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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5435_Библиотеки_им_академика_М_И_Перельмана
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Alam et al. [74] described the synthesis, characterization as well as anticancer evalua-
tion of some pyrazolyl chalcone analogues. Structu ral elucidation of all compounds
was performed with the help of
1
H NMR,
13
C NMR, and ESI-MS spectra. Next, all mole-
cules were screened in silico through docking simulation analysis against colchicine-
binding site of tubulin (PDB: 3E22). The results indicated that compound 6 possessed
superior inhibition of tubulin protein with binding score of −7.002 kcal/mol, which
was comparable to co-crystal ligand colchicine (binding energy = −7.059 kcal/mol).
NMR spectrum for compound 6 explored the characteristic peaks at different δ values
with respect to internal standard tetramethyl silane as shown in Figure 13.8. Further-
more, same analogue 6 exerted most promising inhibition against MCF-7, SiHa, and
PC-3 with IC
50
values of 2.13 ± 0.80, 4.34 ± 0.98, and 4.46 ± 0.53 µM, respectively [74].
Figure 13.7:
1
H NMR and
13
C NMR spectrum of compound 5.
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13.4 Role of mass spectrometry in drug discovery
MS has anticipated as a persuasive and dominant advanced analytical tool in the field
of drug discovery. In the drug discovery process, small molecules, that is, ligands are
intended to being identified and developed as a new ray of hope in the eradication of
specific disorders. Hence, MS is evident with the exact composition of ligands as well as
target protein followed by their interactions and postdocking structural modifications.
This analytical approach guides the researchers about the specific data regarding mo-
lecular configuration of particular synthesized analogues [75]. Whenever the synthesis
of biologically active molecules being apparent to revolutionized, target and lead valida-
tion point strikes in mind of every research professional. There are several analytical
tools including spectroscopy like FTIR and NMR spectroscopy to elucidate the structures
of synthesized compounds. Whereas MS is the only analytical approach that provides
the confirmation for structural composition of synthesized molecule or target protein.
Figure 13.8:
1
H NMR and
13
C NMR spectrum of compound 6.
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As it generates the peaks on the basis of mass by charge ratio for molecular or fragment
ions produced from ruination as a part of entire structural skeleton [76]. After initial
structural characterization through other spectroscopic techniques, structure can only
be validated by knowing the fragmentation pattern and exact molar mass of its possible
metabolites through mass spectrum. In contrast to structural elucidation of synthesized
molecules, MS spectrum confirms the structure of macromolecules by virtue of which
potential therapeutic protein target will be explored. In addition to its role in drug tar-
get identification, MS suggests about the pharmacokinetic as well as pharmacodynamic
assessments and quantitative studies of drugs in biological fluids [77, 78]. As fragment
ions deliver their respective peaks in mass spectrum, pharmacokinetics, that is, drug
metabolism studies, may be explored at significant extent which helps to determine the
half time and biological efficacy. Moreover, MS is a revolutionized technique which
helps at multiple stages of drug discovery ranging from target to lead identification,
ADMET analysis of drug to intensity of pharmacological effect on body. Hence, MS is
ultimately accelerating the generation of safer and highly efficient bioactive compounds
[79]. There are several key points needed to be addressed related to MS with respect to
drug discovery as mentioned below:
1. MS emerged with utmost attention toward the identification of potential therapeutic
targets which possess landscape for owning the ligand inside of active site. Structural
entities allocated in the binding pocket of receptor that may participate in favorable
binding can be elucidated in a more exact manner. MS spectrum helps to predict the
potential drug targets by corroborating the ligand–macromolecule-binding interactions
and directing label-free protein quantification in two or more samples [80].
2. Further, MS spectrum also lends a helping hand to interpret the posttranslational
alterations in the parent structure. Also, coupling of MS with other collective interpre-
tation tools like proteomics, metabolomics, lipidomic, genomics, transcriptomics, and
many more to justify the mechanism of action associated with complicated disorders.
