ACI/EG eutectic mixture mediated synthesis, characterization and: In vitro osteoblast differentiation assessment of spiropyrrolo[1,2- b] isoquinoline analogues

Article abstract of DOI:10.1039/c8ra00646f

An eco-friendly acetylcholine iodide-ethylene glycol (ACI/EG) deep eutectic mixture mediated green protocol has been developed for the synthesis of hitherto unexplored multi-functionalized linear tricyclic spiropyrrolo[1,2-b]isoquinoline analogues. The effects of the synthesized compounds on the osteoblast differentiation of hBMSC-TERT cell lines were investigated and promising results were observed with significant IC₅₀ values. In addition, molecular modeling simulations were also performed with the 3D structure of BMP-2 to reveal binding interactions and orientations of highly potent spiropyrrolo[1,2-b]isoquinoline analogues.

Full text of DOI:10.1039/c8ra00646f

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ACI/EG eutectic mixture mediated synthesis,
characterization and in vitro osteoblast
differentiation assessment of spiropyrrolo[1,2-b]
isoquinoline analogues†
Cite this: RSC Adv., 2018, 8, 16303
Govindasami Periyasami, *ab Natarajan Arumugam,
a
a
Mostafizur Rahaman,
a
c
c
Raju Suresh Kumar, Muthurangan Manikandan, Musaad A. Alfayez,
d
a
Dhanaraj Premnath and Ali Aldalbahi*
An eco-friendly acetylcholine iodide–ethylene glycol (ACI/EG) deep eutectic mixture mediated green
protocol has been developed for the synthesis of hitherto unexplored multi-functionalized linear tricyclic
spiropyrrolo[1,2-b]isoquinoline analogues. The effects of the synthesized compounds on the osteoblast
differentiation of hBMSC-TERT cell lines were investigated and promising results were observed with
significant IC50 values. In addition, molecular modeling simulations were also performed with the 3D
structure of BMP-2 to reveal binding interactions and orientations of highly potent spiropyrrolo[1,2-b]
isoquinoline analogues.
Received 22nd January 2018
Accepted 13th April 2018
DOI: 10.1039/c8ra00646f
rsc.li/rsc-advances
4
enhances the efficiency of osteogenic differentiation. Lee et al.
1
. Introduction
demonstrated that isoquinoline alkaloids induce OB differen-
Healthy bones are important for a safe and happy life. Bone tiation of stromal cells without the loss of cell viability. But, to
growth is controlled by cells called osteoclasts (OCs) and oste- date, there is no report in the literature on the effects of pyr-
oblasts (OBs) during skeletal development throughout the roloisoquinoline and its analogues on osteogenic
human life. Removal of mineralized bone by OCs followed by differentiation.
the formation of the bone matrix through the OBs is the
The pyrroloisoquinoline structural framework is present in
systematic approach taken during bone remodeling. There are a number of alkaloids including the ecteinascidin family, and
various factors that control bone remodeling including insulin- cactus and Hippeastrum genus alkaloids (Fig. 1), and it has
5
like growth factors (IGFs), tumor growth factor-beta (TGF-b), remarkable activities in the central nervous system besides
1
6a
6b
6c
bone morphogenetic proteins (BMPs) and cytokines. Among possessing antitubercular, antihypertensive, anti HIV-1,
6d
6e
the various inductive growth factors of bone morphogenetic anti-leukemic, and anti-cancer activities. Its analogues can
proteins (BMPs) including kidney development, limb forma- be used as radiotracers in positron emission tomography (PET)
tion, angiogenesis, tissue brosis and tumor development, for imaging serotonin uptake sites. The tendency of lycorine
7
osteogenic differentiation is one of the well-known and notable alkaloids to inhibit cell growth and cell division is utilized to
2
regulating processes that involve BMP-2. It increases the study their activity against a number of cancer cell lines,
number of mature OBs by enhancing the differentiation capa- showing a decrease in tumor cell growth and an increase in
bility of various stem cells. Also, biological signal pathways for survival rates with no observable adverse effects in animal
inducing osteogenic differentiation are regulated by the treatment. Isoquinoline analogues containing spiro centers are
3
binding of BMP-2 with microdomains on cell surfaces. Thus, frequently encountered in natural products and they possess
8
BMP-2 is a potent inducer of bone formation and it also diverse pharmacological properties. Also the spiro structural
motif, including polycyclic spiro heterocycles, which interca-
lates easily by its nature into DNA, can be used as a class of
effective anti-tumor pharmacophore units. The structural
a
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia. E-mail: aaldalbahi@ksu.edu.sa; pkandhan@ksu.edu.sa
9
rigidity and complexity of spiro compounds are highly favorable
for increasing affinity towards proteins by reducing the loss of
conformational entropy, which is a topic of interest in drug
b
Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai
6
c
00 025, India
Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University,
Riyadh, Saudi Arabia
10
discovery research.
d
Department of Oral Biology, Dentistry Building-D325, Winnipeg, Manitoba, Canada
Our research aim is to synthesize spiropyrrolo[1,2-b]iso-
quinolines and study their effects on OB differentiation,
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra00646f
1
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mediated synthesis of spiro heterocycles, particularly pyrrolo
[1,2-b]isoquinoline analogues, through azomethine ylide 1,3-
dipolar cycloaddition reactions is yet to be reported. The ret-
rosynthetic strategy explains the construction of regioselective
spiropyrrolo[1,2-b]isoquinoline molecules from easily available
starting materials in a single synthetic step as shown in Fig. 2.
