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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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13.1.2 Defining drug–receptor interactions
Drug–receptor interactions are very important for exerting the biological activity to a
reasonable extent. By using the spectroscopic techniques, researchers may focus over
the drug–target binding pattern to considerate the possible mechanism of action asso-
ciated with their therapeutic potential after administration in body. Drug–receptor in-
teraction studies can be performed at molecular level to observe the binding pose of
drug within the binding domain of target through the incorporation of spectroscopy
[26]. Several spectroscopic techniques including fluorimetry and surface plasmon res-
onance spectroscopy avail the instantaneous analysis of binding pose and interaction
toward the key units of macromolecules available in its active site which is very ad-
vantageous to generate more potent molecules through SBDD. Hence, spectral data
witnesses the actual drug–receptor interactions indeed to display the promising phar-
macological effects [27].
13.1.3 High-throughput screening
High-throughpu t screening is an automated drug discovery technique which sieves
large number of compounds at the same time to recognize the best hits amongst the
screened compound libraries. In this process, millions of designed compounds can be
screened automatically to achieve their target site interactions in the form of biological
activity. In this stage of drug discovery, concurrent investigation of structural parame-
ters can be performed via spectroscopic techniques simultaneously. High-throughput
screening is a pipeline scrutiny analysis which assembled on the basis of IR and NMR
spectroscopy that identifies the possible hits to elicit the desired therapeutic action [28].
13.1.4 Structural characterization of lead molecules
Spectral data is primarily applicable to elucidate the structural composition of any
chemical compound which apply very decisive role in lead identification and optimi-
zation stage of drug discovery. Variou s spectroscopic methods like IR spectroscopy,
NMR spectroscopy, and MS afford the thorough understanding about structural char-
acteristics and 3D atomic arrangement in the parent structure [29]. Hence, scientists
working in research and development section generally incorporate the favorable
substitution over the biologically active already existing scaffold to improve the effi-
cacy. It is essential for them to corroborate the structure of lead molecules to do so in
order to attain the maximal potency with minimum adverse effects [29, 30].
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13.1.5 Quantitative analysis of drug metabolism
Metabolism is another key feature that always linked with biological potency of any
drug molecules. Researcher working in the field of drug discovery always focuses on
metabolic pathway of drug inside the body system. In this context, drug degrades in the
possible fragments on the metabolic site in the body to exhibit the desired pharmacologi-
cal action [31]. During the drug metabolism, parent drug as well as its fragments can be
easily analyzed through MS. MS supports to recognize drug fragments along with their
quantitative analysis. Further, pharmacokinetic investigation may favor through the sig-
nificant information related to quantitative estimation afforded by drug metabolites [32].
13.1.6 Monitoring drug delivery systems
Developing effective drug delivery systems is essential for ensuring targeted and con-
trolled drug release. Spectroscopy plays a significant role in monitoring and characteriz-
ing these delivery systems. Techniques like Raman spectroscopy and near-IR spectroscopy
(NIR) help researchers understand the structural changes in drug carriers and assess
drug release kinetics, leading to the development of more efficient delivery systems [33].
13.2 Role of IR spectroscopy in drug discovery
Spectral characterization of designed moleculesaswellastargetproteinisaveryessential
step in drug discovery. Target identification and validation through suitable spectroscopic
methodology is a must to initiate SBDD. IR spectroscopy is an analytical tool by which func-
tional groups present in entire structure can be determined with higher accuracy [34].
After initial purification through feasible chromatographic techniques, IR spectroscopic
data is the right next step to authenticate the presence of different functional groups or
substituents as a part of whole structure. However, IR spectroscopy bears some limitations
along with it including lesser resolution power and sensitivity by virtue of which impuri-
ties may be apparent as noise signals in the provided IR spectrum [34, 35]. To eliminate
such drawbacks, FTIR is available at present with elevated sensitivity and resolution
power for structural elucidation of different compounds. FTIR spectroscopy requires an ad-
ditional step to increase the resolution power in which radiation from source of light is
changed into time domain which is then consequently be converted into frequency domain
signals [36]. FTIR is a highly advanced analytical technique that provides the higher sensi-
tivity by which complicated structures of biomolecules along with their interactions toward
lead molecules can be corroborated in easy way. All the outcomes afforded through analyz-
ing IR spectrum are required to anticipate the chemical structure and ligand–protein com-
plex stability that aids to initial stages of drug discovery process. IR spectroscopy mainly
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affords qualitative estimation of given sample while somehow exerted both qualitative as
well as quantitative information in a quick, economical, and rapid manner [37, 38]. Also, IR
spectroscopy is an eco-friendly approach which generally requires rock salt or nujol
mull for sample preparation, and hence, nonhazardous chemicals are generally taken in
practice. By considering the feasibility of IR spectroscopy, it becomes a basic unit opera-
tion as a part of quality control department in various industries. Further, this spectros-
copy has a wide range of implications among which it is widely explored to determine
the adulterated content in various food, drug, and biological samples [38–40]. There are
several key points related to IR spectroscopy as deliberated below.