Identification and validation of lead molecules become much easier if get prerational-
ized via utilization of such omics technologies together [81].
3. In addition to the MS, hyphenated techniques are imposed to overcome the limita-
tions of MS alone. It also afforded certain advancements due to conjunction of both
chromatographic as well as spectral techniques together. It favors the isolation of
pure chemical species from a bunch of compounds followed by their structural eluci-
dation in concerted way. In this context, several MS-based hyphenated techniques are
well-versed throughout the research advancements such as liquid chromatography-
MS (LC-MS), gas chromatography-MS (GC-MS), capillary electrophoresis-MS (CE-MS),
tandem mass spectrometry (MS/MS), and LC-electrospray ionization-MS (LC-ESI- MS)
[82, 83].
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4. MS has a significant role in the metabolomics investigation of synthesized mole-
cules. To focus on the identification of various metabolites arose during the phase I
and phase II reactions, MS notices that each and every fragment ion travelling
through mass analyzer and peak for the same will be observed in MS spectrum [84].
5. Pharmacokinetics as well as pharmacodynamics are two major fields in which MS
laid its role to explore the ADME profile of the subjected molecules. Also, toxicological
profile for generated metabolites in various biological samples like tissues, urine, and
blood can also be estimated in both qualitative as well as quantitative manner. MS
helps to recognize the fate of drug in biological system including absorption, distribu-
tion, metabolism, excretion, and toxicity-related data that assists therapeutic potency
and safety profile [83, 84].
6. MS becomes evident as an advanced and sophisticated bioanalytical technique
which is well conversant with quantification tests of drugs in a given sample. As mass
spectrum can notify about the presence of desired molecules as well as trace amount
of impurities too, it is an acquainted tool exploited for bioanalysis to maintain the ab-
solute standards. Mass spectroscopy can easily corroborate a large number of mole-
cules at a time and hence used in high-throughput bioanalysis such as calibration and
quality measurement of multiple samples in limited timeline [85].
7. Beside the hyphenated techniques, MS atta ined the utmost and extreme advance-
ments in itself as in HRMS. A large nu mber of molecules can be subjected via high-
throughput screening which will then be elucidated by HRMS on the basis of chemical
compositions and respective molar mass. Also, ambient ionization techniques are
used like ESI, atmospheric pressure chemical ionization, fast atom bombardment, and
matrix-assisted laser desorption ionization. These forward-looking automation tech-
nologies furnish the new hope or prospective with elevated speed, preciseness, and
sensitivity to develop novel drug candidates [86, 87].
13.4.1 Recent advances in mass spectrometry to develop
biologically active molecules
In this literature, synthesis and anti-inflammatory study of thymol-linked pyrazole ana-
logues as dual COX-2/5-LOX inhibitors was disclosed by El-Miligy et al. [88]. Hereby, all
compounds were generated from multicomponent synthetic strategy and then struc-
tures of synthesized series were corroborated via IR,
1
H NMR,
13
C NMR, and EIMS spec-
trum. Mass spectrum for compound 7 has presented the characteristic molecular ion as
well as fragment ion peaks as shown in Figure 13.9. In vitro anti-inflammatory study
resulted in compound 7 as superior selective COX-2 inhibitor possessing IC
50
value of
0.043 ± 0.001 µM, which was more effective than that of reference selective COX-2 inhib-
itor, that is, celecoxib (IC
50
= 0.045 ± 0.007 µM). In addition, 7 validated in vitro anti-
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inflammatory score by subjecting to in vivo investigation via formalin-induced paw-
oedema method. Moreover, docking study was performed toward COX-2 (PDB Code:
3LN1) and 5-LOX (PDB Code: 3V99) isoenzymes in which again compound 7 elicited top-
most binding affinity with binding score of −10.40 and −8.38 kcal/mol toward COX-2 and
5-LOX, respectively [88].