During the synthesis, the chalcone group is retained in the
spiro adduct which may be due to the stability of the mono
adduct and to avoid the formation of the sterically hindered bis
cycloadduct. One of the C]C bonds in the dipolarophiles,
(
1E,4E)-1,5-p-substituted diphenylpenta-1,4-dien-3-one deriva-
15
tives, may preferably undergo an addition reaction with an
azomethine ylide which leads to the unreacted prop-2-en-1-one
unit in spiro heterocyclic hybrids. Spiropyrroloisoquinoline
with a chalcone group is an excellent candidate in medicinal
chemistry particularly in Alzheimer’s disease treatment, and it
is also capable of inducing apoptosis and uncoupling mito-
16
17
18
chondrial respiration. In general, chalcone compounds did
Fig.
products.
1 Pyrrolo[1,2-b]isoquinoline core and spiro-center natural
not show genotoxic effects and they may be devoid of this
signicant side effect.
19
The spiro heterocycle tethered pyrrolo[1,2-b]quinoline
analogues were obtained in good to excellent yields (85–92%)
through 1,3-dipolar cycloaddition reactions of azomethine
ylides generated in situ from an equimolar amount of a cyclic
amino acid (1), a diketone (2 or 6) and various substituted
particularly the signicant increase in alkaline phosphatase
activity (ALP) of ecteinascidin-743 (alkaloid in the ecteinascidin
11
family), a marker of osteoblastic differentiation.
2. Results and discussion
2
.1. Chemistry
Table 1 Solvent and reaction condition optimization for the synthesis
of pyrrolo[1,2-b]isoquinoline 5a
a
Interesting synthetic methodologies have been reported in the
literature for the synthesis of various pyrrolo[1,2-a]isoquinoline
derivatives including one-pot or multi-step reactions including
solid phase supported, photoredox-catalyzed, metal-cata-
lyzed,
Entry
Solvent system
Time (h)
Yield (%)
12a
12b
1
2
3
4
5
6
7
8
Ethanol
Methanol
Methanol/H
Acetonitrile
Acetonitrile/Et N
Dioxane/MeCN (1 : 1)/Et
ACI/EG, rt
ACI/EG, 40 C
48
48
48
48
48
48
48
12
Trace*
Trace*
Trace*
Trace*
15
10
60
90
1
2c
12d
acid/base catalyzed,
and Pictet–Spengler cycliza-
2
O
1
2e
tion
reactions. However, the syntheses of linear tricyclic
spiropyrrolo[1,2-b]isoquinoline ring systems and their biolog-
ical applications are relatively unexplored. The results of our
recent report on the multicomponent reaction of an inex-
pensive quaternary alkylammonium class of eutectic mixture
mediated heterocyclic molecules encouraged us to synthesize
spiropyrrolo[1,2-b]isoquinoline analogues using
methodology. An inexpensive room temperature eutectic
mixture,
3
2
NH
13
ꢀ
a
General conditions: all reactions were performed under vigorous
stirring at room temperature (except entry 8); optimized reaction
conditions are in bold (entry 8); eutectic mixtures were subjected to
a similar
high vacuum before use (entry 7 and 8); trace*: identifying product R
acetylcholine iodide–ethylene glycol (ACI/EG) by TLC.
f
14
Fig. 2 Synthetic strategy for linear tricyclic spiropyrrolo[1,2-b]isoquinoline cycloadducts.
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Scheme 1 Synthesis of oxindole substituted monospiropyrrolo[1,2-b]
isoquinolines 5a–c.
Fig. 3 Oak Ridge Thermal Ellipsoid Plot (ORTEP) of cycloadduct 5a.
ꢀ
dipolarophiles (4a–c) in ACI/EG at 40 C. The required ACI/EG
eutectic mixture, as a reaction medium, was prepared by mix-
ing acetylcholine iodide and ethylene glycol in a 1 : 9 molar
different temperatures and times of the reaction, the yield of the
product did not change signicantly compared to the reaction
at 40 C for 12 h. Hence, all of the subsequent reactions
ꢀ
20
ratio and then the mixture was heated at 70 C. Although our
group has previously established and reported reaction condi-
tions, we examined the synthesis of our target linear tricyclic
spiropyrrolo[1,2-b]isoquinoline analogues under different
reaction conditions (Table 1). As a result, it is noteworthy that
ꢀ
(Scheme 1) were carried out using the optimized reaction
conditions. Aer completion of the reaction (monitored by
TLC), the pure product was obtained by column
chromatography.
