Glucosamine-6-phosphate synthase inhibiting C3-β-cholesterol tethered spiro heterocyclic conjugates: Synthesis and their insight of DFT and docking study

Article abstract of DOI:10.1016/j.bioorg.2019.102920

A series of novel covalent cholesterol-spiro pyrrolidine/pyrrolizidine heterocyclic hybrids possessing biologically active oxindole, indanedione, and acenaphthylene-1-one have been synthesized by the reaction of C3-β-cholesteroalacrylate with heterocyclic

Full text of DOI:10.1016/j.bioorg.2019.102920

Accepted Manuscript
Glucosamine-6-phosphate synthase inhibiting C3-β-cholesterol tethered spiro
heterocyclic conjugates: Synthesis and their insight of DFT and docking study
Govindasami Periyasami, Subban Kamalraj, Ramanathan Padmanaban,
Santhakumar Yeswanth Kumar, Antony Stalin, Natarajan Arumugam, Raju
Suresh Kumar, Mostafizur Rahaman, Periyan Durairaju, Abdulaziz Alrehaili,
Ali Aldalbahi
PII:
S0045-2068(18)31360-9
https://doi.org/10.1016/j.bioorg.2019.102920
102920
DOI:
Article Number:
Reference:
YBIOO 102920
Bioorganic Chemistry
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Received Date:
Revised Date:
Accepted Date:
21 November 2018
27 March 2019
9 April 2019
Please cite this article as: G. Periyasami, S. Kamalraj, R. Padmanaban, S. Yeswanth Kumar, A. Stalin, N. Arumugam,
R. Suresh Kumar, M. Rahaman, P. Durairaju, A. Alrehaili, A. Aldalbahi, Glucosamine-6-phosphate synthase
inhibiting C3-β-cholesterol tethered spiro heterocyclic conjugates: Synthesis and their insight of DFT and docking
study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102920
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Glucosamine-6-phosphate synthase inhibiting C3-β-cholesterol tethered spiro
heterocyclic conjugates: Synthesis and their insight of DFT and docking study
Govindasami Periyasamia,b,*, Subban Kamalrajc, Ramanathan Padmanaband, Santhakumar
Yeswanth Kumard, Antony Staline,f, Natarajan Arumugama, Raju Suresh Kumara, Mostafizur Rahamana,
Periyan Durairajug, Abdulaziz Alrehailia, Ali Aldalbahia
a Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
b Department of organic chemistry, University of Madras, Guindy campus, Chennai, India.
c Department of microbiology, Indian Institute of Science, Bangalore, Karnataka, India.
d Department of Chemistry, School of Physical, Chemical & Applied Sciences, Pondicherry University,
Puducherry, India.
e
Division of Bioinformatics, Entomology Research Institute, Loyola College, Chennai – 600 034, Tamil
Nadu, India.
f
Centre for Advanced Studies in Botany & Centre for Herbal Sciences, University of Madras, Guindy
Campus, Chennai – 600 025, Tamil Nadu, India.
g Department of Chemistry, Thiruvalluar Government arts college, Periyar University, Raispuram, India.
*Corresponding authors E-mail: pkandhan@ksu.edu.sa; aaldalbahi@ksu.edu.sa
Abstract:
A series of novel covalent cholesterol-spiro pyrrolidine/pyrrolizidine heterocyclic hybrids
possessing biologically active oxindole, indanedione, and acenaphthylene-1-one have been
synthesized by the reaction of C3-β-cholesteroalacrylate with heterocyclic di- and tri- ketones.
All the sixteen compounds were obtained as a single isomer in good yield through a stereo- and
regio- selective 1,3-dipolar cycloaddition methodology. Stereochemistry of the spiranic
cycloadducts has been established by spectroscopic analysis and the regioselectivity outcome of
the spiro adducts has been accomplished by DFT calculations at B3LYP/6-31G (d,p) level study.
In vitro antibacterial activity of the newly synthesized cycloadducts were evaluated against
highly pathogenic Gram-positive and Gram-negative bacteria and the most active compounds 5a,
1

13, and 14 underwent automated in silico molecular docking analysis in order to validate their
effective orientation as a inhibitors bound in the active site of glucosamine-6-phosphate synthase
(1XFF) enzyme by employing AutoDock Tools.
Keywords: Spiro steroid hybrids, 1XFF enzyme, cycloaddition, DFT, HOMO-LUMO,
molecular docking.
1. Introduction
The significant impact on immunity of human body leads to frequent viral infections, which
impairs the function of lymphocytes and also increases the level of infectious diseases such as
AIDS, cancer, clostridium difficile infection, etc. Hence, there is a substantial need for
identification and synthesis of novel substances, which are active against microbes.
Transportation and access of drugs in the metabolic system are two important factors that need to
be considered while synthesizing biologically targeting molecules. Having lipophilic nature of
steroidal analogues including cholesterol plays an important role in medicinal chemistry [1].
Many non-pathogenic diseases like cardiac, brain vascular diseases and dementia have been
directly linked to cholesterol homeostasis [2]. The fused ring structure of cholesterol has also
been shown to directly affect the function of some membrane proteins involved in membrane
trafficking to prevent sodium ion leakage through the plasma membrane [3], bacterial membrane
[4], and cell signaling including the G-protein coupled receptors [5]. Furthermore, cholesterol
embedded heterocyclic molecules are of current research interest due to their diverse biological
applications, structural rigidity and bioavailability. Particularly, C3-β-substituted cholesterol is
2

an exceptional choice to introduce heterocyclic motifs because it does not lose their core
cholesterol structure with the addition of spiropyrrolidine groups [6].
Spiro molecules attract synthetic chemists due to their rigid conformational features and easy
incorporation into biological macromolecules. A number of natural spiro molecules, such as
alkaloids and terpenoids, are isolated from living organisms and plants [7] and the synthesized
spiro pyrrolidine analogues have been identified to possess antimicrobial [8], anti-inflammatory
[9], potential antileukemic [10], antiviral [11], and anticancer [12] activities and act as inhibitors
of acetylcholinesterase (AChE), NK-1 receptor [13], etc. A recent report from Austen et al.
revealed that the spiro heterocycles are potentially valuable compounds for controlling
pathogenesis of Alzheimer’s disease coupled with amyloid precursor proteins in cholesterol
regulation [14]. Inspired by the aforementioned potential biological applications of cholesterol
motif and considering the functionality of spiropyrrolidine nucleus in the present study, we
enthused to synthesis C3-β-cholesterol tethered spiro pyrrolidines through azomethine ylide 1,3-
dipolar cycloaddition methodology and study their biological profiles. Also we intend to
compare the mode of interactions with the glucosamine-6-phosphate synthase (1XFF) enzyme
through in silico study to obtain the better therapeutic values for human antimicrobial diseases.
In silico drug design analysis helps to understand the interactions between the drugs and
receptors in a better way as well as reduces the time needed to achieve the target [15]. The 1XFF
enzyme is present in all kinds of cells and it is responsible for the synthesis of glucosamine-6-
phosphate (Glc-N-6-P) from D-fructose-6-phosphate and L-glutamine, which were involved in
protein synthesis. This indispensable enzyme 1XFF of microorganism and mammal cells is used
3