1. In IR spectroscopy, sample is irradiated with IR radiation, that is, coming from
source of light and then some amount of radiation is absorbed by samples depending
on concentration while remaining radiation transmitted toward detector to provide
the IR spectrum (intensity vs frequency) [41].
2. IR radiation excites the electrons of every functional group to transit from one vibra-
tional state to the other one. Each functional group from entire structural skeleton at-
tributes toward respective IR absorption at particular wavelengths [42].
3. Further, electromagnetic radiations ranging from 4,000 cm
−1
to 400 cm
−1
lie under IR
region. Among the entire region, 4,000 cm
−1
to 1,300 cm
−1
frequency region is denoted as
functionalgroupregioninwhicheveryfunctiongroupofunknowncompoundsexhibits
their respective peak as shown in Figure 13.4. Whereas frequency region ranged be-
tween 1,300 cm
−1
to 400 cm
−1
corresponds to the fingerprint region which represent
unique pattern for specific com pound and mainly display bands for halogens [43].
4. In addition, near IR region, that is, ranging from 14,000 cm
−1
to 4,000 cm
−1
appears to
be due to superimposition of multiple overtone frequencies, that is, higher energy levels.
Basically,mid-IRregion,thatis,4,000–1,300 cm
−1
, is most prominent for different func-
tional groups to display unique and intense peaks at specific wavenumbers [44].
Figure 13.4: IR spectrum with function group and fingerprint regions.
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5. Structural elucidation or idea regarding functional groups in any unknown chemi-
cal compound can be retrieved from specific shape, position as well as intensity of
bands in IR spectrum. In the provided IR spectra, band position indicates about differ-
ent functional groups that may be present in chemical structure of compounds while
band intensity corresponds to the concentration as per Beer-Lambert’s law [45].
6. In contrast to the simple structures, some compounds have somehow complex
structureinwhichmorethanonecompoundsmaybepresentoverthere.Insuch
cases, whole spectrum consisted of overlappe d bands and will be considered as fin-
gerprint for the provided sample and can be correlated in future perspectives by
means of adulterated content or impure at some extent or evaluated through chemo-
metrics tool [46].
7. IR spectroscopy is being exploited by researchers since twentieth century for struc-
tural elucidation, calibration, validation, and quality control or assurance by means
of authentication of adulteration in the given sample [47].
8. To limit several drawbacks associated with IR spectrum such as noise signals due to
absorption by some extent of water molecules (moisture) or impurities in given sam-
ple, FTIR comes in force with higher sensitivity and resolution [48].
9. Apart from FTIR alone, compounds can be subjected to hyphenated FTIR techniques
that furnish several advancements in the aspect of quality. Various hyphenated tech-
niques like LC-FTIR, GC-FTIR, and HPTLC-FTIR are used in the modern era to promote
the quality of drug development process [49].
13.2.1 Recent advances in IR spectroscopy to develop
biologically active molecules
Berhe et al. (2023) explored the synthesis and characterization of novel library of hy-
drazinyl pyrazole derivatives and evaluated them in vitro for antileishmanial poten-
tial against Leishmania aethiopica. Structural elucidation of all synthesized analogues
was performed through FTIR and
1
H NMR spectral data. FTIR spectrum for synthe-
sized compounds possessed higher sensitivity than simple IR to detect functional
groups present in chemical structures. FTIR data validated the structures through
characteristic bands at specific wavenumber like NH appears as single band ranged
between 3,300 cm
−1
and 3,000 cm
−1
while 3° amine elicits no band in the same region,
alkyl C–H stretches to about 2,950 cm
−1
, and C=C band appears to be about 1,650 cm
−1
.