ACI/EG was recovered from the reaction mixture by frac-
tional distillation and dried overnight under high vacuum at
ꢀ
the cycloaddition reaction in ACI/EG at 40 C furnished an
excellent yield of cycloadducts in 12 h (entry 8). Under the
eutectic solvent medium, the interaction of ACI/EG via
hydrogen bonding with the carbonyl group of the substrates,
namely isatin/(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one, would
enhance the polarity of the carbon and oxygen. As a result, the
electrophilicity of the isatin carbonyl would increase which in
turn may inuence the nucleophilic attack by the NH group of
isoquinoline-3-carboxylic acid, then the subsequent dehydra-
tion and decarboxylation furnishes the 1,3-dipolar azomethine
ylide intermediate. Similarly, the polarized enol form of (1E,4E)-
1,5-diphenylpenta-1,4-dien-3-one is presumably activated by the
interaction of the eutectic mixture with the dipolarophile via
hydrogen bonding and then this facilitates the addition of the
azomethine ylide. During this catalytic process, the dipolar-
ophile underwent facile interaction with the intermediate azo-
methine ylide yielded cycloadducts signicantly. Under
Table 2 Reusability in various cycles of recovered ACI/EG in the
ꢀ
synthesis of 5a at 40 C
Yield percentage of isoxazolidine 5a in
experiments
Medium
First
90
Second
88
Third
88
Fourth
87
Fih
Scheme
2 Synthesis of acenaphthoquinone substituted mono-
Recovered ACI/EG
86
spiropyrrolo[1,2-b]isoquinolines 8a–c.
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Scheme 3 Formation of stable anti-ylides in the transition state.
Table 3 Synthesis of linear tricyclic spiropyrrolo[1,2-b]isoquinoline analogues in ACI/EG medium by the reaction of amino acids, diketones and
ꢀ
dipolarophiles through azomethine ylide 1,3-dipolar cycloaddition at 40 C
a,b
c
Entry
Secondary amino acid
Diketone
Dipolarophile
Spiropyrrolo[1,2-b] isoquinoline
Time (h)
Yield (%)
1
2
3
4
5
6
1
1
1
1
1
1
2
2
2
6
6
6
4a
4b
4c
4a
4b
4c
5a
5b
5c
8a
8b
8c
12
12
12
12
12
12
90
92
86
85
86
88
a
ꢀ
b
c
Reaction conditions: vigorous stirring at 40 C in ACI/EG eutectic mixture medium. Completion of the reaction monitored by TLC. Yield of the
isolated product aer ash column chromatography.
ꢀ
4
0 C. It was further utilized for ve consecutive reactions proton. Two broad singlets observed at d 2.89 and 3.94 corre-
and the results revealed that the eutectic mixture is stable sponded to H
d e
and H protons, respectively. The trans vinyl
and recyclable without much signicant loss of activity protons of the olenic system appeared as two doublets at d 6.12
(
Table 2).
(J ¼ 16.2 Hz) and d 7.12 (J ¼ 16.2 Hz). The NH proton of the
13
The FT-IR spectrum of compound 5a showed a band at oxindole ring resonated at d 8.25 as a singlet. In the C NMR
ꢁ
1
1
1
673 cm due to the a,b-unsaturated carbonyl and a band at spectrum, the spiro quaternary carbon displayed a peak at
ꢁ
1
686 cm due to the amide carbonyl functional group. The NH 72.79 ppm. The oxindole carbonyl carbon was observed at
ꢁ
1
1
group stretching was observed at 3382 cm . The H NMR 180.78 ppm. The a,b-unsaturated carbonyl carbon exhibited
spectrum of compound 5a exhibited a doublet at d 4.19 (J ¼ 9.0 a peak at 196.06 ppm. A DEPT-135 study conrmed that the two
a
Hz) for the H proton, a distorted doublet at d 3.53 due to the methylene carbons appeared at 35.30 and 47.90 ppm. The mass
benzylic proton and a multiplet at d 3.95–4.01 due to the H
c
spectrum of 5a exhibited a molecular ion peak at m/z 496.61 and
Fig. 4 Plots (A) and (B) show IC50 values of representative compounds 5a (20 mM) and 8a (8 mM), respectively.
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Fig. 5 Qualitative analysis of alizarin red staining of mineralized nodule formation of 5a and 8a on day 7 in osteogenic differentiation media.
the compound gave acceptable elemental analysis results. with dipolarophiles (4a–c) affording the cycloadducts (8a–c) in
Finally, the structure and stereochemistry of cycloadduct 5a good yields (Scheme 2).
were elucidated unambiguously by single crystal X-ray diffrac-
The structure and regiochemistry of the cycloadducts (8a–c)
tion analysis (Fig. 3). NMR spectra of all synthesized were also unambiguously established by their spectroscopic
compounds are provided in the ESI.† and elemental analysis data.† The stability of formation of the
21
To explore the synthetic utility of the optimized protocol, we azomethine ylide supports the relative stereochemistry of the
have investigated the cycloaddition reaction with a different synthesized novel spiropyrrolo[1,2-b]isoquinolines. In Schemes
23
diketone viz, acenaphthoquinone as this structural motif 1 and 2, formation of syn-ylides 3a and 7a may not be observed
possesses strong antioxidant properties, free radical scavenging due to the unfavorable steric repulsion between the carbonyl
22
activity and the ability to reduce lipid peroxidation. The group of the diketone and the isoquinoline ring. Most probably,
22
generated azomethine ylide (7) readily undergoes cycloaddition anti-ylides 3 and 7, generated from tetrahydroisoquinoline-3-
Fig. 6 (a) Quantitative analysis of osteoimage mineralized nodule formation of 5a and 8a on day 7 in osteogenic differentiation media. (b)
Quantitative mineralization of representative compounds 5a and 8a with DMSO as the standard {data are presented as the means ꢂ SD from
three independent experiments, n ¼ 6; *p < 0.05; **p < 0.01, ***p < 0.001}.