to build macromolecule components for the cell wall assembly in mannoproteins, chitins, and
prokaryotes [16]. A short time inactivation of 1XFF for microorganism is lethal while in
mammals, having longer life span, the depletion of amino sugar pool for a short time is not toxic.
This 1XFF enzyme binds with 16s rRNA of the 30s ribosome subunits in bacteria which
interferes the binding of formyl-methionyl tRNA to the 30s subunit [17]. Therefore, in the
present study, in order to validate the ligand-receptor binding ability with biologically most
active cycloadduct, 1XFF have been chosen.
2. Results and discussions
2.1.Synthesis
A small library of novel cholesterol tethered spiropyrrolidines were accomplished by one pot
azomethine ylide 1,3-dipolar cycloaddition in the presence of isopropyl alcohol as solvent at
reflux conditions. Based on the literature report [18], cholesterol was refluxed with freshly
distilled acryloyl chloride in dry dichloromethane and then recrystallized from ethanol to afford
C3-β-cholesteroalacrylate 4 as colorless crystals in good yield which then was used as
dipolarophile. The required non-stabilized azomethine ylide intermediate was generated from
di/triketone with secondary amino acids, which involves the initial formation of iminium
carboxylate betaine followed by decarboxylation. The generated highly reactive intermediate,
azomethine ylide, undergoes in situ 1,3-dipolar cycloaddition with an electron deficient
dipolarophile.
In the present work, we generate in situ azomethine ylide 3y by reacting isatin 1a and sarcosine 2
which then reacts with the dipolarophile 4 by 1,3-dipolar cycloaddition in various high polar
4

protic solvent media such as, methanol, ethanol, isopropyl alcohol, acetonitrile and
dimethylsulfoxide. The optimized reaction condition was identified in isopropyl alcohol at reflux
condition(Scheme 1). The reaction proceeds stereoselectively affording a single isomer of novel
1-N-methyl spiro[2.3"]oxindolo-3-cholestrylcarboxylate pyrrolidine 5a. This facile one pot
three-component reaction afforded the product in an excellent yield with the generation of two
stereocenters and formation of new C-C and C-N bonds in a single step. Since CO2 and water are
the only by-products during the reaction, it has to be considered as a high atom economy reaction
as per the green chemistry limitations.
Scheme 1. Synthesis of 1-N-methyl spiro[2.3"]oxindolo-3-cholesterylcarboxylate pyrrolidine,
5a
After completion of the reaction (monitoring by TLC), the reaction mixture was poured into ice
cold water and extracted with DCM to obtain the crude product 5a and then was purified by
column chromatography using hexane:EtOAc (7:3) as eluent. Structural elucidation of the
1
13
synthesized cycloadduct 5a was accomplished by FT-IR, H, C NMR and mass spectrometry
5

studies as well as elemental analysis. In the FT-IR spectrum, the amide carbonyl and ester
carbonyl groups exhibited a strong absorption at 1702 cm-1 and 1716 cm-1 respectively. A broad
1
band of NH group stretching was observed at 3365 cm-1. The H NMR spectrum of compound
5a showed a sharp singlet at  2.09 due to N-methyl protons and the CH proton of pyrrolidine
ring as a triplet at δ 3.62. If the other isomer 6 was formed, one would expect a multiplet instead
of a triplet. The Hc proton of methylene carbon of the pyrrolidine ring showed a multiplet in the
range δ 2.23-2.42 and the Hd proton showed a multiplet in the range δ 2.55-2.67. Also, Ha
proton showed a multiplet in the range δ 3.00-3.08 and Hb proton showed a multiplet in the range
δ 3.34-3.41. The C(3)H in cholesterol ring A, tethered with acrylate chain showed a multiplet at 
13
4.12-4.20. The C NMR spectrum of cycloadduct 5a showed two peaks at 168.76 and 177.82
ppm for oxindole and ester carbonyl carbons respectively. The spiro carbon showed a peak at
72.85 ppm. In the mass spectrum analysis, the molecular weight of the cycloadduct was
confirmed by a peak at m/z 614.90 (M+) and the elemental analysis also showed satisfactory
results.
Having optimized the reaction protocol, it is appropriate to utilize the potential protocol to
explore the synthesis of novel spiropyrrolidine and pyrrolizidine tethered cholesterol analogues.
Totally 16 new C3-β-cholesterol tethered spiro pyrrolidine analogues have been synthesized. All
reactions including diketone/triketone and sarcosine/proline proceed smoothly with novel C3-β-
cholesterol acrylate dipoloarphile in isopropyl alcohol medium under reflux for 1−2 h (Scheme
2).
6

Scheme 2. Synthesis of novel C3-β-cholesterol tethered spiro- pyrrolidine/pyrrolizidine
analogues from various substituted diketone/triketone, cyclic and/or acyclic aminoacids with
dipolarophile in i-PrOH medium under reflux conditions for 1–2 h.
3. Computational study
To shed light on the experimental findings, theoretical calculations have been performed for the
1,3 dipolar cycloaddition reactions using the DFT approach. It helps to understand the
selectivity of the cycloadduct 5a formed through the reaction between the azomethine ylide
7

(reactive dipole species 3y) and C3-β-cholesterolacrylate (dipolarophile 4) [cf. Scheme 1].
Initially, the optimized geometries of all compounds were obtained using B3LYP density
functional theory with Pople’s 6-31G (d,p) basis set for all atoms, and the Gaussian 09 quantum
chemistry software [19] is employed for the purpose. Bulk effects of the solvent (i-PrOH) on the
cycloaddition reaction have been taken into account by means of the polarizable continuum
model (PCM) at temperature 356K. The optimized geometry of the azomethine ylide (3y)
reveals that the structure is slightly non-planar due to the N-methyl group and it actually exists in
two diastereomeric syn- and anti- forms (Scheme 3), where the anti-3y form is found to be more
stable than the syn-3y with an energy difference  5.2 kJ/mol and hence the computational
investigation is focused only on the anti-3y configuration.
syn-3y (Z-form)
anti-3y (E-form)
E = -1501289 kJ/mol
E = -1501294 kJ/mol
Scheme 3: (Top) Optimized geometry of the azomethine ylide 3y in its diastereomeric forms
syn- and anti. (Bottom) Dipolar species attacking phases (α & ) of the dipolarophile 4.
8

