Synthesis and molecular modeling studies of indole-based antitumor agents

Article abstract of DOI:10.1039/c6ra07061b

Indole-based compounds 30-63 were synthesized by the multi-component 1,3-dipolar cycloaddition reaction of 1-alkyl-3,5-bis(arylidene)-4-piperidones 11-25 with azomethine ylides (generated by the condensation of isatins 26-28 with sarcosine 29). The single crystal X-ray studies of 46 and 48 supported the regio- and stereoselectivity of the reaction. Most of the synthesized spiro-indoles exhibited potent antitumor properties against the HeLa (cervical cancer) cell line through in vitro sulfo-rhodamine-B bioassay, higher than that of cisplatin. Only compound 54 showed bio-potency against the HepG2 (hepatocellular cancer) cell line, comparable to that of doxorubicin hydrochloride (standard reference). 3D-Pharmacophore and 2D-QSAR studies were used to validate the observed biological data and determine the most important parameters controlling activity. The estimated bio-properties from the computational studies showed high approximations to the experimental data.

Full text of DOI:10.1039/c6ra07061b

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Synthesis and molecular modeling studies of
indole-based antitumor agents†
Cite this: RSC Adv., 2016, 6, 45434
Riham F. George,a Siva S. Panda,b El-Sayed M. Shalaby,c Aladdin M. Srour,d
c
e
*
I. S. Ahmed Farag and Adel S. Girgis
Indole-based compounds 30–63 were synthesized by the multi-component 1,3-dipolar cycloaddition
reaction of 1-alkyl-3,5-bis(arylidene)-4-piperidones 11–25 with azomethine ylides (generated by the
condensation of isatins 26–28 with sarcosine 29). The single crystal X-ray studies of 46 and 48
supported the regio- and stereoselectivity of the reaction. Most of the synthesized spiro-indoles
exhibited potent antitumor properties against the HeLa (cervical cancer) cell line through in vitro sulfo-
rhodamine-B bioassay, higher than that of cisplatin. Only compound 54 showed bio-potency against the
HepG2 (hepatocellular cancer) cell line, comparable to that of doxorubicin hydrochloride (standard
reference). 3D-Pharmacophore and 2D-QSAR studies were used to validate the observed biological data
and determine the most important parameters controlling activity. The estimated bio-properties from
the computational studies showed high approximations to the experimental data.
Received 17th March 2016
Accepted 2nd May 2016
DOI: 10.1039/c6ra07061b
www.rsc.org/advances
Aspergillus fumigates) inhibit the G2/M progression cell division
in mammalian tsFT210 cells9,10 (Fig. 1).
Introduction
Inspired by the above reports and continuation of a research
program directed towards development of novel bio-active
agents with particular interest in cancer chemotherapy,11–20
novel spiro-indoles where, the indolyl nitrogen is attached to
a (cyclic-amino)methylene function were investigated in the
present study. This function provides a positive ionizable tting
our candidate in the pharmacophoric active site. The synthe-
sized spiro-indoles were screened against cervical (HeLa) and
liver (HepG2) carcinoma cell lines. Cervical cancer is the fourth
The indole scaffold is found in many natural and synthetic
biologically active molecules.1,2 The marketed antiviral drugs
delavirdine 1, arbidol 2 and methisazone 3 are indole deriva-
tives.3 Another indole derivative, obatoclax 4 was reported to
overcome myeloid cell leukemia-1 (MCL-1) mediated resistance
to apoptosis.4 Sunitinib 5 has been approved by the US-FDA and
European Agency for the treatment of advanced renal and
gastrointestinal stromal tumors.5,6 Indibulin 6 was reported as
a microtubule inhibitor (phase I/II)7 (Fig. 1). Many naturally
occurring spiro-indoles exhibit biological activity for example;
mitraphylline
7 (from Uncaria tomentosa) exhibits anti-
proliferative activity against brain carcinoma cell lines, neuro-
blastoma SKN-BE(2), and malignant glioma GAMG.8
Spirotryprostatins A 8 and B 9 (from the fermentation broth of
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo,
Egypt
bDepartment of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA
cX-Ray Crystallography Lab, Physics Division, National Research Centre, Dokki, Giza
12622, Egypt
dTherapeutical Chemistry Department, National Research Centre, Dokki, Giza 12622,
Egypt
ePesticide Chemistry Department, National Research Centre, Dokki, Giza 12622, Egypt.
E-mail: girgisas10@yahoo.com; Fax: +202 33370931; Tel: +202 012 20447199
† Electronic supplementary information (ESI) available: Including spectral charts,
gures of structures optimized by different computational techniques,
3D-pharmacophore mapped models, tables of geometrical parameters and
tables of molecular descriptor values of 2D-QSAR model. CCDC 1455157 and
1455158. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c6ra07061b
Fig. 1 Biologically active indole-based compounds.
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leading cause of cancer related death in women.21,22 Infection
with oncogenic human papillomavirus is the main cause for
cervical cancer.23 Although advances have been made in
screening, diagnosis, treatment and management, the mortality
due to cervical carcinoma still approaches 50%. Cervical carci-
noma is treated by radiotherapy or surgery in the early stage and
concurrent chemoradiation in the advanced stages.24 Liver
cancer is the third leading cause of cancer-related death
worldwide.25 Infection with hepatitis virus of either type B or C
(HBV, HCV) increases the risk of liver cancer about 20 folds.26
Other risk factors for hepatocellular carcinoma include alcohol
abuse, diabetes, obesity, and related metabolic syndrome.27 To
date, the chemotherapeutic agents developed for liver cancer
have shown poor efficacy with severe toxicity and side effects.28
Therefore, novel chemotherapeutic agents with a higher
potency and less side effects are still needed. Molecular
modeling studies were undertaken to validate the observed
biological data and determine the important parameters gov-
erning activity.
Scheme 2 Synthesis of indole-based compounds.
1H-NMR spectrum of 30 showed the methylene protons of pyr-
rolidinyl H2C-50 and piperidinyl H2C-200, H2C-600 as diaster-
eotopic protons. The methylene protons attached to the indolyl
N-1 also exhibited diastereotopic (two doublets at dH ¼ 4.16 and
4.54; J ¼ 12.6 Hz). The methylene carbons H2C-600, H2C-50 and
H2C-200 appeared in the 13C-NMR spectrum of 30 at dC ¼ 56.9,
57.3, and 57.5, respectively. The methylene carbon attached to
the indolyl N-1 (NCH2N) appeared at dC ¼ 63.3. The pyrrolidinyl
methine HC-40 exhibited at dC ¼ 45.6. The signals for spiro-
carbons C-30 (C-300) and C-3 (C-20) revealed at dC ¼ 66.5, 75.6,
respectively. Those for the carbonyl carbons C-2 and C-400
showed at dC ¼ 176.5, 198.7, respectively (Fig. S7–S107† show
the spectral data of compounds 30–63).
Results and discussion
Chemistry
The base-catalyzed condensation (KOH/EtOH) of aromatic
aldehydes with 1-alkyl-4-piperidones 10 (in a 2 : 1 molar ratio)
afforded the corresponding 1-alkyl-3,5-bis(arylidene)-4-
piperidones 11–25 (Scheme 1). The 1H-NMR spectra of piper-
idones 14 and 19 showed sharp singlet signals for the olenic
methine protons at dH ¼ 7.72 and 7.76 in the case of 14 and 19,
respectively conrming the E,E0-conguration.29–31 The IR
spectra of compounds 14 and 19 showed unsaturated carbonyl
group at n ¼ 1670, 1678 cmꢀ1, respectively (Fig. S1–S6†).
Multi-component reaction of 1-alkyl-3,5-bis(arylidene)-4-
piperidones 11–25 with azomethine ylides (generated by the
condensation of isatins 26–28 with sarcosine 29)11–15 in reuxing
ethanol afforded the corresponding dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-diones 30–63 (Scheme 2).
The IR spectrum of 30 (a representative example of the family),
showed the ketonic and amidic carbonyls of piperidinyl and
indolyl heterocycles at n ¼ 1699, 1686 cmꢀ1, respectively. The
Single crystal X-ray studies
The single crystal X-ray ORTEP diagrams of compounds 46 and
48 are shown in Fig. 2 and 3, respectively. Both compounds are
Fig. 2 An ORTEP view of compound 46 showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability level
and H atoms are shown as small spheres of arbitrary radii.
Scheme 1 Synthesis of bis(arylidene)piperidones.
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Table 2 Hydrogen-bond geometry (A˚, ꢁ) for compound 48a
D–H/A
D–H
H/A
D/A
D–H/A
C29–H291/F1(i)
C23–H231/F2(ii)
C3–H31/F2(iii)
C10–H101/O2(iv)
0.92
0.94
0.93
0.96
2.54(3)
2.59(3)
2.65(4)
2.71(3)
3.247(6)
3.249(4)
3.312(6)
3.659(5)
135(2)
127(3)
128(2)
169(4)
a
Symmetry codes: (i) x, ꢀ1/2 ꢀ y, 1/2 + z; (ii) 2 ꢀ x, ꢀ1 ꢀ y, ꢀ1 ꢀ z; (iii) x,
ꢀ3/2 ꢀ y, ꢀ1/2 + z; (iv) ꢀ1 + x, y, z.
˚
mean square (rms) deviation ¼ 0.0543 A] in 46 and lies 0.675(4)
˚
A out of the plane of the remaining ve atoms (rms deviation ¼
˚
0.0426 A) in 48. In 46, the sum of the angles around the N1 and
N2 atoms are approximately 334ꢁ and 333ꢁ, respectively; and in
48 are 337ꢁ and 333ꢁ, respectively, consistent with sp3 hybrid-
ization. With respect to the piperidine ring attached to C24, the
C1 occupies an axial position however, the C17 as well as the (4-
uorophenyl)methylidene residue and the N-bound alkyl
substituent occupy equatorial positions.
Fig.
3 An ORTEP view of compound 48 showing the atom-
numbering scheme. Displacement ellipsoids are drawn at the 50%
probability level and H atoms are shown as small spheres of arbitrary
radii.
The most signicant feature of the crystal packing of 46 and
48 is the centrosymmetric eight-membered {/HNCO}2 amide
dimers. In the molecular packing of 46 and 48, molecules are
connected by C–H/F and C–H/O interactions (listed in Tables
1 and 2) resulting in supramolecular layers. These supramo-
lecular layers are parallel to (0 1 1) as illustrated in Fig. 4 for
compound 46. However, the supramolecular layers are parallel
to (1 0 0) as shown in Fig. 5 for compound 48.
crystallized in the monoclinic space group P21/c possessing four
molecules in the unit cell. The asymmetric unit in each
comprises one molecule. Two spiro linkages exist in 46 and 48;
at C1, where the pyrrolidine and indole heterocyclic rings are
attached, and at C24, where the piperidine and pyrrolidine
heterocycles are connected. In general, the bond lengths and
angles of both compounds (Tables S1 and S2†) are in good
agreement with each other and also with the reported structures
possessing similar rings and functional groups.31–38 The (uo-
rophenyl)methylidene moiety is connected to the piperidine
ring at position C26 whereas, the 4-uorophenyl ring is attached
to the pyrrolidine heterocycle at C17. The (1-piperidinyl)meth-
ylene fragment is connected to the indolyl N3. The exocyclic
olenic linkage of compounds 46 and 48 has E-conguration
(C26–C27). The pyrrolidine ring appears as an envelope
Optimized structures
The geometric parameters of the optimized structures of
compounds 46 and 48 by AM1 and PM3 computational chem-
istry techniques were determined and compared with the
experimental X-ray observed data (Tables S1 and S2; Fig. S108–
S111†). Most of the computationally determined bond lengths
and angles derived from the optimized structures are correlated
well with the experimentally observed ones with slight
˚
conformation with N1 being the ap atom which lies 0.594(5) A
˚
and 0.554(3) A out of the plane of the remaining four atoms in
46 and 48, respectively.
The piperidine ring connected to C8 has a chair form with
˚
˚
C11 and N4 deviating by 0.628 A and 0.649 A in 46, and by 0.544
˚
˚
A and 0.652 A in 48, respectively out of the plane of the
remaining four atoms. However, the piperidine ring attached to
C24 adopts a half-chair conguration where, the C35 atom lies
˚
0.682(3) A out of the plane of the remaining ve atoms [root
Table 1 Hydrogen-bond geometry (A˚, ꢁ) for compound 46a
D–H/A
D–H
H/A
D/A
D–H/A
C29–H291/F1(i)
C23–H231/F2(ii)
C3–H31/F2(iii)
C10–H101/O2(iv)
0.96
0.96
0.96
0.96
2.46(2)
2.54(3)
2.60(4)
2.64(3)
3.187(6)
3.258(7)
3.240(6)
3.591(7)
132(2)
132(3)
125(2)
166(4)
Fig. 4 A view of the supramolecular layer parallel to (0 1 1) plane in the
crystal structure of compound 46. The hydrogen-bond interactions
are shown as green dashed lines.
a
Symmetry codes: (i) ꢀ1 ꢀ x, ꢀy, ꢀ2 ꢀ z; (ii) ꢀ1 ꢀ x, ꢀy, ꢀ2 ꢀ z; (iii) x,
ꢀ1/2 ꢀ y, ꢀ1/2 + z; (iv) ꢀ1 + x, y, z.
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deviations. These deviations can be attributed to the fact that
the experimental results are obtained for the solid phase
however; the theoretical calculations are derived from the gas
phase. In the solid state, the existence of the crystal eld along
with the intermolecular interactions connecting the molecules,
are the main reasons for these differences in geometrical
parameters.31,32 The highest difference between the experi-
mental (X-ray) and calculated bond length values for
˚
compounds 46 and 48 are 0.067, 0.075; 0.083, 0.066 A, for AM1
and PM3 methods respectively and the root mean square errors
(RMSE) are 0.0256, 0.0269; 0.0312, 0.0329 for AM1 and PM3
methods, respectively. These statistical observations explain
that the bond length values obtained by any of the computa-
tional technique are strongly correlated with the experimental
(X-ray) ones.
Fig. 5 A view of the supramolecular layer parallel to (1 0 0) plane in the
crystal structure of compound 48. The hydrogen-bond interactions
are shown as green dashed lines.
Regarding the bond angles, the highest difference between
the experimental and theoretical values for compounds 46 and
Table 3 Antitumor properties of the synthesized compounds 30–63
IC50a, mg mlꢀ1 (mM)
ID
Compd
R
R0
X
HeLa
HepG2
1
2
3
4
5
6
7
8
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
Ph
Ph
Ph
Me
Me
Me
Me
Me
Me
Et
Et
n-Pr
n-Pr
CH2Ph
CH2Ph
Me
Me
Me
Me
Et
CH2
O
NMe
CH2
O
NMe
CH2
O
CH2
O
CH2
O
O
CH2
O
NMe
CH2
O
CH2
O
CH2
O
CH2
O
NMe
CH2
O
NMe
CH2
O
CH2
O
CH2
O
—
3.62 (6.46)
3.09 (5.49)
4.46 (7.75)
3.80 (6.04)
3.48 (5.51)
4.02 (6.24)
3.62 (5.62)
3.37 (5.22)
3.37 (5.12)
3.26 (4.94)
3.62 (5.13)
3.37 (4.76)
3.15 (4.50)
3.04 (5.09)
3.15 (5.26)
4.13 (6.75)
3.59 (5.88)
3.30 (5.39)
4.57 (7.32)
3.70 (5.90)
3.30 (4.90)
4.79 (7.10)
3.04 (5.16)
3.48 (5.89)
3.09 (5.12)
3.48 (5.61)
3.15 (5.06)
3.04 (4.78)
3.37 (4.95)
4.24 (6.21)
4.24 (7.40)
3.70 (6.44)
3.91 (6.95)
5.00 (8.85)
4.19 (7.22)
4.19 (7.71)
12.07 (21.52)
12.07 (21.45)
11.20 (19.45)
11.52 (18.30)
15.76 (24.95)
12.17 (18.88)
14.57 (22.64)
14.78 (22.89)
32.17 (48.91)
33.26 (50.42)
22.83 (32.35)
20.87 (29.49)
20.43 (29.16)
8.15 (13.66)
21.63 (36.13)
17.50 (28.61)
14.13 (23.14)
14.13 (23.06)
23.59 (37.76)
17.17 (27.40)
16.52 (24.55)
19.89 (29.48)
19.89 (33.78)
28.04 (47.46)
4.67 (7.73)
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-H3CC6H4
4-H3CC6H4
4-H3CC6H4
4-H3COC6H4
4-H3COC6H4
4-H3COC6H4
2,5-(H3CO)2C6H3
2,5-(H3CO)2C6H3
2-Thienyl
2-Thienyl
3-Pyridinyl
3-Pyridinyl
—
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Et
n-Pr
n-Pr
CH2Ph
CH2Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
—
16.30 (26.26)
19.78 (31.76)
9.02 (14.19)
17.39 (25.54)
12.50 (18.31)
17.61 (30.74)
10.00 (17.40)
47.93 (85.18)
22.28 (39.46)
4.67 (8.05)
62
63
Doxb
Cisplatin
—
—
—
3.58 (11.89)
a
IC50 is the concentration required to produce 50% inhibition of cell growth compared to the control. b Dox is doxorubicin hydrochloride (standard
reference).