Further, in vitro antileishmanial activity demonstrated compound 1 (Figure 13.5) as
most active inhibitor of L. aethiopica promastigotes amongst the series having IC
50
value of 0.018 µM, which was several folds higher than standard antileishmanial
drugs Miltefosine and amphotericin B deoxycholate (IC
50
= 3.130 and 0.047 µM, respec-
tively). Also, most potent analogue 1 showed no significant cytotoxic effec t on Vero
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cell lines. In addition, in vivo antiplasmodial study on synthesized compounds af-
forded 1 as most effective analogue in P. berghei mouse model with 90.4% suppression
of parasitaemia [50].
Design, synthesis, characterization, and antimicrobial evaluation of 1,4-disubstituted
1,2,3-triazole derivatives was performed by Kaushik and Pahwa [51]. Structural characteri-
zation of synthesized derivatives was performed using FTIR,
1
HNMR,
13
CNMR,andhigh-
resolution mass spectrometry (HRMS) spectral data. In this regard, FTIR data indicated
the presence of aliphatic ester via characteristic sharp band in 1,750–1,735 cm
−1
region to
authenticate the chemical structures of synthesized analogues. In contrast to ester, peaks
for other functional groups were analogues as described for compound 1. In the present
article, in vitro antimalarial activity revealed that compound 2 (Figure 13.5) exhibited the
most promising antimalarial activity against Plasmodium falciparum with MIC value of
0.178 μmol/mL as compared to reference drugs chloroquine (MIC = 0.0062 μmol/mL) and
quinine (MIC = 0.0826 μmol/mL). Additionally, compound 2 showed remarkable activity
against all tested bacterial strains with MIC values ranging from 0.0276 to 0.0153 μmol/
mL, which was more potent than ciprofloxacin (MIC = 0.0189 μmol/mL). Moreover, anti-
fungal activity revealed that compound 2 displayedmostpotentactivityagainstCandida
albicans and Aspergillus niger with MIC value of 0.0138 and 0.0139 μmol/mL that was
more potent than standard drug fluconazole (MIC = 0.0204 μmol/mL) [51].
Figure 13.5: Few bioactive analogues with FTIR structural elucidation.
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In this work, Illichachi et al. discussed the synthesis and antifungal evaluation of
novel series of chalcones and their dihydropyrazole carbaldehyde derivatives. At
first, all molecules were structurally elucidated with the help of FTIR, NMR, and
mass spectral data. All peaks in FTIR spectrum were identical as that of compounds
1 and 2,exceptingα ,β-unsaturated carbonyl (C=O) group that appeared as character-
istic band near about 1,690 cm
−1
. All analogues were evaluated in vitro for their an-
tifungal potential toward a panel of eight funga l strains that belong to Candida,
Aspergillus,andTrichophyton species. It wes observed that compound 3 (Figure 13.5)
was found to be the most considerable antifungal compounds amongst the series
with MIC value of 31.25 µg/mL, as that of standard antifungal agents amphotericin B
(MIC = 0.07–12 µg/mL) and te rbinafine (MIC = 0.02–0.05 µg /mL). In addition, only
compound 3 possessed potent inhibition toward Cryptococcus neoformans having
MIC value of 62.5 µg/mL, as that of st andard antifungal agents amphotericin B (MIC
= 0.78 µg/mL) and terbinafine (MIC = 0. 50 µg/mL) [52].
13.3 Role of NMR spectroscopy in drug discovery
NMR appears as much dominant as well as well-explored drug discovery tool by re-
searchers. In the complicated journey of drug discovery, lead candidates can be iden-
tified or validated thoroughly by using NMR spectroscopy which is a nonprotruding
and effective analytical technique. NMR spectroscopy is an essential analytical meth-
odology indeed for corroborating the molecular structure, their target site interac-
tions as well as structure–activity relationship profile [53]. NMR spectroscopy has a
wide range of entanglement with various stages of drug discovery process such as tar-
get selection, lead identifi cation and optimization, and target validation. Apart from
these all stages, NMR spectroscopy exhibited keen association toward the FBDD ap-
proach. Also, recent developments in NMR methodology may handout the fruitful im-
plications in order to expedite the hidden drug discovery outcomes [53, 54]. Further,
NMR spectral data authorize the researcher for having much more insight toward the
drug–target interactions. In addition, NMR spectroscopy is devoid of several limita-
tions that are associated with other instrumental analytical techniques used for mo-
lecular characterization like IR and NIR. Hence, NMR spectroscopy acquires the
several significances throughout the entire drug design and development process
over many other advanced instrumental analytical techniques. Spectral data obtained
from NMR spectroscopy enables the scientists to characterize the structure of most
favorable lead candidates amongst the hit series along with another relevant data in-
cluding target site interactions and molecular dynamics characteristics [55, 56]. There
are many significances of NMR spectroscopy in diverse fields of drug discovery as
given below:
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1. NMR spectroscopy has vital role in the initial stages of drug discovery like hit
screening and lead identification process. In this stage, NMR spectroscopy exerts the
distinct attention toward the ligand as well as macromolecule-based hit screening
methodologies. In the case of ligand-based drug design, previously reported potent
molecules are generally taken into consideration to design the hit series. Whereas
structural features of binding pocket of protein are encountered in the case of SBDD
approach. NMR spectroscopy transverses the key interactions toward the active site
of receptor, fragment-based screening and to explore the best fit molecules possessing
prominent physicochemical data [56, 57].