The -phase attack on anti-3y results in a more favorable cycloadduct 5, whereas the -phase
attack is not a favorable one for the cycloaddition reaction. Moreover, the dipolarophile in -
phase was further stabilized by 1,4-conjugation which resembles the mechanism of the Michael
addition.
Analysis of the Frontier molecular orbitals of the anti-3y (dipole) and 4 (dipolarophile), shown in
Figure 1, clearly reveals that the energy gap values of HOMO(anti-3y) – LUMO(4) is lower (~
120 kJ/mol) than that of HOMO(4) – LUMO(anti-3y). The frontier molecular orbital density
distribution of the respective orbitals strongly supports the HOMO(anti-3y) – LUMO(4)
interaction, and therefore HOMO of the dipole and LUMO of the dipolarophile interaction
controls the cycloaddition reaction under normal condition.
Figure 1: Frontier molecular orbitals of anti-3y and dipolarophile 4 calculated at the B3LYP/6-
31G (d,p) level. The orbital energies and energy gap values (in eV) are indicated in the
respective level.
9

In order to support the favorable cycloadduct by means of regioselectivity, the reaction (Scheme
1) is theoretically investigated by locating all the possible transition states (TS) labeled as TS1,
TS2, and TS3. The meanings of different TS are presented as a Table in Scheme 4. These TS
were energetically optimized under similar level of calculation by using Berny analytical
gradient method, and characterized by saddle point leading to the occurrence of one imaginary
frequency. The stationary point (energy in kJ mol-1) of these TS and the lengths of C-C bond
involved in the cycloaddition reaction are also presented in Scheme 4. For clarity, the relative
energy profile of stationary points of the reaction in solvent phase is also depicted in Figure 2
and the optimized TS geometries are presented in Figure 3. This study confirms that the most
favorable cycloadduct was obtained via the TS2 geometry (anti-3y interacts with C3-β-
cholesterolacrylate 4).
10

Figure 2: Relative energy profile of stationary points of the 1,3 dipolar cycloaddition reaction in
the solvent phase.
Scheme 4: Plausible mechanism for the 1,3-dipolar cycloaddition reaction. The most favorable
regioselective cycloadduct is confirmed via the formation of TS2 geometry as shown in the table.
α-Anti-C1-TS1
11

β-Anti-C1-TS2
α-Anti-C2-TS3
Figure 3. The possible transition states (TS1, TS2, TS3) for the 1,3-dipolar cycloaddition of
HOMOdiene – LUMOdienophile optimized at B3LYP/6-31G(d,p) level
12

4. In vitro anti-bacterial activity
The assessment of antibacterial activity of the synthesized C3--cholesterol tethered spiro-
pyrrolidines/pyrrolizidines was studied against environmentally destructive Gram-negative and
Gram-positive pathogens by agar well diffusion method. The efficacy of antibacterial inhibitory
activity diverges with respect to each compound against a panel of four human pathogens viz. V.
cholera, P. mirabilis, M. luteus and B. subtilis. The data accompanied with the antibacterial
prospective of the synthesized compounds are presented in Tables 1 and 2. The bacterial
inhibition was tested with different concentrations of cycloadducts, i.e., 50 µg/ml, 100 µg/ml,
and 150 µg/ml, against standard drug streptomycin. The results revealed that almost all
synthesized compounds exhibited good to excellent antimicrobial inhibition against the tested
bacteria. Among the cycloadducts, 5a, 5d, 8, 10, 12a, 13 and 14 were found to be more active,
particularly compounds 12a, 13 and 14 showed good activity than the other compounds against
V. cholera and P. mirabilis. Cycloadduct 13 showed potential inhibition activity (18.9 mm at 150
μg/mL) against Gram-negative bacteria and compounds 10 and 14 showed good activity (16.8
mm at 100 μg/mL and 18.2 mm at 150 μg/mL) against Gram-positive bacteria. In general,
acenaphthoquinone and ninhydrin-derived spiropyrrolidine and pyrrolizidines show good activity
against tested bacteria. Even in high concentration at 150 mg/mL, acenaphthoquinone derived
cycloadducts show better inhibition activity than oxindole substituted spiro
pyrrolidines/pyrrolizidines against the tested microbes. C3--Cholesterol tethered
spiropyrrolizidine candidates have high inhibition capability against the harmful microorganisms
than pyrrolidine derived analogues. In order to understand the interaction of drugs with receptor,
as per the drug design concepts, the synthesized cycloadducts (selectively from each derivatives)
13

were subjected to in silico molecular docking studies through docking with Glc-N-6P to obtain
their efficiency to support in vitro anti-bacterial potency against the bacteria.
Table 1. Inhibition effect of C3-β-cholestrylcarboxylate spiropyrrolidines (5a-f, 5, 10) on the
growth of Vibrio cholerae, Proteus mirabilis, Micrococcus luteus and Bacillus subtilis.
Zone of inhibition diameter in mm
Conce.
Compounds
V. cholerae P. mirabilis M. luteus B. subtilis
50 µg/ml
13.4
12.6
13.1
12.8
13.2
13.2
12.5
14.4
14.0
13.8
13.6
14.1
13.3
13.5
13.1
13.4
13.3
13.8
13.6
14.0
13.2
13.1
13.3
14.1
13.0
13.3
13.7
13.8
13.6
13.7
13.6
13.2
5a
5b
5c
5d
5e
5f
8
10
100 µg/ml
15.2
14.8
14.5
15.0
13.1
13.0
14.9
16.8
13.0
13.3
13.6
13.8
13.6
13.2
13.6
16.2
13.0
13.1
13.0
13.5
13.2
13.3
14.2
15.2
13.0
13.6
13.2
13.8
13.5
13.3
13.1
15.8
5a
5b
5c
5d
5e
5f
8
10
150 µg/ml
16.2
15.8
15.5
16.3
15.1
15.6
15.0
17.1
15.4
15.4
15.2
15.8
15.1
15.2
17.3
15.2
15.1
15.3
15.5
15.8
15.5
14.9
16.0
15.3
15.7
15.6
15.8
15.5
15.1
15.1
15.8
14.9
5a
5b
5c
5d
5e
5f
8
10
14