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Table 4 Best fit values and estimated/predicted activity values for the
synthesized compounds 30–63 mapped with the generated 3D-
pharmacophore model due to HeLa (cervical cancinoma) cell line
Antitumor properties
The synthesized indole-based compounds 30–63 were screened
for their antitumor properties utilizing the sulfo-rhodamine-B
standard method11–20 against cervical (HeLa) and hepatocel-
lular (HepG2) carcinoma cell lines. The results (Table 3,
Fig. S114 and S115†) indicate that all the synthesized spiro-
indoles (compounds 32 and 63 are exceptions, IC50 ¼ 7.75,
8.85 mM, respectively) have antitumor properties with potency
higher than that of cisplatin (standard reference, IC50 ¼ 7.71
mM). Cisplatin is the clinically applicable drug either alone or in
combination with topotecan for cervical carcinoma.39 However,
most of the synthesized spiro-alkaloids show mild antitumor
properties against hepatoma cell line (HepG2), compound 54
(IC50 ¼ 7.73 mM) is the only analogue revealing bio-potency
comparable to that of doxorubicin hydrochloride (IC50 ¼ 8.05
mM), the standard reference. Doxorubicin is a typical DNA-
intercalating agent with broad antitumor effect against acute
leukemia, lymphoma, multiple myeloma, breast cancer, osteo-
sarcoma, so tissue sarcoma and liver cancer.40 Structure–
activity relationship (SAR) based on the observed antitumor
bioactivities against HeLa cell explains that, utilization of (4-
morpholiyl)methylene function as a substituent attached to the
indolyl N-1 seems more appropriate for constructing bio-active
hits relative to (1-piperidinyl)methylene residue (compounds
44, 51, 53, 59 and 63 are exceptions). To explore the controlling
parameters governing bio-activity and validate the observed
bioactivity, computational chemistry studies including 3D-
pharmacophore and 2D-quantitative structure activity rela-
tionship (2D-QSAR) were performed.
Observed IC50
Entry Compd mM
,
Estimated IC50,
mM
Errora Fit value
0.62 7.795
1
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
6.46
5.49
7.75
6.04
5.51
6.24
5.62
5.22
5.12
4.94
5.13
4.76
4.50
5.09
5.26
6.75
5.88
5.39
7.32
5.90
4.90
7.10
5.16
5.89
5.12
5.61
5.06
4.78
4.95
6.21
7.40
6.44
6.95
8.85
5.84
5.84
6.59
5.02
5.10
6.07
5.55
5.34
5.56
5.14
5.45
5.05
4.87
7.21
5.57
6.03
6.09
6.54
5.58
6.36
5.64
5.72
5.34
5.17
6.96
5.13
5.38
6.18
4.91
5.09
6.26
5.73
6.85
7.63
2
3
4
5
6
7
8
9
ꢀ0.35 7.795
1.16
1.02
0.41
0.17
0.07
7.743
7.861
7.855
7.778
7.817
ꢀ0.12 7.834
ꢀ0.44 7.817
ꢀ0.20 7.851
ꢀ0.32 7.826
ꢀ0.29 7.858
ꢀ0.37 7.875
ꢀ2.12 7.704
ꢀ0.31 7.816
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0.72
7.782
ꢀ0.21 7.777
ꢀ1.15 7.746
1.74
7.815
ꢀ0.46 7.758
ꢀ0.74 7.811
1.38
7.805
ꢀ0.18 7.834
0.72 7.848
ꢀ1.84 7.719
0.48 7.852
ꢀ0.32 7.831
ꢀ1.40 7.771
0.04
1.12
1.14
0.71
0.10
1.22
7.871
7.855
7.765
7.804
7.726
7.679
Molecular modeling
3D-Pharmacophore. Discovery Studio 2.5 soware (Accelrys
Inc., San Diego, CA, USA) was utilized for performing the 3D-
pharmacophoric studies. The 3D-pharmacophore protocol was
used searching for the best predictive 3D-pharmacophore
hypothesis by aligning different conformations of the synthe-
sized bio-active agents 30–63 in which the molecules are likely
a
Error is the difference between the observed and estimated bio-activity
values.
48 are 6.1, 10.1; 7.4, 8.0ꢁ and the RMSE are 2.0361, 2.3048; to bind with the receptor. The observed HYPOGEN identies
2.1406 and 2.2722 for AM1 and PM3 methods, respectively. a 3D-array of ve chemical features in case of HeLa (cervical
Global comparisons were performed by superimposing the carcinoma) cell line comprising two positive ionizables (PosIon-
molecular skeletons obtained from X-ray diffraction of 46 and 1, PosIon-2), one hydrophobic (H) and one hydrogen bonding
48 with the corresponding ones of AM1 and PM3 techniques acceptor (HBA) (Fig. S116†). Mapping of the generated phar-
(Fig. S112 and S113†). Most of the rings and functional groups macophore with the synthesized bio-active agents indicates that
aligned well with each other. However, the piperidine ring all the compounds have the same alignment in which the
attached to the methylene C8 of the theoretical optimized nitrogen atom of the (cyclic amino)methylene fragment
structures (AM1 and PM3) is aligned in different direction attached to indolyl N-1, aligned with the PosIon-1 (positive
compared to the X-ray one. The torsion angle C7–N3–C8–N4 of ionizable-1), and the piperidonyl nitrogen with PosIon-2 (posi-
compound 46 ¼ ꢀ98.40, 55.97 and 82.18ꢁ for X-ray, AM1 and tive ionizable-2), the indolyl carbonyl oxyen with HBA (hydrogen
PM3, respectively support these observations. Similar words for bonding acceptor) and the aryl group attached to the pyrroli-
torsion angle C7–N3–C8–N4 of compound 48 ¼ ꢀ96.97, 55.90, dinyl heterocycle at C-4 with the H (hydrophobic) function
81.97ꢁ for X-ray, AM1 and PM3, respectively. The lattice form is (Fig. S117†). Although all the bio-active agents synthesized show
the main factor controlling these observations in X-ray struc- the same alignment in the 3D-pharmacophore, variable esti-
ture. However, in the conducted theoretical studies (AM1 or mated bio-properties and consequently variable t values are
PM3) this effect is completely neglected, as the gas phase is observed (Table 4). This is due to the fact that these computed
adopted.
values depend on how close is the effective site of the bio-active
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Table 5 Descriptor of the BMLR-QSAR model for the antitumor indole-based active agents 30, 31, 33–40, 42–49 and 51–63 against HeLa
(cervical cancinoma) cell linea
Entry
ID
Coefficient
s
t
Descriptor
1
2
3
4
5
6
0
ꢀ324.653
0.577
3.069
61.711
0.059
0.480
0.087
0.125
0.008
ꢀ5.261
9.729
6.390
Intercept
D1
D2
D3
D4
D5
Number of double bonds
Max. n–n repulsion for bond C–C
Number of F atoms
Max. e–e repulsion for bond C–O
HBSA H-bonding surface area
(MOPAC PC)
0.341
3.916
ꢀ0.455
ꢀ3.654
ꢀ0.029
ꢀ3.663
a
IC50 (mM) ¼ ꢀ324.653 + (0.577 ꢂ D1) + (3.069 ꢂ D2) + (0.341 ꢂ D3) ꢀ (0.455 ꢂ D4) ꢀ (0.029 ꢂ D5). N ¼ 31, n ¼ 5, R2 ¼ 0.844, R2cvOO ¼ 0.789, R2cvMO
¼ 0.975, F ¼ 27.137, s2 ¼ 0.170.
agent to the centres of the pharmacophoric feature in addition, analysis) soware employing 31 synthesized indole-based
the weight assigned to each of the pharmacophoric feature.
analogues (compounds 30, 31, 33–40, 42–49 and 51–63;
Generally, all the estimated bio-properties correlated well training set) exhibiting variable efficiency against HeLa (cervical
with the experimentally observed properties. In other words, the carcinoma) cell line. Compounds 32, 41 and 50 (i.e. about 10%
experimental potencies exhibited are preserved due to the of the training set) were used as an external test set for vali-
computational studies. Moreover, the major structural frag- dating the attained QSAR model, which represent high potent
ments show alignment in the pharmacophoric features so; they (compounds 41 and 50, IC50 ¼ 4.76, 4.90 mM) and mild
are the main factors affecting the activity/potency. Alignment of (compound 32, IC50 ¼ 7.75 mM) effective agents. The obtained
the nitrogen atom of (cyclic amino)methylene fragment BMLR-QSAR model is statistically signicant. Table 5 exhibits
attached to indolyl N-1 with PosIon-1 is a good indicator for the the QSAR's descriptors, sorted in the descending order based on
importance of this residue in optimizing the bio-properties. The student's t-criterion, which is an acceptable measure of statis-
same words for the aryl group attached to the pyrrolidinyl tical signicance in multiple linear regressions. Fig. 6 shows the
heterocycle at C-4 and the piperidonyl nitrogen (aligned with H QSAR multi-linear model plot of correlations representing the
and PosIon-2, respectively) which are varied substituents. These observed versus predicted IC50, mM values for the antitumor
observations support the rational design adopted for opti- indole-based active agents. The scattered plots are uniformly
mizing bio-effective hits.
distributed, covering ranges, observed 4.50–8.85, estimated/
2D-QSAR. QSAR study was undertaken utilizing CODESSA- predicted 4.37–8.927 (IC50, mM) values. The observed and pre-
Pro (comprehensive descriptors for structural and statistical dicted values of the training set compounds 30, 31, 33–40, 42–49
and 51–63 according to the BMLR-QSAR model are shown in
Table 6.
Number of double bonds, which is
a constitutional
descriptor, is the rst descriptor controlling the obtained
BMLR-QSAR model (t ¼ 9.729). This descriptor participated
positively in the model (coefficient ¼ 0.577), meaning that the
higher double bond number, the lower antitumor efficiency of
the molecule. Max. n–n repulsion for bond C–C is the second
descriptor controlling the 2D-QSAR model based on its t-crite-
rion value (t ¼ 6.390), a semi-empirical descriptor. The elec-
tron–electron repulsion between two given atoms is determined
by eqn (1).41
X X
EeeðABÞ ¼
PmnPlshmnjlsi
(1)
m;n˛Α l;s˛Β
where, A represents a given atomic species, B another atomic
species, PmnPls is the density matrix elements over atomic basis
{mnls} and hmn|lsi is the electron repulsion integrals on atomic
basis hmn|lsi. Number of F atoms (the third descriptor
controlling the obtained BMLR-QSAR model, t ¼ 3.916) is
a constitutional descriptor that characterizes the atomic
constitution and its inductive effect (ꢀI) inuences the aryl
group electrophilicity attached at the position-40 of pyrrolidine
heterocycle. Number of F atoms in this QSAR model, serves as
Fig.
6 BMLR-QSAR model plot of correlations representing the
observed versus predicted IC50, mM values for the antitumor indole-
based active agents against HeLa (cervical carcinoma) cell line.
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Table 6 Observed and estimated/predicted activity values for the
synthesized compounds 30, 31, 33–40, 42–49 and 51–63 according
to the BMLR-QSAR model due to HeLa (cervical cancinoma) cell line
Max. e–e repulsion for bond C–O is a semi-empirical
descriptor determined by eqn (1). HBSA H-bonding surface
area (MOPAC PC) is a charge-related descriptor. This descriptor
has the lowest effect on the QSAR model (coefficient ¼ ꢀ0.029).
For all the training set analogues this descriptor is zero however
for compound 62 ¼ 68.44892 (Table S3† exhibits all the QSAR
descriptor values for each respective training set compound),
which is a considerable value and can explain the enhanced
antitumor properties of this compound relative to its structur-
ally related analogue 63.
Observed IC50
mM
,
Estimated IC50,
mM
Entry
Compd
Errora
1
2
3
4
5
6
7
8
30
31
33
34
35
36
37
38
39
40
42
43
44
45
46
47
48
49
51
52
53
54
55
56
57
58
59
60
61
62
63
6.46
5.49
6.04
5.51
6.24
5.62
5.22
5.12
4.94
5.13
4.50
5.09
5.26
6.75
5.88
5.39
7.32
5.90
7.10
5.16
5.89
5.12
5.61
5.06
4.78
4.95
6.21
7.40
6.44
6.95
8.85
6.42
5.72
5.63
5.04
6.00
5.49
5.65
5.43
5.39
5.42
5.25
5.48
5.35
6.53
5.74
5.98
6.76
6.07
6.77
5.15
5.20
5.60
5.82
4.58
5.00
4.37
5.94
7.07
6.65
6.95
8.93
0.04
ꢀ0.23
0.41
0.47
0.24
0.13
Validation of BMLR-QSAR model. The reliability and statis-
tical relevance of the BMLR-QSAR model were examined by
internal and external validation procedures.
ꢀ0.43
ꢀ0.31
ꢀ0.45
ꢀ0.29
ꢀ0.75
ꢀ0.39
ꢀ0.09
0.22
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Internal validation
CODESSA-Pro is capable for QSAR internal validation including,
(i) Leave-One-Out (LOO) that involves developing number of
models with one training set compound omitted at a time. (ii)
Leave-Many-Out (LMO), which involves developing number of
models with many data points (group of compounds) omitted at
a time (up to 20% of the total data points). The observed
correlations due to the statistical internal validation techniques
are R2cvOO ¼ 0.789, R2cvMO ¼ 0.975. Both of them are corre-
lated with the squared correlation coefficient of the BMLR-
QSAR model (R2 ¼ 0.844). Moreover, the standard deviation of
the regressions (s2 ¼ 0.170) is a measurable value for the QSAR
model together with the Fisher test value (F ¼ 27.137) that
shows the ratio of the variance explained by the QSAR model
and the variance due to their errors. A high value of F-test
compared to the s2 is a signicant validation of the model.
Table 6 exhibits the experimentally observed and estimated/
predicted activities of the training set. The data indicate that the
error values (difference between the observed and estimated
bio-properties) were in the range 0.00–0.75, thus preserving the
potencies of most of the compounds tested.
0.14
ꢀ0.59
0.56
ꢀ0.17
0.33
0.01
0.69
ꢀ0.48
ꢀ0.21
0.48
ꢀ0.22
0.58
0.27
0.33
ꢀ0.21
0.00
ꢀ0.08
a
Error is the difference between the observed and estimated bio-activity
values.
an indicator variable to distinguish between the only uorine
containing compound and among the rest of training set
analogues. 3D-Pharmacophore shows that the aryl group
attached at the pyrrolidinyl C-4 is mapped with the hydrophobic
feature which may exhibit p-interaction with any of the receptor
active site components. Lipophilicity in addition to ꢀI effect of
F atoms can explain not only the impotence of this descriptor
for the 2D-QSAR model but also support the aforementioned
observations due to 3D-pharmacophoric studies.