2. A deep insight toward the structural biology of hits may be well experienced with
the subjection of NMR spectroscopy. In this regard, NMR data sanctions the interpre-
tation of 3D models regarding protein-ligand complexes. Structural features of these
complexes make an informative turn over the best binding modes which ultimately
help the researchers to unveil the most favorable drug–target interactions. There are
many advanced NMR-based analytical techniques that emerged their best aptness for
structural characterization such as STD-NMR, transferred nuclear Overhauser effect,
and many more including chemical shift perturbation studies [58]. By utilizing the
NMR spectroscopy, structural attributes as well as binding interactions can be authen-
ticated by evaluating the different chemical shifts pattern, nuclear Overhauser effect,
and relaxation interval data. Moreover, target site interaction can be encouraged
through supplementary information including molecular dynamics studies that sug-
gests about sturdiness of drug–receptor complexes [58, 59].
3. In order to achieve the maximal binding affinity, FBDD is another useful in silico
tool to design the novel molecules based on the most suitable fragments having best
interactions within the active site of receptor. In this context, FBDD mainly empha-
sizes over the identification and optimization of fragments that enable the develop-
ment of most promising lead candidates. All the fragments are hereby acting like
structural units that tethered to being mature in the form of potent molecule [60].
Now, these potent molecules are already rationalized on behalf of fragments’ interac-
tions toward the target site. NMR-based fragment screening is profound to have sys-
temic master plan to identify and validate the tiny fragments that interact most
smoothly toward the amino acids present in active site of receptor. Further, lead mol-
ecule can be designed with the coexistence of these fragments together which is sup-
posed to present synergistic or higher binding affinity [61].
4. Drug discovery stay very energized with the introduction of ligand-based NMR. In the
ligand-based drug design approach, already appreciated ligand will be chosen with re-
spective macromolecule and then suitable modification may be overlaid onto the core
scaffold to develop a new potent compound. A ligand that is previously reported as po-
tent one will be selected to rationalize the design and development of novel hit series.
These hits are now proposed to have reasonable binding toward the target site as it
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owned the structural features of existing potent compounds [62]. Ligand-based NMR is
utilized to expedite the structural characteristics through structure–activity relationship
data as well as interactions throughout the binding assays. NMR can also contribute to-
ward lead optimization that unveils the prominent interactions within the binding cav-
ity. Further, this structural info rmation for selected molecules suggests researchers
about the necessary modifications in specific part of entire structural skeleton to build
up most potent molecule [63].
5. In contrast to the ligand or target-based drug discovery, NMR also reveals the rele-
vant biological information associated with the structural characteristics of designed
molecules. NMR triumph over the limitations is associated with the ADMET profile of
the designed molecules that later tends to discard the same. In this context, NMR evi-
denced the significant contribution into absorption, distribution, metabolism, excre-
tion, and toxicity studies. Also, NMR reveals the possible metabolites that manifest
during the metabolic pathways that selected molecules must surpass prior to excrete
out from system [64]. The structural information throughout the metabolic pathway
may hint the researchers about possible interactions as well as toxicity issues at early
stage of drug discovery that may be resulted later on. Metabolomics studies endorse
the drug metabolism pathway as well as toxicity profile for tested compounds. NMR
spectroscopy avails the identification and quantification of feasible metabolites and
hence intuited the molecule that has undergone ADMET analysis [65, 66].
