Creatinine recognition using designed synthetic receptors

Article abstract of DOI:10.1002/poc.4066

A series of neutral nonenzymatic receptors have been synthesized for the recognition of creatinine in a nondegrative way. The receptors contain different heterocyclic moieties for better interactions between host and guest. Among these, 1, 4, and 5 are fluorescent receptors for creatinine. From this study, it was found that the receptors 1 and 4 containing the naphthyridine moiety have higher binding affinity to the guest creatinine than receptors containing other heterocyclic moiety. Theoretical studies for the calculation of binding energy were carried out using discrete Fourier transform (DFT) for the hosts and their complexation with creatinine in both gas phase and acetonitrile medium.

Full text of DOI:10.1002/poc.4066

Received: 6 November 2018
DOI: 10.1002/poc.4066
Revised: 8 February 2020
Accepted: 12 February 2020
R E S E A R C H A R T I C L E
Creatinine recognition using designed synthetic receptors
Subrata Jana1
| Sunita Prajapati1 | Kishor Kumar Suryavanshi1
|
Shyamaprosad Goswami2 | Rakesh Parida3 | Santanab Giri4
1Department of Chemistry, Indira Gandhi
Abstract
National Tribal University (Central
University), Amarkantak, Madhya
Pradesh, India
2Department of Chemistry, Indian
Institute of Engineering Science and
Technology, Shibpur, Howrah, West
Bengal, India
3Department of Chemistry, National
Institute of Technology, Rourkela, Odisha,
India
4School of Applied Sciences and
Humanities, Haldia Institute of
Technology, Haldia, West Bengal, India
A series of neutral nonenzymatic receptors have been synthesized for the rec-
ognition of creatinine in a nondegrative way. The receptors contain different
heterocyclic moieties for better interactions between host and guest. Among
these, 1, 4, and 5 are fluorescent receptors for creatinine. From this study, it
was found that the receptors 1 and 4 containing the naphthyridine moiety
have higher binding affinity to the guest creatinine than receptors containing
other heterocyclic moiety. Theoretical studies for the calculation of binding
energy were carried out using discrete Fourier transform (DFT) for the hosts
and their complexation with creatinine in both gas phase and acetonitrile
medium.
Correspondence
K E Y W O R D S
Subrata Jana, Department of Chemistry,
Indira Gandhi National Tribal University
(Central University), Amarkantak,
Madhya Pradesh 484887, India.
Email: jana.s.oc@gmail.com
creatinine, DFT, fluorescence sensor, host-guest, molecular recognition
Funding information
Madhya Pradesh Council of Science and
Technology (MPCST), Bhopal,
Government of Madhya Pradesh, India,
Grant/Award Number: Endt. No
186/CST/R&D/Phy&Engg/2018-19
1 | INTRODUCTION
for creatinine, the enzymatic methods are more
expensive.[5]
Creatinine is a blood metabolite of considerable impor-
tance in clinical chemistry,[1] particularly as indicator of
renal function.[2] It is proved that determination of creati-
nine is more valuable for the detection of renal dysfunc-
tion than that of urea.[3] Generally, about 1.6% of the
body's content of creatine spontaneously breaks down
daily to form creatinine.[4] In medical practice, plasma
and urinary creatinine levels are used on a day-to-day
basis for the detection of renal diseases. Routine clinical
analysis of creatinine in blood is currently conducted
calorimetrically by means of the Jaffe reaction or by
enzymatic method. While the Jaffe reaction is not specific
Its exceptional importance inspired different research
groups to be actively engaged in clinical as well as diag-
nostic research. Hence, recognition of creatinine and its
analytical applications plays a major role in biomedical
research particularly related to renal function.[6] Differ-
ent synthetic receptors have been synthesized to study
the recognition process of creatinine. For instance, a
calix[4]pyrrole with a monophosphonate bridge receptor
has been reported in the recent past, which acts as an
excellent ionophore for the quantification of creatinine
levels in urine or plasma.[7] Another fluorescent chemo-
sensor has been reported that allows detection of
J Phys Org Chem. 2020;e4066.
wileyonlinelibrary.com/journal/poc
© 2020 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/poc.4066

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JANA ET AL.
creatinine by visual inspection, where a dinitrophenolic
moiety is responsible for the colorimetric changes.[8a]
Similarly, a Pd2+-coordinated naphthalimide works as an
efficient fluorescent probe for creatinine in aqueous
buffer at pH 7.2.[8b] Beside these types of chemical sen-
sors, some groups designed electrochemical sensors for
the detection of creatinine.[9] Recently, L-cysteine–
stabilized copper nanoparticles and calix[4]arene-
attached gold nanoparticle-based colorimetric sensors for
creatinine were also reported.[10] Molecularly imprinted
polymer (MIP) containing nickel nanoclusters is also
promising electrochemiluminescence (ECL) emitters
with very high detection limit for creatinine.[11]
We have previously reported on the recognition of dif-
ferent biologically important substrates by designed syn-
thetic receptors.[12] Here, we attempt to recognize
creatinine by neutral synthetic receptors in a non-
degrative protocol. Accordingly, a series of receptors were
designed, synthesized, and tested for the recognition of
creatinine in organic medium. Creatinine has three dif-
ferent tautomeric forms (Scheme 1) as A/B, C, and D,
and among all these forms, C is the energetically more
favorable tautomeric form.[7,13] At comparatively neutral
environment, it can exist in either A/B or C as reported
in literature.[7] We also have studied the donor-acceptor
arrangement of creatinine and its interaction and supra-
molecular behavior with simple monocarboxylic acids in
solid phase, where ionic complexes of the protonated tau-
tomeric form C are involved instead of molecular com-
plexes as in MeOH–CHCl3.[14] Hence, interaction of
synthetic neutral host and creatinine in form D may be
ruled out in nonaqueous environment.
the other is isophathaloyl-naphthyridine amide N─H)
should make two hydrogen bonds with the lactam oxo
group of creatinine. The two naphthyridine ring “N”
atoms will accept two hydrogen bonds from the lactam
and imido N─H groups. Receptor 1 binds creatinine in its
tautomeric form B. Similarly, receptor 5 was designed
and synthesized where ring “N” atoms of both the pyri-
dine and quinoline moiety act as hydrogen bond accep-
tors, whereas two amide protons act as hydrogen bond
donors to the lactam moiety of creatinine. Other recep-
tors may bind creatinine in form A or form B through
three- or four-point interactions, as these are more flexi-
ble compared with the receptor 1. Receptors 2 and 3 con-
tain pyridine and imidazole moieties with the pyridine
2-amide moiety acting as acceptor to the exo imido N─H.
