Synthesis, Biocidal and Antibiofilm Activities of New Isatin–Quinoline Conjugates against Multidrug-Resistant Bacterial Pathogens along with Their In Silico Screening

Abstract

Isatin–quinoline conjugates 10a–f and 11a–f were assembled by the reaction of N-(bromobutyl) isatin derivatives 3a, b with aminoquinolines 6a–c and their corresponding hydrazinyl 9a–c in good yields. The structures of the resulting conjugates were established by spectroscopic tools and showed data consistent with the proposed structures. In vitro antibacterial activity against different bacterial strains was evaluated. All tested conjugates showed significant biocidal activity with lower MIC than the first line drugs chloramphenicol and ampicillin. Conjugates 10a, 10b and 10f displayed the most potent activity against all clinical isolates. The antibiofilm activity for all tested conjugates was screened against the reference drug vancomycin using the MRSA strain. The results revealed that all conjugates had an inhibitory activity against biofilm formation and conjugate. Conjugate 11a showed 83.60% inhibition at 10 mg/mL. In addition, TEM studies were used to prove the mechanism of antibacterial action of conjugates 10a and 11a against (MRSA). Modeling procedures were performed on 10a–f and 11a–f and interestingly the results were nearly consistent with the biological activities. In addition, in silico pharmacokinetic evaluation was performed and revealed that the synthesized compounds 10a–f and 11a–f were considered drug-like molecules with promising bioavailability and high GI absorption. The results confirmed that the title compounds caused the disruption of bacterial cell membranes and could be used as potential leads for the further development and optimization of antibacterial agents.

1. Introduction

Bacterial infections comprise most hospital- and community-acquired infections [1,2]. The emergence and wide spread of drug-resistant pathogens has exacerbated the situation [3]. Finding new antibacterial drugs is one of the challenges that researchers face in overcoming the emergence of antibiotic resistance to various bacterial strains [4]. The misuse and overuse of antibiotics, as well as mutation owing to environmental changes and the gene transfer of bacteria, are the leading causes of antibiotic resistance [5]. From this perspective, searching for new natural or synthetic-based drugs to keep up with the rapid emergence of bacterial resistance is one of the main targets of medicinal chemistry. Nature contains various pharmaceutical compounds with potent biological activity [6,7]. Naturally occurring indole, quinoline and indoloquinoline heterocyclic cores are particularly important due to their diverse biological activities, which inspire many researchers to synthesize series of hybrid systems with different spacers [8,9,10,11]. Natural and synthetic isatin scaffolds have received much attention from many researchers due to their wide pharmaceutical application as antimicrobial, antioxidant, anticancer and antimalarial agents [12,13,14,15]. On the other hand, quinoline’s core structure represents a promising surrogate in drug design and development. This N-containing heterocyclic skeleton is incorporated in many natural and synthetic biologically active molecules [16,17,18,19,20,21,22] such as chloroquine and its analogues, which are well known drugs used to treat malaria [23,24]. Encouraged by the aforementioned facts, it is anticipated that by combining two intrinsically biologically active motifs such as isatin and quinoline moiety a synergistic effect will be obtained for the resulting surrogate’s structure when compared with each moiety independently [25,26,27].

Previous studies have reviewed the biological importance of isatin–quinoline hybrids and illustrated the potent activity of these conjugates [28,29]. Moreover, the presence of linkers between isatin and quinoline have shown a significant role in enhancing the biological activities of these conjugates [30,31]. Therefore, many researchers seek to enhance and optimize the biological activity of isatin–quinoline conjugates. From the above-mentioned information, the present research work emphasizes the synthesis and biological evaluation of some novel isatin–quinoline conjugates with the presence of alkyl spacers aiming to reach the best activity for the synthesized conjugates, Figure 1.

2. Results and Discussion

2.1. Chemistry

The preparation of N-(bromobutyl) isatin derivatives 3a,b was accomplished in good yields according to the method in the literature [32,33], as depicted in Scheme 1.

Moreover, aminoquinolines 6a–c and their corresponding hydrazinyl 9a–c were synthesized in good yields starting from the reaction of 4,7-dichloroquinoline 4 with excess hydrazine or aliphatic diamine 5a–c in DMF under reflux condition according to the method used in the literature [30]. Further reaction of 6a–c with chloroacetyl chloride 7 in DMF in the presence of anhydrous potassium carbonate afforded the corresponding intermediates 8a–c in good yields according to the reported method [32]. Moreover, the reaction of 8a–c with excess hydrazine in DMF at room temperature yielded the corresponding hydrazinyl derivatives 9a–c in good yields as illustrated in Scheme 2.