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representative compounds was also carried out, and the
observed nodule images (Fig. 6a) and the percentage of
mineralization (Fig. 6b) are shown in Fig. 6.
2.2.2. Osteoimage mineralization assay. Inspired by the
initial promising OB differentiation activity of 5a and 8a with
alizarin red S staining, we further assessed the OB differentia-
tion by osteoimage staining to quantify the mineralized nodules
that were induced by synthesized spiropyrrolo[1,2-b]isoquino-
line analogues 5a–c and 8a–c. The results showed that signi-
cant mineralized nodules in the hBMSC-TERT cell line had
formed by day 7 of osteogenic differentiation induction by the
compounds. All of the isatin substituted spiropyrrolo[1,2-b]
isoquinoline analogues 5a–c showed potent effects on OB
differentiation, more than those of acenaphthoquinone
substituted spiropyrrolo[1,2-b]isoquinoline analogues 8a–c. In
both series, the unsubstituted group in the phenyl ring has
Fig. 7 Osteoimage staining mineralization of spiropyrrolo[1,2-b]iso-
quinoline analogues 5a–c and 8a–c with DMSO as the standard. The
data are presented as the means ꢂ SD from three independent a greater OB differentiation effect than the p-substituted deriv-
experiments, n ¼ 6; *p < 0.05; **p < 0.01, ***p < 0.001.
atives. The accessible OB differentiation activities were
observed for halogen substituted (4-bromo, 5c and 4-chloro, 8c)
spiropyrrolo[1,2-b]isoquinolines. Also, the para substituted
carboxylic acid, isatin (2) and acenaphthoquinone (6), respec- electron donating groups, viz methyl and methoxy in 5b and 8b,
tively, are involved in the transition state which undergoes respectively, showed moderate OB differentiation effects
addition to dipolarophiles (4a–c) to give the observed cyclo- compared to the unsubstituted phenyl ring which revealed that
adducts (Scheme 3). In both Schemes 1 and 2, the cycloaddition the electronic factors of the substituted groups may contribute
24
proceeded via an endo-transition state and the possibility of to OB differentiation of hBMSC-TERT cell lines. In general, the
formation of the other isomer via an exo-transition state was good OB differentiation inducing effects of the synthesized
ruled out.
spiropyrrolo[1,2-b]isoquinoline derivatives against hBMSC-
Under the optimized reaction conditions, stirring the ACI/EG TERT stem cells clearly shows that the synthesized cyclo-
ꢀ
medium at 40 C, we obtained linear tricyclic spiropyrrolo[1,2-b] adducts are highly suitable for bone development. Though all of
isoquinoline analogues in good yields. The yields and results the synthesized compounds possessed signicant OB differen-
are summarized in Table 3.
tiation inducing effects, spiropyrrolo[1,2-b]isoquinoline
analogues with the isatin unit are more potent than their ace-
naphthoquinone analogues. Osteoimage quantication of spi-
ropyrrolo[1,2-b]isoquinoline analogues 5a–c and 8a–c is
presented in Fig. 7. The corresponding nodule images are pre-
sented in the ESI.†
2.2. Biology
2.2.1. Alizarin red S staining and osteoblast differentiation.
To evaluate the OB effect of newly synthesized spiropyrrolo[1,2-
b]isoquinoline analogues, 5a and 8a were taken as representa-
tive compounds for each series, based on their IC50 values
2.3. Docking studies
(
Fig. 4; 5a ¼ 20 mM; 8a ¼ 8 mM). Initially, the mineralized matrix
formation was conrmed by alizarin red S staining (Fig. 5).
Molecular docking study results of compounds 5a and 8b (Fig.
In different concentrations of 5a at 15 mM and 8a at 5 mM, 8) clearly reveal that the molecular interactions with the bio-
both the cycloadducts show signicant effects on osteogenic logically proved active site of the BMP-2 protein with docking
differentiation of hBMSC-TERT (Fig. 5). Osteoimage staining of scores of ꢁ2.261 (5a) and ꢁ0.781 (8b) and the energy of ꢁ51.512
Fig. 8 Optimized structure of cycloadducts 5a and 8b using the LigPrep module.
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Fig. 9 Orientation and binding interactions of (a) 5a with the BMP-2 active site and (b) 8b with the BMP-2 active site.