Among the receptors 2 and 3, one extra acceptor unit in
receptor 2 is more flexible and thus stays away from the
pyridine 2-amide moiety compared with the receptor 3,
which was also reflected by the lower binding constants
for receptor 2 than receptor 3. Receptors 1, 4, and 5 may
be used as potential fluorescence sensors as they contain
a fluorophore in their backbone. The receptors are shown
below (Scheme 2).
2 | SYNTHESIS
The receptors were synthesized according to the follow-
ing schemes starting from commercially available
compounds and fully characterized for binding studies
(SI). Receptor 1 was synthesized under high-dilution
conditions from 2-methyl-7-amino-1,8-naphthyridine and
nbutyl amine (Scheme 3). Intermediate naphthyridine
was prepared according to the reported procedure.[15a]
The synthetic receptors were designed with the bind-
ing sites of donors and acceptors being properly arranged
to recognize creatinine molecule efficiently. In receptor 1,
the design is based on the concept that two hydrogen
bond donors (one is isophthaloyl-butyramide N─H, and
Receptors
2
and
3
were prepared using
2-pivaloylamino-6-bromomethyl pyridine (Scheme 4);
2-Pivaloylamino-6-bromomethyl pyridine has been
(A)
SCHEME 1 (A) Tautomeric forms of creatinine (A or
B, C, and D) and (B) optimized structure of tautomers
(B)

JANA ET AL.
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SCHEME 2 Designed receptors (1-5) for the
complexation of creatinine
6-bromomethyl pyridine with imidazole under micro-
wave irradiations. Receptor 4 was synthesized by simple
amidation of the acid chloride of pyrene-1-carboxylic
acid[16] and 2-amino-7-methyl-1,8-naphthyridine.
Receptor 5 was synthesized following a similar proce-
dure as that for receptor 1 using high-dilution conditions
(Scheme 5) from the intermediate isolated by hydrolysis
of compound 6. Compound 6 was prepared by the cou-
pling of intermediate 2-pivaloylamino-6-bromomethyl
pyridine and 8-hydroxyquinoline.
SCHEME 3 Reagents and conditions: Et3N, dry CH2Cl2, high
dilution, 30 hours
3 | THEORETICAL
CALCULATIONS
The binding energy of the host-guest complexation was
calculated using discrete Fourier transform (DFT)[17] at
the B3LYP/6-31G(d,p)[18] level of theory in both gas
phase and acetonitrile as solvent (see Supporting Infor-
mation). Objective of this study is identification of prom-
ising receptors, which would preferably bind with which
tautomer among the possible different tautomers of the
guest creatinine (Scheme 1).[19] Here, binding energy of
the receptors towards both anti and syn orientation of
SCHEME 4 Reagents and conditions: (A) NaH, THF, r.t.,
12 hours; (B) MW, 5 minutes, 450 W; (C) (a) oxalyl dichloride,
DMF, dry CH2Cl2, r.t., 3 hours; (b) 2-amino-
7-methylnaphthyridine, Et3N, dry CH2Cl2, high dilution, r.t.,
20 hours
synthesized using reported procedure.[15b] For receptor 2,
another pyridine moiety was attached by sodium
hydride–mediated
coupling
of
2-hydroxymethyl-
6-methylpyridine with 2-pivaloylamino-6-bromomethyl
pyridine to generate the ether linkage, which makes the
receptor more flexible. In contrast, in receptor 3, one
imidazole moiety was directly attached to pivaloylamino
pyridine by direct coupling of 2-pivaloylamino-
SCHEME 5 Reagents and conditions: (A) 8-hydroxyqinoline,
K2CO3, TBAB, dry acetone, r.t., 2 days; (B) (a) 4M KOH solution
and EtOH (1:1), reflux, 6 hours; (b) isophthaloyldichloride,
nBuNH2, Et3N, dry CH2Cl2, r.t., high dilution, 36 hours

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JANA ET AL.
lactam N─H and imido N─H was calculated along with
other tautomers in gas phase as well as considering aceto-
nitrile as solvent (Table 1). Binding energy of the recep-
tors is further lowered by the complexation with
creatinine in its different forms. All the receptors prefera-
bly bind with guest creatinine in its form B except recep-
tors 1 and 2 in gas phase. But in case of acetonitrile as
medium during calculation, all the receptors except 2 and
3 bind preferably with creatinine in form B. The trends of
the binding energy show that receptor 1 binds with the
tautomeric form C of creatinine with maximum effi-
ciency as reveled by lowest energy (1C: −24.84 kcal/mol)
in comparison with other tautomeric forms. Whereas in
case of receptor 1, the binding energies with the tauto-
meric forms B and C are very close in gas phase, the
binding energy with form B of creatinine in acetonitrile
is higher than all other forms. Receptor 1 binds strongly
with A, B, and C of creatinine as compared with all other
receptors in both gas phase and acetonitrile due to the
four-point interaction. Similarly, receptors 4 and 5 also
bind form B of creatinine with highest efficiency in both
phases. In case of receptors 2, 3, and 5, ring “N” atoms of
imidazole, pyridine, and quinoline moieties, respectively,
represent hydrogen bond acceptor sites for the guest cre-
atinine, separated by either ether linkage or methylene
moiety from the core binding region of pyridine amide
and isothaloyl diamide moiety. The highest binding
energy (3B: −17.65 kcal/mol) was observed for receptor
3 in the gas phase, whereas in acetonitrile, this receptor
prefers to bind form D of creatinine due to polarity of the
solvent. Receptor 4 binds more strongly with form B in
gas phase (−18.16 kcal/mol) than in acetonitrile
(−12.17 kcal/mol). This is due to the perfect orientation
of the hydrogen bonding array between host and guest.