External validation
Compounds 32, 41 and 50 were used as an external test set not
only for validating the obtained BMLR-QSAR model but also for
investigating its predicative ability. The test set analogues
represent high (compounds 41 and 50) and mild (compound 32)
antitumor effective agents against HeLa cell line, relative to
cisplatin (standard reference). This selection provides a good
Table 7 Observed, estimated and molecular descriptor values of the external test set compounds 32, 41 and 50 according to the attained BMLR-
QSAR model
Descriptorsb
Entry
Compd
Observed IC50 (mM)
Estimated IC50 (mM)
Errora
D1
D2
D3
D4
D5
1
2
3
32
41
50
7.75
4.76
4.90
4.62
4.75
5.67
3.13
0.01
ꢀ0.77
3
3
3
136.5313
136.4321
136.3949
0
0
2
200.8148
199.8628
199.0871
0
0
0
a
Error is the difference between the observed and estimated bio-activity values. b D1 ¼ number of double bonds, D2 ¼ max. n–n repulsion for bond
C–C, D3 ¼ number of F atoms, D4 ¼ max. e–e repulsion for bond C–O, D5 ¼ HBSA H-bonding surface area (MOPAC PC).
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idea about the capability of the obtained QSAR model for pre- 8400S spectrophotometer. 1H-NMR spectra were recorded on
dicting analogues of wide potency range. Table 7 reveals the Varian MERCURY 300 (300 MHz), Bruker Avance (300 MHz) and
observed and estimated IC50 values of the test set compounds. Bruker Ascend 400/R (400 MHz) spectrometers. 13C-NMR
The observed data, indicate that compounds 41 and 50, which spectra were recorded on Bruker Avance (75 MHz) and Bruker
are high potent antitumor active agents reveal estimated IC50
¼
Ascend 400/R (100 MHz) spectrometers. Compounds 11–13, 15–
4.75, 5.67 mM with error value ¼ 0.01, ꢀ0.77, respectively. 18, 20–25 42–50 and 26–28 51,52 were prepared according to the
Although the error value due to the estimated biological data of reported procedures.
compound 50 is considered high, the estimated potency of this
high antitumor active agent is still preserved relative to the
standard reference, cisplatin (IC50(observed) 7.71 mM).
Synthesis of 3E,5E-3,5-bis[(aryl)methylidene]-1-(1-propyl)-4-
piperidone (14, 19)
¼
Compound 32 which is considered a mild antitumor active
analogue (IC50(observed) ¼ 7.75 mM), has an estimated IC50 ¼ 4.62
mM with error value ¼ 3.13. Although, this error value is
considered high, the estimated activity of this analogue is
matched with its observed bio-observation which seems so close
to the efficiency of the standard reference. Generally, the net
results of external validation provide a good sign for the predic-
tive capability of the attained BMLR-QSAR model particularly for
optimizing high potent antitumor hits and also support the
previous statement (internal validation technique) concerning its
predictive power as shown by the statistical values.
A mixture of 1-(1-propyl)-4-piperidone (0.75 ml, 5 mmol), and
the appropriate aldehyde (10 mmol) in methanol (10 ml) con-
taining KOH (0.56 g, 10 mmol), was stirred at room temperature
(25 ꢁC) for 3 h. The separated solid aer storing the reaction
mixture overnight at room temperature, was collected, washed
with water, and crystallized from a suitable solvent affording the
corresponding 14 and 19.
3E,5E-3,5-Bis[(4-chlorophenyl)methylidene]-1-(1-propyl)-4-
piperidone (14). Pale yellow microcrystals from ethanol, mp
140–142 ꢁC, yield 1.5 g (78%). IR n (cmꢀ1): 1670, 1612, 1585. 1H-
NMR (300 MHz, CDCl3) d (ppm): 0.85 (t, J ¼ 7.4 Hz, 3H,
NCH2CH2CH3), 1.45 (sextet, J ¼ 7.4 Hz, 2H, NCH2CH2CH3), 2.49
(t, J ¼ 7.5 Hz, 2H, NCH2CH2CH3), 3.76 (s, 4H, 2NCH2), 7.30 (d, J
¼ 8.5 Hz, 4H, arom. H), 7.38 (d, J ¼ 8.6 Hz, 4H, arom. H), 7.72 (s,
2H, 2 olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 11.8,
20.5, 54.8, 59.4, 129.0, 131.7, 133.8, 133.9, 135.2, 187.1.
Elemental analysis: C22H21Cl2NO required C, 68.40; H, 5.48; N,
3.63, found C, 68.55; H, 5.53; N, 3.72.
3E,5E-3,5-Bis[(4-uorophenyl)methylidene]-1-(1-propyl)-4-
piperidone (19). Pale yellow microcrystals from methanol, mp
130–131 ꢁC, yield 1.4 g (79%). IR n (cmꢀ1): 1678, 1612, 1601. 1H-
NMR (300 MHz, CDCl3) d (ppm): 0.86 (t, J ¼ 7.4 Hz, 3H,
NCH2CH2CH3), 1.46 (sextet, J ¼ 7.4 Hz, 2H, NCH2CH2CH3), 2.51
(t, J ¼ 7.6 Hz, 2H, NCH2CH2CH3), 3.78 (s, 4H, 2NCH2), 7.11 (t, J
¼ 8.7 Hz, 4H, arom. H), 7.36–7.41 (m, 4H, arom. H), 7.76 (s, 2H,
2 olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 11.9, 20.6,
54.9, 59.5, 115.8, 116.1, 131.6, 131.7, 132.4, 132.5, 133.27,
133.29, 135.4, 161.4, 164.7, 187.4. Elemental analysis:
Conclusions
From all the above it can be concluded that, indole-based
compounds 30–63 were synthesized in good to excellent yields
(68–86%) through the multi-component reaction of 1-alkyl-3,5-
bis(arylidene)-4-piperidones 11–25 with azomethine ylides
(generated through condensation of isatins 26–28 with sarco-
sine 29). X-ray studies of 46 and 48 provide good support for the
regio- and stereoselectivity of the reaction. Many of the
synthesized spiro-indoles exhibit antitumor properties against
HeLa (cervical cancer) cell line through in vitro sulfo-
rhodamine-B bio-assay with potencies higher than that of
cisplatin (standard reference). Only compound 54 reveals bio-
potency against HepG2 (hepatocellular carcinoma) cell line,
comparable to that of doxorubicin hydrochloride (standard
reference). HYPOGEN of the 3D-pharmacophore studies iden-
ties a 3D-array of ve chemical features in case of HeLa cell
line containing two positive ionizables, one hydrophobic and
one hydrogen bonding acceptor. The estimated bio-properties
owing to the pharmacophic hypothesis are correlated well
with the experimentally observed activities. 2D-QSAR studies
led to a 5 descriptor model validated well by both internal and
C
22H21F2NO required C, 74.77; H, 5.99; N, 3.96, found C, 74.88;
H, 6.08; N, 3.81.
Synthesis of dispiro[3H-indole-3,20-pyrrolidine-30,300-
piperidine]-2(1H),400-diones 30–63 (general procedure)
external validation techniques. The predicated properties ob- A mixture of equimolar amounts of the appropriate 3,5-
tained using the 2D-QSAR technique show high approximations bis(arylidene)-1-alkyl-4-piperidone 11–25 (5 mmol), the corre-
to the experimentally observed data supporting the capability sponding isatines 26–28 and sarcosine 29 in absolute ethanol
for optimizing high potent hits based on the attained model. (25 ml) was boiled under reux for the appropriate time. The
The success of the computational studies although short data separated solid while reuxing was collected and crystallized
points were used, can be attributed to the fact that these studies from a suitable solvent affording the corresponding 30, 34, 41,
utilized a homogeneous data set (non-diverse technique) of 42, 44 and 56. In case of the remaining compounds, the reaction
high structural resemblance (same chemical scaffold).
mixture was concentrated to half of its initial volume and stored
at room temperature overnight, the separated solid was
collected and crystallized from a suitable solvent affording the
corresponding analogues.
Experimental section
Melting points were recorded on a Stuart SMP10 melting point
apparatus. IR spectra (KBr) were recorded on a Shimadzu FT-IR dinyl)methylene]-dispiro[3H-indole-3,20-pyrrolidine-30,300-piperi-
10,100-Dimethyl-40-phenyl-500-[(phenyl)methylidene]-1-[(1-piperi-
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dine]-2(1H),400-dione (30). Obtained from reaction of 11, 26 and
29. Reaction time 10 h, colorless microcrystals from ethanol, mp
(d, J ¼ 12.3 Hz, 1H, upeld H of NCH2N), 4.56 (d, J ¼ 12.3 Hz, 1H,
downeld H of NCH2N), 4.85 (dd, J ¼ 6.9, 11.4 Hz, 1H, pyrroli-
dinyl HC-40), 6.79 (d, J ¼ 7.8 Hz, 1H, arom. H), 6.97–7.41 (m, 14H,
13 arom. H + olenic CH). 13C-NMR (100 MHz, CDCl3) d (ppm):
35.0, 45.1, 45.4, 46.0, 50.7, 54.9, 56.6, 57.1, 57.3, 62.2, 66.4, 75.4,
108.9, 122.0, 126.6, 126.8, 127.3, 128.16, 128.23, 128.5, 128.7,
129.4, 129.9, 133.4, 135.2, 137.4, 138.3, 144.6, 176.2, 198.5.
Elemental analysis: C36H41N5O2 required C, 75.10; H, 7.18; N,
12.16, found C, 75.18; H, 7.22; N, 12.27.
155–157 C, yield 70% (1.96 g). IR n (cmꢀ1): 1699, 1686, 1609,
ꢁ
1
1489, 1466. H-NMR (300 MHz, CDCl3) d (ppm): 1.35–1.43 (m,
2H, piperidinyl H2C-4), 1.51–1.60 (m, 4H, piperidinyl H2C-3/5),
1.63 (d, J ¼ 13.2 Hz, 1H, upeld H of piperidinyl H2C-200), 1.99
(s, 3H, piperidinyl NCH3), 2.17 (s, 3H, pyrrolidinyl NCH3), 2.58
(br s, 4H, piperidinyl NCH2-2/6), 2.86 (dd, J ¼ 2.9, 14.6 Hz, 1H,
upeld H of piperidinyl H2C-600), 3.27–3.36 (m, 3H, downeld
H's of piperidinyl H2C-200, H2C-600, and upeld H of pyrrolidinyl
H2C-50), 3.97 (dd, J ¼ 8.9, 11.3 Hz, 1H, downeld H of pyrroli-
dinyl H2C-50), 4.16 (d, J ¼ 12.6 Hz, 1H, upeld H of NCH2N), 4.54
(d, J ¼ 12.6 Hz, 1H, downeld H of NCH2N), 4.85 (dd, J ¼ 6.9,
11.4 Hz, 1H, pyrrolidinyl HC-40), 6.86 (d, J ¼ 7.8 Hz, 1H, arom.
H), 6.96–7.42 (m, 14H, 13 arom. H + olenic CH). 13C-NMR (75
MHz, CDCl3) d (ppm): 24.3, 26.0, 35.2, 45.3, 45.6, 52.4, 56.9, 57.3,
57.5, 63.3, 66.5, 75.6, 109.2, 122.0, 126.8, 126.9, 127.5, 128.3,
128.4, 128.7, 128.9, 129.6, 130.0, 133.7, 135.5, 137.6, 138.6,
145.2, 176.5, 198.7. Elemental analysis: C36H40N4O2 required C,
77.11; H, 7.19; N, 9.99, found C, 77.11; H, 7.27; N, 10.11.
10,100-Dimethyl-1-[(4-morpholinyl)methylene]-40-phenyl-500-
[(phenyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-30,300-
piperidine]-2(1H),400-dione (31). Obtained from reaction of 11,
27 and 29. Reaction time 12 h, colorless microcrystals from
methanol, mp 169–170 ꢁC, yield 72% (2.02 g). IR n (cmꢀ1): 1694,
1612, 1601, 1491, 1468. 1H-NMR (300 MHz, CDCl3) d (ppm):
1.62 (d, J ¼ 13.2 Hz, 1H, upeld H of piperidinyl H2C-200), 2.00
(s, 3H, piperidinyl NCH3), 2.16 (s, 3H, pyrrolidinyl NCH3), 2.57
(t, J ¼ 4.7 Hz, 4H, morpholinyl 2NCH2), 2.86 (dd, J ¼ 2.7, 14.7
Hz, 1H, upeld H of piperidinyl H2C-600), 3.27–3.36 (m, 3H,
downeld H's of piperidinyl H2C-200, H2C-600 and upeld H of
pyrrolidinyl H2C-50), 3.64 (t, J ¼ 4.4 Hz, 4H, morpholinyl
2OCH2), 3.96 (dd, J ¼ 8.7, 11.1 Hz, 1H, downeld H of pyrro-
lidinyl H2C-50), 4.11 (d, J ¼ 12.6 Hz, 1H, upeld H of NCH2N),
4.52 (d, J ¼ 12.3 Hz, 1H, downeld H of NCH2N), 4.85 (dd, J ¼
6.8, 11.3 Hz, 1H, pyrrolidinyl HC-40), 6.80 (d, J ¼ 7.8 Hz, 1H,
arom. H), 6.97–7.41 (m, 14H, 13 arom. H + olenic CH). 13C-
NMR (75 MHz, CDCl3) d (ppm): 35.1, 45.3, 45.5, 51.3, 56.7,
57.3, 57.5, 62.6, 66.6, 66.9, 75.5, 109.0, 122.3, 126.8, 126.9,
127.6, 128.3, 128.4, 128.7, 128.9, 129.5, 130.0, 133.6, 135.3,
137.6, 138.5, 144.7, 176.5, 198.6. Elemental analysis:
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-10,100-
dimethyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (33). Obtained
from reaction of 12, 26 and 29. Reaction time 12 h, pale yellow
microcrystals from methanol, mp 167–168 ꢁC, yield 76% (2.40
g). IR n (cmꢀ1): 1705, 1686, 1609, 1491, 1466. 1H-NMR (300
MHz, CDCl3) d (ppm): 1.35–1.42 (m, 2H, piperidinyl H2C-4),
1.48–1.56 (m, 4H, piperidinyl H2C-3/5), 1.61 (d, J ¼ 12.6 Hz,
1H, upeld H of piperidinyl H2C-200), 2.00 (s, 3H, piperidinyl
NCH3), 2.15 (s, 3H, pyrrolidinyl NCH3), 2.52 (t, J ¼ 5.1 Hz, 4H,
piperidinyl NCH2-2/6), 2.84 (dd, J ¼ 2.7, 15.0 Hz, 1H, upeld H
of piperidinyl H2C-600), 3.23 (d, J ¼ 13.8 Hz, 2H, downeld H's
of piperidinyl H2C-200, H2C-600), 3.32 (dd, J ¼ 7.1, 8.6 Hz, 1H,
upeld H of pyrrolidinyl H2C-50), 3.91 (dd, J ¼ 8.7, 11.1 Hz, 1H,
downeld H of pyrrolidinyl H2C-50), 4.09 (d, J ¼ 12.3 Hz, 1H,
upeld H of NCH2N), 4.51 (d, J ¼ 12.3 Hz, 1H, downeld H of
NCH2N), 4.79 (dd, J ¼ 6.9, 10.8 Hz, 1H, pyrrolidinyl HC-40),
6.83 (d, J ¼ 7.5 Hz, 1H, arom. H), 6.90–7.42 (m, 12H, 11 arom.
H + olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 23.7,
25.2, 34.3, 44.0, 44.4, 51.4, 55.9, 56.0, 56.3, 62.2, 65.3, 74.6,
109.2, 121.3, 125.6, 126.5, 128.2, 128.3, 128.6, 128.7, 130.6,
131.4, 131.5, 132.1, 133.2, 133.5, 133.6, 135.1, 137.1, 144.7,
175.2, 197.4. Elemental analysis: C36H38Cl2N4O2 required C,
68.67; H, 6.08; N, 8.90, found C, 68.79; H, 6.13; N, 9.04.
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-10,100-
dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (34). Obtained from
reaction of 12, 27 and 29. Reaction time 12 h, colorless micro-
crystals from ethanol, mp 195–197 ꢁC, yield 79% (2.50 g). IR n
(cmꢀ1): 1705, 1682, 1614, 1491, 1466. 1H-NMR (400 MHz, CDCl3)
d (ppm): 1.60 (d, J ¼ 12.7 Hz, 1H, upeld H of piperidinyl H2C-200),
2.01 (s, 3H, piperidinyl NCH3), 2.14 (s, 3H, pyrrolidinyl NCH3), 2.57
(t, J ¼ 4.3 Hz, 4H, morpholinyl 2NCH2), 2.84 (dd, J ¼ 2.2, 14.5 Hz,
1H, upeld H of piperidinyl H2C-600), 3.21–3.25 (m, 2H, downeld
H's of piperidinyl H2C-200 and H2C-600), 3.31 (t, J ¼ 7.8 Hz, 1H,
upeld H of pyrrolidinyl H2C-50), 3.64 (t, J ¼ 4.3 Hz, 4H, mor-
pholinyl 2OCH2), 3.89 (dd, J ¼ 9.1, 10.7 Hz, 1H, downeld H of
pyrrolidinyl H2C-50), 4.09 (d, J ¼ 12.5 Hz, 1H, upeld H of NCH2N),
4.53 (d, J ¼ 12.5 Hz, 1H, downeld H of NCH2N), 4.79 (dd, J ¼ 7.0,
11.0 Hz, 1H, pyrrolidinyl HC-40), 6.79 (d, J ¼ 7.8 Hz, 1H, arom. H),
6.89–7.33 (m, 12H, 11 arom. H + olenic CH). 13C-NMR (100 MHz,
CDCl3) d (ppm): 34.9, 44.7, 45.1, 51.2, 56.6, 57.0, 57.3, 62.5, 66.2,
66.7, 75.3, 108.9, 122.1, 126.4, 127.3, 128.4, 128.5, 128.9, 130.7,
131.0, 132.6, 133.4, 133.7, 134.7, 136.2, 136.8, 144.5, 176.2, 198.1.
Elemental analysis: C35H36Cl2N4O3 required C, 66.56; H, 5.75; N,
8.87, found C, 66.63; H, 5.82; N, 9.01.
C
35H38N4O3 required C, 74.71; H, 6.81; N, 9.96, found C, 74.77;
H, 6.88; N, 10.03.
10,100-Dimethyl-1-[1-(4-methylpiperazinyl)methylene]-40-phenyl-
500-[(phenyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-30,300-
piperidine]-2(1H),400-dione (32). Obtained from reaction of 11, 28
and 29. Reaction time 15 h, colorless microcrystals from ethanol,
mp 142–144 ꢁC, yield 70% (2.01 g). IR n (cmꢀ1): 1694, 1682, 1616,
1601, 1470. 1H-NMR (300 MHz, CDCl3) d (ppm): 1.61 (d, J ¼ 12.6
Hz, 1H, upeld H of piperidinyl H2C-200), 1.99 (s, 3H, piperidinyl
NCH3), 2.16 (s, 3H, pyrrolidinyl NCH3), 2.25 (s, 3H, piperazinyl
NCH3), 2.38 (br s, 4H, piperazinyl 2NCH2), 2.62 (br s, 4H, piper-
azinyl 2NCH2), 2.86 (dd, J ¼ 2.6, 15.2 Hz, 1H, upeld H of
piperidinyl H2C-600), 3.26–3.36 (m, 3H, downeld H's of piper-
idinyl H2C-200, H2C-600 and upeld H of pyrrolidinyl H2C-50), 3.97
(dd, J ¼ 8.7, 11.4 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.11
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-10,100-
dimethyl-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-
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3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (35). Obtained
from reaction of 12, 28 and 29. Reaction time 15 h, colorless
microcrystals from methanol, mp 145–147 ꢁC, yield 69% (2.22 g).
NCH2CH3), 2.56 (br s, 4H, morpholinyl 2NCH2), 2.76 (dd, J ¼
2.7, 14.5 Hz, 1H, upeld H of piperidinyl H2C-600), 3.14 (dd, J ¼
1.8, 12.6 Hz, 1H, downeld H of piperidinyl H2C-200), 3.26 (d, J ¼
14.6 Hz, 1H, downeld H of piperidinyl H2C-600), 3.32 (dd, J ¼
7.4, 8.7 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.64 (t, J ¼ 4.5
Hz, 4H, morpholinyl 2OCH2), 3.87 (dd, J ¼ 8.9, 10.8 Hz, 1H,
downeld H of pyrrolidinyl H2C-50), 4.02 (d, J ¼ 12.6 Hz, 1H,
upeld H of NCH2N), 4.60 (d, J ¼ 12.6 Hz, 1H, downeld H of
NCH2N), 4.77 (dd, J ¼ 7.3, 10.9 Hz, 1H, pyrrolidinyl HC-40), 6.75
(d, J ¼ 7.8 Hz, 1H, arom. H), 6.92–7.35 (m, 12H, 11 arom. H +
olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 10.9, 34.4,
44.3, 50.7, 50.8, 52.6, 55.8, 56.3, 61.4, 64.7, 65.9, 74.9, 109.1,
121.5, 125.3, 126.6, 128.2, 128.4, 128.7, 130.9, 131.4, 131.6,
133.2, 133.4, 133.6, 135.5, 137.2, 144.5, 175.1, 197.8. Elemental
analysis: C36H38Cl2N4O3 required C, 66.97; H, 5.93; N, 8.68,
found C, 67.05; H, 5.97; N, 8.81.
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-10-methyl-
1-[(1-piperidinyl)methylene]-100-(1-propyl)-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (38). Obtained from
reaction of 14, 26 and 29. Reaction time 11 h, colorless micro-
crystals from methanol, mp 134–136 ꢁC, yield 69% (2.27 g). IR n
(cmꢀ1): 1694, 1609, 1491, 1466. 1H-NMR (400 MHz, CDCl3)
d (ppm): 0.73 (t, J ¼ 7.4 Hz, 3H, NCH2CH2CH3), 0.96–1.21 (m, 2H,
NCH2CH2CH3), 1.30–1.38 (m, 2H, piperidinyl CH2-4), 1.51–1.56
(m, 4H, piperidinyl CH2-3/5), 1.71 (d, J ¼ 12.6 Hz, 1H, upeld H
of piperidinyl H2C-200), 1.96 (sextet, J ¼ 5.1, 11.4 Hz, 1H, upeld H
of NCH2CH2CH3), 2.00–2.19 (m, 4H, downeld H of NCH2CH2-
CH3 + pyrrolidinyl NCH3), 2.55 (br s, 4H, piperidinyl 2NCH2), 2.81
(dd, J ¼ 2.6, 14.6 Hz, 1H, upeld H of piperidinyl H2C-600), 3.14
(dd, J ¼ 1.3, 12.7 Hz, 1H, downeld H of piperidinyl H2C-200), 3.26
(d, J ¼ 14.7 Hz, 1H, downeld H of piperidinyl H2C-600), 3.36 (t, J
¼ 8.1 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.90 (dd, J ¼ 9.1,
10.6 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.06 (d, J ¼ 12.8
Hz, 1H, upeld H of NCH2N), 4.62 (d, J ¼ 12.8 Hz, 1H, downeld
H of NCH2N), 4.78 (dd, J ¼ 7.3, 10.8 Hz, 1H, pyrrolidinyl HC-40),
6.80 (d, J ¼ 7.8 Hz, 1H, arom. H), 6.92–7.39 (m, 12H, 11 arom. H +
olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 11.5, 18.7,
23.6, 25.3, 34.4, 44.6, 51.6, 53.3, 56.5, 59.1, 61.3, 62.0, 64.5, 74.9,
109.1, 121.3, 125.2, 126.6, 128.1, 128.4, 128.7, 131.0, 131.4, 131.6,
133.3, 133.6, 135.4, 137.3, 144.8, 175.1, 198.0. Elemental analysis:
C38H42Cl2N4O2 required C, 69.40; H, 6.44; N, 8.52, found C,
69.48; H, 6.48; N, 8.61.
1
IR n (cmꢀ1): 1690, 1611, 1491, 1464. H-NMR (300 MHz, CDCl3)
d (ppm): 1.59 (d, J ¼ 12.3 Hz, 1H, upeld H of piperidinyl H2C-200),
2.00 (s, 3H, piperidinyl NCH3), 2.14 (s, 3H, pyrrolidinyl NCH3),
2.25 (s, 3H, piperazinyl NCH3), 2.37 (br s, 4H, piperazinyl 2NCH2),
2.61 (br s, 4H, piperazinyl 2NCH2), 2.83 (dd, J ¼ 1.8, 14.4 Hz, 1H,
upeld H of piperidinyl H2C-600), 3.21–3.34 (m, 3H, downeld H's
of piperidinyl H2C-200, H2C-600 and upeld H of pyrrolidinyl H2C-
50), 3.89 (t, J ¼ 9.8 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.12
(d, J ¼ 12.6 Hz, 1H, upeld H of NCH2N), 4.55 (d, J ¼ 12.3 Hz, 1H,
downeld H of NCH2N), 4.78 (dd, J ¼ 6.9, 11.1 Hz, 1H, pyrrolidinyl
HC-40), 6.80 (d, J ¼ 7.8 Hz, 1H, arom. H), 6.89–7.34 (m, 12H, 11
arom. H + olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 35.0,
44.8, 45.1, 46.0, 50.8, 54.9, 56.7, 57.0, 57.3, 62.3, 66.3, 75.3, 109.1,
122.0, 126.5, 127.3, 128.4, 128.6, 128.9, 130.7, 131.1, 132.6, 133.5,
133.9, 134.6, 136.1, 136.9, 144.7, 176.2, 198.2. Elemental analysis:
C36H39Cl2N5O2 required C, 67.08; H, 6.10; N, 10.86, found C,
67.21; H, 6.14; N, 11.03.
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-100-ethyl-
10-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (36). Obtained from
reaction of 13, 26 and 29. Reaction time 10 h, colorless micro-
ꢁ
crystals from ethanol, mp 160–162 C, yield 70% (2.26 g). IR n
(cmꢀ1): 1697, 1611, 1491, 1468. 1H-NMR (400 MHz, CDCl3)
d (ppm): 0.72 (t, J ¼ 7.2 Hz, 3H, NCH2CH3), 1.33–1.42 (m, 2H,
piperidinyl H2C-4), 1.50–1.58 (m, 4H, piperidinyl H2C-3/5), 1.69
(d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl H2C-200), 2.07 (sextet, J
¼ 7.1, 12.3 Hz, 1H, upeld H of NCH2CH3), 2.15 (s, 3H, pyrroli-
dinyl NCH3), 2.30 (sextet, J ¼ 7.3, 12.3 Hz, 1H, downeld H of
NCH2CH3), 2.54 (br s, 4H, piperidinyl NH2C-2/6), 2.77 (dd, J ¼ 2.6,
14.6 Hz, 1H, upeld H of piperidinyl H2C-600), 3.16 (dd, J ¼ 1.7,
12.7 Hz, 1H, downeld H of piperidinyl H2C-200), 3.27 (d, J ¼ 14.6
Hz, 1H, downeld H of piperidinyl H2C-600), 3.35 (dd, J ¼ 7.5, 8.6
Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.90 (dd, J ¼ 9.0, 10.7
Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.05 (d, J ¼ 12.7 Hz,
1H, upeld H of NCH2N), 4.61 (d, J ¼ 12.7 Hz, 1H, downeld H of
NCH2N), 4.79 (dd, J ¼ 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-40), 6.80
(d, J ¼ 7.8 Hz, 1H, arom. H), 6.93–7.38 (m, 12H, 11 arom. H +
olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 10.8, 23.6,
25.3, 34.3, 44.4, 50.7, 51.6, 52.7, 55.8, 56.3, 62.0, 64.6, 74.8, 109.1,
121.3, 125.3, 126.5, 128.1, 128.4, 128.6, 130.9, 131.3, 131.6, 133.2,
133.49, 133.5, 135.3, 137.2, 144.7, 175.0, 197.9. Elemental anal-
ysis: C37H40Cl2N4O2 required C, 69.04; H, 6.26; N, 8.70, found C,
69.17; H, 6.29; N, 8.83.
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-10-methyl-
1-[(4-morpholinyl)methylene]-100-(1-propyl)-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (39). Obtained from
reaction of 14, 27 and 29. Reaction time 12 h, colorless micro-
ꢁ
40-(4-Chlorophenyl)-500-[(4-chlorophenyl)methylidene]-100-ethyl-
10-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (37). Obtained
from reaction of 13, 27 and 29. Reaction time 12 h, colorless
microcrystals from ethanol, mp 188–190 ꢁC, yield 77% (2.49 g).
crystals from methanol, mp 187–189 C, yield 73% (2.41 g). IR n
(cmꢀ1): 1699, 1684, 1611, 1489, 1466. 1H-NMR (400 MHz, CDCl3)
d (ppm): 0.74 (t, J ¼ 7.3 Hz, 3H, NCH2CH2CH3), 0.96–1.08 (m, 1H,
upeld H of NCH2CH2CH3), 1.12–1.26 (m, 1H, downeld H of
NCH2CH2CH3), 1.71 (d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl
H2C-200), 1.98, (sextet, J ¼ 5.1, 11.5 Hz, 1H, upeld H of NCH2-
CH2CH3), 2.13–2.19 (m, 4H, pyrrolidinyl NCH3 + downeld H of
NCH2CH2CH3), 2.59 (br s, 4H, morpholinyl 2NCH2), 2.82 (dd, J ¼
2.6, 14.5 Hz, 1H, upeld H of piperidinyl H2C-600), 3.14 (dd, J ¼ 1.4,
12.7 Hz, 1H, downeld H of piperidinyl H2C-200), 3.27 (d, J ¼ 14.6
Hz, 1H, downeld H of piperidinyl H2C-600), 3.35 (t, J ¼ 8.1 Hz, 1H,
1
IR n (cmꢀ1): 1701, 1686, 1611, 1491, 1468. H-NMR (400 MHz,
CDCl3) d (ppm): 0.71 (t, J ¼ 7.1 Hz, 3H, NCH2CH3), 1.66 (d, J ¼
12.6 Hz, 1H, upeld H of piperidinyl H2C-200), 2.09 (sextet, J ¼
7.0, 12.4 Hz, 1H, upeld H of NCH2CH3), 2.12 (s, 3H, pyrroli-
dinyl NCH3), 2.29 (sextet, J ¼ 7.3, 12.4 Hz, 1H, downeld H of
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upeld H of pyrrolidinyl H2C-50), 3.66 (t, J ¼ 4.5 Hz, 4H, mor-
pholinyl 2OCH2), 3.88 (dd, J ¼ 9.1, 10.7 Hz, 1H, downeld H of
pyrrolidinyl H2C-50), 4.05 (d, J ¼ 12.7 Hz, 1H, upeld H of NCH2N),
4.63 (d, J ¼ 12.7 Hz, 1H, downeld H of NCH2N), 4.78 (dd, J ¼ 7.2,
10.8 Hz, 1H, pyrrolidinyl HC-40), 6.77 (d, J ¼ 7.8 Hz, 1H, arom. H),
6.94–7.38 (m, 12H, 11 arom. H + olenic CH). 13C-NMR (75 MHz,
DMSO-d6) d (ppm): 11.5, 18.7, 34.3, 44.4, 50.8, 53.2, 56.4, 59.1,
61.3, 64.5, 65.9, 74.9, 109.0, 121.5, 125.2, 126.6, 128.1, 128.4, 128.7,
130.9, 131.4, 131.6, 133.2, 133.45, 133.5, 135.4, 137.2, 144.5, 175.1,
197.9. Elemental analysis: C37H40Cl2N4O3 required C, 67.37; H,
6.11; N, 8.49, found C, 67.49; H, 6.15; N, 8.54.
