Regio- and stereoselective synthesis of spirooxindole 1′-nitro pyrrolizidines with five concurrent stereocenters under aqueous medium and their bioprospection using the zebrafish (Danio rerio) embryo model

Article abstract of DOI:10.1039/c3ob41302k

A highly regio- and stereoselective method has been developed and expanded for the synthesis of a 20-membered library of spirooxindole 1′-nitro pyrrolizidines via 1,3-dipolar cycloaddition of azomethine ylides, generated in situ by a decarboxylative route from a common set of diverse isatins and l-proline derivatives, with substituted β-nitrostyrenes under aqueous medium. Among various reaction conditions, water proved to be necessary for the interaction of the reagents as well as heating the reaction at 90 °C for one hour, during which time the desired products were obtained in good yields and with excellent regio- and stereoselectivities. We subsequently applied in silico drug discovery computational methods to (i) identify the ADME properties, based on Lipinski's rule, (ii) screen the toxicological profile, and (iii) predict the penetration through the blood brain barrier (BBB) of the synthesized compounds. Next, the LC₅₀ values of all these spirocyclic oxindoles were determined in zebrafish embryos cultured individually in buffer solutions of each compound and, finally, the phenotypes induced by these molecules in the zebrafish embryos at concentrations below their LC₅₀ were analyzed at 48, 72 and 96 hours post fertilization.

Full text of DOI:10.1039/c3ob41302k

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Organic & Biomolecular Chemistry
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PAPER
Regio- and stereoselective synthesis of spirooxindole 1’-nitro
pyrrolizidines with five concurrent stereocenters under aqueous media
and their bioprospection using the zebrafish (Danio rerio) embryo
model†
5
Carlos E. Puerto Galvis and Vladimir V. Kouznetsov*
Received (in XXX, XXX) Xth XXXXXXXXX 2013, Accepted Xth XXXXXXXXX 2013
DOI: 10.1039/b000000x
A highly regio- and stereoselective method has been developed and expanded for the synthesis of a 20-
membered library of spirooxindole 1’-nitro pyrrolizidines via 1,3-dipolar cycloaddition of azomethine
ylides, generated in situ by a decarboxylative route from a common set of diverse isatins and L-proline
derivatives, with substituted β-nitrostyrenes under aqueous media. Among various reaction conditions,
water proved to be necessary for the interaction of the reagents as well as heating the reaction at 90 °C for
one hour, time in which the desired products were obtained in good yields and with excellent regio- and
stereoselectivities. Then, we have applied in silico drug discovery computational methods to i) identify
the ADME properties, based on the Lipinski’s rule, ii) screening the toxicological profile and iii) predict
the penetration through the Blood Brain Barrier (BBB) of the synthesized compounds. Next, the LC50 of
all these spirocyclic oxindoles were determined in zebrafish embryos cultured individually in buffer
solutions of each compound and finally, the phenotypes induced by these molecules in the zebrafish
embryos, at concentrations below their LC , were analyzed at 48, 72 and 96 hours post fertilization.
1
0
1
5
5
0
2
0
Introduction
Among the various classes of organic small molecules (SMs),
spirocyclic-3,3’-oxindoles have become an important synthetic
target because this structural framework is commonly present in
wide a number of synthetic and naturally occurring bioactive
1
,2
2
3
3
5
0
5
compounds.
Undoubtedly, the natural spirotryprostatin A 1, and its related
alkaloids, which have been identified as novel inhibitors of
microtubule assembly, have encouraged the design and synthesis
of novel spirocyclic oxindoles with relevant and notable
3
4
medicinal properties. Among them, agent NITD609 2 highlights
with nanomolar activity on the blood stage of Plasmodium
5
falciparum, while the synthetic derivatives 3 and 4 exhibit
micromolar activities against bacterial cell division and
Mycobacterium tuberculosis H37Rv, respectively (Figure 1).
The biological activities displayed by those spirooxindoles
emphasize the need to develop efficient strategies to access to
these scaffolds, and knowing that each expanded work results in a
6
,7
Fig. 1 Natural (1) and synthetic (2-4) spirocyclic oxindoles with
significant biological activities.
8
50
different types of spiroheterocycles, new methodologies to
afford this complex core are highly desired.
In recent years, the 1,3-dipolar cycloaddition of azomethine
ylides has played a key role in the construction of varied
spirooxindoles. This multi-component reaction constitutes a
*
Laboratorio de Química Orgánica y Biomolecular, Universidad
4
4
0
5
Industrial de Santander, Cra 27 calle 9, Bucaramanga A.A. 678,
Colombia. Fax/Tel: +57 76 349069; E-mail: kouznet@uis.edu.co
†
9
Electronic Supplementary Information (ESI) available: Characterization 55 highly effective one pot procedure that has many advantages
1
1
3
1
3
data, including copies of H NMR, C NMR, DEPT-135, COSY,
C
including wide structural diversity, high atom economy, excellent
bond-forming efficiency and high regio- and stereoselectivity in a
HMBC, HSQC and HSQC charts, of all synthesized spirooxindoles and
selected photographic record of the phenotypic changes during the
zebrafish embryo development. See DOI: 10.1039/b000000x/
1
0
single-step process. Although these two last statements has been
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addressed in elegant studies using organocatalysts and transition 50 knowledge that there are no reports on the synthesis of
1
1
metal-catalyzed reactions,
a
straightforward and flexible
spirooxindole 1’-nitro pyrrolizidines containing the both 6’-
hydroxypyrrolizidine and 3,4,5-trimethoxyphenyl fragments, our
research was focused in: i) establishing the optimal conditions for
the reaction of isatin, L-proline and β-nitrostyrenes, according to
55 the variables: solvent, reaction times, temperature, yield and
regio- and stereoselectivities in which the desired product is
obtained. ii) with the established conditions, prepare a 20-
membered library of new spirooxindole 1’-nitro pyrrolizidines
through a 1,3-dipolar cycloaddition of azomethine ylides,
approach in which selective stereoisomers can be obtained,
through a routing changes of the reactions conditions and
avoiding as possible the use of any catalyst, is a current challenge
to the organic chemistry.
Another important class of compounds that are synthesized
through a 1,3-dipolar cycloaddition of azomethine ylides are the
pyrrolizidine alkaloids, which are the common constituents of
hundreds of vegetable species and it has been reported that they
are present in about 3% of the world flowering plants. Within 60 generated in situ from diverse isatins and L-proline derivatives,
5
0
5
0
5
1
2
1
1
2
2
1
3
the most simple members of this family, casuarine 5 is a potent
with substituted β-nitrostyrenes. iii) identify their safety or
toxicity profiles (ADME-Tox properties) through in silico
1
4
glycosidase inhibitor, as the other pyrrolizidine alkaloids;
bioactivity that is overshadowed by the acute hepatotoxicity
2
9
methods based on the Lipinski’s rule (drug-likeness) and drug-
1
5
30
exhibited by these derivatives, but fortunately only one of these
score criterion. iv) predict their in silico penetration through the
alkaloids, thesinine 6, not have shown this side effect and in 65 Blood Brain Barrier (BBB). v) determine the toxicity (LC ) of
5
0
1
6
contrast it had maintained their biological properties (Figure 2).
Additionally, the 3,4,5-trimethoxyphenyl moiety, present in
some of the synthesized compounds illustrated in Figure 1, is also
one of the main structurally fragments in which this study is
the prepared spirooxindoles in the zebrafish embryos and vi)
analyse the phenotypes induced by these SMs at concentrations
below their LC , in the zebrafish embryos at 24, 48, 72 hours
5
0
post chemical exposure. All this, are in order to contribute in
based. The most known natural derivatives possessing this moiety 70 future SAR studies of this interesting class of molecules.