Table 2. Inhibition effect of C3-β-cholestrylcarboxylate spiropyrrolizidine (12a-f, 13, 14) on the
growth of Vibrio cholerae, Proteus mirabilis, Micrococcus luteus and Bacillus subtilis.
Zone of inhibition diameter in mm
Conce.
Compounds
V. cholerae P. mirabilis M. luteus B. subtilis
50 µg/ml
14.4
13.9
12.8
13.1
14.2
12.8
13.8
15.1
15.4
14.1
13.1
13.8
13.6
13.2
13.3
15.1
13.3
13.6
14.2
14.1
14.0
14.0
13.0
13.9
14.1
13.1
12.7
13.2
13.4
13.9
13.5
13.9
12a
12b
12c
12d
12e
12f
13
14
100 µg/ml
16.2
15.0
14.8
15.0
14.6
14.9
15.9
17.4
15.4
14.6
14.7
15.0
14.6
15.2
15.5
17.6
15.5
15.0
14.8
14.6
15.2
14.7
16.3
15.4
15.3
14.9
14.7
14.8
15.5
15.0
15.0
16.3
12a
12b
12c
12d
12e
12f
13
14
150 µg/ml
16.7
15.7
15.6
16.0
15.5
16.0
18.9
18.5
15.9
15.6
15.3
15.7
15.9
16.3
18.3
17.3
16.0
15.0
15.7
15.9
15.7
16.7
17.1
17.4
15.3
15.4
16.1
16.1
15.7
15.9
16.9
17.4
12a
12b
12c
12d
12e
12f
13
14
15

5. In silico screening and the mode of receptor interaction
To validate the ligand-receptor associations, pronounced bioactive cycloadducts such as 5a, 13,
and 14 were selected for docking studies as representative compounds of the series with the
receptor Glc-N-6P synthase.
The molecular docking of selected pyrrolidine/pyrrolizidine derivatives with Glc-N-6P
aminotransferase revealed significant interactions with the active amino acids in the active sites.
3D picture and 2D interaction diagram of the corresponding binding conformations has been
presented in Figures 4-6 and the docking results are shown in Table 3. Compound 5a showed
binding interaction with aliphatic nonpolar amino acid GLYʹ78/HN/O (-6.51 kcal/mol) whereas
compound 13 showed binding interaction with one aromatic nonpolar amino acid TRPʹ74/HE1,
and one aliphatic nonpolar amino acid ALAʹ75/O (-6.96 kcal/mol). The cycloadduct 14 showed
interaction with one cyclic nonpolar amino acid PROʹ62/O (-6.74 kcal/mol).
The spiro pyrrolizidine 13 showed good binding (TRPʹ74/HE1, ALAʹ75/O) with the active
amino acids in the embedded active site of the GlcN-6-P synthase. It is in agreement with the
original crystallographic structure of GlcN-6-P synthase, which has been docked with glutamic
acid in the same active region [20]. TRPʹ74 acts as a gate mediator, which is the key path to
transfer the ammonia from glutaminase domains to synthase domains. In the absence of
glutamine from the glutaminase domain, the channel will be closed and it prevents the enzyme to
use ammonia for sugar amination. Upon glutamine hydrolysis, the channel will be open and is
separated from the solvent so that the ammonia intermediate product is transferred to the second
active site [21].
16

Based on the interaction, the activity of the adduct 13 may due to the inhibition of enzyme GlcN-
6-P synthase through the binding with TRPʹ74 and don’t allow to open the ammonia channel for
the activation of the enzyme glutaminase function. At the same time, CYSʹ1 is also present in the
hydrophobic region and it might catalyze a complex reaction involving ammonia transfer from
L-glutamine to fructose-6-phosphate, followed by isomerization of the formed fructosamine-6-
phosphate to glucosamine-6-phosphate. These results are in agreement with the previous studies
where they have analyzed the inhibition of enzyme GlcN-6-P synthase using chemically
synthesized motifs and natural plant molecules such as (3-alkoxy-5-nitrobenzofuran-2-yl)
(phenyl) methanone derivatives, entagenic acid, and 3,4,4a,5,8,8a-hexahydro-6-
methylisochromen-1-one (HMIC) [22]. The other derivatives 5a and 14 showed binding in the
similar domain of GlcN-6-P synthase as 13, but their binding energy, inhibition constant and
ligand efficiency are significantly different and may not be deciphered when compared to 13.
Based on our results, we conclude that all synthesized spiro pyrrolidine/pyrrolizidine derivatives
might act as a prominent antimicrobial agent through inhibition of GlcN-6-P synthase. Further
studies may be helpful to identify the complete antimicrobial properties.
17

(a)
(b)
Figure 4. (a) 3D interaction diagram and (b) 2D interaction diagram of compound 5a with active
site of target receptor protein 1XFF. The hydrogen bond interactions are shown as dotted yellow
and black lines and the hydrophobic regions are shown in green coloured lines.
(a)
(b)
Figure 5. (a) 3D interaction diagram and (b) 2D interaction diagram of compound 13 with active
site of target receptor protein 1XFF. The hydrogen bond interactions are shown as dotted yellow
and black lines and the hydrophobic regions are shown in green coloured lines.
18

(a)
(b)
Figure 6. (a) 3D interaction diagram and (b) 2D interaction diagram of compound 14 with active
site of target receptor protein 1XFF. The hydrogen bond interactions are shown as dotted yellow
and black lines and the hydrophobic regions are shown in green coloured lines.
Table 3. In Silico molecular docking of compounds 5a, 13, and 14 with glucosamine-6-
phosphate synthase (1XFF)
Ligand
Protein
(PDB ID)
Binding amino acid
residues
Binding
energy
(kcal/mol)
Inhibition
constant
(μM)
VDW_HB
desolv_energy efficiency
(kcal/mol)
Ligand
1XFF
1XFF
1XFF
Glyʹ78/Hn/O
Trpʹ74/He1, Alaʹ75/O
Proʹ62/O
-6.51
-6.96
-6.74
16.78
15.62
8.16
-8.51
-8.52
-8.76
0.14
0.15
0.14
5a
13
14
5. Conclusions
A series of C3-β-cholesterol tethered spiro pyrrolidines/pyrrolizidines as Glc-N-6-P inhibitor
were designed and synthesized through a straightforward one pot three-component 1,3-dipolar
cycloaddition methodology. A convergent DFT conceptual strategy has been developed to
19

account the regioselectivity of spiro cycloadducts during the in situ cyclization. It reveals that,
having lowest energy anti-ylide underwent cyclization through the β-phase of HOMO of
cholesterol tethered diene which yield selectively one regioisomeric cycloadduct. The
synthesized compounds were tested against highly pathogenic bacteria and all sixteen steroidal
spiro pyrrolidine and pyrrolizidines showed good anti-bacterial activity and the highly potent
compounds 5a, 13 and 14 were selected to validate the inhibition mode of Glc-N-6-P synthase
inhibitor by in silico docking analysis using AutoDock Tools Version 1.5.6. The docking results
showed the good binding ability with active amino acids of GlcN-6-P synthase and it effectively
inhibit the glutaminase domain The present study revealed that the newly synthesized novel C3-
β-cholesterol tethered spiro pyrrolidines/pyrrolizidines are the best target for further development
as wide spectrum antibacterial agents.
6. Materials and methods
6.1. Antibacterial assay
The test bacteria were subcultured in the muller-Hinton broth (overnight) from which 1 ml of
cell suspension was taken and OD was adjusted to 0.5, after which it was spread as a thin film
over the muller-Hinton agar plates. The synthetic compounds were loaded onto the disc at 50 g,
100 g, and 150 g concentrations and air-dried. This was placed on the inoculated muller-
Hinton agar plates, after which they were incubated at 37 ˚C for 48 h. After incubation, the zone
of inhibition was measured. A streptomycin disc (10 g / disc) was used as the standard. A disc
of 150 ml of DMSO served as the control.
6.2. In silico screening by molecular docking studies
20