Finally, the new conjugates 10a–f and 11a–f bearing isatin and quinoline motifs were achieved in good yields by the reaction of 3a,b with 6a–c or 9a–c in the presence of triethyl amine in DMF under reflux condition for 4h until the consumption of the reactants as monitored by TLC chromatography as depicted in Scheme 3.

For the structural elucidation of 10a–f, the FT-IR showed υ_(NH) absorption ranging from 3397 to 3312 cm⁻¹. In addition, for υ _(>N-C=O) of C-2 the isatin core appeared from 1675 to 1653 cm⁻¹ and 1731 to 1703 cm⁻¹for υ _(>C=O) of C-3 in the isatin core. The ¹H-NMR spectra performed in (CDCl₃) confirmed the formation of isatin—quinoline conjugates by a noticeable increase in the number of chemical shifts in the aliphatic and aromatic ranges, the appearance of the (N-CH₂) signal ranging from δ: 3.79 to 4.28 ppm and the characteristic peak quinoline (CH=N) was reported by signals at a range from δ: 8.54 to 8.82 ppm as singlet. Moreover, the ¹³C-NMR spectra performed in (CDCl₃) showed an aliphatic chain ranging from δ: 24.07 to 53.44 ppm. Furthermore, the quinoline (CH=N) showed signals ranging from δ: 150.23 to 158.24 ppm and the (>N-C=O) group of the isatin core recorded for position C-2 from δ: 151.02 to 162.55 ppm and C-3 from δ: 181.95 to 184.55 ppm as illustrated in

Table 1. Spectral data for conjugates 10a–f and 11a–f.

	FT-IR υ (cm−1)					1H-NMR δ (ppm)			13C-NMR δ (ppm)
Com.	NH	υ (CH) Aliphatic	υ (C=O) Isatin	υ (N-C=O) Isatin	υ (C=N) Quinoline	NH-CH2 Quinoline	N-CH2 Isatin	CH=NAr Quinoline	CH=NAr Quinoline	N-C=O Isatin	C=O (C-3) Isatin	C=O (C-2) hydrazide
10a	3374	2930	1710	1675	1562	3.79		8.54	153.10	167.35	183.20
10b	3319	2936	1703	1665	1577	3.78	3.88	8.77	150.61	158.17	183.40	--------
10c	3397	2931	1713	1654	1559	3.77	4.28	8.55	155.49	162.34	184.53	--------
10d	3360	2933	1732	1655	1587	3.26	3.91	8.82	158.24	162.55	183.29	--------
10e	3312	2949	1731	1657	1555	3.45	3.81	8.81	151.02	167.17	181.95	--------
10f	3343	2934	1705	1653	1587	3.75	3.84	8.65	158.41	165.41	183.40	--------
11a	3405	2935	1718	1668	1577	3.46	3.79	8.79	152.24	152.82	184.52	171.88
11b	3297	2933	1721	1664	1577	3.42	3.79	8.77	151.12	162.62	184.31	176.13
11c	3343	2933	1705	1676	1566	3.47	3.86	8.78	152.50	152.94	182.65	170.29
11d	3352	2937	1710	1657	1564	3.77	4.20	8.38	157.75	160.91	181.96	178.31
11e	3324	2938	1715	1654	1556	3.49	3.79	8.77	149.08	150.71	184.52	170.14
11f	3365	2937	1731	1651	1555	3.78	4.31	8.77	150.55	157.75	183.90	178.83

.

Meanwhile, the structure characterization of isatin—quinoline conjugates of type 11a–f confirmed the formation of target conjugates as the FT-IR absorption bands showed the presence of υ _(NH) from 3405 to 3293 cm⁻¹ and the υ _(>C=O) of C-2 was reported as ranging from 1657 to 1653 cm⁻¹, and for the other υ _(>C=O) of C-3 of the isatin core from 1731 to 1705 cm⁻¹. Furthermore, the ¹H-NMR spectra showed the (N-CH₂) for 11a–f ranging from δ: 3.79 to 4.31 ppm and for quinoline (CH=N) showed at a range from δ: 8.54 to 8.82 ppm. In ¹³C-NMR, for the (CH=N) signals were reported at a range from δ: 149.08 to 150.55 ppm and the (>N-C=O) group of the isatin core recorded for position C-2 from δ: 150.71 to 162.62 ppm, while the (>C=O) group-bearing hydrazine group appeared from δ: 170.19 to 178.83 ppm and C-3 from δ: 181.96 to 184.53 ppm as shown in Table 1.