1
7,18
are the combretastatin A-4 7 and colchicine 8,
these SMs are
recognized for their potent tubulin polymerization inhibitory
Results and Discussion
1
9
activity and several structure-activity relationship (SAR) studies
performed on these alkaloids, have concluded the importance of
Preparation of the 20-membered library of spirooxindoles
2
0
We initiated our investigation by exploring the reaction
conditions that would lead to the synthesis of the spirooxindole
1’-nitro pyrrolizidines through a 1,3-dipolar cycloaddition in
which three new bonds and five concurrent stereogenic centres,
including one quaternary carbon, are incorporated simultaneously
in a single step. Isatin 9a, L-proline 10a and 3,4,5-trimethoxy-β-
nitrostyrene 11a were selected as a simple model substrates to
study the feasibility of this reaction according to previously
the 3,4,5-trimethoxyphenyl moiety (Figure 2).
7
5
80
3
1
established conditions for this type of reaction (Scheme 1).
Fig. 2 Naturally occurring small molecules containing the pyrrolizidine
fragment (5 and 6) and the 3,4,5-trimethoxyphenyl moiety (7 and 8).
3
3
4
4
0
5
0
5
The zebrafish (Danio rerio) embryo model, one of the best-
described, less cost-intensive and less time- and space-consuming
2
1
vertebrate model, offers the possibility to perform small-scale
high-throughput screens of the acute toxicity of SMs in the
embryos and at the same time, identifies the phenotypical
perturbations or development disorders in a defined biological
2
2
pathway that correspond or that are induced by the SMs.
Among other mammalian models (e.g. mice and rabbits), the
zebrafish organism has shown a high degree of homology to
human genomes, strong similarity to mammalian tissue structures
Scheme 1 Synthesis of spirooxindole-pyrrolizidines 12a and 12a’ using
ethanol as a solvent at 90 °C for 4 h.
2
3
and equal responses to external threats, and within the
remarkable features of this model, the size of the zebrafish
embryo/larvae (1-1.5 mm in diameter), their rapid development
outside mother’s body, transparency during early developmental
stages and their high fecundity (a single spawning produces 300-
400 embryos), allowed their visual inspection, their study as a
single and individual system and the rapid screening through the
easy administration of drugs and a libraries of SMs diluted in the
8
5
Using ethanol as a solvent, the reaction was monitored by TLC
analysis and the starting materials were consumed after 4 hours.
Afterward column chromatography, a mixture of regioisomers
that could not be separated due to their similar R values through
f
this technique, were isolated as a white solid in 75 % yield and
1
90
their regiochemistry was analyzed through
H
NMR
spectroscopy. The regioisomer 12a was recognized as the major
product, showing the signal of proton H-1 at 6.26 ppm as a triplet
24-28
surrounding water through their membrane, skin and gills.
(
J = 9.9 Hz, CHNO ) due to its coupling with protons H-2 and H-
2
According to the statements described above and with the
2
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3
2
3
, while the minor product 12a’ presented the signal of proton
H-2’ at 5.67 ppm as a doublet (J = 11.0 Hz, CHNO ) due to its
2
interaction with proton H-1’.
With four stereocenters, compound 12a has eight pairs of
potential diastereoisomers, but since this reaction is also
stereospecific, the defined stereochemistry of amino acid 10a and
the trans-configuration of the β-nitrostyrene 11a will determine
the stereochemistry of stereocenters C-1, C-2 and C-3 in the
adduct 12a. Consequently, six of those diastereoisomeric pairs
would not be obtained due to that the hydrogen in stereogenic C-
5
0
5
0
5
1
1
2
2
3
centre cannot be spatially located above the plane of symmetry
and the substituents in the stereocenters C-1 and C-2 cannot be
found in the Z-configuration (Scheme 1).
With respect to the stereochemistry of the spiro-quaternary
chiral carbon centre, the discussion has to be focused on the first
steps of the reaction mechanism, which involves the formation of
the corresponding azomethine ylide. Various authors have
described the in situ formation of the azomethine ylide as a
50
Scheme 3 Formation of S-shaped ylide I3 and W-shaped ylide I3’ and
their possible reaction with the dipolarophile 11a.
Having determined the stereochemistry of the quaternary
stereocenter, the possible formation of one additional pair of
diastereoisomers is rejected and the next task was to determine
the stereochemistry of the substituents at positions C-1’ and C-2’
of cycloadduct 12a, whose stereoselectivity will be determined
by the 1,3-dipolar cycloaddition between the azomethine ylide I3
and the dipolarophile 11a. It should be noted that in this process
the structure of the ylide plays the key role due to its cyclic nature
that provides a rather rigid ring template, resulting in a better
diastereofacial approach between the ylide and the β-nitrostyrene,
substrate that can be approximated to the this 1,3-dipole by two
possible pathways. In the first, the exo-approach, the steric
hindrance between the bulky phenyl moiety of nitrostyrene and
the core of isatin will result in an electrostatic repulsion within
these fragments, increasing the free energy of activation for this
transition state. Instead, in the endo-approach the phenyl moiety
can get sufficiently away from the isatin core and this more
stabilized transition state will be required less free energy for its
activation. Thus, the formation of the cycloadduct 12a will be
highly favoured through the endo-approach pathway while the
stereoisomer I3b’, derived from the exo-approach, was not
observed (Scheme 4).
3
a,7,33,34
simple decarboxylation route of an iminium salt,
here we
5
6
6
7
5
0
5
0
suggest that this could be a three-step process in which the first
condensation of isatin 9a with the amino acid 10a would lead the
formation of the intermediate I1, with a new C-N bond that can
freely rotate in its axis, then in a second step one molecule of
water is lost due to an intramolecular rearrangement generating
two possible intermediates I2 and I2' (Scheme 2).
Scheme 2 Probable intermediates I2 and I2’, prior to the formation of the
corresponding azomethine ylide, from derivatives 9a and 10a.
We proposed that the hydrogen atom from the carboxilic acid
function is transferred to promote the loss of water and the
intramolecular cyclization of I1. Probably, the way in which the
new oxazolidinone ring is closed in this second step would
determine the stereochemistry of the spiro-quaternary carbon, and
it is clear that the influence of the carbonyl function, present in
the isatin core, would mediate this process through some kind of
hydrogen-bond interactions, therefore the intermediate I2 will be
generated exclusively. Another additional fact that will prove this
statement, is that if the two species I2 and I2’ are formed, after
the decarboxylation process two different types of ylides would
be formed, the S-shaped ylide I3 and W-shaped ylide I3’ (Scheme
3
3
4
4
0
5
0
5
3
5
3
), and the reaction of each of them with β-nitrostyrene 11a will
produce two different stereoisomers. Accordingly to the obtained
results and the analysis of 12a through NMR spectroscopy, the
stereochemistry of the spiro-quaternary chiral carbon centre is
consistent with the structure of S-shaped ylide I3, the more
contributing structure to the resonance energy and the most stable
specie, which is derived exclusively from intermediated I2. While
the cycloadduct I3a’, corresponding to the reaction of ylide I3’
and nitroalkene 11a, was never observed (Scheme 3).
75
Scheme 4 Favored transition state for the stereoselective 1,3-dipolar
cycloaddition between azomethine ylide I3 and nitrostyrene 11a.
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The high stereoselectivity of this reaction was deduced on the
1
basis of H NMR spectra where the diastereomer 12a was
exclusively obtained, by analyzing the coupling constants of the
proton H-1 and H-7a', whose stereochemistry was known from
the L-proline 10a, and thus establishing the stereochemistry of
the substituents in positions C-1 and C-2 (Scheme 4).
With the determination of the complete stereochemistry for the
compound 12a, the versatility and high stereoselectivity of the
5
0
5
1
,3-dipolar cycloaddition stands out when the adequate reagents
1
are used. However, the poor regioselective ratio displayed by this
reaction needed to be improved. For this reason, two additional β-
nitrostyrenes were tested as a dipolarophiles in order to evaluate
their effect on the regioselectivity. Unfortunately, our attempt
failed proving that the chemical and electronic nature of the
dipolarophile did not have any effect in the regioselectivity of the
reaction (Table 1).