The binding energy of receptor 5 with tautomeric form
B (5B: −19.99 kcal/mol) is higher in comparison with all
the tautomeric forms in gas phase, whereas it is
−12.70 kcal/mol in case of acetonitrile. The binding
behavior of receptor 2 is completely different with respect
to all the other receptors. It prefers to bind the tautomeric
form D in both gas and acetonitrile medium, possibly
due to basic nature of the alkyl pyridine moiety of the
receptor. The binding energy with the tautomeric form
D (2D: −14.55 kcal/mol) is higher in the gas phase than
in acetonitrile (−11.12 kcal/mol). The optimized struc-
ture of receptors and their mode of binding with guest
creatinine in different forms in gas phase are as follows
(Figure 1). All the theoretical calculations are carried out
by G09[20] software.
4 | BINDING STUDIES
4.1 | UV-Vis studies
The binding behavior of the receptors with creatinine
was studied by means of UV-Vis titrations. The titration
experiments were carried out with known solutions of
receptors 1, 2, 3, 4 (1 × 10−6M), and 5 (1 × 10−5M) in
CH3CN. The solution of guest creatinine was also pre-
pared in either approximately 1 × 10−4M or 1 × 10−5
M
order in CH3CN. Each titration was performed using
2 mL of the stock solution of receptors and the solution
of guest substrates by judicious choice
(see Supporting Information). Receptor 1 showed a
strong absorption at approximately 335 nm, which gradu-
ally decreased upon addition of the guest solution during
titration. The other absorption peak appeared at
TABLE 1 Binding energya (kcal/mol) of the complexes in gas phase and in acetonitrile between receptors and creatinine in different
forms
Receptors
Tautomeric Forms of Creatinine
1
2
3
4
5
Binding energy of the complexes in gas phase
A
−21.23
−24.16
−24.84
−14.07
−12.99
−12.58
−14.07
−14.55
−11.35
−17.65
−17.02
−14.35
−15.44
−18.16
−6.02
−17.14
−19.99
−17.64
−14.67
B
C
D
−15.00
Binding energy of the complexes in acetonitrile
A
B
C
D
−14.23
−16.65
−15.60
−10.86
−8.91
−7.98
−5.86
−10.78
−7.72
−10.48
−12.70
−5.38
−12.57
−13.67
−12.34
−10.82
−9.34
−11.12
−10.94
−11.24
aBinding energy (EComplex – [EHost + EGuest]).

JANA ET AL.
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FIGURE 1 Calculated energy minimized structure in gas phase of receptors and their different mode of complexation with tautomeric
forms A/B, C, and D of creatinine using B3LYP/6-31+G(d,p) method
approximately 321 nm with reduced intensity. Receptor
2 showed absorption at approximately 273 nm, which
also gradually decreased upon addition of guest solution.
The presence of the imidazole instead of a pyridine moi-
ety slightly changed the absorption spectra of receptor 3,
where strong absorption was observed at approximately
279 nm. In this case, the spectral change upon addition
of creatinine solution was more prominent with respect
to the other two receptors. The absorption spectra of
receptor 4 contained two humps (λmax = 341, 276 nm) of
very close intensity. In this case, decrease of intensity at
341 nm was more prominent than at 276 nm during titra-
tion with creatinine. The absorption spectra of the recep-
tor 5 (λmax = ~287 nm) also gradually decreased upon
addition of creatinine solution.
The binding constant values were calculated using
the data of UV-Vis titration experiments (Figure 2A).[21]
The binding constant values depend on the donor-
acceptor array of the receptors. The receptors 1 and
4 show higher association constant values due to the
presence of the naphthyridine moiety in which two ring
“N” atoms act as hydrogen bond acceptors. The other

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JANA ET AL.
FIGURE 2 (A) Binding constant
calculation curves and (B) JOB plots of
the receptors with creatinine in CH3CN
by UV-Vis method
two receptors 2 and 3 bind weakly with guest creatinine.
The association constant of receptor 3 with creatinine is
higher due to the presence of two ring “N” atoms in close
proximity compared with receptor 2. Receptor 5 shows a
lower association constant with respect to receptors con-
taining naphthyridine moiety (1 and 4) due to the higher
flexibility of the quinoline moiety.
The stoichiometry of the host-guest interaction has
been derived from JOB plots of the receptors with creati-
nine in CH3CN (Figure 2B). A 1:1 stoichiometry in case
of the receptors 1, 2, 3, and 5 was observed, whereas in
case of receptor 4, it slightly deviated due to the dynamic
nature of complex.
the binding behavior.[22] The receptor 5 was excited at
287 nm during binding studies, and the emission inten-
sity was decreased (λmax = ~393 nm) regularly upon addi-
tion of increasing amount of guest solution (SI).
All the receptors (1, 4, and 5) behave as fluorescent
probes towards creatinine.[23] The emission intensity in
all the cases was quenched during complexation. The
receptors 1 and 4 differ slightly probably due to the
strength of the host-guest interaction as well as the
change of order of the concentration of the receptors dur-
ing titration experiment. The higher extent of quenching
of receptor 1 during titration also indicates the higher
interaction with creatinine. But in the case of receptor 5,
the extent of quenching is higher than that of the other
receptors due to the presence of different fluorophore as
well as different mode of interaction with creatinine.
The binding constants (Figure 3) were determined by
the fluorescence titration method.[24] The association
constants of the receptors 1 and 4 having naphthyridine
moiety as binding motif are higher with respect to those
of receptors with the quinoline moiety. The association
4.2 | Fluorescence studies
The binding behavior of the receptors 1, 4, and 5 was also
studied by the fluorescence titration method at the same
concentration as for the UV-Vis titration except for recep-
tor 4. The solution of receptor 4 was further diluted
10 times for the measurement of emission intensity due
to the presence of strong fluorescent pyrene moiety.