Ligand preparation: the selected synthesized prominent bioactive cycloadducts 5a, 13, and 14
were drawn and optimized in ChemDraw Ultra version 12.0 with three-dimensional orientation
and the connection errors have been checked. After optimization, the low energy confirmation of
the selected ligands was docked using AutoDock Tools v 1.5.6. (autodock and autogrid) 4.2
software (http://www.scripps.edu/mb/olson/doc/autodock) and followed by the reported
methodology by Stalin et al. [23].
Receptor preparation: in the series of synthesized cycloadducts, each ligand from C3-β-
substituted cholesterol tethered spiro pyrrolidine/pyrrolizidine derivatives was docked with the
active sites of target protein glucosamine-6-phosphate synthase (PDB ID: 1XFF) to predict the
binding interaction and conformational orientation. The three dimensional X-ray crystal structure
of the enzyme was obtained from the protein data bank (https://www.rcsb.org/structure/1XFF).
The active sites of the target protein were identified using the CASTp (Computed Atlas of
Surface Topography of proteins) server. All hydrogens with no bond order were added to the
target proteins; Kollman and Gasteiger-type charges were assigned and the nonpolar hydrogens
were merged.
Docking: in the docking analysis, protein molecules were considered as a rigid body and the
ligands were flexible. The electrostatic maps were calculated in a 0.375 Å grid space and the
docking computed based on the Lamarckian Genetic Algorithm. The population has been set to
150 individuals and the docking confirmation was calculated for 10 confirmations. The finest
confirmation of the ligand has been identified based on the binding energy, inhibition constant,
and ligand efficiency. The ligand-protein interaction was developed by the PyMOL visualization
21

tool (PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger) and during docking all
torsion angles were allowed to rotate.
7. Experimental
7.1. Preparation of Cholesterylacrylate, 4
A stirred solution of cholesterol (1 mmol) in dry dicholoromethane (75 mL) was kept in 20 °C
and freshly distilled acryloyl chloride (1.5 mmol) dissolved in 10 mL dry dichloromethane was
added dropwise with constant stirring. After stirring for 1 h, a white precipitate was formed. The
temperature was raised to reflux and the mixture was stirred for another 6 h. After the
completion of reaction as indicated by TLC, the organic phase was washed with 50 mL saturated
sodium hydrogen carbonate solution and 50 mL water. The organic layer was dried over
anhydrous Na2SO4, and evaporated under reduced pressure. The crude product 4 was purified by
column chromatography using hexane as eluent (yield: 85%).
7.2. General procedure for the synthesis of monospiropyrrolidines
To a solution of C3-β-cholestrylcarboxylate (4) (1 mmol) in aqueous methanol (20 mL),
sarcosine (2) (1 mmol) and a mixture of various substituted isatin (1a-f)/acenapthenequinone
(7)/ninhydrin (9) (1 mmol) stirred for 1-2 h at reflux temperature. After the completion of
reaction as indicated by TLC, methanol was evaporated under reduced pressure. The crude
product was purified by column chromatography using hexane:EtOAc (7:3) as eluent.
22

7.2.1. 1-N-methyl spiro[2.3"]oxindolo-3-cholestrylcarboxylate pyrrolidine (5a). Yield 0.55 g,
76%; white solid, mp 226-228 ˚C; IR (KBr) 3365, 1716, 1702 cm•1. 1H (300 MHz, CDCl3) 0.62-
1.61 (m, 43H); 2.09 (s, 3H, NMe); 2.23-2.42 (m, 1H, Hc); 2.55-2.67 (m, 1H, Hd); 3.00-3.08 (m,
1H, Ha); 3.34-3.41 (m, 1H, Hb); 3.59-3.65 (t, 1H, He); 4.12-4.20 (m, 1H, C(3)H); 5.07-5.25 (two
13
doublets, 1H, C(6)H); 6.87-7.24 (s, 4H, ArH); 8.39 (s, 1H, NH). C (75 MHz, CDCl3) 10.26,
12.52, 17.14, 17.62, 19.40, 20.97, 21.22, 22.25, 22.68, 24.76, 25.88, 26.43, 27.78, 28.12, 30.24,
30.35, 33.78, 34.20, 34.62, 34.87, 35.84, 36.21, 37.95, 40.72, 48.38, 50.91, 51.81, 54.58, 55.09,
72.85, 108.42, 112.48, 120.93, 124.13, 125.50, 127.59, 137.98, 139.93, 168.76, 177.82. EI-MS
m/z 614.90 (M+). Anal. Calcd for C40H58N2O3: C, 78.13; H, 9.51; N, 4.56; found: C, 78.26; H,
9.43; N, 4.65%.
7.2.2. 1-N-Methyl spiro[2.3"]-(N-methyl)-oxindolo-3-cholestrylcarboxylate pyrrolidine (5b).
1
Yield 0.49 g, 70%; white solid, mp 202-204 ˚C; IR (KBr) 1716, 1707 cm•1. H (300 MHz,
CDCl3) 0.59-1.60 (m, 43H); 1.98 (s, 3H, NMe); 2.21-2.39 (m, 1H, Hc); 2.48-2.51 (m, 1H, Hd);
2.79 (s, 3H, NMe); 2.96-3.01 (m, 1H, Ha); 3.30-3.38 (m, 1H, Hb); 3.53 (t, 1H, He); 4.09-4.16 (m,
1H, C(3)H); 5.10-5.32 (two doublets, 1H, C(6)H); 6.92-7.31 (s, 4H, ArH). 13C (75 MHz, CDCl3)
10.11, 11.98, 17.13, 17.62, 19.36, 20.12, 21.63, 22.42, 22.66, 24.71, 25.79, 26.39, 27.72, 28.09,
29.62, 30.19, 30.52, 33.21, 34.74, 34.58, 34.85, 36.26, 37.95, 40.67, 48.14, 50.89, 51.84, 54.50,
55.14, 74.51, 109.10, 112.43, 121.02, 124.15, 125.53, 127.48, 137.66, 139.95, 168.92, 178.37.
EI-MS m/z 628.93 (M+). Anal. Calcd for C41H60N2O3: C, 78.30; H, 9.62; N, 4.45; found: C,
78.21; H, 9.76; N, 4.36%.
7.2.3. 1-N-Methyl spiro[2.3"]-(N-phenyl)-oxindolo-3-cholestrylcarboxylate pyrrolidine (5c).
1
Yield 0.56 g, 72%; white solid, mp 218-220 ˚C; IR (KBr) 1714, 1708 cm•1. H (300 MHz,
CDCl3) 0.66-1.82 (m, 43H); 2.16 (s, 3H, NMe); 2.00-2.25 (m, 1H, Hc); 2.41-2.46 (m, 1H, Hd);
23