1
Table 1 Regioselectivity of 1,3-dipolar cycloaddition between isatin 9a,
a
L-proline 10a and diverse trans-β-nitrostyrenes 11a-c
50
Scheme 5 Influence of HOMO-controlled and hydrogen bond interactions
in the regioselectivity of 1,3-dipolar cycloaddition between azomethine
ylide I3 and nitrostyrene 11a.
Table 2 Highly regioselective 1,3-dipolar cycloaddition between isatin
a
9
b, L-proline 10a and diverse trans-β-nitrostyrenes 11a-c
b
Regioisomer
ratio (12:12’)
64:36
Entry
R
1
R
2
R
3
Time, h Yield, %
c
1
2
3
OMe OMe OMe
4
4
75
74
87
OMe OMe
-OCH
H
O-
74:26
62:38
H
2
3.5
b
a
Entry
R1
OMe
OMe
H
R2
OMe
OMe
R3
OMe
H
Time, h
4
3.5
Yield, %
The reaction was performed on a 2.50 mmol scale using 1 equiv of 9a,
b
1
2
3
82
78
87
2
0
1.3 equiv of 10a and 1 equiv of 11a-c. Combined yield of the two
isolated cycloadducts 12 and 12’. Ratio determined by H-NMR
spectroscopy (H-1/H-2’).
c
1
30
-OCH O-
3.5
2
a
55
The reaction was performed on a 2.50 mmol scale using 1 equiv of 9b,
.3 equiv of 10a and 1 equiv of 11a-c. Isolated yield of the respective
b
1
With the results of Table 1 and after having described a
mechanistic proposal in Schemes 2-4. Our next supposition
settled that the N-H bond present in the core of I3 could act as a
hydrogen bond donor and its interaction with the oxygen of the
nitro group would direct the orientation of the β-nitrostyrene from
the highly expected disposition controlled by the high-lying
HOMO of I3 which overlaps with LUMO of 11a, where by
allopolarization the partial charges induced in the double bond of
the dipolarophile by the nitro group oriented the β-nitrostyrene to
regioisomer.
2
5
After established the structural requirements of the reagents
needed to guide the synthesis of the desired compounds in a high
regio- and stereoselective way, we focused our attention in vary
the reaction conditions in order to determine if the regio- and
stereoselectivity will be affected under other conditions and,
mainly, to establish a green method for the selective preparation
65 of the desired spirooxindoles. Choosing N-methyl isatin 9b, L-
proline 10a and 3,4,5-trimethoxy-β-nitrostyrene 11a as a simple
model substrates for studied different reaction conditions, we
found that i) experiments carried at room temperature did not
gave the desired product after 24 hours. This agrees with the
6
0
3
3
4
4
0
5
0
5
3
6
form 12a, instead of the orientation promoted by the hydrogen
bonding interaction where the oxygen acts as an hydrogen bond
acceptor, decreasing in that way the regioselectivity of the
reaction (Scheme 5). To verify this last hypothesis, we encourage
our efforts in using N-methyl isatin 9b, instead of isatin 9a, as a
precursor of the corresponding azomethine ylide, expecting in
this order avoid the hydrogen bonding interaction proposed in
Scheme 5. Fortunately, when N-methyl isatin 9b, L-proline 10a
and β-nitrostyrenes 11a-c were used as reagents, the reaction took
place in similar reaction times and in slightly higher yields.
Nonetheless, the most interesting aspect of these experiments
7
7
8
0
5
0
statement depicted in Scheme 3, where moderate temperatures are
needed to promote the decarboxylation process to generate the
reactive azomethine ylide, ii) all the experiments performed at 90
°C gave in similar yields the same product 12a as a stable white
solid, whose NMR analysis determined that the regio- and stereo-
selectivities were not affected, and iii) this reaction can be carried
out smoothly in polyethylene glycol 400 (PEG-400) or in
propylene carbonate at 90 °C, observing a significant reduction in
reaction times (Table 3).
With the pursuit of the most safe and environmentally friendly
protocol for the synthesis of the desired compounds and in
agreement with the current environmental concerns, designing
1
resulted from H NMR analysis that revealed, in all cases, that the
reaction was oriented to the exclusive formation of regioisomers
1
2d-f derived from the HOMO-controlled pathway, while the
corresponding regioisomer from the hydrogen bond interaction
pathway were not observed, not even in traces (Table 2).
4
| Org. Biomol. Chem., 2013, [vol], 00–00
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Organic & Biomolecular Chemistry
Table 3 Criteria for the selection of the best conditions for the reaction of
isatin 9b, L-proline 10a and trans-β-nitrostyrene 11a
Now, having optimized the reaction conditions, a 20-
membered library of a novel and diverse spirooxindole 1’-nitro
pyrrolizidines 12a-t (Table 5) were easily prepared though a 1,3-
dipolar cycloaddition of azomethine ylides, generated in situ by a
decarboxylative process from diverse isatins 9a-d and L-proline
derivatives 10a and 10b, with substituted β-nitrostyrenes 11a-c
selecting as a standard conditions: a mixture of water and
propylene carbonate, in relation 1:0.2, as a solvent and 90 °C as
the optimal temperature.
4
5
Temperature, Conversion, Time, Yield of
Entry
Solvent
a
b
c
°
r.t.
90 °C
90 °C
90 °C
C
%
0
100
100
100
h
24
4
12d, %
n.r.
82
84
81
d
1
2
3
4
EtOH
EtOH
PEG-400
2.5
1.5
e
P.C.
50
a
Reaction performed on a 2.50 mmol scale using 1 equiv of 9b, 1.3 equiv
b
c
d
of 10a and 1.1 equiv of 11a. Monitored by TLC. Isolated yield. n.r. =
no reaction. P.C.= Propylene carbonate
e
5
Table 5 Regio- and stereoselective synthesis of spirooxindole 1’-nitro
a
pyrrolizidines 12a-t under aqueous media
and developing economically and environmentally benign
synthesis, propylene carbonate (Table 3, entry 4) was chosen as a
solvent, over ethanol and PEG-400, due to its valuable physical
properties (such as low viscosity, anhydrous, noncorrosive,
biodegradable and nontoxic) that have lead its widespread use in
1
1
2
2
3
3
0
5
0
5
0
5
3
7
many organic transformations.
However, when trans-4-hydroxy-L-proline 10b was replaced
for the derivative 10a, surprisingly the reaction between N-
methyl isatin 9a, this amino acid 10b and β-nitrostyrene 11a did
not yield the expected product after 24 hours. Posterior solubility
tests were negative for 10b, at room temperature and even at 90
°C, in propylene carbonate, as well as in ethanol and PEG-400,
but were positive in water under this range of temperatures; these
factors encouraged again our efforts in search the optimal
reaction conditions for the formation of the corresponding
cycloadduct. Selecting N-methyl isatin 9b, trans-4-hydroxy-L-
proline 10b and 3,4,5-trimethoxy-β-nitrostyrene 11a as a simple
model substrates for studied different reaction conditions under
aqueous media (Table 4), we found that i) under the conditions
depicted in Table 3 the reaction, employing the amino acid 10b
did not run, ii) using water as a solvent, the reaction took place
after 1 hour but in lower yields due to the formation of an
inhomogeneous mixture that can be stirred for long periods of
time (entry 4), iii) the reaction was carried out under ultrasonic
irradiation (u/s) conditions at 30 °C. Our attempt to homogenise
the system failed and the product was isolated in a very poor
yield after 5 hours (entry 5), iv) by using propylene carbonate as a
solvent and water as a co-solvent in relation 1:0.5, the reaction
took place in 1 hour and in good yield (entry 6), and finally v)
using water as a solvent and propylene carbonate as a co-solvent
in relation 1:0.2, the reaction finished after 1 hour and without the
formation of any inhomogeneous mixture, leading the formation
of the desired compound in excellent yield (entry 7).