100-Benzyl-40-(4-chlorophenyl)-500-[(4-chlorophenyl)methylidene]-
10-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (40). Obtained from
reaction of 15, 26 and 29. Reaction time 10 h, pale yellow
microcrystals from ethanol, mp 154–156 ꢁC, yield 73% (2.57 g). IR
n (cmꢀ1): 1699, 1682, 1605, 1491, 1466. 1H-NMR (400 MHz, CDCl3)
d (ppm): 1.31–1.40 (m, 2H, piperidinyl H2C-4), 1.43–1.52 (m, 4H,
piperidinyl H2C-3/5), 1.85 (d, J ¼ 12.7 Hz, 1H, upeld H of
piperidinyl H2C-200), 2.15 (s, 3H, pyrrolidinyl NCH3), 2.47–2.54 (m,
4H, piperidinyl H2CN-2/6), 2.72 (dd, J ¼ 2.6, 14.7 Hz, 1H, upeld
H of piperidinyl H2C-600), 3.19–3.36 (m, 3H, downeld H of
piperidinyl H2C-200, H2C-600 + upeld H of PhCH2), 3.40 (t, J ¼ 8.2
Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.49 (d, J ¼ 14.0 Hz, 1H,
downeld H of PhCH2), 3.92 (dd, J ¼ 9.2, 10.5 Hz, 1H, downeld
H of pyrrolidinyl H2C-50), 4.00 (d, J ¼ 12.8 Hz, 1H, upeld H of
NCH2N), 4.50 (d, J ¼ 12.8 Hz, 1H, downeld H of NCH2N), 4.78
(dd, J ¼ 7.5, 10.6 Hz, 1H, pyrrolidinyl HC-40), 6.80–7.43 (m, 18H,
17 arom. H + olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm):
23.6, 25.3, 34.4, 45.0, 51.7, 53.0, 56.5, 56.7, 60.9, 62.3, 64.6, 75.0,
109.2, 121.6, 125.2, 126.7, 126.9, 127.9, 128.15, 128.2, 128.3,
131.0, 131.3, 131.5, 133.2, 133.5, 135.4, 135.7, 137.3, 144.7, 175.3,
197.9. Elemental analysis: C42H42Cl2N4O2 required C, 71.48; H,
6.00; N, 7.94, found C, 71.56; H, 5.98; N, 7.98.
40-(2,4-Dichlorophenyl)-500-[(2,4-dichlorophenyl)methylidene]-
10,100-dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-
3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (42). Obtained
from reaction of 16, 27 and 29. Reaction time 9 h, colorless
microcrystals from ethanol, mp 205–207 ꢁC, yield 80% (2.80 g).
IR n (cmꢀ1): 1701, 1611, 1483, 1466. 1H-NMR (300 MHz, CDCl3)
d (ppm): 1.73 (d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl H2C-
200), 1.90 (s, 3H, piperidinyl NCH3), 2.09 (s, 3H, pyrrolidinyl
NCH3), 2.63 (br s, 4H, morpholinyl 2NCH2), 2.82–2.93 (m, 2H,
upeld H of piperidinyl H2C-600 + downeld H of piperidinyl
H2C-200), 3.06 (d, J ¼ 15.0 Hz, 1H, downeld H of piperidinyl
H2C-600), 3.49 (t, J ¼ 8.6 Hz, 1H, upeld H of pyrrolidinyl H2C-50),
3.70 (br s, 4H, morpholinyl 2OCH2), 3.86 (t, J ¼ 9.0 Hz, 1H,
downeld H of pyrrolidinyl H2C-50), 4.20 (d, J ¼ 12.6 Hz, 1H,
upeld H of NCH2N), 4.56 (d, J ¼ 12.3 Hz, 1H, downeld H of
NCH2N), 5.08 (t, J ¼ 8.3 Hz, 1H, pyrrolidinyl HC-40), 6.83–7.47
(m, 10H, 9 arom. H + olenic CH), 7.96 (d, J ¼ 8.7 Hz, 1H, arom.
H). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 34.2, 44.9, 50.7, 56.3,
56.9, 57.4, 57.5, 61.6, 63.1, 65.9, 75.8, 109.3, 122.0, 124.7, 126.5,
127.2, 128.4, 128.6, 129.1, 131.2, 131.3, 131.7, 132.0, 132.1,
134.1, 134.4, 134.6, 135.4, 135.7, 144.5, 175.3, 195.9. Elemental
analysis: C35H34Cl4N4O3 required C, 60.01; H, 4.89; N, 8.00,
found C, 60.13; H, 4.94; N, 8.17.
10,100-Dimethyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methyl-
idene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyrro-
lidine-30,300-piperidine]-2(1H),400-dione (43). Obtained from
reaction of 17, 26 and 29. Reaction time 12 h, pale yellow
microcrystals from ethanol, mp 170–172 ꢁC, yield 86% (2.57 g).
1
IR n (cmꢀ1): 1697, 1686, 1595, 1582, 1508, 1462. H-NMR (400
MHz, CDCl3) d (ppm): 1.33–1.40 (m, 2H, piperidinyl H2C-4), 1.47–
1.53 (m, 4H, piperidinyl H2C-3/5), 1.59 (d, J ¼ 12.7 Hz, 1H,
upeld H of piperidinyl H2C-200), 1.99 (s, 3H, piperidinyl NCH3),
2.15 (s, 3H, pyrrolidinyl NCH3), 2.52 (br s, 4H, piperidinyl NCH2-
2/6), 2.84 (d, J ¼ 14.5 Hz, 1H, upeld H of piperidinyl H2C-600),
3.20–3.25 (m, 2H, downeld H's of piperidinyl H2C-200 and H2C-
600), 3.32 (t, J ¼ 7.9 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.90
(t, J ¼ 9.9 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.10 (d, J ¼
12.6 Hz, 1H, upeld H of NCH2N), 4.51 (d, J ¼ 12.6 Hz, 1H,
downeld H of NCH2N), 4.80 (dd, J ¼ 7.1, 10.9 Hz, 1H, pyrroli-
dinyl HC-40), 6.82 (d, J ¼ 7.6 Hz, 1H, arom. H), 6.96–7.14 (m, 10H,
9 arom. H + olenic CH), 7.36 (t, J ¼ 6.8 Hz, 2H, arom. H). 13C-
NMR (100 MHz, CDCl3) d (ppm): 24.1, 25.8, 34.9, 44.7, 45.1,
52.2, 56.9, 57.1, 57.4, 63.1, 65.9, 75.5, 109.1, 114.9, 115.1, 115.3,
115.5, 121.8, 126.4, 127.2, 128.7, 130.8, 130.9, 131.2, 131.3, 131.4,
131.7, 131.8, 133.15, 133.16, 134.0, 134.1, 136.3, 145.0, 160.6,
161.4, 163.0, 163.8, 176.3, 198.4. Elemental analysis:
C36H38F2N4O2 required C, 72.46; H, 6.42; N, 9.39, found C, 72.52;
H, 6.53; N, 9.48.
100-Benzyl-40-(4-chlorophenyl)-500-[(4-chlorophenyl)methylidene]-
10-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (41). Obtained from
reaction of 15, 27 and 29. Reaction time 11 h, pale yellow
microcrystals from ethanol, mp 198–200 ꢁC, yield 82% (2.90 g).
1
IR n (cmꢀ1): 1697, 1686, 1605, 1491, 1462. H-NMR (400 MHz,
CDCl3) d (ppm): 1.82 (d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl
H2C-200), 2.12 (s, 3H, pyrrolidinyl NCH3), 2.48–2.58 (m, 4H,
piperidinyl 2NCH2), 2.70 (dd, J ¼ 2.6, 14.6 Hz, 1H, upeld H of
piperidinyl H2C-600), 3.20–3.34 (m, 3H, downeld H of piper-
idinyl H2C-200, H2C-600 + upeld H of PhCH2), 3.38 (dd, J ¼ 7.6, 8.7
Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.48 (d, J ¼ 14.0 Hz,
1H, downeld H of PhCH2), 3.60 (t, J ¼ 4.6 Hz, 4H, morpholinyl
2OCH2), 3.89 (dd, J ¼ 9.1, 10.5 Hz, 1H, downeld H of pyrroli-
dinyl H2C-50), 3.95 (d, J ¼ 12.8 Hz, 1H, upeld H of NCH2N), 4.48
(d, J ¼ 12.8 Hz, 1H, downeld H of NCH2N), 4.76 (dd, J ¼ 7.5,
10.7 Hz, 1H, pyrrolidinyl HC-40), 6.77–7.40 (m, 18H, 17 arom. H
+ olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 35.2, 46.0,
51.5, 53.8, 57.3, 57.5, 61.9, 62.6, 65.6, 66.9, 76.1, 109.2, 122.5,
126.2, 127.3, 127.7, 128.3, 128.4, 128.5, 128.7, 129.1, 131.1,
131.3, 132.9, 133.5, 133.7, 134.8, 136.0, 136.6, 137.1, 144.7,
176.4, 198.8. Elemental analysis: C41H40Cl2N4O3 required C,
69.58; H, 5.70; N, 7.92, found C, 69.69; H, 5.68; N, 8.06.
10,100-Dimethyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methyl-
idene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (44). Obtained from
reaction of 17, 27 and 29. Reaction time 14 h, almost colorless
microcrystals from ethanol, mp 176–178 ꢁC, yield 73% (2.18 g).
1
IR n (cmꢀ1): 1705, 1684, 1612, 1508, 1489, 1464. H-NMR (400
MHz, CDCl3) d (ppm): 1.60 (d, J ¼ 12.8 Hz, 1H, upeld H of
piperidinyl H2C-200), 2.01 (s, 3H, piperidinyl NCH3), 2.14 (s, 3H,
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pyrrolidinyl NCH3), 2.57 (br s, 4H, morpholinyl 2NCH2), 2.84 (d, Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.07 (d, J ¼ 12.7 Hz,
J ¼ 14.4 Hz, 1H, upeld H of piperidinyl H2C-600), 3.20–3.26 (m, 1H, upeld H of NCH2N), 4.61 (d, J ¼ 12.7 Hz, 1H, downeld H of
2H, downeld H's of piperidinyl H2C-200 and H2C-600), 3.32 (t, J ¼ NCH2N), 4.80 (dd, J ¼ 7.3, 10.8 Hz, 1H, pyrrolidinyl HC-40), 6.81 (d,
8.0 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.63 (br s, 4H, J ¼ 7.8 Hz, 1H, arom. H), 6.93–7.14 (m, 10H, 9 arom. H + olenic
morpholinyl 2OCH2), 3.89 (t, J ¼ 9.9 Hz, 1H, downeld H of CH), 7.41 (dd, J ¼ 5.6, 8.3 Hz, 2H, arom. H). 13C-NMR (75 MHz,
pyrrolidinyl H2C-50), 4.11 (d, J ¼ 12.5 Hz, 1H, upeld H of CDCl3) d (ppm): 11.3, 24.2, 26.0, 35.1, 45.3, 51.6, 52.5, 53.4, 56.5,
NCH2N), 4.53 (d, J ¼ 12.5 Hz, 1H, downeld H of NCH2N), 4.80 57.5, 63.2, 65.4, 75.9, 109.2, 115.0, 115.2, 115.4, 115.7, 121.9,
(dd, J ¼ 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-40), 6.80 (d, J ¼ 7.6 Hz, 126.3, 127.4, 128.9, 131.2, 131.3, 131.9, 132.0, 133.27, 133.29,
1H, arom. H), 6.96–7.14 (m, 10H, 9 arom. H + olenic CH), 7.35 134.3, 134.4, 136.7, 145.2, 160.4, 161.1, 163.6, 164.4, 176.2, 199.1.
(t, J ¼ 6.8 Hz, 2H, arom. H). 13C-NMR (100 MHz, CDCl3) d (ppm): Elemental analysis: C37H40F2N4O2 required C, 72.76; H, 6.60; N,
34.9, 44.6, 45.1, 51.2, 56.9, 57.1, 57.3, 62.5, 66.1, 66.7, 75.4, 9.17, found C, 72.84; H, 6.64; N, 9.28.
108.9, 115.0, 115.2, 115.3, 115.5, 122.1, 126.5, 127.4, 128.8,
100-Ethyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methylidene]-
130.7, 130.8, 131.15, 131.18, 131.7, 131.8, 133.06, 133.08, 133.90, 10-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-
133.93, 136.4, 144.5, 160.6, 161.4, 163.1, 163.9, 176.3, 198.3. pyrrolidine-30,300-piperidine]-2(1H),400-dione (47). Obtained
Elemental analysis: C35H36F2N4O3 required C, 70.22; H, 6.06; N, from reaction of 18, 27 and 29. Reaction time 11 h, colorless
ꢁ
9.36, found C, 70.29; H, 6.17; N, 9.43.
microcrystals from methanol, mp 191–193 C, yield 73% (2.23
10,100-Dimethyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methyl- g). IR n (cmꢀ1): 1707, 1686, 1607, 1508, 1468. 1H-NMR (400
idene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole- MHz, CDCl3) d (ppm): 0.73 (t, J ¼ 7.2 Hz, 3H, NCH2CH3), 1.68 (d,
3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (45). Obtained J ¼ 12.7 Hz, 1H, upeld H of piperidinyl H2C-200), 2.09 (sextet, J
from reaction of 17, 28 and 29. Reaction time 15 h, yellow ¼ 7.0, 12.4 Hz, upeld H of NCH2CH3), 2.14 (s, 3H, pyrrolidinyl
microcrystals from ethanol, mp 180–182 ꢁC, yield 82% (2.51 g). NCH3), 2.32 (sextet, J ¼ 7.3, 12.3 Hz, 1H, downeld H of
1
IR n (cmꢀ1): 1686, 1605, 1585, 1508, 1462. H-NMR (400 MHz, NCH2CH3), 2.59 (br s, 4H, morpholinyl 2NCH2), 2.79 (dd, J ¼
CDCl3) d (ppm): 1.59 (d, J ¼ 12.7 Hz, 1H, upeld H of piperidinyl 2.6, 14.5 Hz, 1H, upeld H of piperidinyl H2C-600), 3.15 (dd, J ¼
H2C-200), 2.00 (s, 3H, piperidinyl NCH3), 2.14 (s, 3H, pyrrolidinyl 1.9, 12.7 Hz, 1H, downeld H of piperidinyl H2C-200), 3.28 (d, J ¼
NCH3), 2.24 (s, 3H, piperazinyl NCH3), 2.37 (br s, 4H, piper- 14.4 Hz, 1H, downeld H of piperidinyl H2C-600), 3.36 (dd, J ¼
azinyl 2NCH2), 2.62 (br s, 4H, piperazinyl 2NCH2), 2.84 (d, J ¼ 7.4, 8.7 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.66 (t, J ¼ 4.5
14.4 Hz, 1H, upeld H of piperidinyl H2C-600), 3.21–3.26 (m, 2H, Hz, 4H, morpholinyl 2OCH2), 3.89 (dd, J ¼ 8.9, 10.9 Hz, 1H,
downeld H's of piperidinyl H2C-200 and H2C-600), 3.32 (t, J ¼ 7.8 downeld H of pyrrolidinyl H2C-50), 4.06 (d, J ¼ 12.6 Hz, 1H,
Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.89 (t, J ¼ 9.9 Hz, 1H, upeld H of NCH2N), 4.62 (d, J ¼ 12.6 Hz, 1H, downeld H of
downeld H of pyrrolidinyl H2C-50), 4.12 (d, J ¼ 12.5 Hz, 1H, NCH2N), 4.80 (dd, J ¼ 7.3, 10.9 Hz, 1H, pyrrolidinyl HC-40), 6.78
upeld H of NCH2N), 4.55 (d, J ¼ 12.6 Hz, 1H, downeld H of (d, J ¼ 7.6 Hz, 1H, arom. H), 6.95–7.41 (m, 12H, 11 arom. H +
NCH2N), 4.80 (dd, J ¼ 7.0, 10.9 Hz, 1H, pyrrolidinyl HC-40), 6.80 olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 11.4, 35.2,
(d, J ¼ 8.0 Hz, 1H, arom. H), 6.96–7.15 (m, 10H, 9 arom. H + 45.2, 51.5, 51.7, 53.4, 56.5, 57.5, 62.6, 65.6, 67.0, 75.9, 109.0,
olenic CH), 7.35 (t, J ¼ 6.8 Hz, 2H, arom. H). 13C-NMR (100 115.1, 115.4, 115.5, 115.8, 122.3, 126.4, 127.6, 129.0, 131.2,
MHz, CDCl3) d (ppm): 35.0, 44.6, 45.1, 46.0, 50.8, 54.9, 56.9, 131.3, 131.5, 132.0, 132.1, 133.18, 133.2, 134.2, 134.3, 136.8,
57.1, 57.3, 62.2, 66.1, 75.4, 109.0, 114.9, 115.1, 115.2, 115.5, 144.8, 160.4, 161.2, 163.7, 176.2, 185.4, 199.1. Elemental anal-
122.0, 126.5, 127.3, 128.7, 128.8, 130.75, 130.83, 131.18, 131.22, ysis: C36H38F2N4O3 required C, 70.57; H, 6.25; N, 9.14, found C,
131.7, 131.8, 133.2, 133.95, 133.98, 136.3, 144.7, 160.6, 161.3, 70.71; H, 6.28; N, 9.21.