Receptors 1 and 4 showed emission due to the presence
of naphthyridine moiety, which acted as a binding motif
for creatinine as well as a fluorophore to study the photo-
physical behavior.[15] The emission spectra of receptor
1 showed one peak at approximately 402 nm, when
excited at 335 nm. Upon addition of creatinine, the emis-
sion intensity was gradually decreased due to the forma-
tion of host-guest complex. But the emission spectra of
receptor 4 are different due to the presence of both naph-
thyridine and pyrene moieties. In this case, the emission
spectra consisted of two bands (λmax = 392, 409 nm)
when excited at 341 nm. Between these two bands, the
peak at the higher wavelength is of higher intensity. The
receptor 5 contains one quinoline moiety attached
through an ether linkage, which acts as both acceptor to
the imido N─H of creatinine and fluorophore to study
FIGURE 3 Binding constant calculation curves of the
receptors 1, 4, and 5 with creatinine in CH3CN by fluorescence
titration method

JANA ET AL.
7 of 11
TABLE 3 Association constants (Ka [M−1]) for receptors (1
and 5) determined by UV-Vis method under different water
constants determined by fluorescence method are almost
double with respect to the association constants deter-
mined by UV-Vis method in case of the receptor 5.
concentration in acetonitrile
15% H2O in
Receptors CH3CN
10% H2O in
CH3CN
5% H2O in
CH3CN
5 | DISCUSSION
1
5
1.25 × 104
2.39 × 104
3.87 × 104
1.14 × 104
1.131 × 104
ND
The association constant values determined by UV-Vis
and fluorescence methods are summarized (Table 2). All
the receptors are of mainly three types. Receptors 1 and
4 are of the same type where naphthyridine amide moi-
ety is the basic binding motif and this type of receptors
binds better compared with other type of receptors due to
matching of the donor-acceptor array between receptors
and creatinine of form B. The association constant value
in case of receptor 1 is higher than receptor 4 in both the
methods due to the presence of one extra donor site. This
may be attributed by the arrangement of two donor
amide protons directed inward to the cavity of the recep-
tor 1, which enhance the binding efficiency to the guest.
The other types of receptors contain one pyridine unit
(receptor 2) and one imidazole unit (receptor 3) as extra
hydrogen acceptor motifs, which are attached with the
same pyridine 2-amide moiety. In this case, receptor
2 shows higher association constant than receptor 3 due
to flexible ether linkage by which the pyridine ring “N”
interacts with the guest more predominantly, whereas
the imidazole moiety is oriented away from the pyridine
amide binding moiety due to repulsive forces. The recep-
tor 5 contains a quinoline moiety linked to the pyridine
amide unit through an ether linkage. The association
Abbreviation: ND, not determined.
constant of this receptor is lower as that of the receptors
1 and 4 but comparable with that of receptors 2, 3, and 5.
This is probably due to the distance among the two
hydrogen bond acceptors ring “N,” which is higher and
also due to the free movement of the quinoline moiety in
solution phase.
As creatinine is an important renal function indicator
and usually determined from urine samples, hence, rec-
ognition behavior of the receptors in aqueous environ-
ment has been tested. For this purpose, receptors 1 and
5 were used that have shown solubility up to 15% and
10% H2O in acetonitrile, respectively. The association
constant for receptor 1 is maximum at 10% H2O in aceto-
nitrile, which is even twofold higher than the value in
pure acetonitrile (Tables 2 and 3). In all other cases, asso-
ciation constants are almost close to each other. An inter-
esting feature for receptor 5 is that the association
constant is enhanced by approximately 10-fold in the 10%
H2O in acetonitrile solvent system in comparison with
pure acetonitrile. For receptor 5, the association constant
in the acetonitrile with more than 10% H2O was not
determined due to insolubility. Overall, receptor 1 showed
slight enhancement in binding effect in water-acetonitrile
system in comparison with the pure acetonitrile solvent,
whereas receptor 5 showed prominent enhancement in
water-acetonitrile system over pure acetonitrile system.
TABLE 2 Association constants (Ka [M−1])a and the free
energy changes (ΔG [kcal/mol]) at 25ꢀC for receptors determined
by UV-Vis (1-5) and fluorescence titration methods (1, 4, and 5)
Methods
UV-Vis Methodb
Fluorescence Methodc
Receptors
Ka
ΔG
Ka
ΔG
−6.03
-
6 | CONCLUSION
1
2
3
4
5
1.47 × 104
3.79 × 103
1.32 × 103
1.24 × 104
3.03 × 103
−5.75
−4.32
−5.36
−6.62
−5.10
2.14 × 104
-
In this work, a series of different receptors including fluo-
rescent ones were designed and synthesized, and their
binding behavior with creatinine was studied. All the
experimental binding results were compared with theo-
retical calculation values. In the receptors, one pyridine
amide moiety is fixed to bind the lactam moiety of creati-
nine, whereas the other acceptor unit of the receptors
was carefully changed for better binding. The receptors
contain fused pyridines (1 and 4), isolated pyridine (2),
imidazole (3), and quinoline (5) with the basic pyridine
amide moiety. Among all the receptors, 1 and 5 contain
one extra hydrogen bond donor site to enhance the
-
-
1.74 × 104
1.66 × 103
−6.92
−5.54
aAll the errors are 15%.
bFor receptor 1: λmax = 335 nm; receptor 2: λmax = 273 nm; receptor 3:
λmax = 279 nm; receptor 4: λmax = 341 nm; receptor 5: λmax = 287 nm.
cFor receptor 1: λmax (ex) = 335 nm, λmax (em) = 402 nm, emission slit
width = 5.5 nm, excitation slit width = 8.0 nm. For receptor 4: λmax
(ex) = 341 nm, λmax (em) = 409 nm, emission slit width = 2.7 nm, excitation
slit width = 14.0 nm. For receptor 5: λmax (ex) = 287 nm, λmax
(em) = 393 nm, emission slit width = 4.0 nm, excitation slit
width = 10.0 nm. Scan rate is 500 nm/min in all cases.