2.2. In Vitro Antibacterial Screening

The antibacterial properties of conjugates 10a–f and 11a–f at a concentration of 40 mg/mL were assessed against clinical isolates. In this study, the most resistant bacterial strains such MRSA, Streptococcus mutans, Klebsiella pneumoniae and Serratia marcescens were used against first line drugs ampicilin and chloramphnicol as a reference. The results revealed that all conjugates had bactericidal activity higher than the reference drugs (ampicilin and chloramphnicol) as well as the mother cores of synthesized conjugates (isatin derivatives and 4.7 dichloroquinoline).

2.2.1. Antibacterial Screening of Conjugates 10a–f and 11a–f against Methicillin-Resistant Staphylococcus aureus (MRSA)

As stated in

Table 2. Antibacterial activity of conjugates 10a–f and 11a–f against MRSA clinical isolate.

Conjugates	Zone of Inhibition ZOI (mm) 1	MRSA			MRSA ATCC 43300
		Minimum Bactericidal Concentration MBC 2	Minimum Inhibitory Concentration MIC 3	MBC/MIC	MBC	MIC	MBC/MIC
1a	13 ± 1	10	2.5	4(+)	10	5	2(+)
1b	13.3 ± 0.6	10	2.5	4(+)	10	5	2(+)
4	12.3 ± 0.6	20	2.5	8(+)	20	5	4(+)
10a	24.31 ± 0.60	0.05	0.006	8 (+)	0.05	0.025	2(+)
10b	25.00 ± 1.00	0.05	0.0125	4 (+)	0.05	0.025	2(+)
10c	23.00 ± 1.00	0.05	0.025	2(+)	0.05	0.025	2(+)
10d	23.75 ± 0.60	0.05	0.025	2(+)	0.05	0.0125	4 (+)
10e	24.73 ± 0.60	0.05	0.025	2(+)	0.05	0.025	2(+)
10f	24.78 ± 0.60	0.05	0.025	2(+)	0.05	0.0125	4 (+)
11a	47.33 ± 0.60	0.625	0.156	4 (+)	0.156	0.08	2 (+)
11b	24.00 ± 1.00	2.5	1.25	2(+)	5	2.5	2 (+)
11c	30.72 ± 1.00	0.312	0.156	2(+)	0.625	0.312	2 (+)
11d	20.00 ± 1.00	2.5	1.25	2(+)	5	2.5	2 (+)
11e	18.31 ± 0.60	2.5	1.25	2(+)	5	2.5	2 (+)
11f	16.34 ± 0.60	5	1.25	4 (+)	5	2.5	2 (+)
Ampicilin	14.77 ± 0.60	6.25	3.13	2(+)	6.25	1.56	4 (+)
Chloramphnicol	14.32 ± 0.60	3.13	0.78	4(+)	12.5	8	1.6(+)

¹: Zone of inhibition, ²: Minimum bactericidal concentration, ³: minimum inhibitory concentration.

and Figure S1, all conjugates were screened against MRSA clinical isolate MRSA ATCC 43300 and displayed higher activity than the controls. It is noticeable that 11a had the maximum zone of inhibition against MRSA with inhibition zone (47.33 ± 0.60 mm), followed by conjugate 11c with inhibition zone (30.72 ± 1.00 mm), while the other conjugates showed variable activity ranging from 20 to 25 mm and the ones with least effect were 11e and 11f with zones of inhibition 18.31 ± 0.60 and 16.34 ± 0.60 mm, respectively. In addition, the MIC values of 10 and 11 ranged from 0.006 mg/mL to 2.5 mg/mL and the MBC values ranged from 0.05 mg/mL to 5 mg/mL for 10a–f and 11a–f.

2.2.2. Antibacterial Screening of Conjugates 10a–f and 11a–f against Streptococcus Mutans

As reported in

Table 3. Antibacterial activity of conjugates 10a–f and 11a–f against Streptococcus mutans clinical isolate.

Conjugates	Zone of Inhibition ZOI (mm) 1	Streptococcus mutans			Streptococcus mutans ATCC 35668
		Minimum Bactericidal Concentration MBC 2	Minimum Inhibitory Concentration MIC 3	MBC/MIC	MBC	MIC	MBC/MIC
1a	14 ± 1	10	1.25	8(+)	10	2.5	4(+)
1b	13 ± 1	5	1.25	4(+)	10	2.5	4(+)
4	14.3 ± 0.6	20	2.5	8(+)	20	5	4(+)
10a	23 ± 1	0.0008	0.0004	1.9 (+)	0.003	0.0015	2 (+)
10b	20.3 ± 0.6	0.003	0.0008	4 (+)	0.003	0.0008	4 (+)
10c	18 ± 1	0.0016	0.0008	2 (+)	0.003	0.0015	2 (+)
10d	17.3 ± 0.6	0.0004	0.0002	2 (+)	0.0008	0.0004	2 (+)
10e	20.7 ± 0.6	0.0004	0.0002	2 (+)	0.0004	0.0002	2 (+)
10f	20 ± 1	0.0004	0.0002	2 (+)	0.0004	0.0002	2 (+)
11a	46.3 ± 1	0.156	0.08	1.9 (+)	0.625	0.312	2 (+)
11b	22 ± 1	1.25	0.312	4 (+)	2.5	1.25	2(+)
11c	24 ± 1	0.625	0.156	4 (+)	1.25	0.625	2 (+)
11d	21.7 ± 0.6	0.625	0.156	4 (+)	1.25	0.625	2 (+)
11e	20.3 ± 0.6	0.312	0.156	2 (+)	0.156	0.08	2 (+)
11f	14.3 ± 0.6	5	2.5	2 (+)	10	5	2(+)
Ampicilin	12 ± 1	3.13	1.565	2 (+)	6.25	3.13	2(+)
Chloramphnicol	12.7 ± 0.6	6.25	1.56	4 (+)	12.5	6.25	2(+)