Table 4 Criteria for the selection of the best conditions for the reaction of
isatin 9b, L-proline 10b and trans-β-nitrostyrene 11a
4
0
Temperature, Conversion, Time, Yield of
Entry
Solvent
a
b
c
°C
%
h
12g, %
1
2
3
4
5
EtOH
PEG-400
PC
90 °C
90 °C
90 °C
90 °C
30 °C
0
0
0
90
70
24
24
24
1
n.r.
n.r.
n.r.
40
H
2
O
a
Reactions performed on a 2.50 mmol scale using 1 equiv of 9a-d, 1.3
H
2
O, u/s )))
PC/H
1:0.5)
O/PC
1:0.2)
5
10
b
55
equiv of 10a-b and 1.1 equiv of 11a-c. Isolated yield after SiO
chromatography. Combined yield of the isolated mixture of regioisomers
2
column
2
O
c
6
7
90 °C
90 °C
100
100
1
1
75
80
(
H
2
(
We examined also the substrate scope and limitations under
the optimized reaction conditions; found that the reaction
60 proceeded smoothly to give the desired products in high yields,
a
Reaction performed on a 2.50 mmol scale using 1 equiv of 9b, 1.3 equiv
of 10b and 1.1 equiv of 11a. Monitored by GC. Isolated yield.
b
c
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Page 6 of 14
3
8
high regio- and stereoselectivities and with four or five
development of the pharmaceutical industry. The methods
concurrent stereocenters. Also, it was found that this reaction 50 commonly used for the in silico predictions are recognized for
could be broad in scope without been affected by the mixture of
solvents or by the thermal conditions used.
being built based in two principles: those that used literature data
of structural fragments linked to adverse outcomes, and those that
are based upon statistical analysis of relationships between
5
0
5
0
5
0
5
Through the judicious choice of the isatin derivative employed,
we demonstrated that this reaction could be readily adapted to
predominantly afford the desired regioisomer, for example, the
use of N-methyl isatin 9b resulted in the exclusively formation of
products 12d-t while a mixture of regioisomers were obtained
when isatin 9a was used.
3
9
structure and/or property descriptors and toxicity endpoints. In
this sense, we attempt to characterize the risk and safety profiles
of the synthesized spirooxindoles using both approaches.
5
6
6
5
0
5
4
0
First, Using the Molinspiration Cheminformatics software,
all synthesized molecules were subjected to the virtual screening,
1
1
2
2
3
3
4
1
mainly, Lipinski’s Rule of Five analysis, which indicates that a
chemical substance can be orally active for humans if the tested
compound has: i) an octanol/water partition coefficient (LogP) ≤
All the N-methyl isatin derivatives 9b-d, as well as the β-
nitrostyrenes 11a-c, were well tolerated and gave the
corresponding products 12d-t with excellent regio- and
stereoselectivities regardless the electronic nature or position of
the substituents on the aromatic ring. However, the variation of
R in the L-proline ring led to moderate yields when R = OH,
5
; ii) a molecular weight (MW) ≤ 500 Daltons; iii) a number of
hydrogen bond acceptors (nNO) ≤ 10; iv) a number of hydrogen
bond donors (nNHOH) ≤ 5; v) a number of rotable bonds (nRB) ≤
10 and vi) topological polar surface area (TPSA) ≤ 140 (Table 6).
6
6
contrary when R = H, but still keeping the excellent regio- and
6
Table 6 Molecular descriptors predicted for molecules 12a-t according to
the Lipinski’s Rule using the Molinspiration Cheminformatics software.
stereoselectivities.
The relative stereochemistry of compounds 12a-t was deduced
from the NOESY correlations as depicted on the proposed
conformation of 12j (Figure 3). The NOESY spectrum clearly
shows the cross peaks that correspond to the following
interactions: H-5b/H-6b, H-5b/H-7b and H-6b/H-7b, suggesting
that protons H-5b, H-6b and H-7b have an equatorial orientation,
while interactions between H-5a/H-6a and H-6a/H-7a, indicates
that protons H-5a, H-6a and H-7b have an axial orientation. The
two five-membered rings of the pyrrolizidine moiety have
envelope conformations, where the NOESY correlation identify
the C-7 and the C-3 centers as the flap of the A and B rings,
respectively. In addition, the NOESY correlations, through the
interaction of H-2 with H-5b and H-7b, also indicates that both
pyrrolidine rings of the pyrrolizidine core are not in the same
plane, an angle of 130 degrees between these two five-membered
rings is form, having the C-7a’-N bond as a vertex. Finally, the
cross peak corresponding to the interaction H-1/H-7a’ defines the
stereochemistry of the protons H-1and H-7a’, as well as H-2’, and
thereby elucidate the stereochemistry and the favorable
conformation compound 12j, complement the results of NOESY
experiments with the COSY, HMBC and HSQC data (See ESI†).
Molecular descriptors
Comp.
a, b
c
LogP Molweight
nNO nNHOH nRB TPSA
1
1
2a
2b
2.078
2.723
2.967
2.953
2.968
3.212
2.037
2.052
2.296
3.607
3.622
3.866
2.691
2.706
2.950
2.888
2.903
3.146
1.972
2.231
439
409
393
453
423
407
469
439
423
487
457
9
8
8
9
8
8
10
9
9
9
8
8
10
9
9
12
11
11
13
12
1
1
1
0
0
0
1
1
1
0
0
0
1
1
1
0
0
0
1
1
5
4
2
5
4
2
5
4
2
5
4
2
5
4
2
6
5
3
6
3
105.862
96.628
96.628
97.073
87.839
1
2c
12d
2e
2f
1
1
87.839
1
1
2g
2h
117.301
108.067
108.067
97.073
87.839
87.839
12i
2j
2k
2l
2m
12n
1
1
1
441d
1
503
117.301
108.067
108.067
142.897
133.663
133.663
473
457
498
468
452d
514
468
12o
12p
12q
d
d
d
d
d
d
1
2r
12s
2t
d
163.125
153.891
d
1
a
b
Parameter expressed in g/mol. Data confirmed by HRMS technique,
†
c
2 d
see ESI . Descriptor expressed Å . Descriptor that violates the rule.
7
7
8
8
0
5
0
5
As shown in Table 6, not all the physicochemical descriptors
calculated for spirooxindole 1’-nitro pyrrolizidines 12a-t are
within the range set by the Lipinski’s rule of five. Since, this rule
states that the bioavailability profile of any molecule can be
consider as good if no more than one descriptor is violated, this
profile will be favourable and reliable for compounds 12a-o and
1
2q, while the bioavailability profile predicted for the series of
derivatives 12p-t, containing the nitro group in position C-5, not
were as good as spirooxindoles without any substituent or with
chlorine at this position, mainly due to an increase in the MW and
in the presence of more than 10 hydrogen bond acceptors. In
addition to lipophilicity, described by the Log P parameter, the
TPSA value is a descriptor that appears to be a complement to
link the lipophilicity of a SM with its ability to access to tissue
and cell compartments through membrane permeability and
lipophilicity driven non-specific binding to hydrophobic protein
sites or membranes. We found that in general, the value of TPSA
descriptor increases with the molecular volume and with the
4
0
Fig. 3 Key NOESY correlations and relative stereochemistry of 12j (ring
A: red; ring B: green).