8 of 11
JANA ET AL.
interaction between receptors and creatinine. From the
detailed study of the recognition process of creatinine by
different methods, it is found that receptors containing
naphthyridine moiety have a higher tendency to interact
with the guest creatinine. Another important aspect from
this study is that creatinine molecule binds better in
water-acetonitrile than in pure acetonitrile. Receptors
1 and 5 are not able to recognize creatinine at higher con-
centration of water than 15% in acetonitrile due to solu-
bility problems. Besides, the binding behavior of the
receptors with different forms of creatinine has been
studied theoretically using DFT at the B3LYP/6-31G(d,p)
level of theory in both gas phase and acetonitrile as sol-
vent. The overall trend remains the same with higher
binding in the gas phase than in acetonitrile, but no
explicit solvent molecules were used in this study. From
the trend of the calculated data, the experimental results
can be justified by the nature of binding.
ΔI vs [G]/[H] (ΔI is change of absorbance; [G] and
[H] are the concentration of creatinine and receptors,
respectively). Association constants were calculated by
plotting 1/ΔI vs 1/[G].[21,24]
7.3 | General procedure for fluorescence
titration
The same stock solutions of receptors and guest creati-
nine in CH3CN were used for fluorescence titration
experiment. Then, the guest solution is added to the
receptor solution (taking 2 mL in the cell), and continu-
ous decrease of intensity of emission spectra was
recorded for each time. Titration curves were obtained by
plotting ΔI vs [G]/[H]. Association constants were calcu-
lated by plotting I0/I0 − I vs 1/[G] (I0 and I are the initial
and final intensity of the receptor solution after each
addition during titration).[24]
7 | EXPERIMENTAL
7.1 | General
7.4 | N-Butyl-N0-(7-methyl-[1,8]
naphthyridin-2-yl)-isophthalamide (1)
Chromatographic separations were performed on silica
gel (100-200 meshes). For preparative thin-layer chro-
matographic (TLC) (PTLC) purification, the layer was
formed on a glass plate using water gel–GF 254 silica gel.
The petroleum ether used has a boiling range of 40ꢀC to
60ꢀC. All the melting points were determined on hot-coil
2-Amino-7-methyl-1,8-naphthyridine was synthesized
according to the literature procedure.[15] 2-Amino-
7-methyl-1,8-naphthyridine (200 mg, 1.26 mmol) in dry
n
CH2Cl2 (15 mL) and butylamine (92 mg, 1.26 mmol) in
dry CH2Cl2 (15 mL) containing Et3N (0.1 mL) were added
with isophthaloyl dichloride (255 mg, 1.26 mmol) in dry
CH2Cl2 (20 mL) using high-dilution technique for 4 hours
under N2 atmosphere. The reaction was continued for
another 30 hours. CH2Cl2 was distilled out, and the
gummy crude product was extracted with CH2Cl2
(20 mL × 4) after washing with saturated NaHCO3 solu-
tion, which was dried over anhydrous Na2SO4 and con-
centrated under vacuum. The crude compound was
purified by preparative TLC using CHCl3 (2% MeOH) as
eluant and afforded an off-white solid substance (100 mg,
22%).
1
stage melting point apparatus and are uncorrected. H
NMR spectra were recorded on 500- or 300-MHz spec-
trometers. 13C NMR spectra were recorded on a 500-MHz
spectrometer. For NMR spectra, CDCl3 and DMSO-d6
were used as solvents using transcranial magnetic stimu-
lation (TMS) as an internal standard. Chemical shifts are
1
1
1
expressed in δ units and H─ H, H─C coupling con-
stants in hertz. Infrared (IR) spectra were recorded using
KBr disks.
Mp. 149ꢀC-151ꢀC.
7.2 | General procedure for UV-Vis
titration
1H NMR (CDCl3, 500 MHz): δ (ppm): 9.12 (bs, 1H),
8.63 (d, 1H, J = 8.9 Hz), 8.33 (s, 1H), 8.23 (d, 1H,
J = 8.8 Hz), 8.11-8.05 (m, 3H), 7.63 (t, 1H, J = 7.7 Hz),
7.32 (d, 1H, J = 8.2 Hz), 6.31 (bs, 1H), 3.52 (q, 2H,
J = 7.1 Hz), 2.78 (s, 3H), 1.65 (qnt, 2H), 1.45 (sxt, 2H),
0.99 (t, 3H, J = 7.4 Hz).
Stock solution of the receptors (1, 2, 3, and 5) was pre-
pared in the order of 1 × 10−5M in CH3CN (receptor
4 was prepared in the order of 1 × 10−6M). The solution
of guest creatinine was prepared in either 1 × 10−4M or
1 × 10−5M order in CH3CN by adding individual host
solutions. Then, the guest solution is added to the recep-
tor solution (taking 2 mL in the UV cell), and continuous
decreases of absorbance in UV spectra were recorded
each time. Titration curves were determined by plotting
FT-IR (KBr): 3420, 1683, 1636, 1508, 1324, 1025,
713 cm−1.
MS (ESI): m/z (%): 385 ((M
+
Na)+, 27),
363 ((M + H)+, 100).
Anal. Calcd. for C21H22N4O2: C, 69.59; H, 6.12; N,
15.46. Found: C, 69.73; H, 6.05; N, 15.71.

JANA ET AL.
9 of 11
7.5 | 2,2-Dimethyl-N-[6-(6-methyl-
pyridin-2-ylmethoxymethyl)-pyridin-2-yl]-
propionamide (2)
1H NMR (CDCl3, 500 MHz): δ (ppm): 8.19 (d, 1H,
J = 8.3 Hz), 7.97 (bs, 1H), 7.82 (s, 1H), 7.67 (t, 1H,
J = 7.9 Hz), 7.14 (s, 1H), 6.99 (s, 1H), 6.77 (d, 1H,
J = 7.5 Hz), 5.14 (s, 2H), 1.33 (s, 9H).