(
5a) and ꢁ47.780 (8b). The molecular interaction studies LEU 84 amino acid. Compound 8b has shown a good hydrogen
demonstrate that ligands were bound on the same active site bonding backbone interaction with positively charged amino
cavity in the molecule, as biologically proved and deposited in acids. Moreover, compound 5a has shown surface molecular
the scientic database. This is evident from the superposition of interactions with strong hydrogen bonding with good energy
residues in active site molecules (12–114) and amino acid resi- scores. Computational docking study results reveal that the
dues of the BMP-2 protein, in Fig. 9 and 10. At a distance of 4 A˚ , molecular biological activities shown are based on the molec-
THR 58, ASN 56, SER 85, LEU 84, GLN 83, THR 82, PRO 81, VAL ular interaction and energy and structural orientation between
80, ARG 114, GLN 109, ASP 105, and GLN 104 amino acids were the compounds and the active site amino acid polymer.
found in the active sites and involved in biochemical interac-
tions with compounds 5a and 8b. Docking of 5a with the BMP-2 2.4. Evaluation of BMP-2 gene expression of spiropyrrolo
protein is shown with two hydrogen bonding interactions with [1,2-b]isoquinoline analogues
the respective active site molecules of ARG 114 (H–O–C) along
To authenticate the interactions between synthesized cyclo-
with p–p stacking. Similarly 8b has also shown moderate
adducts and the BMP-2 protein, based on the docking studies,
hydrogen bonding interactions with the active site and has
the cycloadducts 5a–c and 8a–c have been subjected to BMP-2
shown one hydrogen bonding interaction with ARG 114 with p–
expression evaluation aer OS differentiation in OS differenti-
p stacking and another hydrogen bonding interaction with the
ation media. In the spiroindolin-2-one series 5a–c, compound
Fig. 10 In silico packing illustration of representative compounds (a) 5a and (b) 8b with their appropriate binding packets of BMP-2.
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ꢁ
1
Eagle’s medium (DMEM) supplemented with 4.5 g L
D-
ꢁ1
glucose, 4 mM L-glutamine, 110 mg L sodium pyruvate, 10%
fetal bovine serum (FBS), 1ꢃ penicillin–streptomycin (Pen–
Strep), and non-essential amino acids.
4.2. In vitro osteoblast differentiation
Cells were grown in 48-well plates in standard DMEM at
5
a density of 1 ꢃ 10 cells per mL. When cells reached 70–80%
conuence, they were cultured in DMEM supplemented with
osteogenic induction supplements containing 10% FBS, 1%
ꢁ
1
Pen–Strep, 50 mg mL L-ascorbic acid (Wako Chemicals, Ger-
many), 10 mM b-glycerophosphate (Sigma-Aldrich), 10 nM cal-
citriol (1a, 25-dihydroxyvitamin D3; Sigma), and 10 nM
dexamethasone (Sigma-Aldrich). The medium was replaced
three times per week. Compounds 5a and 8a were mixed with
osteogenic differentiation media at 15 mM and 5 mM, respec-
tively, to evaluate their osteogenic differentiation potential.
Fig. 11 BMP-2 gene expression pattern of compounds 5a–c and 8a–c
using qRT-PCR.
4.3. Cytochemical staining
4
.3.1. Alizarin red S staining of mineralized matrix. Cell
5
5
a maintained BMP-2 expression equal to the control, whereas
b and 5c showed subdued expression. In the acenaphthylen-1-
layers were washed with PBS and then xed with 4% para-
formaldehyde (Sigma-Aldrich) for 15 min at room temperature.
Aer removal of the xative, each cell layer was rinsed with
distilled water and then stained with 2% alizarin red S (Alizarin
Red S Staining Kit; Cat. no. 0223; Sciencell Research Laborato-
ries, Carlsbad, CA, USA) for 20–30 min at room temperature.
Excess dye was washed away with water.
one series 8a–c, cycloadduct 8a resulted in lower expression of
BMP-2, while 8b and 8c maintained BMP-2 expression level
equal to the control. The results are presented in Fig. 11.
3
. Conclusion
4
.3.2. Osteoimage mineralization assay. The mineralized
In conclusion, two series of spiropyrrolo[1,2-b]isoquinoline
analogues were synthesized employing an ACI/EG eutectic
mixture as an eco-friendly reaction medium through 1,3-dipolar
cycloaddition methodology. To the best of our knowledge, this
is the rst report of the synthesis of spiropyrrolo[1,2-b]iso-
quinoline analogues in the ACI/EG benign reaction medium
and their effects on OB differentiation of the hBMSC-TERT cell
line. All of the synthesized compounds possessed the capability
of enhancing the osteoblast differentiation of hBMSC-TERT cell
lines with signicant IC50 values of less than 20 mM. The
structural and functional insights of spiropyrrolo[1,2-b]iso-
quinoline analogues with the BMP-2 protein were studied by
molecular docking simulations using the Schr ¨o dinger Glide
matrix was quantied using the Osteoimage Mineralization
Assay Kit (Cat. no. PA-1503; Lonza, Allendale, NJ, USA). Aer
removal of the culture medium, cells were washed once with
PBS and then xed with 70% cold ethanol for 20 min. The
appropriate amount, as recommended by the manufacturer, of
diluted staining reagent was added, and the plates were incu-
bated in the dark for 30 min at room temperature. Aerwards,
the cells were washed, and the bound reagent was quantied
using a uorescence plate reader (excitation wavelength
4
92 nm; emission wavelength 520 nm).