¹: Zone of inhibition, ²: Minimum bactericidal concentration, ³: minimum inhibitory concentration.

and Figure S2, all the compounds of 10a–f and 11a–f were screened against Streptococcus mutans. Compound 11a showed the highest zone of inhibition against Streptococcus mutans (46.3 ± 1mm), others showed variable activity ranging from 18 to 24 mm and the least effective one was 11f (14.3 ± 0.6 mm). As shown in Table 2, we compared the Streptococcus mutans clinical isolate and Streptococcus mutans ATCC 35668. The MIC values ranged from 0.0002 mg/mL to 2.5 mg/mL and MBC values ranged from 0.0004 mg/mL to 5 mg/mL for 10a–f and 11a–f.

2.2.3. Antibacterial Screening of Conjugates 10a–f and 11a–f against Klebsiella pneumoniae

As shown in

Table 4. Antibacterial activity of conjugates 10a–f and 11a–f against Klebsiella pneumoniae clinical isolate.

Conjugates	Zone of Inhibition ZOI (mm) 1	Klebsiella pneumoniae			Klebsiella pneumoniae ATCC 700603
		Minimum Bactericidal Concentration MBC 2	Minimum Inhibitory Concentration MIC 3	MBC/MIC	MBC	MIC	MBC/MIC
1a	13.7 ± 0.6	5	1.25	4(+)	10	5	2
1b	12 ± 1	10	2.5	4(+)	10	5	2
4	11 ± 1	40	5	8(+)	20	5	4
10a	23 ± 1	0.003	0.0008	4(+)	0.006	0.003	2(+)
10b	17 ± 1	0.0125	0.006	2(+)	0.025	0.0125	2(+)
10c	22 ± 1	0.625	0.156	4(+)	0.625	0.312	2(+)
10d	19 ± 1	0.0125	0.006	2(+)	0.0125	0.006	2(+)
10e	21 ± 1	0.003	0.0008	4(+)	0.006	0.003	2(+)
10f	21.3 ± 0.6	0.006	0.003	2(+)	0.0125	0.006	2(+)
11a	41.6 ± 0.6	0.156	0.04	4 (+)	0.312	0.156	2(+)
11b	23 ± 1	1.25	0.625	2(+)	2.5	1.25	2(+)
11c	25.3 ± 0.6	0.312	0.08	4(+)	0.625	0.312	2(+)
11d	23.7 ± 0.6	0.625	0.156	4(+)	1.25	0.625	2(+)
11e	25 ± 1	0.312	0.08	4(+)	0.312	0.156	2(+)
11f	16 ± 1	5	1.25	4(+)	5	2.5	2(+)
Ampicilin	14.3 ± 0.6	8	4	2(+)	6.25	3.125	2(+)
Chloramphnicol	8.7 ± 0.6	3.13	1.565	2 (+)	6.25	1.56	4 (+)

¹: Zone of inhibition, ²: Minimum bactericidal concentration, ³: minimum inhibitory concentration.

and Figure S3, conjugate 11a had the best zone of inhibition against Klebsiella pneumoniae (41.6 ± 0.6mm), while the other conjugates showed variable activity ranging from a 19 to 25 mm zone of inhibition. On the other hand, the least potent analogues were 10b and 11f with ZOI (17 ± 1, 16 ± 1, respectively). As shown in Table 4, we compared the Klebsiella pneumoniae clinical isolate and Klebsiella pneumoniae ATCC 700603. The MIC values ranged from 0.0008 mg/mL to 2.5 mg/mL and the MBC values ranged from 0.006 mg/mL to 5 mg/mL for 10a–f and 11a–f.

2.2.4. Antibacterial screening of conjugates 10a–f and 11a–f against Serratia marcescens

As shown in

Table 5. Antibacterial activity of conjugates 10a–f and 11a–f against the Serratia marcescens clinical isolate.