2.92-2.99 (m, 1H, Ha); 3.11-3.21 (m, 1H, Hb); 3.49 (t, 1H, He); 4.36-4.51 (m, 1H, C(3)H); 5.06-
5.29 (two doublets, 1H, C(6)H); 6.95-8.12 (m, 9H, ArH). 13C (75 MHz, CDCl3) 11.19, 11.96,
17.16, 17.54, 19.33, 20.14, 21.61, 22.39, 22.61, 23.95, 25.68, 26.84, 27.57, 28.46, 30.24, 30.94,
34.14, 34.57, 34.85, 36.74, 37.26, 40.65, 47.94, 50.83, 51.97, 53.42, 54.98, 73.82, 109.58,
110.43, 112.51, 121.57, 123.14, 123.95, 124.15, 124.61, 125.97, 126.35, 127.44, 133.16, 136.52,
139.51, 140.83, 169.14, 179.53. EI-MS m/z 691.00 (M+). Anal. Calcd for C46H62N2O3: C, 79.96;
H, 9.04; N, 4.05; found: C, 79.85; H, 9.11; N, 4.13%.
7.2.4. 1-N-Methyl spiro[2.3"]-(4",7"-dichloro)-oxindolo-3-cholestryl carboxylate pyrrolidine
1
(5d). Yield 0.60g, 78%; white solid, mp 196-198 ˚C; IR (KBr) 3321, 1717, 1704 cm•1. H (300
MHz, CDCl3) 0.59-1.82 (m, 43H); 2.01 (s, 3H, NMe); 1.98-2.16 (m, 1H, Hc); 2.16-2.32 (m, 1H,
Hd); 3.04-3.11 (m, 1H, Ha); 3.41-3.48 (m, 1H, Hb); 4.01 (t, 1H, He); 4.12-4.20 (m, 1H, C(3)H);
5.16-5.38 (two doublets, 1H, C(6)H); 6.98 (d, 1H, ArH); 7.14 (d, 1H, ArH); 8.68 (s, 1H, NH).
13C (75 MHz, CDCl3) 10.65, 11.12, 12.16, 14.51, 16.23, 18.61, 20.31, 22.64, 23.95, 25.68, 26.47,
27.34, 28.96, 30.14, 30.68, 33.16, 34.85, 35.38., 37.84, 39.57, 40.65, 47.84, 50.64, 53.65, 54.98,
72.01, 75.52, 110.43, 113.95, 121.57, 123.95, 124.15, 124.61, 127.44, 131.52, 133.16, 139.51,
140.83, 163.61, 176.83. EI-MS m/z 682.79 (M+). Anal. Calcd for C40H56Cl2N2O3: C, 70.26; H,
8.25; N, 4.10; found: C, 70.33; H, 8.36; N, 3.92%.
7.2.5. 1-N-Methyl spiro[2.3"]-(5"-bromo)-oxindolo-3-cholestrylcarboxylate pyrrolidine (5e).
Yield 0.62 g, 80%; pale yellow solid, mp 206-208 ˚C; IR (KBr) 3308, 1710, 1702 cm•1. 1H (300
MHz, CDCl3) 0.49-1.99 (m, 43H); 1.98 (s, 3H, NMe); 2.00-2.15 (m, 1H, Hc); 2.17-2.48 (m, 1H,
Hd); 3.00-3.24 (m, 1H, Ha); 3.46-3.52 (m, 1H, Hb); 4.11 (t, 1H, He); 4.12-4.22 (m, 1H, C(3)H);
24

13
5.07-5.41 (two doublets, 1H, C(6)H); 6.86-7.35 (m, 3H, ArH); 8.85 (s, 1H, NH). C (75 MHz,
CDCl3) 11.35, 12.17, 13.52, 16.98, 18.46, 21.11, 22.28, 23.84, 24.68, 26.57, 26.61, 28.68, 31.11,
31.68, 33.16, 33.01, 34.25, 34.34, 36.35, 36.98, 37.81, 39.34, 40.67, 41.69, 45.20, 48.74, 49.63,
53.84, 54.91, 73.27, 111.14, 120.97, 123.65, 124.61, 127.91, 130.60, 133.74, 140.14, 168.54,
178.23. EI-MS m/z 693.80 (M+). Anal. Calcd for C40H57BrN2O3: C, 69.25; H, 8.28; N, 4.04;
found: C, 69.36; H, 8.20; N, 4.11%.
7.2.6. 1-N-Methyl spiro[2.3"]-(5",7"-bromo)-oxindolo-3-cholestrylcarboxylate pyrrolidine
1
(5f). Yield 0.75 g, 86%; pale yellow solid, mp 180-182 ˚C; IR (KBr) 3310, 1712, 1704 cm•1. H
(300 MHz, CDCl3) 0.50-2.11 (m, 43H); 2.12 (s, 3H, NMe); 2.13-2.18 (m, 1H, Hc); 2.20-2.41
(m, 1H, Hd); 3.12-3.32 (m, 1H, Ha); 3.40-3.46 (m, 1H, Hb); 3.89 (t, 1H, He); 4.11-4.29 (m, 1H,
C(3)H); 5.31-5.65 (two doublets, 1H, C(6)H); 7.21-7.52 (m, 2H, ArH); 8.36 (s, 1H, NH). 13C (75
MHz, CDCl3) 13.22, 14.81, 14.99, 15.81, 16.36, 18.57, 20.47, 21.10, 23.14, 26.96, 27.58, 28.35,
31.84, 32.11, 33.82, 34.75, 34.86, 36.27, 36.91, 37.88, 39.01, 40.37, 41.62, 46.18, 47.47, 50.12,
51.35, 54.54, 71.68, 73.27, 112.85, 122.54, 123.18, 124.60, 127.86, 131.49, 134.62, 141.11,
169.50, 176.92. EI-MS m/z 772.68 (M+). Anal. Calcd for C40H56Br2N2O3: C, 62.18; H, 7.30; N,
3.63; found: C, 62.30; H, 7.41; N, 3.51%.
7.2.7. 1-N-Methyl spiro[2.2"]-acenaphthyleno-3-cholestrylcarboxylate pyrrolidine (8). Yield
1
0.48 g, 66%; pale yellow solid, mp 124-126 ˚C; IR (KBr) 1716, 1706 cm•1. H (300 MHz,
CDCl3) 0.47-1.87 (m, 43H); 1.96 (s, 3H, NMe); 2.38-2.46 (m, 1H, Hc); 2.67-2.73 (m, 1H, Hd);
3.11-3.15 (m, 1H, Ha); 3.40-3.50 (m, 1H, Hb); 3.69 (t, 1H, He); 3.77-3.80 (m, 1H, C(3)H); 4.77-
5.06 (two doublets, 1H, C(6)H); 7.41-8.16 (m, 6H, ArH). 13C (75 MHz, CDCl3) 11.76, 14.09,
25