4
.4. Molecular docking
program. In addition, the hypothesis has been proved by aRT- Molecular docking studies were performed using the
PCR studies. These results emphasized the role of spi- Schr ¨o dinger Glide program (2017-4, Schr ¨o dinger LLC, New York
ropyrrolo[1,2-b]isoquinolines in OB differentiation and could 2014). The selected compounds 5a and 8b structures are drawn
have myriad applications in bone biology.
using ChemSketch soware. In order to prepare ligand mole-
cules with high quality, all-atom 3D structures for large
numbers of drug-like molecules, starting with the 3D structures
in SD Maestro format, LigPrep was used (Fig. 8). LigPrep
produced a single, low-energy, 3D structure with corrected
4
. Materials and methods
4.1. Cell culture
We employed hBMSC-TERT cell lines created from primary chiralities for each successfully processed input structure. The
normal human bone marrow BMSCs by over-expressing the optimised ligands were docked into the bone morphogenetic
human telomerase reverse transcriptase gene (hTERT). We are protein-2 (BMP-2) structure using the Glide (SP & XP) module.
grateful to our collaborator Prof. Moustapha Kassem, University The required typical structure le of BMP-2 protein structure
of Odense, Southern Denmark for providing the hBMSC-TERT was downloaded from the protein data bank (PDB). The struc-
25
cell line. These cells have been extensively characterized and tural le from experimental techniques may not be suitable for
exhibit cellular and molecular phenotypes similar to those of immediate use in molecular modelling calculations. PDB
primary hBMSCs and were cultured in Dulbecco’s modied structures may miss information on connectivity that has been
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assigned along with bond orders and formal charges using the stirred in an ACI/EG eutectic mixture for 1 hour. Aer the
Protein Preparation Wizard. However, the receptor grid gener- completion of the reaction as evidenced by TLC, in an adequate
ation requires a “prepared” structure with all atoms and test, the eutectic mixture was removed by vacuum distillation
appropriate bond orders with formal charges. The shape and and the residue was chromatographed over silica gel with the
properties of receptor BMP-2 is generated on a grid by several hexane–ethyl acetate mixture (8 : 2) to give novel mono-
different sets of elds around BMP-2 binding sites that provide spiropyrrolo[1,2-b]isoquinolines (5a–c) in good to excellent
progressively more accurate scoring of the ligand presence. To yields.
0
0
0
0
conrm the best molecular interaction and validate the docking
score, the protocol was evaluated by re-docking.
5.2.1. 3-Phenyl-4-[3 -phenyl-(E)-prop-2 -en-1 -one]spiro[5.3 ]-
oxindolo-pyrrolo[1,2-b]isoquinoline, 5a.
4.5. Real time qRT-PCR
Total RNA was extracted from differentiating cells with a series
of cycloadducts 5a–c and 8a–c using the innuPREP RNA Mini
Kit (Cat no: 845-KS-2040250, Analytik Jena, Berlin, Germany) as
recommended by the manufacturer. Total RNA was quantied
using a Nanodrop spectrophotometer (Nanodrop 2000, Thermo
Scientic). cDNA was synthesized from 1 mg of the RNA samples
using a High Capacity cDNA Reverse Transcription kit (Applied
Biosystems, Foster City, CA) using a ProFlex PCR System,
Applied Biosystems according to the manufacturer’s instruc-
tions. Relative levels of mRNA were determined from cDNA
using real time PCR (Applied Biosystem-Real Time PCR Detec-
tion System) with a Power SYBR Green PCR kit (Applied Bio-
systems, UK), according to the manufacturer’s instructions.
Following normalisation to the reference gene GAPDH, quanti-
ꢀ
Yellow solid, mp 136 C; yield: 90%; IR (KBr) 3382, 1686,
ꢁ1 1
1
673 cm ; H (300 MHz, CDCl ) d 2.89 (br s, 2H, H ), 3.53 (dis
d, 1H, H ), 3.94 (br s, 2H, H ), 3.95–4.01 (m, 1H, H ), 4.19 (d, 1H,
3 d
b
e
c
H
a
, J ¼ 9.0 Hz), 6.12 (d, 1H, J ¼ 16.2 Hz), 6.72–7.58 (m, 18H,
13
ArH), 7.12 (d, 1H, J ¼ 16.2 Hz), 8.25 (s, 1H, NH). C (75 MHz,
CDCl ) 35.30, 47.90, 52.38, 62.42, 63.71, 72.79, 109.30, 123.39,
25.67, 126.15, 126.47, 126.87, 127.11, 127.18, 127.82, 128.04,
3
1
1
1
28.65, 128.73, 128.75, 129.03, 129.31, 130.35, 133.58, 133.76,
34.30, 139.89, 140.58, 142.46, 180.78, 196.06. EI-MS m/z 496.61

cation of gene expression was carried out using a comparative
C
t
method where DC
t
is the difference between the C
t
values of
+
0
(M ). Anal. calcd for C34
H
28
N
2
O
2
: C, 82.23; H, 5.68; N, 5.64%.
the target and reference gene. (Primer sequence: GAPDH – F 5
CTGGTAAAGTGGATATTGTTGCCAT 3 ,
TATTGGAACATGTAAACC 3 , BMP2
CATTCGGTCCTT 3 , R 5 CACCATGGTCGACCTTTAGGA 3 ).
0
0
0
Found: C, 82.31; H, 5.59; N, 5.71%.
R
F
5
5
TGGAATCA-
GGAACGGA-
00 0 00
0
5.2.2. 3-(4 -Methylphenyl)-4-[(3 -(4 -methoxyphenyl))-(E)-
–
0
0
0
0
0
0
prop-2 -en-1 -one]-spiro[5.3 ]oxindolopyrrolo[1,2-b]isoquino-
line, 5b.