163.0, 163.8, 176.2, 198.3. Elemental analysis: C36H39F2N5O2
required C, 70.68; H, 6.43; N, 11.45, found C, 70.72; H, 6.48; N, 1-[(1-piperidinyl)methylene]-100-(1-propyl)-dispiro[3H-indole-3,20-
11.53.
pyrrolidine-30,300-piperidine]-2(1H),400-dione (48). Obtained from
40-(4-Fluorophenyl)-500-[(4-uorophenyl)methylidene]-10-methyl-
100-Ethyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methylidene]- reaction of 19, 26 and 29. Reaction time 15 h, colorless micro-
ꢁ
10-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyr- crystals from ethanol, mp 191–193 C, yield 87% (2.70 g). IR n
rolidine-30,300-piperidine]-2(1H),400-dione (46). Obtained from (cmꢀ1): 1697, 1682, 1609, 1508, 1466. 1H-NMR (300 MHz, CDCl3)
reaction of 18, 26 and 29. Reaction time 10 h, colorless micro- d (ppm): 0.73 (t, J ¼ 7.4 Hz, 3H, NCH2CH2CH3), 1.02–1.18 (m, 2H,
ꢁ
crystals from ethanol, mp 194–196 C, yield 85% (2.59 g). IR n NCH2CH2CH3), 1.35–1.62 (m, 6H, piperidinyl 3CH2), 1.72 (d, J ¼
(cmꢀ1): 1699, 1684, 1609, 1508, 1466. 1H-NMR (400 MHz, CDCl3) 12.9 Hz, 1H, upeld H of piperidinyl H2C-200), 1.98 (sextet, J ¼ 5.1,
d (ppm): 0.72 (t, J ¼ 7.1 Hz, 3H, NCH2CH3), 1.35–1.44 (m, 2H, 10.5 Hz, 1H, upeld H of NCH2CH2CH3), 2.08–2.16 (m, 4H,
piperidinyl H2C-4), 1.49–1.56 (m, 4H, piperidinyl H2C-3/5), 1.69 (d, pyrrolidinyl NCH3 + downeld H of NCH2CH2CH3), 2.58 (br s,
J ¼ 12.7 Hz, 1H, upeld H of piperidinyl H2C-200), 2.08 (sextet, J ¼ 4H, piperidinyl 2NCH2), 2.83 (dd, J ¼ 2.7, 14.7 Hz, 1H, upeld H
7.0, 12.4 Hz, 1H, upeld H of piperidinyl NCH2CH3), 2.15 (s, 3H, of piperidinyl H2C-600), 3.14 (dd, J ¼ 2.0, 12.8 Hz, 1H, downeld H
pyrrolidinyl NCH3), 2.31 (sextet, J ¼ 7.3, 14.5 Hz, 1H, downeld H of piperidinyl H2C-200), 3.27 (d, J ¼ 14.7 Hz, 1H, downeld H of
of NCH2CH3), 2.54 (br s, 4H, piperidinyl NH2C-2/6), 2.80 (dd, J ¼ piperidinyl H2C-600), 3.37 (dd, J ¼ 7.5, 9.0 Hz, 1H, upeld H of
2.4, 14.5 Hz, 1H, upeld H of piperidinyl H2C-600), 3.15 (dd, J ¼ pyrrolidinyl H2C-50), 3.90 (dd, J ¼ 9.0, 10.8 Hz, 1H, downeld H of
1.5, 12.7 Hz, 1H, downeld H of piperidinyl H2C-200), 3.27 (d, J ¼ pyrrolidinyl H2C-50), 4.10 (d, J ¼ 12.6 Hz, 1H, upeld H of
14.5 Hz, 1H, downeld H of piperidinyl H2C-600), 3.36 (t, J ¼ 8.1 NCH2N), 4.64 (d, J ¼ 12.6 Hz, 1H, downeld H of NCH2N), 4.80
Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.91 (dd, J ¼ 9.0, 10.7 (dd, J ¼ 7.5, 10.8 Hz, 1H, pyrrolidinyl HC-40), 6.80–7.44 (m, 13H,
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12 arom. H + olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm):
11.9, 19.6, 24.2, 26.0, 35.2, 45.4, 52.6, 54.0, 57.0, 57.6, 60.1, 63.1,
65.3, 76.0, 109.2, 115.0, 115.3, 115.5, 115.8, 122.0, 126.3, 127.4,
128.9, 131.3, 131.4, 131.5, 131.54, 132.0, 132.1, 133.3, 134.4,
136.7, 145.3, 160.4, 161.1, 163.6, 164.5, 176.2, 199.3. Elemental
analysis: C38H42F2N4O2 required C, 73.05; H, 6.78; N, 8.97, found
C, 73.19; H, 6.82; N, 9.08.
100-Benzyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methylidene]-
10-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (51). Obtained from
reaction 20, 27 and 29. Reaction time 12 h, pale yellow micro-
ꢁ
crystals from methanol, mp 172–174 C, yield 83% (2.80 g). IR n
1
(cmꢀ1): 1705, 1684, 1607, 1508, 1466. H-NMR (400 MHz, CDCl3)
d (ppm): 1.85 (d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl H2C-200),
2.14 (s, 3H, pyrrolidinyl NCH3), 2.53–2.57 (m, 4H, morpholinyl
2NCH2), 2.75 (d, J ¼ 14.5 Hz, 1H, upeld H of piperidinyl H2C-600),
3.15–3.45 (m, 4H, downeld H's of piperidinyl H2C-200, H2C-600,
upeld H of pyrrolidinyl H2C-50 and upeld H of PhCH2), 3.50 (d, J
¼ 14.0 Hz, 1H, downeld H of PhCH2), 3.62 (t, J ¼ 4.5 Hz, 4H,
morpholinyl 2OCH2), 3.91 (dd, J ¼ 9.2, 10.4 Hz, 1H, downeld H of
pyrrolidinyl H2C-50), 3.98 (d, J ¼ 12.8 Hz, 1H, upeld H of NCH2N),
4.51 (d, J ¼ 12.8 Hz, 1H, downeld H of NCH2N), 4.79 (dd, J ¼ 7.5,
10.6 Hz, 1H, pyrrolidinyl HC-40), 6.80–7.46 (m, 18H, 17 arom. H +
olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 34.4, 44.9,
50.9, 53.0, 56.7, 60.9, 61.6, 64.6, 65.9, 75.1, 109.1, 114.8, 115.1,
115.2, 115.5, 121.7, 125.3, 126.7, 126.9, 127.9, 128.3, 128.8, 131.0,
131.1, 131.9, 132.0, 134.3, 134.4, 135.7, 144.5, 159.5, 160.3, 162.7,
163.6, 175.4, 197.9. Elemental analysis: C41H40F2N4O3 required C,
72.98; H, 5.97; N, 8.30, found C, 73.13; H, 6.02; N, 8.46.
40-(4-Fluorophenyl)-500-[(4-uorophenyl)methylidene]-10-methyl-
1-[(4-morpholinyl)methylene]-100-(1-propyl)-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (49). Obtained from
19, 27 and 29. Reaction time 16 h, colorless microcrystals from
methanol, mp 186–188 C, yield 83% (2.60 g). IR n (cmꢀ1): 1705,
ꢁ
1688, 1607, 1508, 1468. 1H-NMR (400 MHz, CDCl3) d (ppm): 0.71
(t, J ¼ 7.3 Hz, 3H, NCH2CH2CH3), 0.94–1.06 (m, 1H, upeld H of
NCH2CH2CH3), 1.10–1.20 (m, 1H, downeld H of NCH2CH2CH3),
1.68 (d, J ¼ 12.7 Hz, 1H, upeld H of piperidinyl H2C-200), 1.98
(sextet, J ¼ 5.1, 11.1 Hz, 1H, upeld H of NCH2CH2CH3), 2.11–2.16
(m, 4H, pyrrolidinyl NCH3 + downeld H of NCH2CH2CH3), 2.58
(br s, 4H, morpholinyl 2NCH2), 2.80 (dd, J ¼ 2.6, 14.5 Hz, 1H,
upeld H of piperidinyl H2C-600), 3.11 (dd, J ¼ 1.8, 12.7 Hz, 1H,
downeld H of piperidinyl H2C-200), 3.25 (d, J ¼ 14.5 Hz, 1H,
downeld H of piperidinyl H2C-600), 3.35 (dd, J ¼ 7.4, 8.7 Hz, 1H,
upeld H of pyrrolidinyl H2C-50), 3.64 (t, J ¼ 4.6 Hz, 4H, mor-
pholinyl 2OCH2), 3.86 (dd, J ¼ 8.9, 10.8 Hz, 1H, downeld H of
pyrrolidinyl H2C-50), 4.04 (d, J ¼ 12.7 Hz, 1H, upeld H of NCH2N),
4.61 (d, J ¼ 12.7 Hz, 1H, downeld H of NCH2N), 4.77 (dd, J ¼ 7.3,
10.8 Hz, 1H, pyrrolidinyl HC-40), 6.75 (d, J ¼ 8.0 Hz, 1H, arom. H),
6.92–7.39 (m, 12H, 11 arom. H + olenic CH). 13C-NMR (75 MHz,
DMSO-d6) d (ppm): 11.5, 18.7, 34.4, 44.4, 50.9, 53.3, 56.4, 56.7,
59.2, 61.4, 64.4, 66.0, 75.1, 109.1, 114.8, 115.1, 115.3, 115.7, 121.5,
125.4, 126.7, 128.7, 130.9, 132.2, 134.4, 135.7, 144.6, 175.3, 198.0.
Elemental analysis: C37H40F2N4O3 required C, 70.91; H, 6.43; N,
8.94, found C, 71.03; H, 6.41; N, 9.13.
10,100-Dimethyl-40-(4-methylphenyl)-500-[(4-methylphenyl)methyl-
idene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyrro-
lidine-30,300-piperidine]-2(1H),400-dione (52). Obtained from
reaction of 21, 26 and 29. Reaction time 10 h, almost colorless
microcrystals from ethanol, mp 177–178 ꢁC, yield 71% (2.09 g).
1
IR n (cmꢀ1): 1701, 1686, 1609, 1512, 1487, 1466. H-NMR (300
MHz, CDCl3) d (ppm): 1.41 (br s, 2H, piperidinyl H2C-4), 1.55–
1.70 (m, 5H, piperidinyl H2C-3/5 and upeld H of piperidinyl
H2C-200), 2.01 (s, 3H, piperidinyl NCH3), 2.16 (s, 3H, pyrrolidinyl
NCH3), 2.32 (s, 3H, ArCH3), 2.33 (s, 3H, ArCH3), 2.64 (br s, 4H,
piperidinyl NH2C-2/6), 2.87 (dd, J ¼ 2.6, 14.3 Hz, 1H, upeld H of
piperidinyl H2C-600), 3.23–3.33 (m, 3H, downeld H's of piper-
idinyl H2C-200, H2C-600 and upeld H of pyrrolidinyl H2C-50), 3.92
(dd, J ¼ 8.7, 11.1 Hz, 1H, downeld H of pyrrolidinyl H2C-50),
4.23 (br s, 1H, upeld H of NCH2N), 4.58 (d, J ¼ 13.2 Hz, 1H,
downeld H of NCH2N), 4.82 (dd, J ¼ 7.2, 11.4 Hz, 1H, pyrroli-
dinyl HC-40), 6.88–7.29 (m, 13H, 12 arom. H + olenic CH). 13C-
NMR (75 MHz, DMSO-d6) d (ppm): 20.5, 20.8, 23.7, 25.2, 34.3,
44.2, 44.5, 51.4, 65.3, 74.7, 109.0, 121.2, 125.9, 128.6, 128.8,
128.9, 129.8, 131.6, 132.1, 135.0, 135.6, 138.6, 144.6, 175.2, 197.4.
Elemental analysis: C38H44N4O2 required C, 77.52; H, 7.53; N,
9.52, found C, 77.61; H, 7.59; N, 9.59.
10,100-Dimethyl-40-(4-methylphenyl)-500-[(4-methylphenyl)methyl-
idene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,20-pyrroli-
dine-30,300-piperidine]-2(1H),400-dione (53). Obtained from reaction
of 21, 27 and 29. Reaction time 12 h, colorless microcrystals from
ethanol, mp 198–200 ꢁC (lit. mp 198–200 ꢁC 38), yield 75% (2.21 g).
