V.K.R. Tangadanchu et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 128030
Fig. 5. UV absorption spectra of DNA with different concentrations of com-
pound 7a at pH 7.4 and room temperature. c(DNA) = 5 × 10-5 mol/L, and c
(compound 7a) = 0–2.0 × 10-5 mol/L for curves a–h respectively at an incre-
ment of 0.25 × 10-5 mol/L. Inset: comparison of absorption at 260 nm between
the 7a–DNA complex and the sum values of free DNA and free compound 7a.
Fig. 4. Comparison of MIC and MBC/MFC values for compound 7a against
E. faecalis, E. coli ATCC 25922, C. tropicals and C. parapsilosis ATCC 22019.
derivative 7b (MIC = 8 µg/mL), being equipotent to norfloxacin. Be-
sides, 4-methyl benzene isatin conjugate 7c could suppress the growth of
S. aureus ATCC 25923 with MIC of 8 µg/mL. The results indicated that
enone derivatives were favorable for enhancing antibacterial potency.
Prominently, molecule 7a endowed optimal antibacterial behavior with
broad spectrum and special inhibition towards E. coli ATCC 25922.
Although the antifungal potency was not satisfactory, there is
something worthy to be mentioned (Table S1). Various azole-substituted
isatins 3–6 exhibited weak inhibition against the tested fungi as well,
including antibacterial precursor 5a. Surprisingly, the antifungal po-
tency of active molecule 7a was also desirable with MICs of 8–32 µg/mL,
in which the C. tropicals was sensitive to 7a comparably with flucona-
zole. Nevertheless, when methyl group was replaced with phenyl frag-
ments, the antifungal ability of the phenyl conjugates 7b–e reduced
obviously. These results might be explained that the larger conjugate
systems were unfavorable for enhancing antifungal potentiality. Hence,
the antimicrobial evaluations selected an active molecule 7a to be
further studied.
To investigate whether the active molecule 7a exerted bactericidal
and fungicidal ability, the minimal bactericidal concentration (MBC)
and minimal fungicidal concentration (MFC) against bacteria
(E. faecalis, E. coli ATCC 25922) and fungi (C. tropicals, C. parapsilosis
ATCC 22019) were determined and subsequently compared to respec-
tive MIC values. According to CLSI, the ratio of MBC or MFC/MIC in the
range of 1 to 2 is indicative of killing behavior, while a ratio ≥ 8 is
contrastively determined as inhibitory action.17 As shown in Fig. 4,
compound 7a not only demonstrated a weak bactericidal activity
against clinical resistant E. faecalis, but also exerted fungi eradicating
effect on C. tropicals. However, the highly suspectable E. coli ATCC
25922 could not be killed by hybrid 7a in spite of the low suppressive
concentration. Conjugate 7a also presented a 4-fold difference between
MIC and MFC against C. parapsilosis ATCC 22019, indicating a fungi-
static behavior. These implied that isatin derivative 7a had potentiality
to be developed as bactericidal or fungicidal agent against some resis-
tant strains.
Fig. 6. Molecular modeling of compound 7a and E. coli DNA polymerase III
(PDB code: 5fkv).
DNA is widely utilized to rationally design and construct potential
new antimicrobial agents, being a target with multiple active sites. Isatin
is a planar structure, so it might be able to intercalate into DNA. In vitro
binding mode of bioactive conjugate 7a with DNA was studied through
UV–Vis absorption spectroscopy, and calf thymus DNA was used as a
model with medical importance, low cost and easy availability.19 The
maximum absorption peak of DNA at 260 nm in Fig. 5 got proportionally
increased along with slightly red shift under the enhanced concentra-
tions of 1,2,4-triazole isatin 7a. Meanwhile, the measured absorbance of
7a-DNA complex was higher than the simple sum of dissociative DNA
and 7a, which revealed a hyperchromism between 7a and DNA. This
consequence intrinsically demonstrated conformational changes in DNA
duplex, where the DNA helix was quite possibly broken, and base-pairs
were exposed. Based on the variations in the absorption spectra of DNA
upon binding to 7a, equation could be utilized to calculate the binding
constant (K) (Fig. S2).
Preventing bacteria from developing resistance is an important
quality of potential antimicrobial agents.18 Therefore, the drug resis-
tance test was carried out to evaluate the probability of hybrid 7a to
elicit resistance towards E. coli ATCC 25922. Fig. S1 indicated that the
susceptibility of E. coli ATCC 25922 to molecule 7a remained nearly
unchanged even after 10 passages, while the MIC values of norfloxacin
towards E. coli ATCC 25922 got quickly enhanced after 6 passages. This
consequence manifested that 1,2,4-triazole isatin 7a developed unde-
tectable resistance towards E. coli ATCC 25922.
Neutral red (NR) NR, a planar dye with a confirmed intercalative
binding mode with DNA, was used to probe into the action mode be-
tween 7a and DNA.20 The absorption spectrum (Fig. S3) manifested a
competitive binding between NR and 7a with DNA. With the increasing
concentration of 7a, the maximum absorption around 530 nm
decreased, presenting a reverse process in comparison with the ab-
sorption of free NR in the presence of the increasing concentrations of
4