In silico drug-likeness evaluation
With the growing understanding of the use of in silico methods,
as a tool to link the structure or the overall physicochemical
properties of a SM with their safety or toxicity endpoints, the
optimal design of compounds, directed to a specific and different
biological targets, has been of wide utility during the
4
5
6
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Organic & Biomolecular Chemistry
number of functional groups that were introducing in the
spirooxindole 1’-nitro pyrrolizidine core (e.g. polar fragments
evaluated against four toxic potential risks: as mutagenic,
tumorigenic, irritant and reproductive toxicity, and the results
like -OH, -NO , Cl, and OMe). Taking into account that 40 indicated that all the evaluated molecules tested positive as
2
2
compounds with TPSA values less than 140 Å will present good
membrane permeability profiles, almost all the tested
spirooxindoles, excepting the bulky derivatives 12p, 12s and 12t,
irritant, pointing out that these molecules would cause acute
inflammation, a painful reaction to some allergy or cell-lining
damage, while any of the synthesized compounds tested positive
for the other potential risks. Another values obtained through the
5
0
5
0
5
would be able to cross several biological membranes. However,
2
since compounds with TPSA values less than 60 Å could be 45 Osiris Property Explorer software for the spirooxindoles 12a-t
classified as agents that specifically permeate the Blood Brain
Barrier (BBB), it is expected that none of the spirooxindole 12a-t
have a biological activity or take action in the CNS.
Within the toolkit of the Molinspiration Cheminformatics
software, there is an application that focus on predict the specific 50 (Table 8). Additionally, the drug-likeness, an accumulating
activity by calculating the drug-likeness score of the tested
molecules towards G protein-coupled receptors (GPCR) ligands,
ion channel modulators, kinase inhibitors, nuclear receptor
ligands, protease inhibitors and other enzyme targets based on
were the drug-score, parameter that combines all the predictions
described into one grand total, which in average is 0.36 for the
series 12a-t, a relative low value because in some cases one or
two parameters described by the Lipinski’s Rule is violated
1
1
2
2
parameter of the bioactive sub-structure fragments present in
trade drugs, were positive in almost cases, reaching values
between 1 and 4.82, which indicates their highly similitude with
known drugs. To pointing out, the drug-likeness values of
comparison of the typical structures of active molecules with the 55 compounds derived from 3,4-methylenedioxy-β-nitrostyrene 11c
running structure in the platform. Having evaluated the series of
spirooxindole 1’-nitro pyrrolizidines 12a-t, the negative and
lower bioactivity scores for all these compounds indicates that
these molecules will not be active in any of those bioactivities.
However, the less negative bioactivity outcomes of
spirooxindoles 12a-t toward GPCR ligands, especially for
compound 12o, suggest the need to perform advanced in vitro
and in vivo studies to evaluate this hypothesis and establish if
these molecules could act as GPCR agonists (Table 7).
were two and three time more lower than the spirooxindoles
derived from nitrostyrenes 11a and 11b, suggesting their poor
structural relation with known molecules used in the
pharmaceutical industry (Table 8).
6
0
Table 8 Drug-score and the drug-likeness parameters calculated for
molecules 12a-t using the Osiris Property Explorer software.
a
Molecular parameters
Drug-score Drug-likeness
Comp.
2a
b
c
1
0.42
0.42
0.34
0.41
0.42
0.35
0.42
0.44
0.40
0.34
0.35
0.29
0.36
0.38
0.34
0.34
0.33
0.23
0.37
0.29
3.46
2.42
0.68
3.84
2.84
1.1
4.81
3.83
2.09
3.85
2.8
1.07
4.82
3.79
2.05
2.06
1.0
-0.75
3.03
0.23
6
7
7
5
0
5
12b
2c
2d
12e
2f
12g
Table 7 Bioactivity scores calculated for molecules 12a-t using the
Molinspiration Cheminformatics software.
1
1
a
Bioactivity Scores
1
Nuclear
Comp. GPCR Ion channel Kinase
ligand modulator inhibitor
Protease Enzyme
inhibitor inhibitor
Receptor
ligand
-0.49
-0.47
-0.51
-0.49
-0.47
-0.51
-0.41
-0.39
-0.42
-0.50
-0.47
-0.51
-0.42
-0.39
-0.43
-0.50
-0.48
-0.52
-0.43
-0.44
1
2h
1
2i
1
2a -0.31
1
2b -0.33
-0.28
-0.30
-0.31
-0.30
-0.31
-0.33
-0.27
-0.28
-0.29
-0.29
-0.30
-0.32
-0.27
-0.27
-0.28
-0.34
-0.32
-0.34
-0.34
-0.30
-0.44
-0.47
-0.51
-0.46
-0.49
-0.53
-0.37
-0.40
-0.43
-0.47
-0.50
-0.54
-0.38
-0.41
-0.45
-0.50
-0.54
-0.58
-0.42
-0.49
-0.25
-0.25
-0.24
-0.26
-0.26
-0.25
-0.15
-0.14
-0.12
-0.29
-0.29
-0.28
-0.18
-0.17
-0.16
-0.33
-0.33
-0.32
-0.22
-0.20
-0.35
-0.38
-0.37
-0.32
-0.34
-0.34
-0.21
-0.22
-0.22
-0.33
-0.30
-0.35
-0.23
-0.24
-0.24
-0.36
-0.38
-0.38
-0.26
-0.27
1
2j
1
2k
12l
2m
1
1
1
1
2c -0.29
2d -0.24
2e -0.25
2f -0.22
1
1
2n
2o
2p
2q
1
1
1
1
1
2g -0.13
2h -0.13
1
1
2i -0.10
2j -0.24
1
1
2r
2s
1
1
1
2k -0.24
2l -0.21
2m -0.13
1
2t
a
b
c
All compounds were identified as Irritant. Scale within 0-1. Positive
1
1
1
1
2n -0.13
2o -0.09
2p -0.32
2q -0.33
values states high grade in which fragments, that are frequently present in
commercial drugs, are contained in the tested molecule, While negative or
low values states poor similitude with trade drugs.
80
1
1
2r -0.30
2s -0.21
Finally, knowing that the ability of a molecule to cross the
BBB barrier is one of the key factors that have to be taken into
account during the chemical toxicological studies and in drug
design, Martins et al. recently used an approach based on
Bayesian statistics and coupled with state-of-the-art machine
learning methods to develop a robust model to predict the
penetration of SMs through the BBB, and in this order foretell
how this barrier could protect the brain against the foreign
1
2t -0.19
a
3
0
Negative values predict poor bioactivities while the magnitude of the
scores are related with the potency of the predicted activity.
85
4
2
Then, the Osiris Property Explorer software was also used to
predict the potential risk, the drug-likeness and the drug-score of
the obtained spirooxindole 1’-nitro pyrrolizidines 12a-t. The
calculations of this virtual package are based in the structural
comparison of the tested compounds with the main molecular
fragments frequently present in trade drugs. Each molecule was
4
3
3
5
spirooxindoles once they enter into the circulatory system.
90
Fortunately, this model was adapted into a Web based tool that
was used to predict the permeability of compounds 12a-t and
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Page 8 of 14
according to this model, the derivatives containing the hydroxyl 40 Determination of zebrafish embryo LC50
group at position C-5’ should not be able to cross the BBB,
Having obtained and characterized the new 20-membered library
of spirooxindole 1’-nitro pyrrolizidines 12a-t, we continued our
pharmacological investigation conducting the toxicity assays
unlike to compounds that not have this functionality, which their
prediction established that they could permeate the BBB with
more facility than the hydroxylated compounds (Table 9).
The measure of the probability of each spirooxindole to
penetrate the BBB is expressed by the magnitude of the
5
0
5
0
5
0
(
lethal concentration - LC , μmol/L) in zebrafish embryos. For
50
4
5
this purpose, we adapted and followed in our laboratory the
4
4
protocol described by Ali et al. with some modifications. In first
place, independent experiments were made in order to find a
series of concentrations, for each spirooxindole compound, in
which the mortality of the embryos after chemical exposure were
P(BBB |MW) value in Table 9. This parameter will be dependent
+
on the molecular weight (MW) of the tested compound, and thus
this is the reason why the magnitude of this parameter is too low
1
1
2
2
3
for all the spirooxindoles 12a-t, indicating that is not expected 50 among 0 % and 100 %. With this preliminary study, a geometric
that these molecules permeate efficiently this biological barrier
since their MW, expressed in g/mol, is on the range 400-500.