18.69, 18.89, 20.87, 22.53, 22.78, 23.79, 24.21, 24.92, 27.00, 27.98, 28.15, 29.68, 31.69, 35.85,
36.21, 37.28, 39.49, 42.22, 49.77, 52.80, 54.11, 56.09, 56.57, 74.05, 121.91, 122.32, 125.23,
126.91, 128.10, 128.40, 130.72, 131.17, 131.88, 132.85, 139.20, 142.64, 170.05, 204.85. EI-MS
m/z 649.94 (M+). Anal. Calcd for C44H59NO3: C, 81.31; H, 9.15; N, 2.16; found: C, 81.24; H,
9.26; N, 2.22%.
7.2.8. 1-N-Methyl spiro[2.2"]-indane-1",3"-dione-3-cholestrylcarboxylate pyrrolidine (10).
Yield 0.48 g, 68%; white solid, mp 136-139 ˚C; IR (KBr) 1741, 1712, 1703 cm•1. 1H (300 MHz,
CDCl3) 0.63-1.94 (m, 43H); 1.98 (s, 3H, NMe); 2.35-2.40 (m, 1H, Hc); 2.40-2.48 (m, 1H, Hd);
3.78-3.85 (m, 1H, Ha); 3.96-3.98 (m, 1H, Hb); 3.65 (t, 1H, He); 4.28-4.30 (m, 1H, C(3)H); 5.01-
5.35 (two doublets, 1H, C(6)H); 7.53-8.05 (m, 4H, ArH). 13C (75 MHz, CDCl3) 11.78, 18.68,
19.05, 20.90, 22.54, 22.80, 23.80, 24.22, 27.02, 27.99, 28.17, 28.55, 31.76, 31.98, 33.48, 35.75,
36.17, 36.35, 36.59, 36.82, 39.49, 42.24, 47.85, 49.82, 55.08, 56.08, 56.56, 65.84, 74.71, 117.16,
122.53, 122.73, 123.51, 135.99, 139.03, 141.91, 169.21, 184.34, 192.05. EI-MS m/z 627.90
(M+). Anal. Calcd for C41H57NO4: C, 78.43; H, 9.15; N, 2.23; found: C, 78.51; H, 9.03; N,
2.31%.
7.3. General procedure for the synthesis of monospiropyrrolizidines
A solution of C3-β-cholestrylcarboxylate (4) (1 mmol), proline (11) (1 mmol) and isatin (1a-
f)/acenaphthenequinone (7)/ninhydrin (9) (1 mmol) was stirred in aqueous methanol for 1-2 h at
reflux temperature. After the completion of reaction as indicated by TLC, methanol was
evaporated under reduced pressure. The crude product was purified by column chromatography
using hexane:EtOAc mixture (2:1) as an eluent.
26

7.3.1. Spiro[2.3"]-oxindolo-3-cholestrylcarboxylate pyrrolizidine (12a)
1
Yield 0.50g, 70%; white solid, mp 258-260 ˚C; IR (KBr) 3372, 1711, 1703 cm•1. H (300 MHz,
CDCl3) 0.64-1.86 (m, 43H); 2.05-2.47 (m, 7H, pyrrolizidine H); 2.58-2.63 (m, 1H, Hc); 3.79-
3.85 (dq, 1H, Hd); 4.01-4.07 (m, 1H, Hb); 4.12-4.16 (m, 1H, C(3)H); 5.09-5.23 (two doublets,
1H, C(6)H); 6.87-7.24 (s, 4H, ArH); 8.35 (s, 1H, NH). 13C (75 MHz, CDCl3) 11.50, 18.38, 18.88,
20.64, 22.21, 22.46, 23.50, 23.93, 26.04, 27.01, 27.68, 27.86, 28.31, 29.59, 31.48, 32.59, 33.32,
35.44, 35.86, 37.38, 36.12, 36.40, 39.19, 39.38, 41.96, 43.09, 46.88, 49.62, 55.61, 55.81, 56.33,
64.56, 72.33, 74.10, 109.78, 121.91, 122.07, 125.63, 126.48, 129.00, 139.18, 141.42, 169.26,
180.41. EI-MS m/z 640.94 (M+). Anal. Calcd for C42H60N2O3: C, 78.70; H, 9.44; N, 4.37; found:
C, 78.61; H, 9.57; N, 4.43%.
7.3.2. Spiro[2.3"]-(N-methyl)-oxindolo-3-cholestrylcarboxylate pyrrolizidine (12b). Yield 0.53
1
g, 72%); white solid, mp 232-234 ˚C; IR (KBr) 3355, 1712, 1705 cm•1. H (300 MHz, CDCl3)
0.61-1.82 (m, 43H); 2.10-2.56 (m, 7H, pyrrolizidine H); 2.59-2.68 (m, 1H, Hc); 2.76 (s, 1H,
NMe); 3.75-3.82 (dq, 1H, Hd); 4.00-4.05 (m, 1H, Hb); 4.15-4.19 (m, 1H, C(3)H); 4.99-5.21 (two
doublets, 1H, C(6)H); 6.98-7.35 (m, 4H, ArH). 13C (75 MHz, CDCl3) 11.48, 18.32, 18.68, 20.51,
22.26, 22.51, 23.54, 23.86, 25.97, 27.00, 27.66, 27.91, 28.36, 30.52, 31.26, 32.64, 33.84, 34.17,
34.41, 35.67, 35.71, 36.24, 36.62, 39.16, 39.29, 41.85, 44.62, 46.53, 49.58, 55.51, 55.78, 56.23,
64.48, 72.29, 74.52, 108.69, 121.86, 122.05, 125.59, 129.13, 139.15, 141.50, 150.30, 169.23,
180.54. EI-MS m/z 654.96 (M+). Anal. Calcd for C43H62N2O3: C, 78.85; H, 9.54; N, 4.28; found:
C, 78.71; H, 9.62; N, 4.37%.
27