6. Target-specific drug discovery may also investigate in more reproductive and imme-
diate manner through the NMR spectroscopy. Three-dimensional structure of macro-
molecule or protein can be evaluated easily by using NMR spectroscopic data. Although
X-ray crystallographic methodology is well adopted for crystalline structures of pro-
teins, solution NMR spectroscopy enables the structural characterization of samples
which are not acceptable to crystallize. It can comfortably corroborate the protein flexi-
bility and thermodynamics data. On the other hand, it suggests researchers about con-
formational alterations in receptor prevailed by binding of lead molecule [66 ]. By
considering the NMR data, allosteric interaction inside the macromolecules can also
easily corroborated which affords the idea regarding the preferable binding pocket for
selected ligand. Additionally, NMR spectroscopy reveals the molecular dynamics studies
for the same compound to disclose the bonding strength toward the key amino acid
residues present in the active site. These features allow the researchers to demonstrate
the allosteric binding mode for particular compounds which ultimately help to promote
the target-specific drug delivery. The compounds possessing target-specific conforma-
tions elicit the negligible or no adverse effects along with maximal desired pharmaco-
logical response [65–67].
7. NMR may also exercise for target selection and optimization stage of drug discov-
ery. Structural proteomics leads to validate the structure of macromolecule, that is,
receptor to explore the amino acids present over there. Electronic behavior like elec-
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tron attracting as well as releasing nature of amino acid residues present in macro-
molecule suggests that the structural features must be included in designed molecule
to present the remarkable biological response. Target-based drug design contributes
toward the generation of large number of lead candidates in minimum timelines and
hence may bring into play noteworthiness in pandemic-like consequences [68].
8. Spectroscopy tends to explore the structural characterization point of view while
its potential is now promoted in synergistic manner by incorporating advancements
through hyphenated techniques. Structural purification is one of the major challenges
which are being tackled at present by researchers through subjection of sample in
NMR-based hyphenated analytical techniques including LC-NMR/LC-CD-NMR and
HPLC-NMR/HPLC-SPE-NMR. Further, potential of NMR spectroscopy in drug discovery
may encourage the association of other biophysical techniques like MS, X-ray crystal-
lography, and cryo-electron microscopy. These advancements in spectroscopic techni-
ques through intermixing the advantages of more than one analytical technique lead
to give a new hope in the drug discovery [69, 70].
9. At present, NMR is now advanced as high-throughput NMR which corroborates the
structural information of a number of molecules in alignment with the protein at a
single time. This automation offered advanced analytical tool that explore the most
potent molecules in limited time and with less effort. Moreover, X-ray crystallization
is not possible for membrane proteins by which solution NMR spectroscopy intense to
characteri ze its structural features and finally plays a vital role in the discovery of
new therapeutic targets [71].
13.3.1 Recent advances in NMR spectroscopy to develop
biologically active molecules
Although there are several disputes that generally experienced while dealing with
NMR spectroscopy in drug discovery including sample preparation and sensitivity is-
sues, it affords the most legitimate discernment regarding the prominence in struc-
tural confirmation during early stages of drug discovery. NMR spectroscopy becomes
a strategist tool for researchers to develop the lead molecules with minimal toxicities
in lesser time.
Amado et al. [72] reported the synthesis and characterization of a series of novel
endoperoxide substituted pyrazole analogs. All of the synthesized analogs were struc-
turally characterized by using X-ray crystallography, 1D NMR as well as 2D NMR tech-
niques viz. COSY, HSQC, and HMBC. Further, in vitro antileishmanial study resulted in
compound 4 as most significant inhibitor of Leishmania tropica and Leishmania infan-
tum bearing IC
50
values of 161 ± 19 µM and 219 ± 44 µM, respectively. Also, 1D
1
H and
13
C NMR spectrum for compound 4 has presented the characteristic peaks as shown in
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Figure 13.6. Moreover, same compound, that is, 4, exhibited most appreciable cyto-
toxic effect against monocytic THP-1 cells with CC
50
value of 202 ± 116 µM [72].
In this chapter, a library of new pyrazole carbaldehydes from galloyl hydrazide
were synthesized and evaluated for antibacterial potential by Nashaan and Al-Ra wi
[73]. All the proposed pyrazole carbaldehydes were successfully generated with the
help of Vilsmeier-Haack reaction followed by their structural confirmation by consid-
ering IR,
1
HNMR,
13
C NMR, and mass spectrum. Furthermore, in vitro antibacterial
study suggested compound 5 as the best inhibitor of Staphylococcus aureus amongst
the series with zone of inhibition value of 28 mm. The structure of the most active
analogue 5 was corroborated through characteristics peaks in NMR spectra at diverse
δ values (ppm) as mentioned in Figure 13.7. In vitro observation concluded that com-
pound 5 is superior antibacterial potency than that of standard antibacterial agent
Ampicillin toward S. aureus [73].
Figure 13.6:
1
H NMR and
13
C NMR spectrum of compound 4.
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