Actually, all the spirooxindoles with the substitution at position
C-5’ by the hydroxyl group were rejected as possible molecules
that would be able to cross the BBB, perhaps by the interaction
between these molecules and some biological targets present in
the BBB (e.g. proteins), preventing their diffusion through this
barrier.
series of seven concentrations, in which each concentration was
50 % greater than the next lowest value (starting from 19.53 μM
and finishing in 1250 μM), was well established.
For this toxicity testing, we started the chemical exposure at
24 hpf in order to avoid the natural early mortality during the first
stages of development that could affect the interpretations of the
5
6
6
5
0
5
4
5
obtained results. Thereby, having selected the live, healthy and
well developed embryos at 24 hpf, ensuring that the survival of
the controls will be 100 %, they were individually distributed in a
96-well plate and then the embryos were exposed three times at
the geometric series of concentrations of each spirooxindole 1’-
nitropyrrolizidine. As a range for the acute toxicity assessment,
the embryos were carefully examined after 72 hours of chemical
exposure (96 hpf) and were classified as death if they present: i)
coagulation, ii) lack of somite formation, iii) lack of detachment
of the tail-bud from the yolk sac (YS), iv) their heart was not
beating, and v) they were no longer moving after external
stimulation. Finally, the data recollected from three independent
experiments were statistically analyzed and the determined LC50
In the other hand, even though the prediction for some
molecules to penetrate the BBB was assured, their P(BBB |MW)
+
value was still lower than the value for the molecules that should
not be able to cross this barrier, this fact can be explained by the
presence of three methoxy groups that make the molecule too
bulky for permeate easily the BBB. However, it is expected that
these spirooxindoles permeate different kind of tissues and cells.
Interestingly, the low P(BBB |MW) values for molecules bearing
+
the hydroxyl group, the nitro group in C-5 position or the bulky
derivatives, can be correlated with the results presented in Table
6 where were discussed that most of these analogues presented 70 values are shown in Table 10.
2
high TPSA values, in some cases above 140 Å , which indicates
Table 10 Determined zebrafish embryo LC50 values for spirooxindole 1’-
nitro pyrrolizidines.
that the prediction of libraries of SMs to permeate specifically the
BBB could be study from the P(BBB |MW) and TPSA values.
+
Zebrafish embryo LC50
Compound
2a + 12a’
a
Table 9 Drug-score and the drug-likeness parameters calculated for
molecules 12a-t using the Osiris Property Explorer software.
(
μmol/L ± SEM)
110.83 ± 0.17
84.43 ± 7.68
70.96 ± 8.87
71.68 ± 7.68
94.82 ± 8.09
432.30 ± 2.23
219.52 ± 0.08
280.46 ± 0.04
222.77 ± 9.27
53.10 ± 3.39
56.12 ± 0.09
419.59 ± 9.92
211.09 ± 8.27
219.53 ± 0.09
414.51 ± 9.00
56.24 ± 0.14
94.77 ± 9.88
297.11 ± 9.05
219.92 ± 1.04
292.56 ± 8.02
3
5
1
1
2b + 12b’
12c + 12c’
2d
2e
2f
Molecular parameters
P(BBB |MW) P(BBB |MW)
+
Comp.
a
b
_
1
1
1
2a
2b
0.0118
0.0147
0.0160
0.0105
0.0135
0.0148
0.0093
0.0118
0.0135
0.0079
0.0102
0.0116
0.0065
0.0091
0.0102
0.0070
0.0094
0.0106
0.0057
0.0094
0.9882
0.9853
0.9840
0.9895
0.9865
0.9852
0.9907
0.9882
0.9865
0.9921
0.9898
0.9884
0.9935
0.9909
0.9898
0.9930
0.9906
0.9894
0.9943
0.9906
1
1
1
1
1
1
2c
2d
2e
2f
1
1
2g
2h
1
1
2i
2j
1
1
2g
2h
1
2k
2l
2m
1
1
1
1
2i
2j
1
1
1
1
2n
2o
2p
2q
1
2k
2l
2m
1
1
1
1
1
1
2n
2o
2p
2q
1
1
2r
2s
1
2t
a
1
1
1
2r
2s
2t
LC50 values are the mean ± SEM of three different experiments in
triplicate.
a
Probability of the molecule to cross the BBB according to their
75
From the results obtained it is clearly observed that the
mixture of regioisomers 12a-12a’, 12b-12b’ and 12c-12c’
displayed an acute toxicity, pointing out that derivatives 12c and
b
molecular weight (MW). Non probability of the molecule to cross the
BBB according to their molecular weight (MW).
1
2c’, where the 3,4-methylenedioxy moiety could be identified as
the main toxicophore, resulted to be more toxic than the mixture
8
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Organic & Biomolecular Chemistry
of regioisomers 12a and 12a', in which the presence of the 3,4,5- 60 important; the dead embryos by any cause, would not affect the
trimethoxyphenyl fragment decrease the toxicity. However, these
results could be ambiguous since it is known that the zebrafish
embryos have different biological responses when they are
development of the others in the well, generating ambiguous
interpretations.
As well as for the determination of zebrafish embryo LC , the
5
0
4
6
5
0
5
0
5
0
5
0
exposed, individually, to a pure enantiomers.
phenotyping assays started at 24 hpf in order to correlate both
Then, when in the structure of derivatives 12d-t, the N-H 65 tests. By starting the chemical exposure at 24 hpf, many
group of the isatin core was methylated to afford the desired
regioisomer, the molecules bearing the 3,4,5-trimethoxyphenyl
fragment exhibited the most lowest values of LC , followed by
discussions and debates can arise in relation with the biological
process and toxic properties of the compounds that we are not
taking into account during the early stages of development (e.g.
oblong, epiboly and somite stages), but this approach is useful
5
0
1
1
2
2
3
3
4
the derivatives possessing the 3,4-dimethoxyphenyl moiety. We
found that in contrast to the results obtained for the mixtures of 70 during the manipulation and selection of healthy embryos and to
regioisomers, in this case the main toxicophores were the 3,4,5-
trimethoxyphenyl and 3,4-dimethoxyphenyl moiety, increasing
the toxicity of the N-methyl derivatives, while in those
derivatives that possess the 3,4-methylenedioxy fragment the
ensure the control survival (100 %) after having finished the test.
It is also known that the presence of two outer membranes at
early stages of development that would act as a barrier for the
4
9
penetration of SMs. However in this case, the permeation of
toxicity decreases significantly, showing LC50 values above 400 75 molecules 12a-t through this barrier is not affected because the
μmol/L and identifying compound 12f as the less toxic compound
among the synthesized spirooxindoles.
thickness of these membranes is about 10 μm and within of them,
the chorion, with a pore size of 0.17 μm , imposes the size
2
With respect to the substituents of the N-methylisatin core, the
inclusion of chlorine atom at the position C-5 of the isatin greatly
increases the toxicity of 12j and 12k, with the 3,4,5- 80 membrane.
restrictions for the uptake of SMs into the embryo, leading that
molecules that exceed 3 kDa would not be able to penetrate this
5
0
trimethoxyphenyl
and
3,4-dimethoxyphenyl
fragments
After starting the chemical exposure at 24 hpf, during the test
the morphology of the embryos, exposed at nine different
concentrations below the LC50 of each compound, were observed
at various stages from 48 to 96 hours post fertilization (hpf) at 24-
respectively, these molecules resulted to be the most lethal
compounds, however the derivative 12l, with the chlorine
substituent and the 3,4-methylenedioxy moiety, was eight times
less toxic than derivatives 12j and 12k. Similar results were 85 hours intervals using a dissecting stereomicroscope.
obtained from the substitution at the position C-5 of the isatin
core by the nitro group, where the derivative 12p has like toxicity
to their chlorinated analogues 12j and 12k. Nonetheless, despite
the present of the 3,4-methylendioxy moiety, the toxicity of
In general, all the embryos treated with compounds 12a-t, at
two or three concentrations below the LC50 of each derivative, did
not manifested any visual phenotype and each treated embryo
reached its corresponding development stage after 96 hpf,
derivative 12r increased, been only five times less toxic than 90 without any visual evidence that might indicate that the
compound 12p, concluding that the presence of another nitro
group bounded to the aromatic ring of isatin, is a potent
toxicophore that increases the toxicity of these spirooxindoles and
in the same way decreases their bioavailability profiles according
to the Lipinski’s rule (Tables 6-9).
morphology of these treated embryos differed from the control
embryo morphology (see ESI†).