(6-Methyl-pyridin-2-yl)-methanol was obtained from the
corresponding aldehyde by the reduction with NaBH4 in
dry EtOH. The conversion was almost quantitative, and it
was directly used for the next step without further purifi-
cation. (6-Methyl-pyridin-2-yl)-methanol (320 mg,
2.6 mmol) was taken in a two-neck r.b. and dried well
under vacuum and filled with N2. NaH (216 mg, 10 mmol)
was added carefully and stirred for 30 minutes in dry
THF (5 mL) at r.t. Then, N-(6-bromomethyl-pyridin-2-yl)-
2,2-dimethyl-propionamide (704 mg, 2.6 mmol) in dry
THF (5 mL) was added dropwise for 30 minutes, and the
reaction was continued by stirring overnight at r.t. THF
was distilled off under reduced pressure, and the brown
gummy crude was extracted with EtOAc (20 mL × 5)
after washing with water, which was dried over anhy-
drous Na2SO4 and concentrated under vacuum. This
crude product was purified by column chromatography
using silica gel (100-200 meshes) and EtOAc (15%) in
petroleum ether as eluant to afford light yellow dense liq-
uid (530 mg, 65%).
13C NMR (CDCl3, 125 MHz): 177.5, 154.5, 151.9,
139.9, 138.1, 130.0, 119.9, 117.3, 113.6, 52.5, 40.2, 27.9.
FT-IR (KBr): 3440, 2970, 1672, 1577, 1541, 1457, 1081,
794 cm−1.
MS (ESI): m/z (%): 281 ((M
+
Na)+, 25),
259 ((M + H)+, 100), 191 (87).
Anal. Calcd. for C14H18N4O; C, 65.09; H, 7.02; N,
21.69. Found: C, 65.21; H, 6.96; N, 21.75.
7.7 | Pyrene-1-carboxylic acid (7-methyl-
[1,8]naphthyridin-2-yl)-amide (4)
2-Amino-7-methyl-1,8-naphthyridine and pyrene carbox-
ylic acid were synthesized according to the literature pro-
cedure.[16] This pyrene carboxylic acid (155 mg,
0.63 mmol) was converted to its acid chloride with
oxaloyl dichloride (0.15 mL, 1.6 mmol) and a catalytic
amount of DMF in dry CH2Cl2 at r.t. After stirring for
3 to 4 hours, excess CH2Cl2 was distilled out, and dry
CH2Cl2 (4 mL) was added. This solution was added with
2-amino-7-methyl-1,8-naphthyridine (100 mg, 0.63) in
dry CH2Cl2 (5 mL) dropwise under N2 atmosphere. This
reaction was continued for 20 hours. After completion of
the reaction, CH2Cl2 was distilled out and extracted with
CHCl3 (20 mL × 3) after washing with saturated NaHCO3
solution, which was dried over anhydrous Na2SO4 and
concentrated under vacuum. This crude product was
purified by column chromatography using silica gel
(100-200 meshes) and EtOAc (15%) in petroleum ether as
eluant to afford a deep yellow solid (173 mg, 71%).
Mp. 194ꢀC-197ꢀC.
1H NMR (CDCl3, 500 MHz): δ (ppm): 8.18 (d, 1H,
J = 8.3 Hz), 8.16 (bs, 1H), 7.75-7.69 (m, 2H), 7.41 (d, 1H,
J = 7.6 Hz), 7.20 (d, 1H, J = 7.5 Hz), 7.14 (d, 1H,
J = 7.7 Hz), 4.85 (s, 2H), 4.66 (s, 2H), 2.58 (s, 3H) 1.33 (s,
9H).
13C NMR (CDCl3, 125 MHz): δ (ppm): 177.5, 158.1,
157.7, 156.7, 151.5, 139.3, 137.3, 122.5, 118.8, 117.8, 113.0,
74.1, 73.6, 40.2, 27.8, 24.7.
FT-IR (KBr): 3435, 1684, 1578, 1522, 1456, 1153, 1026,
798 cm−1
.
MS (ESI): m/z (%): 336 ((M
+
Na)+, 95),
314 ((M + H)+, 17), 207 (100), 191 (57.5), 122 (67.5).
Anal. Calcd. for C18H23N3O2; C, 68.98; H, 7.40; N,
13.41. Found: C, 68.75; H, 7.43; N, 13.59.
1H NMR (CDCl3, 300 MHz): δ (ppm): 8.89 (d, 1H,
J = 8.6 Hz), 8.48 (d, 1H, J = 9.2 Hz), 8.39 (d, 1H,
J = 7.9 Hz), 8.33-8.18 (m, 2H), 8.13 (d, 1H, J = 7.9 Hz),
8.10-8.05 (m, 2H), 7.99-7.92 (m, 1H), 7.84 (d, 1H,
J = 9.3 Hz), 7.76-7.66 (m, 2H), 7.48 (d, 2H, J = 8.9 Hz),
2.71 (s, 3H).
7.6 | N-(6-Imidazol-1-ylmethyl-pyridin-
2-yl)-2,2-dimethyl-propionamide (3)
13C NMR (CDCl3 + 2 drops DMSO-d6, 125 MHz): δ
(ppm): 196.9, 139.6, 138.2, 136.8, 132.0, 131.4, 130.9,
130.6, 129.5, 129.3, 129.1, 127.5, 126.9, 126.7, 126.5, 126.4,
126.3, 125.3, 124.8, 124.5, 124.2, 124.1, 123.9, 122.0, 114.7,
27.2.
N-(6-Bromomethyl-pyridin-2-yl)-2,2-dimethyl-
propionamide (270 mg, 1.0 mmol) and imidazole (70 mg,
1.0 mmol) were taken in an open-mouth conical flask
and were mixed thoroughly. This mixture was irradiated
at 450 W for 5 minutes in a microwave oven. Then, this
mixture was directly used for purification by column
chromatography (100-200 meshes) and EtOAc (15%) in
petroleum ether as eluant to afford an off-white solid
compound (210 mg, 81%).
FT-IR (KBr): 3420, 3047, 2921, 1668, 1558, 1539, 1506,
1278, 1224, 847, 713 cm−1
.
MS (HRMS-FAB): m/z (%): Calculated for C26H18N3O
is 388.1450 (M + H)+; found 388.1447.
Anal. Calcd. for C26H17N3O; C, 80.60; H, 4.42; N,
10.85. Found: C, 80.48; H, 4.45; N, 10.78.
Mp. 114ꢀC-116ꢀC.