Conjugates	Zone of Inhibition ZOI (mm) 1	Serratia marcescens			Serratia marcescens ATCC13880
		Minimum Bactericidal Concentration MBC 2	Minimum Inhibitory Concentration MIC 3	MBC/MIC	MBC	MIC	MBC/MIC
1a	14.7 ± 0.6	5	2.5	2(+)	10	5	2(+)
1b	14.5 ± 0.6	5	1.25	4(+)	5	2.5	2(+)
4	13.3 ± 0.6	20	5	4(+)	20	10	2(+)
10a	21.7 ± 0.6	0.0008	0.0004	2(+)	0.0018	0.0008	2(+)
10b	26.3 ± 0.6	0.003	0.0008	4(+)	0.006	0.003	2(+)
10c	28 ± 1	0.0004	0.0002	2(+)	0.0008	0.0004	2(+)
10d	23.7 ± 0.6	1.25	0.62	2(+)	0.006	0.003	2(+)
10e	22.7 ± 0.6	0.05	0.0125	4(+)	0.05	0.025	2(+)
10f	28 ± 1	0.003	0.0008	4(+)	0.003	0.0008	4(+)
11a	43 ± 1	0.08	0.04	2(+)	0.156	0.08	2(+)
11b	15.3 ± 0.6	1.25	0.312	4(+)	2.5	1.25	2(+)
11c	32.3 ± 0.6	1.25	0.312	4(+)	2.5	1.25	2(+)
11d	23.3 ± 0.6	5	1.25	4(+)	5	2.5	2(+)
11e	17.3 ± 0.6	5	1.25	4(+)	5	2.5	2(+)
11f	10.7 ± 0.6	160	40	4(+)	160	80	2(+)
Ampicilin	8.7 ± 0.6	12.5	6.25	2(+)	6.25	1.56	4 (+)
Chloramphnicol	13.3 ± 0.6	3.13	1.565	2 (+)	3.13	1.565	2 (+)

¹: Zone of inhibition, ²: Minimum bactericidal concentration, ³: minimum inhibitory concentration.

and Figure S4, 11a had the maximum zone of inhibition against Serratia marcescens (43 ± 1mm), followed by 11c (32.3 ± 0.6 mm) and other conjugates varying between 21.7–28 mm; the least effective ones were 11f, 11b and 11e (10.7 ± 0.6 mm, 15.3 ± 0.6 mm and 17.3 ± 0.6 mm, respectively). All the conjugates were higher than the antibiotics that were used as positive control except 11f. As shown in Table 4, we compared the Serratia marcescens clinical isolate and Serratia marcescens ATCC13880. The MIC values ranged from 0.0004 mg/mL to 80 mg/mL and the MBC values ranged from 0.0008 mg/mL to 160 mg/mL for 10a–f and 11a–f.

2.3. Time-Kill Assay

All conjugates had a MBC/MIC ratio ≤ 4, (bactericidal), as illustrated in

Table 6. MIC, MBC and MBC/MIC ratio for 10a–f and 11a–f against MRSA.

Conjugates	MBC	MIC	MBC/MIC Ratio
10a	0.025	0.006	4 (+)
10b	0.05	0.0125	4(+)
10c	0.05	0.025	2(+)
10d	0.05	0.025	2(+)
10e	0.05	0.025	2(+)
10f	0.05	0.025	2(+)
11a	0.625	0.156	4 (+)
11b	2.5	1.25	2(+)
11c	0.312	0.156	2(+)
11d	2.5	1.25	2(+)
11e	2.5	1.25	2(+)
11f	5	1.25	4(+)

. Therefore, they were tested for the time-kill assay [34,35]. The results obtained for the time-kill study of MRSA are shown in Figure 2 and Figure 3 for 10a and 11a, respectively, as they are the best conjugates in the two series. The MBC values for 10a and 11a were consistent with the cidal concentration as displayed in the growth curve of this time-kill study. This study supported that the antibacterial activity is dose dependent. The data in Table 6 revealed that the activity depends on structure variations as seen from the value of the MIC among the tested conjugates. The lowest MIC values (0.006 and 0.025 mg/mL) were in 10a, 10c, 10d, 10e and 10f against MRSA, and 10b had a MIC of (0.0125 mg/mL); also, 11a and 11c had a MIC value of (0.156 mg/mL) and finally the highest MIC values were in 11b, 11d, 11e and 11f. The difference in MIC of conjugates 10 and 11 was due to the variation in their chemical structure. In general, conjugates of 10 had lower MIC values than conjugates of 11, as they could affect multiple target sites against bacterial cells.