5. Experimental
5.1. General procedure for the preparation of (1E,4E)-1,5-
bis(p-substituted-phenyl)penta-1,4-dien-3-one, (4a–c)
A solution of p-substituted benzaldehyde (2.0 mol) and acetone
ꢀ
(
1.0 mol) was stirred at 15–20 C in a 250 mL round bottom

ask. 10% sodium hydroxide solution prepared and maintained
ꢀ
at 15–20 C was added slowly. The rate of addition was adjusted
to make sure that the reaction temperature did not exceed 25–
ꢀ
3
0 C. The reaction mixture was vigorously stirred and then the
ꢀ
Yellow solid, mp 144 C; yield: 92%; IR (KBr) 3384, 1686,
precipitate was formed in 5 min. The stirring was continued for
a further 1 hour. Aer completing the reaction as evidenced by
TLC, dilute hydrochloric acid was added to neutralize the
reaction mixture. The resulting precipitate was ltered and
washed with cold water. The crude product was dried and
recrystallized in an ethyl acetate/hexane mixture yielding 90–
ꢁ
1 1
1
3
1
6
674 cm ; H (300 MHz, CDCl
H, Me), 3.48 (dis d, 1H, H ), 3.91 (br s, 2H, H
H, H ), 4.23 (d, 1H, H
, J ¼ 9.0 Hz), 6.09 (d, 1H, J ¼ 16.0 Hz),
.64–7.53 (m, 16H, ArH), 7.14 (d, 1H, J ¼ 16.0 Hz), 8.29 (s, 1H,
3
) d 2.81 (br s, 2H, H
d
), 3.16 (s,
b
e
), 3.96–4.10 (m,
c
a
1
3
NH). C (75 MHz, CDCl
3
) 29.81, 34.28, 39.64, 56.38, 61.31,
6
1
1
1
2.69, 72.22, 110.52, 122.38, 126.49, 126.52, 126.59, 126.76,
27.09, 127.27, 127.86, 127.99, 128.29, 128.64, 128.70, 129.41,
29.49, 130.51, 133.58, 133.69, 134.48, 138.92, 140.11, 142.43,
80.99, 197.60. EI-MS m/z 556.65 (M ). Anal. calcd for
36 32 2 2
C H N O : C, 82.41; H, 6.15; N, 5.35%. Found: C, 82.66; H,
9
5% of pure (1E,4E)-1,5-bis(p-substituted-phenyl)penta-1,4-
dien-3-one (4a–c).
+
5
.2. General procedure for the synthesis of
monospiropyrrolo[1,2-b]isoquinolines (5a–c)
6
.22; N, 5.28%.
A mixture of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1)
1.2 equiv.), isatin (2) (1.2 equiv.) and (1E,4E)-1,5-bis(p-
substituted-phenyl)penta-1,4-dien-3-one (4a–c) (1.0 equiv.) was
00
0
00
5
.2.3. 3-(4 -Bromophenyl)-4-[(3 -(4 -bromophenyl))-(E)-prop-
-en-1 -one]spiro[5.3 ]oxindolopyrrolo[1,2-b]isoquinoline, 5c.
(
0
0
0
2
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ꢀ
ꢀ
Pale yellow solid, mp 122 C; yield: 86%; IR (KBr) 3382, 1683, Colourless solid, mp 160 C; yield: 86%; IR (KBr) 1716,
ꢁ1
1
ꢁ1
1
1
676 cm ; H (300 MHz, CDCl
d, 1H, H ), 3.87 (br s, 2H, H ), 3.91–4.19 (m, 1H, H
, J ¼ 9.0 Hz), 6.21 (d, 1H, J ¼ 16.2 Hz), 6.98–8.16 (m, 16H,
3
) d 2.88 (br s, 2H, H
), 4.35 (d, 1H, d, 1H, H
), 4.11–4.14 (m, 1H, H
ArH), 7.20 (d, 1H, J ¼ 16.2 Hz), 8.16 (s, 1H, NH). C (75 MHz, (m, 19H, ArH with alkene H). C NMR (75 MHz, CDCl
CDCl ) 34.29, 39.02, 49.62, 58.31, 61.64, 70.22, 111.12, 121.00, 46.21, 52.33, 56.62, 62.95, 64.19, 73.01, 120.85, 123.60, 125.42,
d
), 3.51 (dis 1678 cm ; H (300 MHz, CDCl
), 3.68 (s, 3H, OMe), 3.98 (d, 1H, H
), 5.55 (d, 1H, J ¼ 16.2 Hz), 6.61–8.13
3
) d 2.96 (br s, 2H, H
d
), 3.13 (dis
b
a
), 4.00–4.03 (m, 2H,
b
e
c
H
a
H
d
c
1
3
13
) 32.52,
3
3
1
1
1
25.09, 126.31, 126.38, 126.86, 127.12, 127.89, 128.22, 128.29, 125.39, 125.90, 126.33, 126.44, 127.09, 127.67, 127.91, 128.49,
28.58, 128.66, 128.89, 129.39, 129.59, 129.91, 133.19, 133.64, 128.65, 128.71, 129.00, 129.09, 130.05, 130.53, 131.17, 132.46,
34.84, 138.89, 140.56, 142.73, 181.52, 197.85. EI-MS m/z 654.36 133.61, 133.68, 134.12, 137.66, 140.28, 141.68, 142.98, 196.19,
+
+
(
M ). Anal. calcd for C34
H
26Br
2
N
2
O
2
: C, 62.40; H, 4.00; N, 4.28%. 210.00. EI-MS m/z 591.69 (M ). Anal. calcd for C40
4
H33NO : C,
Found: C, 62.59; H, 3.89; N, 5.01%.