10 of 11
JANA ET AL.
7.8 | 2,2-Dimethyl-N-[6-(quinolin-
8-yloxymethyl)-pyridin-2-yl]-propionamide
(6)
J = 8.3 Hz), 8.15 (d, 1H, J = 8.3 Hz), 8.09 (d, 1H,
J = 7.8 Hz), 8.06 (d, 1H, J = 7.8 Hz), 7.73 (t, 1H,
J = 7.9 Hz), 7.59 (t, 1H, J = 7.8 Hz), 7.45 (dd, 1H, J = 8.3,
4.2 Hz), 7.43-7.38 (m, 2H), 7.32 (d, 1H, J = 7.5 Hz), 7.01
(dd, 1H, J = 7.0, 1.8 Hz), 6.64 (bs, 1H), 5.34 (s, 2H), 3.46
(q, 2H), 1.57 (qnt, 2H), 1.38 (sxt, 2H), 0.92 (t, 3H,
J = 7.4 Hz).
N-(6-Bromomethyl-pyridin-2-yl)-2,2-dimethyl-
propionamide (500 mg, 2.5 mmol), 8-hydroxyquinoline
(336 mg, 2.5 mmol), K2CO3 (860 mg, 6.25 mmol), and
TBAB (80 mg, 0.25 mmol) were taken in an r.b. Dry ace-
tone (7 mL) was added to it and stirred for 2 days at
r.t. Acetone was distilled out, and the crude product was
extracted with CHCl3 (20 mL × 4) after washing with sat-
urated brine and with water, which was then dried over
anhydrous Na2SO4 and concentrated under vacuum. This
crude product was purified by column chromatography
using silica gel (100-200 meshes) and EtOAc (15%) in
petroleum ether as eluant to afford an off-white solid
(561 mg, 67%).
13C NMR (CDCl3, 125 MHz): δ (ppm): 166.9, 165.3,
155.2, 154.5, 151.5, 149.8, 140.7, 139.6, 136.6, 135.5, 134.4,
131.9, 130.9, 129.0, 129.6, 127.1, 125.5, 122.3, 120.7, 118.6,
113.8, 109.9, 71.4, 40.4, 31.9, 20.5, 14.1.
FT-IR (KBr): 3326, 3063, 2952, 2920, 2868, 1684, 1668,
1573, 1558, 1539, 1506, 1457, 1378, 1307, 1261, 1107,
820, 784 cm−1
.
MS (ESI-HRMS): m/z (%): Calculated for
C27H26N4O3Na is 477.1897; found 477.1905.
Anal. Calcd. for C27H26N4O3; C, 71.35; H, 5.77; N,
12.33. Found: C, 71.18; H, 5.73; N, 12.42.
Mp. 67ꢀC-69ꢀC.
1H NMR (CDCl3, 300 MHz): δ (ppm): 8.99 (d, 1H,
J = 4.0 Hz), 8.18-8.13 (m, 2H), 8.08 (bs, 1H), 7.67 (t, 1H,
J = 7.9 Hz), 7.48-7.38 (m, 3H), 7.31 (d, 1H, J = 7.5 Hz),
7.00 (d, 1H, J = 6.6 Hz), 5.42 (s, 2H), 1.35 (s, 9H).
FT-IR (KBr): 3359, 2950, 2916, 2870, 1695, 1589, 1558,
ACKNOWLEDGEMENTS
S.J. and S.P. thank to Madhya Pradesh Council of Science
and Technology (MPCST), Bhopal, Government of
Madhya Pradesh, India, for the financial support (vide
project no: Endt. No 186/CST/R&D/Phy&Engg/2018-19).
K.K.S. thanks to Indira Gandhi National Tribal Univer-
sity (Central University), Amarkantak, Madhya Pradesh,
India, for the research fellowship.
1455, 1114, 795 cm−1
.
MS (ESI-HRMS): m/z (%): Calculated for C20H22N3O2
is 336.1707 (M + H)+; found 336.1710 (100), 358.1530
((M + Na)+, 84).
ORCID
7.9 | N-Butyl-N0-[6-(quinolin-
8-yloxymethyl)-pyridin-2-yl]-
isophthalamide (5)
Subrata Jana https://orcid.org/0000-0002-7533-3745
REFERENCES
[1] N. N. Turner, N. Lameire, D. J. Goldsmith, C. G. Winearls,
J. Himmelfarb, G. Remuzzi, W. G. Bennet, M. E. de Broe,
J. R. Chapman, A. Covic, V. Jha, N. Sheerin, R. Unwin,
A. Woolf, Oxford Textbook of Clinical Nephrology, fourth ed.,
Oxford University Press, Oxford 2015.
[2] N. W. Tietz, W. B. Saunders, P. A. Philadelphia, Textbook of
Clinical Chemistry, 3rd ed. Elsivier; 1059 1999.
[3] M. Gutierrez, S. Alegret, M. Del Valle, Biosens. Bioelectron.
2008, 23, 795.
[4] G. J. L. De Azevedo, L. H. Canani, T. Zelmanovitz, Diabetes
Care 2005, 28, 164.
[5] a) J. R. Delanghe, M. M. Speeckaert, NDT Plus 2011, 4, 83;
b) R. M. Jacobs, J. H. Lumsden, J. A. Taylor, E. Grift, Can.
J. Vet. Res. 1991, 55, 150.
[6] a) R. Arts, S. Katrina, J. Ludwig, B. C. B. van Gerven,
E. M. Estirado, L.-G. Milroy, M. Merkx, ACS Sens. 2017, 2;
b) C. S. Pundir, S. Yadav, A. Kumar, TrAC Trends Anal. Chem.