2.4. Assessment of Anti-Biofilm Assay

This method allowed the determination of the anti-biofilm property of tested conjugates versus vancomycin using the 96-well plate. The biomass of bacterial cells was quantitatively analyzed on the microplate reader at an absorbance of 570 nm, showing significant reduction of biofilm when an increasing concentration of the tested conjugates was used when compared to the control (TSB only). The results showed that tested conjugates at 10 mg/mL exhibited the most significant anti-biofilm activity, ranging from 79.4 ± 0.22 to 62.9 ± 0.07% in conjugate 10, while ranging in conjugate 11 from 83.6 ± 0.11 to 50.2 ± 0.11%. Meanwhile, the anti-biofilm activity of the positive control vancomycin was measured at 43 ± 0.17% at the higher concentration of 100 mg/mL [36] as reported in

Table 7. Anti-biofilm assay of conjugate 10 towards MRSA ATCC 43300. Experiment evaluated based on quadruplicate results with standard deviation. (n = 4).

Concentration (mg/mL)	10a	10b	10c	10d	10e	10f
10	79.4 ± 0.22	65.4 ± 0.05	68.3 ± 0.07	71.8 ± 0.06	77.4 ± 0.07	62.9 ± 0.07
5	75.5 ± 0.11	59.8 ± 0.08	61.3 ± 0.08	65.3 ± 0.05	70.9 ± 0.06	58.8 ± 0.07
2.5	57.2 ± 0.07	48.8 ± 0.07	46.8 ± 0.05	43.8 ± 0.07	61.4 ± 0.07	43.9 ± 0.07
1.25	38.5 ± 0.07	33.4 ± 0.05	32.8 ± 0.06	31.7 ± 0.06	46.4 ± 0.07	34.6 ± 0.08

and

Table 8. Anti-biofilm assay of conjugates of 11 towards MRSA ATCC 43300. Experiment evaluated based on quadruplicate results with standard deviation. (n = 4).

Concentration (mg/mL)	11a	11b	11c	11d	11e	11f
10	83.6 ± 0.11	72.7 ± 0.06	77.7 ± 0.08	67.4 ± 0.11	58.8 ± 0.08	50.2 ± 0.11
5	78.4 ± 0.08	58.6 ± 0.08	62.5 ± 0.08	54.2 ± 0.11	47.5 ± 0.13	41.9 ± 0.06
2.5	62.6 ± 0.09	43.3 ± 0.08	53.8 ± 0.08	39.3 ± 0.11	32.3 ± 0.11	24.7 ± 0.14
1.25	48.8 ± 0.06	34.7 ± 0.07	44.4 ± 0.09	28.5 ± 0.09	24.9 ± 0.09	16.4 ± 0.11

and Figures S5–S7.

2.5. Transmission Electron Microscopy (TEM) Assay

The structural appearance of untreated MRSA as depicted in Figure 4 with treated 10a and 11a as shown in Figure 5 and Figure 6, respectively, was analyzed using TEM. The figures of treated MRSA show an altered MRSA cell membrane and dead bacterial cells after exposure to conjugates. The figures also show vacuoles created in the cytoplasm. These micrographs demonstrate that the compounds disrupt the MRSA bacterial cell wall, resulting in cell death; these effects were amplified and can be described as bactericidal. The figure also depicts increased cell death because of fewer cells, which is consistent with the observations made with untreated MRSA in Figure 4.

2.6. Molecular Docking

Molecular docking studies were performed on the prepared compounds 10a–f and 11a–f. The target enzyme crystal structure bound to its co-crystallized ligand was downloaded from Protein Data Bank (PDB:4DKI) [37]. An in silico screening study of the prepared structures was carried out at the transpeptidase enzyme active site in comparison with the co-crystalized ligand.

A redocking step was performed for validation of results, Figure 7. Docking results of the synthesized compounds, including binding affinity score and root mean square deviation RMSD in addition to the ligand interactions with the active site residues, either hydrogen bonding or hydrophobic interactions, are tabulated below,

Table 9. Molecular modeling data regarding binding affinity, RMSD values and receptor residues involved in interactions.