81.20; H, 5.62; N, 2.37%. Found: C, 81.26; H, 5.58; N, 2.42%.
00 0 00
.3.3. 3-(4 -Chlorophenyl)-4-[(3 -(4 -chlorophenyl))-(E)-prop-
5
0
0
0
0
5.3. General procedure for the synthesis of
2 -en-1 -one]spiro[5.2 ]acenapthen-1 -ono-pyrrolo[1,2-b]isoquino-
monospiropyrroloisoquinolines (8a–c)
line, 8c.
A mixture of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1)
(1.2 equiv.), acenaphthoquinone (6) (1.2 equiv.) and (1E,4E)-1,5-
bis(p-substituted-phenyl)penta-1,4-dien-3-one (4a–c) (1.0 equiv.)
was stirred in an ACI/EG eutectic mixture for 1 hour. Aer the
completion of the reaction as evidenced by TLC, in an adequate
test, the eutectic mixture was removed by vacuum distillation
and the residue was chromatographed over silica gel with the
hexane–ethyl acetate mixture (8 : 2) to give novel mono-
spiropyrrolo[1,2-b]isoquinolines (8a–c) in good yields.
0
0
0
0
5
.3.1. 3-Phenyl-4-[3 -phenyl-(E)-prop-2 -en-1 -one]-spiro[5.2 ]-
0
ꢀ
acenapthen-1 -ono-pyrrolo[1,2-b]isoquinoline, 8a.
Colourless solid, mp 172 C; yield: 88%; IR (KBr) 1720,
ꢁ1 1
1
679 cm ; H (300 MHz, CDCl ) d 2.98 (br s, 2H, H ), 3.32 (dis
3 d
d, 1H, H ), 3.89 (d, 1H, H ), 4.01–4.03 (m, 2H, H ), 4.09–4.13 (m,
b
a
d
1
H, H
c
), 5.62 (d, 1H, J ¼ 16.2 Hz), 6.76–8.16 (m, 19H, ArH with
13
alkene H). C NMR (75 MHz, CDCl
3
) 31.25, 45.95, 52.13, 62.91,
4.18, 73.09, 120.83, 123.44, 125.38, 125.54, 125.89, 126.30,
26.41, 126.98, 127.59, 127.88, 128.52, 128.64, 128.73, 129.07,
29.10, 130.00, 130.42, 131.24, 132.50, 133.61, 133.68, 134.17,
6
1
1
1
37.68, 140.30, 141.72, 142.96, 196.23, 210.15. EI-MS m/z 600.53
+
(M ). Anal. calcd for C H Cl NO : C, 76.00; H, 4.53; N, 2.33%.
3
8
27
2
2
ꢀ
ꢁ1
1
Found: C, 76.07; H, 4.59; N, 2.29%.
Yellow solid, mp 186 C; yield: 85%; IR (KBr) 1716, 1678 cm ; H
(
300 MHz, CDCl ) d 2.93 (br s, 2H, H ), 3.42 (dis d, 1H, H ), 3.94 (d,
3
d
b
1
H, H ), 4.08–4.09 (m, 2H, H ), 4.13–4.19 (m, 1H, H ), 5.69 (d, 1H,
a d c
Conflicts of interest
13
J ¼ 16.2 Hz), 6.65–8.05 (m, 21H, ArH with alkene H). C (75 MHz,
CDCl ) 35.44, 48.00, 52.67, 63.17, 64.25, 72.81, 121.09, 123.82,
25.02, 125.62, 126.13, 126.35, 126.42, 127.09, 127.67, 127.95,
There are no conicts to declare.
3
1
1
1
1
28.44, 128.70, 128.75, 129.07, 129.12, 130.09, 130.23, 131.50,
32.44, 133.67, 133.78, 134.00, 137.64, 140.23, 141.84, 142.53,
96.42, 210.06. EI-MS m/z 531.64 (M ). Anal. calcd for C38
Acknowledgements
+
H29NO
2
:
The authors express their appreciation to the Deanship of
C, 85.85; H, 5.50; N, 2.63%. Found: C, 85.91; H, 5.43; N, 2.70%.
Scientic Research at King Saud University for funding this
00 0 00
5
.3.2. 3-(4 -Methoxyphenyl)-4-[(3 -(4 -methoxyphenyl))-(E)- work through research group no. RG-1438-038. Also, GPS
0
0
0
0
prop-2 -en-1 -one]spiro[5.2 ]acenapthen-1 -ono-pyrrolo[1,2-b] thanks the Council of Scientic and Industrial Research (CSIR),
isoquinoline, 8b. New Delhi.
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1
2 (a) T. E. Nielsen and M. Meldal, J. Comb. Chem., 2005, 7, 599–
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