2013, 50, 42; c) A. C. Sharma, T. Jana, R. Kesavamoorthy,
L. Shi, M. A. Virji, D. N. Finegold, S. A. Asher, J. Am. Chem.
Soc. 2004, 126, 2971; d) M. Subat, A. S. Borovik, B. Knig,
J. Am. Chem. Soc. 2004, 126, 3185.
Compound 6 was hydrolyzed to obtain the corresponding
amine by refluxing in 4M KOH (1:1) in ethanol for
6 hours, which was directly used for the mixed amide
synthesis after usual workup. These amine (200 mg,
0.8 mmol) in dry CH2Cl2 (20 mL) and nbutylamine
(58 mg, 0.8 mmol) in dry CH2Cl2 (20 mL) containing
Et3N (0.12 mL) were added with isophthaloyl dichloride
(160 mg, 0.8 mmol) in dry CH2Cl2 (25 mL) using high-
dilution technique for 5 hours under N2 atmosphere. The
reaction was continued with stirring for another 36 hours
at r.t. CH2Cl2 was distilled out, and the gummy crude
product was extracted with CH2Cl2 (20 mL × 4) after
washing with saturated NaHCO3 solution, which was
dried over anhydrous Na2SO4 and concentrated under
vacuum. The product was purified by preparative TLC
using CHCl3 (2% MeOH) as eluant to afford an off-white
solid substance (112 mg, 31%).
Mp. 84ꢀC-86ꢀC.
1H NMR (CDCl3, 500 MHz): δ (ppm): 8.96 (d, 1H,
J = 4.1 Hz), 8.89 (bs, 1H), 8.45 (s, 1H), 8.25 (d, 1H,
[7] a) T. Guinovart, D. Hernandez-Alonso, L. Adriaenssens,
P. Blondeau, M. Martinez-Belmonte, F. X. Rius, F. J. Andrade,

JANA ET AL.
11 of 11
P. Ballester, Angew. Chem. Int. Ed. 2016, 55, 2435;
different mode of complexation with tautomeric form of creat-
inine was calculated using B3LYP/6-31G(d,p) method.
[18] a) P. C. Hariharan, J. A. Pople, Theor. Chem. Acc. 1973, 28,
213; b) P. Hohenberg, W. Kohn, Phys. Rev. 1964, 136, B864.
[19] J. Gao, Y. Hu, S. Li, Y. Zhang, X. Chen, Chem. Phys. 2013,
410, 81.
[20] M. J. Frisch, G. W. Trucks, H. B. Schlegel, et al., Gaussian
09, Revision B.01, Gaussian, Inc, Wallingford, CT 2010.
[21] a) K. A. Connors, Binding Constant—The Measurement of
Molecular Complex Stability, John Wiley & Sons, New York
1987; b) H. Benesi, J. H. Hildebrand, J. Am. Chem. Soc. 1949,
71, 2703.
b) P. Buhlmann, W. Simon, Tetrahedron 1993, 49, 7627;
c) P. Buhlmann, W. Simon, M. Badertscher, Tetrahedron 1993,
49, 595.
[8] a) T. W. Bell, Z. Hou, Y. Luo, M. G. B. Drew, E. Chapoteau,
B. P. Czech, A. Kumar, Science 1995, 269, 671; b) S. Pal,
S. Lohar, M. Mukherjee, P. Chattopadhyay, K. Dhara, Chem.
Commun. 2016, 52, 13706.
[9] a) I. Pandey, P. K. Bairagi, N. Verma, Sensors & Actuators:
B. Chemical 2018, 277, 562; b) C.-H. Chen, M. S. Lin, Biosens.
Bioelectron. 2012, 31, 90.
[10] a) U. Sivasankaran, T. C. Jos, K. G. Kumar, Anal. Biochem.
2018, 544, 1; b) P. G. Sutariya, A. Pandya, A. Lodha,
S. K. Menon, Talanta 2016, 147, 590; c) S. Ellairaja,
K. Shenbagavalli, V. S. Vasantha, ChemistrySelect 2017, 2,
1025; d) M. M. Rahman, J. Ahmed, A. M. Asiri, Sens. Actuators
B 2017, 242, 167.
[22] G. Sivaraman, M. Iniya, T. Anand, N. G. Kotla, O. Sunnapu,
S. Singaravadivel, A. Gulyani, D. Chellappa, Coord. Chem. Rev.
2018, 357, 50.
[23] S.
Ellairaja,
V.
Subramanian,
K.
Velayutham,
R. Gomathinayagam, V. S. Vasantha, ACS Sens. 2018, 3, 763.
[24] a) A. E. Hargrove, Z. Zhong, J. L. Sessler, E. V. Anslyn, New
J. Chem. 2010, 34, 348; b) P. Thordarson, Chem. Soc. Rev. 2011,
40, 1305.
[11] B. Babamiri, A. Salimi, R. Hallaj, Biosens. Bioelectron. 2018,
107, 272.
[12] a) S. Goswami, S. Dey, J. Org. Chem. 2006, 71, 7280;
b) S. Goswami, R. Mukherjee, J. Roy, Org. Lett. 2005, 7, 1283.
[13] a) A. R. Butler, C. Glidewell, J. Chem. Soc. Perkin Trans. 1985,
11, 1465; b) W. L. Jorgensen, J. Peanata, J. Am. Chem. Soc.
1990, 112, 2008; c) D. Kotsyubynskyy, S. Molchanov, A. Gryff-
Keller, Pol. J. Chem. 2004, 78, 239.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[14] S. Goswami, S. Jana, A. Hazra, H.-K. Fun, S. Anjum,
A.-U. Rahman, Crst Eng Comm 2006, 8, 712.
[15] a) M.-M. Yu, Z.-X. Li, L.-H. Wei, D.-H. Wei, M.-S. Tang, Org.
Lett. 2008, 10, 5115; b) S. Goswami, K. Ghosh, R. Mukherjee,
A. K. Adak, A. K. Mahapatra, J. Heterocyclic Chem. 2001,
38, 173.
[16] A. Kumar, P. S. Chae, Dyes Pigm. 2018, 156, 307.
[17] a) S. Schlund, C. Schmuck, B. Engels, J. Am. Chem. Soc. 2005,
127, 11115; b) Energy minimization of receptors and their
How to cite this article: Jana S, Prajapati S,
Suryavanshi KK, Goswami S, Parida R, Giri S.
Creatinine recognition using designed synthetic
receptors. J Phys Org Chem. 2020;e4066. https://
doi.org/10.1002/poc.4066