IR n (cmꢀ1): 1703, 1686, 1609, 1512, 1487, 1466. 1H-NMR (400 MHz,
CDCl3) d (ppm): 1.62 (d, J ¼ 12.8 Hz, 1H, upeld H of piperidinyl
H2C-200), 1.98 (s, 3H, piperidinyl NCH3), 2.12 (s, 3H, pyrrolidinyl
NCH3), 2.29 (s, 3H, ArCH3), 2.30 (s, 3H, ArCH3), 2.54 (t, J ¼ 4.6 Hz,
4H, morpholinyl 2NCH2), 2.84 (dd, J ¼ 2.6, 14.5 Hz, 1H, upeld H
of piperidinyl H2C-600), 3.23–3.30 (m, 3H, downeld H's of piper-
idinyl H2C-200, H2C-600 and upeld H of pyrrolidinyl H2C-50), 3.62 (t, J
100-Benzyl-40-(4-uorophenyl)-500-[(4-uorophenyl)methylidene]-
10-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,20-pyr-
rolidine-30,300-piperidine]-2(1H),400-dione (50). Obtained from
reaction of 20, 26 and 29. Reaction time 10 h, pale yellow
microcrystals from ethanol, mp 143–144 ꢁC, yield 77% (2.59 g). IR
n (cmꢀ1): 1699, 1680, 1601, 1508, 1466. 1H-NMR (400 MHz, CDCl3)
d (ppm): 1.32–1.40 (m, 2H, piperidinyl H2C-4), 1.45–1.54 (m, 4H,
piperidinyl H2C-3/5), 1.86 (d, J ¼ 12.7 Hz, 1H, upeld H of
piperidinyl H2C-200), 2.16 (s, 3H, pyrrolidinyl NCH3), 2.47–2.55 (m,
4H, piperidinyl NH2C-2/6), 2.74 (dd, J ¼ 2.4, 14.6 Hz, 1H, upeld
H of piperidinyl H2C-600), 3.19–3.34 (m, 3H, downeld H's of
piperidinyl H2C-200, H2C-600 and upeld H of PhCH2), 3.42 (t, J ¼
8.2 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.49 (d, J ¼ 13.9 Hz,
1H, downeld H of PhCH2), 3.92 (t, J ¼ 9.8 Hz, 1H, downeld H of
pyrrolidinyl H2C-50), 4.00 (d, J ¼ 12.8 Hz, 1H, upeld H of
NCH2N), 4.50 (d, J ¼ 12.8 Hz, 1H, downeld H of NCH2N), 4.79
(dd, J ¼ 7.6, 10.6 Hz, 1H, pyrrolidinyl HC-40), 6.80–7.47 (m, 18H,
17 arom. H + olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm):
23.6, 25.3, 34.4, 45.0, 51.6, 53.0, 56.7, 60.9, 62.2, 64.6, 75.0, 109.2,
114.8, 115.0, 115.2, 115.5, 121.5, 125.3, 126.6, 126.8, 127.9, 128.2,
128.7, 130.8, 130.9, 131.0, 131.1, 131.9, 132.0, 132.7, 134.4, 135.6,
135.8, 144.7, 159.5, 160.3, 162.7, 163.6, 175.3, 198.0. Elemental
analysis: C42H42F2N4O2 required C, 74.98; H, 6.29; N, 8.33, found
C, 75.12; H, 6.34; N, 8.51.
45446 | RSC Adv., 2016, 6, 45434–45451
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10,100-Dimethyl-40-(4-methoxyphenyl)-500-[(4-methoxyphenyl)
methylidene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-
3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (56). Obtained
from reaction of 22, 27 and 29. Reaction time 15 h, colorless
microcrystals from ethanol, mp 202–204 ꢁC, yield 71% (2.21 g).
¼ 4.6 Hz, 4H, morpholinyl 2OCH2), 3.90 (dd, J ¼ 8.8, 11.2 Hz, 1H,
downeld H of pyrrolidinyl H2C-50), 4.07 (d, J ¼ 12.5 Hz, 1H,
upeld H of NCH2N), 4.50 (d, J ¼ 12.5 Hz, 1H, downeld H of
NCH2N), 4.78 (dd, J ¼ 7.0, 11.3 Hz, 1H, pyrrolidinyl HC-40), 6.75 (d, J
¼ 8.0 Hz, 1H, arom. H), 6.87 (d, J ¼ 8.0 Hz, 2H, arom. H), 6.96–7.26
(m, 10H, 9 arom. H + olenic CH). 13C-NMR (100 MHz, CDCl3)
d (ppm): 21.1, 21.4, 35.0, 45.0, 45.1, 51.2, 56.7, 57.3, 62.5, 66.3, 66.8,
75.5, 108.7, 122.1, 126.7, 127.4, 128.7, 128.9, 129.2, 130.0, 132.4,
132.6, 135.2, 136.3, 137.6, 138.9, 144.5, 176.4, 198.4. Elemental
analysis: C37H42N4O3 required C, 75.23; H, 7.17; N, 9.48, found C,
75.30; H, 7.26; N, 9.61.
1
IR n (cmꢀ1): 1701, 1682, 1607, 1510, 1489, 1464. H-NMR (300
MHz, CDCl3) d (ppm): 1.66 (d, J ¼ 12.6 Hz, 1H, upeld H of
piperidinyl H2C-200), 2.02 (s, 3H, piperidinyl NCH3), 2.16 (s, 3H,
pyrrolidinyl NCH3), 2.61 (br s, 4H, morpholinyl 2NCH2), 2.89
(dd, J ¼ 2.4, 14.1 Hz, 1H, upeld H of piperidinyl H2C-600), 3.21–
3.37 (m, 3H, downeld H's of piperidinyl H2C-200, H2C-600, and
upeld H of pyrrolidinyl H2C-50), 3.66 (t, J ¼ 4.4 Hz, 4H, mor-
pholinyl 2OCH2), 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.90 (t,
J ¼ 9.9 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.14 (d, J ¼
12.6 Hz, 1H, upeld H of NCH2N), 4.55 (d, J ¼ 12.6 Hz, 1H,
downeld H of NCH2N), 4.82 (dd, J ¼ 6.9, 10.8 Hz, 1H, pyrro-
lidinyl HC-40), 6.80–7.32 (m, 13H, 12 arom. H + olenic CH).
13C-NMR (75 MHz, DMSO-d6) d (ppm): 34.3, 43.8, 44.6, 50.7,
54.9, 55.1, 65.2, 65.9, 74.8, 99.4, 108.9, 113.6, 113.9, 121.3,
125.9, 126.5, 127.0, 128.4, 129.7, 129.9, 130.7, 131.8, 136.5,
144.4, 157.9, 159.7, 175.4, 197.3. Elemental analysis:
10,100-Dimethyl-40-(4-methylphenyl)-500-[(4-methylphenyl)methyl-
idene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-3,20-
pyrrolidine-30,300-piperidine]-2(1H),400-dione (54). Obtained from
reaction of 21, 28 and 29. Reaction time 15 h, colorless micro-
ꢁ
crystals from methanol, mp 201–202 C, yield 71% (2.14 g). IR n
1
(cmꢀ1): 1692, 1609, 1512, 1489, 1462. H-NMR (300 MHz, CDCl3)
d (ppm): 1.64 (d, J ¼ 12.9 Hz, 1H, upeld H of piperidinyl H2C-200),
2.01 (s, 3H, piperidinyl NCH3), 2.15 (s, 3H, pyrrolidinyl NCH3), 2.24
(s, 3H, piperazinyl NCH3), 2.32 (s, 3H, ArCH3), 2.33 (s, 3H, ArCH3),
2.37 (br s, 4H, piperazinyl 2NCH2), 2.62 (br s, 4H, piperazinyl
2NCH2), 2.86 (dd, J ¼ 2.7, 14.7 Hz, 1H, upeld H of piperidinyl H2C-
600), 3.26–3.33 (m, 3H, downeld H's of piperidinyl H2C-200, H2C-600
and upeld H of pyrrolidinyl H2C-50), 3.93 (dd, J ¼ 8.7, 11.1 Hz, 1H,
downeld H of pyrrolidinyl H2C-50), 4.10 (d, J ¼ 12.3 Hz, 1H, upeld
H of NCH2N), 4.55 (d, J ¼ 12.3 Hz, 1H, downeld H of NCH2N), 4.81
(dd, J ¼ 6.8, 11.3 Hz, 1H, pyrrolidinyl HC-40), 6.77–7.29 (m, 13H, 12
arom. H + olenic CH). 13C-NMR (75 MHz, CDCl3) d (ppm): 21.3,
21.5, 35.2, 45.3, 46.2, 51.0, 55.1, 57.0, 57.5, 62.5, 66.5, 75.7, 109.1,
122.2, 127.0, 127.5, 128.8, 129.1, 129.5, 130.2, 132.7, 132.9, 135.5,
136.5, 137.7, 139.0, 144.9, 176.5, 198.6. Elemental analysis:
C38H45N5O2 required C, 75.59; H, 7.51; N, 11.60, found C, 75.71; H,
7.59; N, 11.68.
C
37H42N4O5 required C, 71.36; H, 6.80; N, 9.00, found C, 71.43;
H, 6.87; N, 9.14.
10,100-Dimethyl-40-(4-methoxyphenyl)-500-[(4-methoxyphenyl)
methylidene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-
indole-3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione
(57).
Obtained from reaction of 22, 28 and 29. Reaction time 20 h,
colorless microcrystals from ethanol, mp 186–188 ꢁC, yield 69%
(2.19 g). IR n (cmꢀ1): 1703, 1680, 1607, 1510, 1464. 1H-NMR (400
MHz, CDCl3) d (ppm): 1.63 (d, J ¼ 12.6 Hz, 1H, upeld H of
piperidinyl H2C-200), 2.00 (s, 3H, piperidinyl NCH3), 2.14 (s, 3H,
pyrrolidinyl NCH3), 2.24 (s, 3H, piperazinyl NCH3), 2.38 (br s,
4H, piperazinyl 2NCH2), 2.62 (br s, 4H, piperazinyl 2NCH2), 2.87
(dd, J ¼ 2.3, 14.2 Hz, 1H, upeld H of piperidinyl H2C-600), 3.21–
3.32 (m, 3H, downeld H's of piperidinyl H2C-200, H2C-600, and
upeld H of pyrrolidinyl H2C-50), 3.78 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 3.90 (dd, J ¼ 8.9, 11.0 Hz, 1H, downeld H of pyrrolidinyl
H2C-50), 4.11 (d, J ¼ 12.5 Hz, 1H, upeld H of NCH2N), 4.56 (d, J
¼ 12.6 Hz, 1H, downeld H of NCH2N), 4.78 (dd, J ¼ 7.0, 11.1
Hz, 1H, pyrrolidinyl HC-40), 6.76–7.12 (m, 11H, 10 arom. H +
olenic CH), 7.31 (d. H, J ¼ 9 Hz, 2H, arom.). 13C-NMR (100
MHz, CDCl3) d (ppm): 35.1, 44.7, 45.2, 46.0, 50.8, 54.9, 55.20,
55.24, 56.9, 57.3, 57.4, 62.2, 66.1, 75.7, 108.8, 113.6, 113.7, 121.9,
126.7, 127.3, 127.9, 128.6, 130.3, 130.4, 131.5, 131.9, 137.3,
144.6, 158.4, 159.9, 176.3, 198.4. Elemental analysis:
10,100-Dimethyl-40-(4-methoxyphenyl)-500-[(4-methoxyphenyl)
methylidene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-
3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (55). Ob-
tained from reaction of 22, 26 and 29. Reaction time 12 h,
ꢁ
almost colorless microcrystals from ethanol, mp 175–177 C,
yield 82% (2.55 g). IR n (cmꢀ1): 1701, 1682, 1607, 1510, 1489,
1
1464. H-NMR (300 MHz, CDCl3) d (ppm): 1.35–1.42 (m, 2H,
piperidinyl H2C-4), 1.50–1.59 (m, 4H, piperidinyl H2C-3/5),
1.64 (d, J ¼ 12.6 Hz, 1H, upeld H of piperidinyl H2C-200),
2.01 (s, 3H, piperidinyl NCH3), 2.15 (s, 3H, pyrrolidinyl NCH3),
2.55 (t, J ¼ 5.0 Hz, 4H, piperidinyl NH2C-2/6), 2.88 (dd, J ¼ 2.7,
14.1 Hz, 1H, upeld H of piperidinyl H2C-600), 3.20–3.34 (m,
3H, downeld H's of piperidinyl H2C-200, H2C-600, and upeld
H of pyrrolidinyl H2C-50), 3.79 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.91 (dd, J ¼ 8.7, 11.4 Hz, 1H, downeld H of pyrro-
lidinyl H2C-50), 4.12 (d, J ¼ 12.6 Hz, 1H, upeld H of NCH2N),
4.54 (d, J ¼ 12.9 Hz, 1H, downeld H of NCH2N), 4.79 (dd, J ¼
7.1, 11.3 Hz, 1H, pyrrolidinyl HC-40), 6.79–7.34 (m, 13H, 12
arom. H + olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm):
23.7, 25.3, 34.3, 43.9, 44.6, 51.5, 54.9, 55.2, 56.1, 56.7, 56.8,
62.2, 65.1, 74.8, 109.0, 113.6, 113.9, 121.2, 126.0, 126.5, 127.0,
128.4, 129.7, 130.0, 130.8, 131.8, 136.5, 144.7, 158.0, 159.7,
175.3, 197.4. Elemental analysis: C38H44N4O4 required C,
73.52; H, 7.14; N, 9.02, found C, 73.59; H, 7.22; N, 9.12.
C
38H45N5O4 required C, 71.79; H, 7.13; N, 11.01, found C, 71.82;
H, 7.21; N, 11.09.
40-(2,5-Dimethoxyphenyl)-500-[(2,5-dimethoxyphenyl)methyl-
idene]-10,100-dimethyl-1-[(1-piperidinyl)methylene]-dispiro[3H-
indole-3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione (58).
Obtained from reaction of 23, 26 and 29. Reaction time 16 h,
ꢁ
yellow microcrystals from ethanol, mp 139–140 C, yield 85%
(2.89 g). IR n (cmꢀ1): 1694, 1607, 1593, 1495, 1466. 1H-NMR
(300 Hz, CDCl3) d (ppm): 1.35–1.43 (m, 2H, piperidinyl H2C-
4), 1.50–1.59 (m, 4H, piperidinyl H2C-3/5), 1.78 (d, J ¼ 12.6 Hz,
1H, upeld H of piperidinyl H2C-200), 1.88 (s, 3H, piperidinyl
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NCH3), 2.14 (s, 3H, pyrrolidinyl NCH3), 2.55 (t, J ¼ 4.8 Hz, 4H, 8.7, 11.1 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.14 (d, J ¼
piperidinyl NH2C-2/6), 2.82 (dd, J ¼ 2.7, 14.7 Hz, 1H, upeld H 12.6 Hz, 1H, upeld H of NCH2N), 4.61 (d, J ¼ 12.3 Hz, 1H,
of piperidinyl H2C-600), 2.99 (dd, J ¼ 2.0, 12.5 Hz, 1H, downeld downeld H of NCH2N), 5.07 (dd, J ¼ 7.5, 10.8 Hz, 1H, pyrroli-
H of piperidinyl H2C-200), 3.15 (d, J ¼ 14.7 Hz, 1H, downeld H dinyl HC-40), 6.82–7.54 (m, 11H, 10 arom. H + olenic CH). 13C-
of piperidinyl H2C-600), 3.33 (dd, J ¼ 7.5, 8.7 Hz, 1H, upeld H NMR (100 MHz, CDCl3) d (ppm): 24.1, 25.8, 34.8, 40.9, 45.2,
of pyrrolidinyl H2C-50), 3.67 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 52.3, 56.0, 57.1, 58.3, 63.1, 65.3, 75.8, 108.9, 121.9, 123.9, 126.0,
3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.06 (dd, J ¼ 8.9, 10.7 126.2, 126.7, 127.1, 127.8, 128.7, 129.5, 129.8, 130.4, 132.7, 138.5,
Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.11 (d, J ¼ 12.3 141.3, 145.1, 176.1, 197.0. Elemental analysis: C32H36N4O2S2
Hz, 1H, upeld H of NCH2N), 4.53 (d, J ¼ 12.9 Hz, 1H, down- required C, 67.10; H, 6.34; N, 9.78, found C, 67.36; H, 6.31; N,
eld H of NCH2N), 4.88 (dd, J ¼ 7.5, 10.8 Hz, 1H, pyrrolidinyl 9.89.