Compound No.	Binding Affinity	Root Mean Square Deviation (RMSD)	Amino Acids Involved in Interactions at the Active Site
10a	−7.181	1.674	THR600(H-donor), HIS583(H-acceptor), THR600(pi-H)
10b	−6.501	1.616	THR 600(H-donor), HIS583(H-acceptor), THR600(pi-H)
10c	−6.805	1.873	GLN521(H-donor), GLU447(pi-H)
10d	−6.992	1.498	MET641(H-acceptor)
10e	−7.191	1.184	THR 600(H-donor), THR 600(H-acceptor), THR 600(H-acceptor)
10f	−7.171	2.030	SER462(H-acceptor), SER598(H-acceptor), LYS639(pi-H)
11a	−7.254	1.464	THR 600(H-donor), THR 600(H-acceptor), ASN464(H-donor), ASN464(H-acceptor)
11b	−7.323	1.300	THR 600(H-donor), GLN521(pi-H)
11c	−8.098	1.784	GLU523(H-donor), TYR519(H-donor), GLN521 (H-donor), GLU447(H-donor), SER403(H-acceptor), SER462(pi-H)
11d	−7.115	2.134	TYR446 (pi-H)
11e	−7.444	1.987	THR 600(H-donor), TYR446(pi-H), TYR446(pi-H)
11f	−7.741	1.825	THR 600(H-acceptor), THR 600(H-acceptor), GLY640(H-donor)
RB6	−6.9862	1.0736	THR 600(H-acceptor), ASN464(H-acceptor), GLU447(H-donor), TYR519(H-donor)

, Figure 8. The receptor residues that were mainly involved in most interactions between the synthesized ligands and the active site were “THR 600, HIS583, GLN521, SER462 and TYR446”, Table 9.

2.7. Structure Activity Relationships Study (SARs)

From the previous biological study for the synthesized conjugates, 10 and 11 against Gram-positive and Gram-negative bacteria with variable concentration were all shown to have significant activity compared to the reference drugs ampicillin and chloramphenicol as well as higher activity than isatin and quinoline scaffolds, which affirmed the synergistic effect of these conjugates.

For conjugates of type 10, it was noticeable that 10b showed the best activity against MRSA where the two-carbon spacer between quinoline and isatin moieties is attached; the same activity with 10e was seen with C-5 of the isatin core bearing the bromine atom with the same two-carbon spacer. On the other hand, conjugate 10a showed the best activity against Streptococcus mutans and Klebsiella pneumoniae compared to the other analogues of 10. It is worthy of note that 10a containing a hydrazine spacer has higher activity than 10d having the same skeleton but containing the bromo atom at C-5 of the isatin core. Meanwhile, conjugates 10c and 10f, which have a three-carbon spacer with or without a bromo atom at C-5 of the isatin core, respectively, have more significant activity than the other conjugates of type 10 against Serratia marcescens. For the series of conjugates 11, it is noticeable that 11a has significant activity against all Gram-positive and Gram-negative bacteria compared to the other conjugates of type 11. Comparing the conjugates of 10 and 11 with each other, it is worthy of note that 11a, having Acyl-methyl hydrazinyl without the carbon spacer, is effective against all tested bacteria stains compared to 10a containing the hydrazine spacer.

2.8. In silico Pharmacokinetic Assessment

The assessment of pharmacokinestic properties was performed on the prepared compounds 10a–f and 11a–f, using both Molsoft and Swiss ADME [38]. In reference to Lipinski’s rule of five, they all had no Lipinski’s rule violations except in the molecular weight of compounds 10e, 10f and 11c–f, which exceeded 500. The lipophililicity of the tested compounds recorded ilog p < 5, which indicated their strong tolerance by cell membranes [39]. Topological polar surface area (TPSA) values ranged from 74 to 115. The number of hydrogen bond donors (HBD) was either two or four, and the number of hydrogen bond acceptors (HBA) ranged from four to six acceptors. Interestingly, all compounds showed high GI absorption ability. Moreover, the blood brain barrier (BBB) score was between zero and six as referenced [40], recording a minimum value of 2.25 and a maximum of 4.01,

Table 10. Results recorded by compounds 10a–f and 11a–f upon in silico pharmacokinetic screening.

Molecule	MWt	HBA	HBD	TPSA	iLogP	GI Absorption	BBB Score	Drug Likeness Score	Bioavailability Score
10a	394.85	4	2	74.33	2.98	High	3.84	0.53	0.55
10b	422.91	4	2	74.33	3.36	High	3.39	0.47	0.55
10c	436.93	4	2	74.33	3.55	High	3.76	0.47	0.55
10d	473.75	4	2	74.33	3.27	High	4.01	0.31	0.55
10e	501.8	4	2	74.33	3.61	High	3.54	0.26	0.55
10f	515.83	4	2	74.33	3.85	High	3.89	0.25	0.55
11a	466.92	6	4	115.46	2.41	High	2.36	0.66	0.55
11b	494.97	6	4	115.46	3.1	High	2.34	0.59	0.55
11c	509	6	4	115.46	3.15	High	2.30	0.57	0.55
11d	545.82	6	4	115.46	2.53	High	2.31	0.44	0.55
11e	573.87	6	4	115.46	2.69	High	2.30	0.39	0.55
11f	587.9	6	4	115.46	3.45	High	2.25	0.37	0.55

. Analyzing the drug likeness of the compounds, good drug-like candidates are considered to express positive drug-likeness scores [41], and as illustrated below in Table 10, positive values ranging from 0.25 to 0.66 were recorded by all the investigated compounds, among which compound 11a was the most promising, recording a score of 0.66. All the investigated compounds showed good bioavailability scores and can be considered promising “drug-like” molecules, Table 10.