In this article, da Silva et al. [89] demonstrated the synthesis as well as antileish-
manial effect of some tetrasubstituted pyrazole analogues. Initially, structural infor-
mation for all molecules was gathered from
1
HNMR,
13
C NMR, and HRMS spectral
data. Further, all molecules were subjected in vitro for antileishmanial evaluation in
which compound 8 resulted in topmost antileishmanial agent toward Leishmania am-
azonensis and Trypanosoma cruzi having IC
50
values of 3.8 ± 0.1 µM and 4.5 ± 0.7 µM,
respectively. The structure of the most active analogue 8 was corroborated through
characteristics peaks apparent to m/z ratio as mentioned in Figure 13.9 [89].
In this article, design and synthesis of thiophene- and furan-linked 4-amino-7-
chloroquinoline hybrid derivatives and their in vitro and in vivo antimalarial evaluation
was reported by Opsenica et al. [90]. Structural characterization of synthesized deriva-
tives was performed using IR,
1
HNMR,
13
CNMR,andGC-MSspectraldata.Allcom-
pounds were evaluated for in vitro antimalarial potential against chloroquin e
sensitive (CQ
S
, D6), chloroquine-resistant (CQ
R
, W2) and multidrug-resistant (MDR)
Figure 13.9: Some bioactive heterocyclic derivatives corroborated through mass spectral data.
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C235 strain of P. falciparum. It was revealed that compound 9 displayed most potent
antimalarial potential against chloroquine-sensitive (CQ
S
, D6) and chloroquine-
resistant (CQ
R
,W2)strainwithIC
50
values of 2 and 8 nM, respectively, which was
equal to more potent than chloroquine (IC
50
=595 nM against CQ
S
,D6,and15nM
against CQ
R
,W2)andmefloquine(IC
50
= 7 nM against CQ
S
,D6,and23nMagainst
CQ
R
, W2). Mass spectrum for compound 9 explored the characteristic peaks for mo-
lecular ion as well as fragment ions as shown in Figure 13.9. Also, compound 9 is
foundtobemorepotentagainstMDRC235strainwithIC
50
value of 5 nM as com-
pared to chloroquine (IC
50
= 206 nM) and mefloquine (IC
50
= 55 nM ) [90].
13.5 Role of X-ray crystallography in drug discovery
X-ray crystallography remains a divergent analytical methodology in the various fields
of drug discovery. In this particular technique, more focused or highly resolute struc-
tural characteristics are procured regarding different receptor macromolecules or bind-
ing interactions of selected molecule within binding cavity of target protein. It enables
the researchers to validate the best fit binding pose of ligand toward the active site
amino acid residues. It attributes toward the extensive knowledge about various inter-
molecular electrostatic interactions which are essential to design the analogues series
of ligand in the search of potent therapeutic agents [91]. Pharmacophore can be mod-
eled with the help of fragments afforded clues to being docked perfectly within binding
pocket of receptor to deliver remarkable biological potency. It also suggests about the
suitable orientation of ligand to best fit onto target site with minimal binding energy. In
modern drug discovery scenario, high-resolution structural confirmation is a key point
by virtue of which one can adopt the knowledge about the drawbacks of racemic mix-
tures [91, 92]. In this context, X-ray crystallography provides the better understanding
about more justifiable binding affinity of enantiomeric pure form which definitely re-
sults in lack of side effects as compared to its racemic mixture. Hence, X-ray crystallogra-
phy is a well-used analytical methodology in order to corroborate the atomic structure
of macromolecules followed by finding the drug–receptor interactions. As biological tar-
gets are emerged to being understood prior of generating novel lead molecules that
may interact most perfectly with it, X-ray crystallographic studies are profound to
bear milestone handout to transfigure the pharmaceutical research [91–94]. There are
several essential involvements of X-ray crystallography that cannot be compromised
with another analytical approach in aspect of quality or high-resolution outcomes as
deliberated below:
1. X-ray crystallography raised as a great reward in the drug development process
as it is tangled to supreme history with it. Initially, X-ray crystallography was em-
ployed for one and only small and tiny molecules, that is, ligands alone while at
present, it has developed versatility for a range of ligands to macromolecules,
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that is, proteins. The only drawback related to selection criteria adopted in the
case of X-ray crystallography is to have a crystallized form of sample only. Apart
of such limitation associated with sample type, it is time to time exploited by re-
searchers to obtain the knowledge regarding atomic arrangement in ligand as
well proteins till date [95].