HC-40), 6.41 (d, J ¼ 2.1 Hz, 1H, arom. H), 6.72–7.55 (m, 10H, 9
10,100-Dimethyl-1-[(4-morpholinyl)methylene]-40-(2-thienyl)-500-
arom. H + olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): [(2-thienyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-30,300-
23.7, 25.3, 34.3, 45.0, 51.5, 55.2, 55.4, 55.8, 56.0, 56.7, 56.9, piperidine]-2(1H),400-dione (61). Obtained from reaction of 24, 27
62.2, 63.1, 75.7, 109.0, 111.0, 111.2, 112.1, 114.9, 115.2, 115.5, and 29. Reaction time 12 h, pale yellow microcrystals from
121.4, 124.2, 125.6, 126.8, 128.1, 128.4, 131.5, 132.5, 144.8, methanol, mp 210–212 ꢁC, yield 71% (2.04 g). IR n (cmꢀ1): 1694,
151.8, 151.9, 152.3, 153.0, 175.4, 195.7. Elemental analysis: 1676, 1612, 1578, 1468. 1H-NMR (300 MHz, CDCl3) d (ppm): 1.89
C
40H48N4O6 required C, 70.57; H, 7.11; N, 8.23, found C, 70.68; (d, J ¼ 12.9 Hz, 1H, upeld H of piperidinyl H2C-200), 2.10 (s, 3H,
H, 7.31; N, 8.37.
piperidinyl NCH3), 2.14 (s, 3H, pyrrolidinyl NCH3), 2.61 (t, J ¼ 4.7
40-(2,5-Dimethoxyphenyl)-500-[(2,5-dimethoxyphenyl)methyl- Hz, 4H, morpholinyl 2NCH2), 2.94 (dd, J ¼ 2.6, 15.5 Hz, 1H,
idene]-10,100-dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H- upeld H of piperidinyl H2C-600), 3.29–3.36 (m, 2H, downeld H's
indole-3,20-pyrrolidine-30,300-piperidine]-2(1H),400-dione
(59). of piperidinyl H2C-200, and H2C-600), 3.44 (dd, J ¼ 7.2, 8.7 Hz, 1H,
Obtained from reaction of 23, 27 and 29. Reaction time 15 h, upeld H of pyrrolidinyl H2C-50), 3.68 (t, J ¼ 4.7 Hz, 4H, mor-
pale yellow microcrystals from ethanol, mp 181–183 ꢁC, yield pholinyl 2OCH2), 3.90 (dd, J ¼ 8.6, 11.0 Hz, 1H, downeld H of
71% (2.42 g). IR n (cmꢀ1): 1699, 1605, 1578, 1493, 1464. 1H-NMR pyrrolidinyl H2C-50), 4.15 (d, J ¼ 12.9 Hz, 1H, upeld H of
(300 MHz, CDCl3) d (ppm): 1.78 (d, J ¼ 12.9 Hz, 1H, upeld H of NCH2N), 4.61 (d, J ¼ 12.6 Hz, 1H, downeld H of NCH2N), 5.08
piperidinyl H2C-200), 1.89 (s, 3H, piperidinyl NCH3), 2.13 (s, 3H, (dd, J ¼ 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-40), 6.80–7.50 (m, 11H,
pyrrolidinyl NCH3), 2.60 (t, J ¼ 4.5 Hz, 4H, morpholinyl 2NCH2), 10 arom. H + olenic CH). Elemental analysis: C31H34N4O3S2
2.82 (dd, J ¼ 2.7, 14.7 Hz, 1H, upeld H of piperidinyl H2C-600), required C, 64.78; H, 5.96; N, 9.75, found C, 64.83; H, 6.04; N,
2.99 (dd, J ¼ 2.1, 12.6 Hz, 1H, downeld H of piperidinyl H2C- 9.90.
200), 3.15 (d, J ¼ 14.4 Hz, 1H, downeld H of piperidinyl H2C-600),
10,100-Dimethyl-1-[(1-piperidinyl)methylene]-40-(3-pyridinyl)-500-
3.34 (dd, J ¼ 7.5, 8.4 Hz, 1H, upeld H of pyrrolidinyl H2C-50), [(3-pyridinyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-30,300-
3.67–3.69 (m, 7H, morpholinyl 2OCH2 + OCH3), 3.71 (s, 3H, piperidine]-2(1H),400-dione (62). Obtained from reaction of 25, 26
OCH3), 3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.05 (dd, J ¼ 8.7, and 29. Reaction time 16 h, colorless microcrystals from methanol,
10.8 Hz, 1H, downeld H of pyrrolidinyl H2C-50), 4.12 (d, J ¼ 12.3 mp 119–120 ꢁC, yield 72% (2.03 g). IR n (cmꢀ1): 1701, 1686, 1611,
1
Hz, 1H, upeld H of NCH2N), 4.54 (d, J ¼ 12.6 Hz, 1H, downeld 1481, 1466. H-NMR (400 MHz, CDCl3) d (ppm): 1.32 (br s, 2H,
H of NCH2N), 4.88 (dd, J ¼ 7.4, 10.7 Hz, 1H, pyrrolidinyl HC-40), piperidinyl H2C-4), 1.50 (br s, 4H, piperidinyl H2C-3/5), 1.57 (d, J ¼
6.41 (d, J ¼ 2.4 Hz, 1H, arom. H), 6.73–7.55 (m, 10H, 9 arom. H + 12.7 Hz, 1H, upeld H of piperidinyl H2C-200), 1.92 (s, 3H, piper-
olenic CH). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 34.4, 45.0, idinyl NCH3), 2.09 (s, 3H, pyrrolidinyl NCH3), 2.50 (br s, 4H,
50.8, 55.2, 55.3, 55.4, 55.9, 56.8, 56.9, 61.6, 63.2, 65.9, 75.7, piperidinyl NH2C-2/6), 2.81 (dd, J ¼ 2.2, 14.6 Hz, 1H, upeld H of
108.9, 111.0, 111.2, 112.2, 114.9, 115.2, 115.4, 121.6, 124.1, piperidinyl H2C-600), 3.16 (t, J ¼ 12.6 Hz, 2H, downeld H's of
125.5, 126.9, 128.2, 128.4, 131.6, 132.4, 144.6, 151.8, 151.9, piperidinyl H2C-200 and H2C-600), 3.30 (t, J ¼ 7.8 Hz, 1H, upeld H of
152.3, 153.0, 175.5, 195.7. Elemental analysis: C39H46N4O7 pyrrolidinyl H2C-50), 3.85 (t, J ¼ 9.8 Hz, 1H, downeld H of pyr-
required C, 68.60; H, 6.79; N, 8.21, found C, 68.82; H, 6.74; N, rolidinyl H2C-50), 4.11 (d, J ¼ 13.1 Hz, 1H, upeld H of NCH2N),
8.35.
4.46 (d, J ¼ 12.7 Hz, 1H, downeld H of NCH2N), 4.74 (dd, J ¼ 7.0,
10,100-Dimethyl-1-[(1-piperidinyl)methylene]-40-(2-thienyl)-500- 10.7 Hz, 1H, pyrrolidinyl HC-40), 6.82–8.18 (m, 10H, 9 arom. H +
[(2-thienyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-30,300- olenic CH), 8.42 (dd, J ¼ 4.4, 10.9 Hz, 2H, arom. H), 8.49 (s, 1H,
piperidine]-2(1H),400-dione (60). Obtained from reaction of 24, 26 arom. H). 13C-NMR (75 MHz, DMSO-d6) d (ppm): 23.7, 25.2, 34.3,
and 29. Reaction time 14 h, pale yellow microcrystals from 42.5, 44.4, 51.4, 56.0, 56.2, 57.1, 62.2, 65.2, 74.6, 109.3, 121.4, 123.3,
methanol, mp 147–148 ꢁC, yield 68% (1.95 g). IR n (cmꢀ1): 1694, 125.4, 126.6, 128.7, 130.2, 132.9, 133.8, 134.9, 136.3, 136.4, 144.7,
1676, 1614, 1578, 1470. 1H-NMR (300 MHz, CDCl3) d (ppm): 1.35– 148.1, 149.2, 150.1, 150.4, 175.2, 197.3. Elemental analysis:
1.45 (m, 2H, piperidinyl H2C-4), 1.51–1.60 (m, 4H, piperidinyl C34H38N6O2 required C, 72.57; H, 6.81; N, 14.93, found C, 72.69; H,
H2C-3/5), 1.89 (d, J ¼ 13.2 Hz, 1H, upeld H of piperidinyl H2C- 6.74; N, 15.06.
200), 2.09 (s, 3H, piperidinyl NCH3), 2.15 (s, 3H, pyrrolidinyl
10,100-Dimethyl-1-[(4-morpholinyl)methylene]-40-(3-pyridinyl)-
NCH3), 2.57 (t, J ¼ 5.1 Hz, 4H, piperidinyl NH2C-2/6), 2.94 (dd, J ¼ 500-[(3-pyridinyl)methylidene]-dispiro[3H-indole-3,20-pyrrolidine-
2.6, 15.5 Hz, 1H, upeld H of piperidinyl H2C-600), 3.28–3.34 (m, 30,300-piperidine]-2(1H),400-dione (63). Obtained from reaction of
2H, downeld H's of piperidinyl H2C-200 and H2C-600), 3.45 (dd, J 25, 27 and 29. Reaction time 14 h, colorless microcrystals from
¼ 7.1, 8.6 Hz, 1H, upeld H of pyrrolidinyl H2C-50), 3.92 (dd, J ¼ methanol, mp 192–194 ꢁC, yield 78% (2.20 g). IR n (cmꢀ1): 1699,
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1680, 1607. 1H-NMR (400 MHz, CDCl3) d (ppm): 1.58 (d, J ¼ 12.4 washed with acetic acid. The attached stain was recovered with
Hz, 1H, upeld H of piperidinyl H2C-200), 1.93 (s, 3H, piperidinyl Tris–EDTA buffer. Cell survival and drug activity were deter-
NCH3), 2.09 (s, 3H, pyrrolidinyl NCH3), 2.52 (br s, 4H, morpho- mined by measuring the color intensity spectrophotometrically
linyl 2NCH2), 2.82 (dd, J ¼ 2.3, 14.4 Hz, 1H, upeld H of piper- at 564 nm using an ELISA microplate reader (Meter tech. S 960,
idinyl H2C-600), 3.15 (t, J ¼ 14.4 Hz, 2H, downeld H's of USA). Data are collected as mean values for experiments that
piperidinyl H2C-200, and H2C-600), 3.32 (t, J ¼ 7.8 Hz, 1H, upeld H performed in three replicates for each individual dose which
of pyrrolidinyl H2C-50), 3.58 (t, J ¼ 4.2 Hz, 4H, morpholinyl measured by SRB assay. Control experiments did not exhibit
2OCH2), 3.84 (t, J ¼ 9.7 Hz, 1H, downeld H of pyrrolidinyl H2C- signicant change compared to the DMSO vehicle. Doxorubicin
50), 4.07 (d, J ¼ 12.6 Hz, 1H, upeld H of NCH2N), 4.47 (d, J ¼ 12.6 hydrochloride and cisplatin were used as standard references
Hz, 1H, downeld H of NCH2N), 4.75 (dd, J ¼ 7.0, 10.5 Hz, 1H, during the present in vitro bioactivity screening assay. The
pyrrolidinyl HC-40), 6.76–7.85 (m, 9H, 8 arom. H + olenic CH), percentage of cell survival was calculated according to eqn (2).
8.16 (s, 1H, arom. H), 8.43 (dd, J ¼ 4.2, 16.0 Hz, 2H, arom. H),
8.52 (s, 1H, arom. H). 13C-NMR (100 MHz, CDCl3) d (ppm): 34.9,
43.1, 45.1, 51.1, 56.5, 56.8, 57.5, 62.4, 66.1, 66.6, 75.3, 109.1,
122.3, 123.2, 123.6, 126.0, 127.5, 129.2, 130.8, 133.8, 134.5, 135.1,
136.5, 137.6, 144.5, 147.6, 149.4, 150.0, 150.5, 176.1, 197.6.
Elemental analysis: C33H36N6O3 required C, 70.19; H, 6.43; N,
14.88, found C, 70.34; H, 6.42; N, 15.03.
Surviving fraction ¼ optical density (O.D.) of treated cells/
O.D. of control cells
(2)
The IC50 (concentration required to produce 50% inhibition
of cell growth compared to control experiment) was determined
using Graph-Pad PRISM version-5 soware. Statistical calcula-
tions for determination of the mean and standard error values
were determined by SPSS 16 soware. The observed antitumor
properties were presented in Table 3 (Fig. S114 and S115†).
Single crystal X-ray
Intensity data for 46 and 48 were collected at room temperature
(298 K) on an Enraf-Nonius 590 diffractometer with a Kappa
CCD detector using graphite monochromated Mo-Ka (l ¼
Computational chemistry
Structure optimization. Computational chemistry calcula-
tions using both AM1 and PM3 methods were undertaken for
structural optimization. The geometry of the bio-active
compounds 30–63 was optimized by the molecular mechanics
force eld (MM+), followed by either semi-empirical AM1 or
PM3 methods implemented in the HyperChem 8.0 package. The
structures were fully optimized without constraining any
parameters, thus bringing all geometric variables to their
equilibrium values. The energy minimization protocol
employed the Polake–Ribiere conjugated gradient algorithm.
Convergence to a local minimum was achieved when the energy
gradient was #0.01 kcal molꢀ1. The RHF (Restricted Hartree–
Fock) method was used in the spin pairing for the two semi-
empirical tools.31,32,38,61
0.71073 A) radiation.53 Data were recorded in the rotation mode
˚
using the 4 and u scan technique with 2qmax ¼ 34.9ꢁ and 29.5ꢁ
for 46 and 48, respectively. Data were corrected for absorption
effects using the multi-scan method SADABS.54 The structures
were solved by direct methods using SHELX55 implemented in
the CRYSTALS program suit.56 The renement was carried out
by the full-matrix least-squares method with anisotropic
displacement parameters for all non-hydrogen atoms based on
F2 using CRYSTALS. The hydrogen atoms were positioned
geometrically in their idealized positions.57 The general-
purpose crystallographic tool PLATON58 was used for the
structure analysis and presentation of the results. ORTEP-3 for
Windows59 and MERCURY60 programs were used for molecular
graphics representations. Details of the data collection and
renement are given in ESI† Table S4.
2D-QSAR. QSAR study was undertaken utilizing compre-
hensive descriptors for structural and statistical analysis
(CODESSA-Pro) soware. Bio-active indole-based analogues
(compounds 30, 31, 33–40, 42–49 and 51–63) against HeLa
Antitumor activity screening
Antitumor properties of the synthesized compounds were (cervical carcinoma) cell line were used as a training set for
screened by the National Cancer Institute, Cairo University, constructing the QSAR model. Compounds 32, 41 and 50 (i.e.
Egypt, using the reported in vitro Sulfo-Rhodamine-B (SRB) about 10% of the training set) were used as an external test set
standard method adopting HeLa (cervical) and HepG2 (liver) for validating the attained QSAR model. Geometry of both
carcinoma cell lines.11–20 Cells were seeded in 96 well microtiter training and test set compounds was initially optimized by AM1
plates at a concentration of 5 ꢂ 104 to 105 cell per well in a fresh technique then, exported to CODESSA-Pro that includes
medium and le for 24 h before treatment with the tested MOPAC capability for the nal geometry optimization.
compounds to allow attachment of cells to the wall of the plate. CODESSA-Pro calculated 714 molecular descriptors (constitu-
The tested compounds were dissolved in dimethylsulfoxide tional, topological, geometrical, charge-related, semi-empirical,
(DMSO) and diluted 1000 fold in the assay. Different concen- molecular-type, atomic-type and bond-type descriptors) for the
trations of the compounds under test (0, 5, 12.5, 25, and 50 mg exported 31 training set antitumor active agents. Different
mlꢀ1) were added to the cell monolayer. Triplicate wells were mathematical transformations [including property, 1/property,
prepared for each individual dose. The monolayer cells were log(property) and 1/log(property)] of the experimentally
incubated with the tested compounds for 48 h at 37 ꢁC, in observed antitumor property/activity (IC50, mM) of the training
atmosphere of 5% CO2. Aer 48 h, the cells were xed, washed set compounds were utilized searching for the best QSAR
and stained with sulfo-rhodamine-B stain. Excess stain was model. The best multi-linear regression (BMLR) technique was
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