However, at one or two concentrations below the LC50 of each
compound, developmental delay, curved bodies (CB) and severe
95 edemas or big yolk sac (YS), whose size depended of the
administrated compound, were identified as the major visual
phenotypes observed for the series of spirooxindoles 1’-nitro
pyrrolizidines, suggesting the level of toxicity of these derivatives
and their possible mode of action (Table 7).
Compound 12d (Table 7: D, E and F) induced a slight delay in
the development of the embryos, visualized after 24 hours of
chemical exposure, however at 72 and 96 hpf the examination of
the embryos revealed that they seem to be normal in comparison
with the control embryos and where the nutrients present in YS
05 are consumed during the early stages of development. Moreover,
compound 12g (Table 7: G, H and I) significantly delays the
development of the treated embryos and they did not break the
embryonic membrane after 96 hpf.
Finally, all compounds derived from trans-4-hydroxy-L-
proline 10b exhibited in general
a
moderate toxicity
independently of the kind of substituent at 5-position of the isatin
core and of the chemical nature of the substituents present in the
phenyl fragment from the β-nitrostyrenes.
100
In vivo zebrafish phenotyping
Once the LC50 for compounds 12a-t was determined, a range of
concentrations, below this value of LC , were establishing in
5
0
1
1
1
1
4
5
5
5
0
5
order to screening our molecules using the zebrafish embryos and
studying how the activity and normal expression of endogenous
genes and proteins are subtly manipulated by the exogenous
4
7
molecules 12a-t, putting forward that these expressions (data)
could be correlated with the results of Tables 6-9 and/or with
clinical situations and disorders in humans.
The examination of embryos treated with compound 12j
10 (Table 7: J, K and L) revealed a characteristic phenotype that can
be identified as an edema or big YS, and that could be correlated
with the phenotypes expressed by compounds 12d and 12g. After
Taking into account that each fertilized zebrafish embryo
represents macroscopic single cell, and in contrast to previous
reports in which three embryos were distributed in a 96-well plate
7
2 hours of chemical exposure (96 hpf), probably the larvae was
unable to absorb the nutrients, minerals and vitamins from the
15 intestinal tract into the bloodstream, and this considerably
decreases the utilization of endogenous reserves from the YS,
mainly phospholipids and triaglycerols. This would be the reason
(
putting at least 3 embryos/well, sometimes more), where each
4
8
compound is evaluated one time. In this phenotyping assay we
evaluated each SM, at the range of established concentrations, in
two independent experiments putting a single embryo peer well,
this allows the examination and tracking of the embryos during
their development until the end of the experiment, and more
51
why these embryos have wider YS than those of the control.
The visual evidence of this phenotype cannot be assigned to a
20 probable teratogenic effects because these genetic damages would
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DOI: 10.1039/C3OB41302K
Organic & Biomolecular Chemistry
Page 10 of 14
not have been evidenced since the first hours of development. In
addition, a few development abnormalities were also observed in
the treated embryo after 96 hpf like curved bodies (CB) and
20
Finally, compound 12t (Table 7: P, Q and R) displayed as well
the same phenotypes in the YS and the CB, but the edema
induced by this compound was less severe than that one observed
for molecule 12o. Then, it is clear that the presence of the
hydroxyl group in the skeleton of the prepared compounds could
affect the metabolism of the zebrafish larvae in a significant way
than the derivatives that not possess this hydroxyl group in their
structure. Additionally, it has to be noted that these phenotypes
were visual at these tested concentrations, and at concentrations
below them, they did not exhibit this similar patron and that
larvae reached posterior stages of development at the same rate
that the control did and the same morphology of the control was
observed, leading to suggest that the exposed embryos at lower
concentrations would also been affected by these SMs, but the
ability of the larvae to recover from the hepatotoxic effects of
these SMs or their capacity to metabolized them into some non-
toxic metabolites would explain this observation, however
additional studies are needed to prove this hypothesis.
5
2
disorders or abnormalities in the middle intestine (MI).
5
0
5
As it was mentioned, one of the major problems of
pyrrolizidine alkaloids in clinical trials is their hepatotoxicity, and
even more when hydroxyl groups are present in their structures.
Analyzing the embryos treated with compound 12o (Table 7: M,
N and O), we found that the embryos displayed severe edemas or
big YS after 96 hpf. This would indicate that the metabolism of
the larvae has been affected by compound 12o, with one hydroxyl
group in the pyrrolizidine ring. Possibly, the organ affected by
this compound will be the liver, which primary morphogenesis
starts after the first 48 hpf and is fully formed and functional at
72 hpf, metabolizing the 70 % of the neutral lipids containing in
2
3
3
5
0
5
1
1
5
3
the YS. However, this compound exhibited one of the most
highest LC50 values among the series of synthesized
spirooxindoles, indicating that the liver failure is not lethal for the
larvae during the 96 hpf.
a
Table 7 Visual phenotypes induced by the selected spirooxindoles 1’-nitro pyrrolizidines at concentrations below their LC50
8 hpf 72 hpf
4
96 hpf
Control
Wild-type (WT)
a
(A-C) Microscope photographs of the zebrafish embryos non-treated with spirooxindole 1'-nitro pyrrolizidines 12a-t; (D-F) Microscope photographs of
4
0
the zebrafish embryos treated with compound 12d at 50 μM, YS = yolk sac; (G-I) Microscope photographs of the zebrafish embryos treated with
compound 12g at 150 μM; (J-L) Microscope photographs of the zebrafish embryos treated with compound 12j at 50 μM, CB = curved body, a = yolk sac
edema (YSE), b = abnormal development of middle intestine (MI); (M-O) Microscope photographs of the zebrafish embryos treated with compound 12o
at 200 μM; (P-Q) Microscope photographs of the zebrafish embryos treated with compound 12t at 200 μM.
1
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Organic & Biomolecular Chemistry
5
4a
Conclusions
K CO to afford 9b in 99 % yield. N-methyl isatin 9b was
2 3
chlorinated with trichloroisocyanuric acid (TCCA) using sulfuric
acid as a catalyst to afford 9c in 97 %. N-methyl isatin 9b was
nitrated by using a nitration minture (KNO + H SO ) to afford
d in 99 % yield. β-Nitrostyrenes 11a-11b were prepared from
the respective benzaldehyde using ethanolamine supported on
SiO2 as a catalyst in yields up to 98 %.
compounds were purified and crystallized by using appropriate
solvents and the acquired spectroscopy and spectrometric data
were in agreement with the literature.
Unless otherwise noted, all reactions have been carried out
with distilled and dried solvents and under atmosphere pressure.
All work-up and purification procedures were carried out with
reagent grade solvents (purchased from Aldrich and Merck) in
air. Thin-layer chromatography (TLC) was performed using E.