2. Drug discovery always began with the choice and availability of suitable target to
which drug must bin d over for eliciting the pharmacological response. In this
way, scientists utilize the X-ray crystallography for identifying as well as optimiz-
ing the drug target. The atomic conformation of protein or macromo lecule indi-
cates the binding affinity of ligand whenever it will be docked in the active site
present in the particular receptor [96].
3. By keeping in mind the crystallized structure of protein, SBDD approach basically
comes in the front to demonstrate the structural features of protein that may play
role during the molecular docking within the binding pocket of receptor. It gener-
ally considers the electronic nature of all the amino acid residues present over
the specific position where ligand is proposed to bind over. Further, it allows the
researchers to design the molecule in accordance of selected amino acids that
contribute to possess the key interactions to deliver the biological potency. In con-
trast to higher potency, side effects may also overcome due to increased specific-
ity toward the single active site and negligible allosteric binding [97].
4. In addition, X-ray crystallography can help to predict the most probable mecha-
nism endowed with desired biological effect. Drug–receptor interactions along
with their type of binding and molecular dynamics studies explored by X-ray
crystallography are followed by spelling out the possible mechanism of action for
particular pharmacological response [98].
5. In contrast to advanced features of X-ray crystallography, it exhibits the several
challenges or obstacles during the entire methodology like difficulty in sampling
(not easy to get precise quality crystals) and compromises molecular binding
strength toward target protein (altered molecular dynamics). Beside these points,
it is not possible to shape every protein target in the crystalline form and hence
could not be a suitable sample for being evaluated under X-ray crystallographic
analysis [99].
6. Nowadays, X-ray crystallography is coupled with various other analytical techni-
ques like 1D or 2D NMR spectroscopy, serial crystallography, electron microscopy,
and microcrystallography to achieve the maximal efficacy. On the other hand,
same technique is tethered with molecular dynamics simulations analysis to get
knowledge about drug–target interactions. In order to gather the more justifiable
data with respect to drug–target interaction, X-ray spectroscopy is appended with
computational molecular modeling techniques. These computational approaches
retrieve the data from X-ray spectroscopy and elucidate it with more accuracy
regarding drug interactions and dynamics analysis [100].
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As researchers subjected many tiny ligands to huge macromolecules in X-ray crystal-
lographic analysis time to time for getting the precise atomic conformation. Also,
there are some limitations of same technique like sampling difficulties, radiations
hazard with data retrieval, and issues with elucidation of flexible and dynamic con-
formations of crystals. Along with such drawbacks, several advancements have been
achieved with time including coexistence of other analytical technique to accelerate
the potential in synergistic manner. X-ray crystallography is intended to provide the
more close-up view of 3D structure of protein as well as their keen interactions of li-
gand and amino acids present in protein [101, 102].
13.5.1 Recent advances in X-ray crystallography to develop
biologically active molecules
In this literature, design, synthesis, characterization, and in vitro antibacterial evaluation
of a Schiff base endowed imidazole scaffold (10, Figure 13.10) was described by Slassi
et al. [103]. Here, X-ray diffraction study was employed to get a crystal structure of syn-
thesized derivative 10. In addition, the structure of compound 10 was validated through
various structural as well as electronic parameters with the help of density functional
theory (DFT) along with time-dependent (TD-DFT) technique. Results of this analysis con-
firmed the effective relationship with crystal structure obtained from X-ray analysis. In
vitro assay observed compound 10 as topmost potent compound against S. aureus with
zone of inhibition diameter value of 20 mm (MIC value = 29 µg/mL) which was less effec-
tive as compared to standard antibacterial drug chloramphenicol (MIC= 11.65 µg/mL).
