 1H), 3.89 (s, 3H), 3.63ꢀ3.61 (m, 2H), 3.03ꢀ
2.96 (m, 1H), 2.82ꢀ2.81 (m, 1H), 2.69ꢀ2.50 (m, 1H); 13C NMR (100 MHz, DMSOꢀd6) δ172.3,
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137.6, 134.4, 128.0, 127.8, 127.0, 126.8, 126.6, 125.6, 122.8, 122.5, 119.3, 119.1, 118.5, 111.3,
111.1, 109.8, 109.7, 58.7, 57.8, 51.5, 48.1, 44.0, 42.7, 40.2, 35.7, 31.7, 29.0, 28.7, 19.5, 19.3. Qꢀ
Tof LCMS (MS ESI): [M + H]+ calcd for C25H22F3N3O2 454.1737, found 454.2038.
11ꢀmethylꢀ11bꢀ(trifluoromethyl)ꢀ1,5,6,11bꢀtetrahydroꢀ1'Hꢀspiro[indolizino[8,7ꢀb]indoleꢀ2,3'ꢀ
quinoline]ꢀ2',3(4'H,11H)ꢀdione (23). Compound 17 (100 mg, 0.26 mmol) was dissolved in THF
(10mL), cooled to ꢀ0°C and was treated with NaH (60% dispersion in mineral oil) (15 mg, 0.3
mmol). The resulting mixture was stirred for 0.5h and was treated with 2ꢀnitrobenzyl bromide
(56 mg, 0.26 mmol). The reaction was warmed to rt and when LCMS indicated complete
conversion to the desired compound, the reaction was quenched with brine and was extracted
with ethyl acetate. The organic layer was further washed with brine and was dried over
magnesium sulphate. The solvent was evaporated and the crude was taken subjected to
hydrogenation.
To an EtOAc (10 mL) solution of the above crude, was added Pd/C (10% w/w) followed by
ammonium formate and stirring was continued at ambient temperature. Once TLC indicates
completion of the reaction, the reaction mixture was filtered over celite, washed the celite with
EtOAc (2x20mL). The filtrate collected was dried over anhydrous Na2SO4 and concentrated
under reduced pressure to afford the crude, which was purified by 20% EtOAcꢀhexane as eluent
1
to generate the desired compound 23. Yield: 50 mg, 44%; colorless solid. H NMR (400 MHz,
CDCl3) δ 8.23 (s, 1H), 7.58ꢀ7.56 (d, J = 7.88 Hz, 1H), 7.35ꢀ7.34 (d, J = 3.24 Hz, 2H), 7.23ꢀ7.21
(d, J = 6Hz, 2H), 7.02ꢀ6.96 (m, 2H), 6.84ꢀ6.82 (d, J = 7.8 Hz, 1H), 4.68ꢀ4.63 (dd, J1 = 7.2 Hz, J2
= 6.64 Hz, 1H), 3.75 (s, 3H), 3.70ꢀ3.49 (m, 1H), 3.43ꢀ3.39 (d, J = 15.8 Hz, 1H), 3.23ꢀ3.19 (d,
13
1H), 3.02ꢀ2.85 (m, 2H), 2.64ꢀ2.60 (d, J = 16 Hz, 1H), 2.41ꢀ2.39 (d, J = 16 Hz, 1H); C NMR
(400 MHz, DMSOꢀd6) δ 171.9, 168.7, 138.4, 136.6, 128.7, 128.6, 128.4, 126.7, 125.9, 123.9,
26

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123.8, 123.7, 120.9, 121.0, 119.2, 115.6, 111.6, 109.7, 63.3, 63.0, 51.0, 37.6, 37.0, 36.1, 32.1,
32.0, 30.3, 29.8, 29.7, 29.5, 29.3, 22.8, 20.4, 14.34, 14.3, 1.2. LCMS (MS ESI): [M + Na]+ calcd
for C24H20F3N3O2 462.1402, found 462.1686.
ASSOCIATED CONTENT
Supporting Information. 1H, 13C, LCMS and single crystal Xꢀray structures of all new
compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Subhabrata.Sen: subhabrata.sen@snu.edu.in
Author Contributions
CB and PD have contributed equally for the manuscript
ACKNOWLEDGMENT
We acknowledge Department of Biotechnology for funding through BT/2/2015ꢀIFD. We thank
Dr. A. Roychoudhury, Department of Chemical Sciences, Indian Institute of Science Education
and Research, Mohali, India for helping us with the Xꢀray diffraction data collection.
ABBREVIATIONS
SNAr, Aromatic nucleophilic substitution; CAD, caspase activated deoxyribonuclease; NBS, Nꢀ
Bromosuccinimide; MCFꢀ7, Michigan Cancer Foundationꢀ7.
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