Merck silica gel 60 F254 precoated plates (0.25 mm). Column
chromatography was performed using silicagel 60 (0.063 - 0.200
mm) 70-230 mesh.
With the developed protocol based on the 1,3-dipolar
cycloaddition reaction between azomethine ylides, generated in
situ from substituted isatins and L-prolines, and different β-
nitrostyrenes in aqueous media, diverse 20-compound pilot-scale
library of novel heterocyclic hybrids with the spirooxindole,
pyrrolizidine and 3,4,5-trimethoxyphenyl pharmacophoric
moieties has been effectively constructed in good to excellent
yields and with perfect regio- and stereoselectivities.
Performing LC50 toxicity assays in zebrafish embryos for all
compounds obtained, three molecules 12j, 12k and 12p, were
identified as the most toxic derivatives with a LC50 below 56 μM.
In addition, the in vivo biological activity of the 1’-nitro
pyrrolizidines 12a-t was evaluated, observing that several edemas
and big YS were expressed by the series of spirooxindoles
containing the hydroxyl group in the pyrrolizidine ring,
suggesting that the delivery of nutrients and hormones, contained
in the YS into the circulatory system is slow or that several
damage are induce in the liver. Noteworthy, results of their
bioprospection showed a close correlation between in silico and
in vivo assessment. Despite the evidence of hepatotoxicity,
further research is needed to confirm the level of liver damage
and how other developmental processes could be affected by the
spirooxindoles 12a-t at the administrated doses.
54b
6
6
7
7
0
5
0
5
3
2
4
54c
9
5
0
5
0
5
0
54d
All the above
1
1
2
2
3
General procedure for the synthesis of the spirooxindoles 1’-nitro
pyrrolizidines 12a-t
Into a 15 mL round bottom flask equipped with a condenser, a
gas bubbler and a stir bar was added the corresponding isatin 9a-
d (2.50 mmol, 1 equiv), the respective β-nitrostyrene 11a or 11b
8
8
9
0
5
0
Finally, taking as a start point the results shown in this
research, in vitro anti-herpetic, antifungal and cytotoxic assays
are under way in order to establish their possible biological
mechanism of action and to identify novel biological targets
involved in vertebrate embryonic development, these novel
results will be published elsewhere as soon as possible.
(
1.1 equiv), 1 mL of distilled water and 0.2 mL of propylene
carbonate. The reaction mixture was stirred at 90 °C for 15 min.
prior to addition of the appropriate L-proline 10a-b (1.3 equiv)
and then the mixture was stirred at this temperature for one hour
controlling the bubbling of CO . TLC monitoring confirmed the
2
end of the reaction, then the reaction mixture was cooled at room
temperature and quenched with saturated sodium bicarbonate
solution (10 mL, 1 M) and was extracted three times with CH Cl
Experimental section
2
2
(
3 x 20 mL). The organic layer was separated, dried with Na SO ,
2 4
Chemistry
and concentrated to afford the crude product, which was purified
by silica gel flash chromatography to yield the corresponding
spirooxindole 1’-nitro pyrrolizidines 12a-t.
Infrared (FT-IR) spectra were recorded on a Lumex Infralum FT-
-
1
0
2 spectrometer, νmax in cm . Bands are characterized according
1
3
4
4
5
5
5
0
5
0
5
to the functional group. H NMR spectra were recorded on a
Bruker Avance-400 (400 MHz) spectrometer. Chemical shifts are
reported in ppm with the solvent resonance as the internal
Bioinformatics
standard (CDCl : δ 7.26 ppm). Data were reported as follows:
3
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd
= doublet of doublets, br = broad, m = multiplet), coupling
constants (Hz) and integration. C NMR spectra were recorded
on a Bruker Avance-400 (400 MHz) spectrometer with complete
proton decoupling. Chemical shifts are reported in ppm from
95
In silico analysis for the calculation of the molecular
descriptors and the predicted bioactivities were performed using
the Molinspiration Cheminformatics software, free available
online at [http://www.molinspiration.com/cgi-bin/properties],
while the toxicity prediction as well as the calculation of the
13
solvent resonance as the internal standard (CDCl : δ 77.00 ppm). 100 drug-score and drug-likeness were performed using the Osiris
3
On DEPT-135 spectra, the signals of CH and CH carbons are
Property Explorer software, free available online at
[http://www.organic-chemistry.org/prog/peo/], and finally, the
Blood-Brain Barrier penetration prediction was performed using
3
shown as positive (+) and CH carbons are shown negative (-).
2
Quaternary carbons are not shown. A Hewlett Packard 5890a
Series II Gas Chromatograph interfaced to an HP 5972 Mass
Selective Detector (MSD) with an HP MS ChemStation Data 105 [http://b3pp.lasige.di.fc.ul.pt].
system was used for MS identification at 70 eV using a 60 m
the
B3PP
model,
free
available
online
at
capillary column coated with HP-5 [5%-phenylpoly
dimethylsiloxane)]. Accurate mass data were obtained on
Toxicity testing and phenotypic screening of
spirooxindoles 1’-nitro pyrrolizidines 12a-t using the
zebrafish embryo model
(
Micromass Q-TOF by electrospray ionisation (ESI). Melting
points were measured on a Fisher Johns melting point apparatus
and are uncorrected.
Wild-type adult zebrafish of both sexes were separated in two
10 tanks (30 L each), according to their gender, at 26 ± 2 °C under
natural light-dark photoperiods. The fish were feed twice daily
1
Isatin 9a was methylated with methyl iodide in presence of
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DOI: 10.1039/C3OB41302K
Organic & Biomolecular Chemistry
Page 12 of 14
and the water quality was recorded weekly, in order to acclimate
Acknowledgments
the fishes for at least two weeks before experiments begin. For
the reproduction of the adult fishes, small breeding tanks were set
up in the evening previous to experiment, each containing three
males and one female specimen. The tanks were isolated until
next morning when the lights switch on and the natural mating
occurs, without any perturbation.
The adult fishes were returned to their corresponding tank and
the embryos were collected, pooled and washed with E3 medium
and transferred into a 92 mm glass Petri dish. Further, dead,
delayed, malformed and unfertilized embryos were identified
under a dissecting microscope and removed by select aspiration
with a pipette. This last procedure was repeated at 12 and 20 hpf
in order to remove the unfit embryos. Throughout this period of
time, the embryos were kept at 28 ± 2 °C in an incubator under
natural light-dark photoperiods.
The selected embryos of 24 hpf from the petri dish were
gently distributed into 96-well plates, placing a single embryo
and 200 μL of E3 medium per well.
Adult zebrafish were care and used according to the Guide of
the National Institute of Health for Care and Use of Laboratory
Animals, keep them healthy and free of any signs of disease. The
Ethics and Research Committee of the Heart Institute of
Bucaramanga approved the protocol under the Acta Number 050
of May 26 of 2012.
5
6
6
5
0
5
This work was financially supported by the Colombian
Institute for Science and Research (COLCIENCIAS) under the
project No. RC-366-2011. We wish to thank to Prof. Joel Jones
Junior, Prof. Flavia Martins da Silva and Josué S. Bello Forero,
from the Environmental Organic Synthesis Group (SOA),
Institute of Chemistry of Federal University of Rio de Janeiro,
Brazil, for their generous donation of L-proline derivative 10b
and for their helpful discussions and opinions in the preliminary
stages of this research.
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5
0
5
1
1
2
2
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Org. Biomol. Chem., 2013, [vol], 00–00 | 13

Organic & Biomolecular Chemistry
Page 14 of 14
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DOI: 10.1039/C3OB41302K
Table of contents/Graphical abstract
The synthesis of spirooxindolo 1’-nitro pyrrolizidines with five concurrent stereocenters under
aqueous media is described as well as their bioprospection using the zebrafish embryo model