Moreover, compound 10 exerted the weak inhibition against Escherichia coli and Klebsi-
ella pneumoniae with zone of inhibition of 13 and 15 mm, respectively (MIC = 42 and
68 µg/mL toward E. coli and K. pneumoniae, respectively) [103].
Latha et al. [104] disclosed the synthesis, structural characterization, and antican-
cer potential of di(p-chlorobenzyl) (dibromo) (1,10-phenanthroline) tin (IV) complex
(11, Figure 13.10). After synthesis, complex 11 was introduced to FTIR, Raman spectros-
copy,
1
H NMR,
13
C NMR,
119
Sn NMR, DFT, and X-ray crystallography. In this, theoreti-
cally obtained data was correlated through DFT analysis which was performed with
B3LYP/LanL2DZ. X-ray crystallographic analysis was used to characterize the crystal-
lized 2D interactions via hydrogen bonding like C–H–Br (Sn–Br–H-phenyl) interaction
in complex. Atomic conformation was evaluated through XRD which described about
regular octahedral configuration of crystal structure of the complex 11. Finally, anti-
cancer evaluated indicated the identical anticancer effect of both complex 11 as well
as standard anticancer agent cisplatin toward U87 cells at the drug concentration of
15.6 g/mL [104].
By keeping in view already existing antileishmanial potential of reference antifun-
gal compound Ketoconazole, structural alteration was carried out at some extent to
limit its lesser water solubility. de Queiroz et al. [105] explored the design and synthe-
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sis of p-sulfonic acid-based complex of Ketoconazole (12, Figure 13.10) to improve the
respective antileishmanial effect. Initially, proposed ketoconazole complex was synthe-
sized successfully followed by their structural elucidation which was performed using
FTIR, solid-state
13
C NMR, TGA, DSC, and X-ray diffraction data. In this case, FTIR sug-
gested the functional groups that are present in entire structural skeleton while NMR
data represented atomic conformation of molecule where exact configuration was eval-
uated through XRD due to complicated chemical nature of complex 12.Further,phase
solubility diagram of complex 12 indicated good water solubility power at 120.00 μM
which was almost 90 –100 times more than that of ketoconazole alone. Moreover,
in vitro antileishmanial study highlighted complex 12 as most remarkable inhibitor of
both Leishmania amazonesis and Leishmania infantum having IC
50
values of 3.95 and
6.74 µM, respectively, which was against highly potent than ketoconazole alone (IC
50
=
14.35 and 17.47 µM toward L. amazonesis and L. infantum, respectively) [105].
13.6 Conclusions
Spectroscopy is proved as an indispensable tool to transfigure the drug discovery pro-
cess via pushing the interest of researchers into structures and fundamentals of mole-
cules, ligand–receptor interactions, molecular dynamics as well as ADMET analysis.
Spectroscopy is active since twentieth century and fixed in early stages of drug discov-
ery like target identification and validation, lead discovery followed by scrutiny of
their interactions together. Spectroscopic techniques become more advanced with
time and hence, indisputably endure itself as nondisplaceable technique in numerous
fields of pharmaceutical research. In this era, novel drug candidates are intended to
being developed time to time for combating the resistance against available drug regi-
men. In this context, different spectroscopic methodologies are technologically ad-
Figure 13.10: Novel bioactive molecules with structural characterization by XRD.
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vanced up to a significant level to generate best fit ligands to acquire the potent thera-
peutic response. By surpassing several limitations, spectroscopy transverses through
the ongoing research at a molecular level to advance the drug discovery process in-
cluding hyphenated spectroscopic techniques like GC-FTIR, HPTLC-FTIR, HPLC-SPE-
NMR, GC-MS, and LC-ESI-MS. These advanced spectroscopic techniques tackled all is-
sues related to sensitivity or resolution strength and aids the drug discovery process
in a more productive way.
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