7..3.3 Spiro[2.3"]-(N-phenyl)-oxindolo-3-cholestrylcarboxylate pyrrolizidine (12c). Yield 0.55
1
g, 68%); white solid, mp 218-220 ˚C; IR (KBr) 3351, 1718, 1708 cm•1. H (300 MHz, CDCl3)
0.59-1.82 (m, 43H); 1.98-2.46 (m, 7H, pyrrolizidine H); 2.50-2.59 (m, 1H, Hc); 3.72-3.80 (dq,
1H, Hd); 4.09-4.13 (m, 1H, Hb); 4.14-4.19 (m, 1H, C(3)H); 5.14-5.31 (two doublets, 1H, C(6)H);
13
6.81-8.32 (m, 9H, ArH). C (75 MHz, CDCl3) 11.10, 17.99, 18.53, 20.58, 22.16, 22.43, 23.61,
23.82, 26.12, 27.11, 26.83, 27.47, 28.53, 31.52, 32.54, 32.62, 33.86, 34.54, 35.46, 35.90, 36.25,
36.38, 36.46, 39.20, 39.41, 41.95, 46.82, 49.58, 55.63, 55.83, 56.30, 64.53, 72.30, 74.00, 108.85,
121.96, 122.12 125.69, 129.00, 139.20, 141.53, 150.64, 169.31, 179.86. EI-MS m/z 717.00 (M+).
Anal. Calcd for C48H64N2O3: C, 80.40; H, 9.00; N, 3.91; found: C, 80.32; H, 9.09; N, 3.97%.
7.3.4. Spiro [2.3"]-(4".7"-dichloro)-oxindolo-3-cholestrylcarboxylate prrolizi dine (12d). Yield
1
0.62 g, 77%; white solid, mp 212-214 ˚C; IR (KBr) 3368, 1712, 1705 cm•1. H (300 MHz,
CDCl3) 0.59-1.91 (m, 43H); 1.98-2.46 (m, 7H, pyrrolizidine H); 2.56-2.61 (m, 1H, Hc); 3.80-
3.86 (dq, 1H, Hd); 4.05-4.11 (m, 1H, Hb); 4.14-4.17 (m, 1H, C(3)H); 4.99-5.18 (two doublets,
1H, C(6)H); 6.99 (d, 1H, ArH); 7.31 (d, 1H, ArH); 8.40 (s, 1H, NH). 13C (75 MHz, CDCl3)
11.10, 18.21, 18. 69, 20.50, 22.09, 22.48, 23.46, 23.95, 26.10, 27.00, 27.59, 27.79, 28.36, 31.41,
32.61, 33.81, 34.50, 35.43, 35.47, 35.82, 36.18, 36.37, 39.20, 39.37, 41.96, 46.83, 49.60, 55.65,
55.76, 56.38, 65.55, 72.28, 74.16, 109.01, 121.96, 122.12, 125.60, 129.10, 139.09, 141.48,
150.22, 169.57, 180.29. EI-MS m/z 709.83 (M+). Anal. Calcd for C42H58Cl2N2O3: C, 71.07; H,
8.24; N, 3.95; found: C, 71.15; H, 8.32; N, 3.88%.
7.3.5. Spiro[2.3"]-(5"-bromo)-oxindolo-3-cholestrylcarboxylate pyrrolizidine (12e). Yield 0.62
1
g, 76%; white solid, mp 266-268 ˚C; IR (KBr) 3365, 1716, 1702 cm•1. H (300 MHz, CDCl3)
28

0.61-1.88 (m, 43H); 1.96-2.43 (m, 7H, pyrrolizidine H); 2.50-2.58 (m, 1H, Hc); 3.86-3.91 (dq,
1H, Hd); 3.99-4.16 (m, 1H, Hb); 4.17-4.29 (m, 1H, C(3)H); 4.98-5.18 (two doublets, 1H, C(6)H);
6.79-7.27 (m, 3H, ArH); 8.38 (s, 1H, NH). 13C (75 MHz, CDCl3) 11.02, 18.32, 18.58, 20.33,
22.11, 22.62, 23.41, 23.89, 26.16, 27.04, 27.48, 27.80, 28.32, 31.39, 32.60, 33.86, 34.65, 35.41,
35.41, 35.48, 36.20, 36.36, 39.18, 39.35, 41.93, 46.79, 49.58, 55.61, 55.74, 56.32, 65.48, 72.23,
74.20, 109.21, 121.85, 122.19, 125.56, 129.16, 139.11, 141.43, 150.26, 169.42, 181.01. EI-MS
m/z 719.83 (M+). Anal. Calcd for C42H59BrN2O3: C, 70.08; H, 8.26; N, 3.89; found C, 70.15; H,
8.19; N, 3.96%.
7.3.6. Spiro[2.3"]-(5".7"-dibromo)-oxindolo-3-cholestrylcarboxylate pyrrolizi dine (12f). Yield
1
0.68 g, 76%; white solid, mp 252-254 ˚C; IR (KBr) 3371, 1718, 1706 cm•1. H (300 MHz,
CDCl3) 0.53-1.86 (m, 43H); 1.89-2.41 (m, 7H, pyrrolizidine H); 2.49-2.53 (m, 1H, Hc); 3.84-
3.89 (dq, 1H, Hd); 4.06-4.15 (m, 1H, Hb); 4.13-4.20 (m, 1H, C(3)H); 4.99-5.18 (two doublets,
1H, C(6)H); 6.98-7.32 (m, 2H, ArH); 8.31 (s, 1H, NH). 13C (75 MHz, CDCl3) 10.85, 17.35,
18.58, 20.31, 22.51, 22.46, 23.40, 23.91, 26.15, 27.16, 27.42, 27.76, 28.35, 31.42, 32.58, 33.83,
34.61, 35.46, 35.48, 36.21, 36.40, 39.21, 39.37, 41.96, 46.81, 49.62, 55.63, 55.77, 56.28, 65.50,
72.20, 74.21, 109.22, 121.84, 122.20, 125.53, 129.19, 139.13, 141.41, 150.23, 169.40, 180.96.
EI-MS m/z 798.73 (M+). Anal. Calcd for C42H58Br2N2O3: C, 63.16; H, 7.32; ; N, 3.51; found C,
63.24; H, 7.26; ; N, 3.60%.
7.3.7. Spiro[2.2"]-acenaphthyleno-3-cholestrylcarboxylate pyrrolizidine (13).
1
Yield 0.51 g, 67%; white solid, mp 240-242 ˚C; IR (KBr) 1710, 1703 cm•1. H (300 MHz,
CDCl3) 0.47-1.03 (m, 43H); 1.85-2.53 (m, 7H, pyrrolizidine H); 2.22-2.26 (m, 1H, Hc); 3.75-
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