3. Materials and Methods

3.1. General

All NMR analyses with the Bruker magnet system 400’54 Ascend/R (USA) 400 MHz and 100 MHz for ¹H-NMR and ¹³C-NMR, respectively, as well as FT-IR spectroscopy, were performed with Alpha-Bruker ATR mode (USA) at Zagazig University. The chemical shift of CDCl₃ was recorded at 7.26 ppm. Mass spectrum was performed on Direct Inlet part to mass analyzer in GCMS model with ISQ single quadrupole thermoscientific Electron Impact mode (UK) at the Regional Center for Mycology and Biotechnology, Al-Azhar University. Melting points (m.p.) were determined using the Stuart scientific melting point apparatus and are uncorrected. The in vitro antibacterial screening was executed at the Department of Botany and Microbiology, Faculty of Science, Menoufia University. Without further purification, the solvents were used as received. Commercially available starting materials as isatin derivatives 1a,b, free diamines 5a–c and 4.7-dichloro quinoline 4 were purchased from Acros and Sigma Aldrich, Germany. Synthetic starting materials such as N-(bromobutyl) isatin derivatives 3a–c, aminoquinoline derivatives 6a–c and intermediates 8a–c were synthesized as illustrated [32,33,42,43]. Mueller Hilton broth (Becton Dickinson, Sparks, MD, USA), nutrient broth, Tryptic soy broth (TSB) and nutrient agar were purchased from (Bacto), DMSO was purchased from R&M Marketing, Essex, UK. Iodonitrotetrazolium chloride (INT), as well as the first line antibiotics chloramphenicol, ampicillin and vancomycin hydrochloride were purchased from Sigma Aldrich, Germany. Other materials included the 96-wells microplate reader, 96-wells microtiter, 15 mL centrifuge tubes, which contained glucose and stimulate biofilm formation especially with Methicillin-resistant Staphylococcus aureus, Aqueous crystal violet (0.1% w/v), Glacial acetic acid (30% v/v), 1 × Phosphate buffer saline, pipette, as the positive control in antibiofilm test.

3.1.1. Bacterial Strains

Clinical bacterial isolates were obtained from the National Research Center. Four different microbial (ATCC reference) cultures and four clinical isolates were used in all of the experiments.

3.1.2. Reference Strains

Methicillin-resistant Staphylococcus aureus, MRSA (ATCC 43300), Streptococcus mutans ATCC 35668, Klebsiella pneumoniae ATCC 700603 and Serratia marcescens ATCC13880.

3.1.3. Clinical Isolates

Staphylococcus aureus, MRSA, Streptococcus mutans, Klebsiella pneumonia and Serratia marcescens.

3.2. Chemistry

3.2.1. General Procedure for Synthesis of 4-Bisamino Quinoline Hydrazine Derivatives 9

To (1 mmol) of 8a–c was added excess amount (3 mmol) of hydrazine, with stirring for 3h at room temperature (25 °C) in presence of (1 mL) of DMF until the consumption of the reactants, monitoring throughout by TLC. After the completion of the reaction, the reaction mixture was poured into ice water. The obtained products 9a–c were filtered, washed off and recrystallized from methanol.

N-(6-chloronaphthalen-1-yl)-2-hydrazineylacetohydrazide (9a)

Pale yellow solid, yield (0.42 g, 71%), m.p = 160–162 °C, FT–IR (KBr) cm⁻¹ υ: 3277 (NH), 3123 (NH₂), 2940 (CH), 1725 (C=O), 1628 (C=C_Ar), 1578 (C=N), 812 (C-Cl). ¹H NMR (CDCl₃, 400 MHz) ppm δ: 3.77 (s, 2H, CH₂), 6.82–7.84 (m, 4H, CH_Ar), 8.38 (br.m, 1H, CH=N_Ar), 8.52 (br.m, 2H, NH₂), 8.95 (br.m, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 62.88, 111.85, 116.86, 118,70, 119.36, 121.33, 138.64, 149.11, 157.75, 178.31. EI–MS m/z (C₁₂H₁₃ClN₄O): calcd., 265; found: 265 [M]⁺, 267 [M+2H]⁺.

N-(2-((6-chloronaphthalen-1-yl)amino)ethyl)-2-hydrazineylacetamide (9b)

Pale yellow solid, yield (0.48 g, 72%), m.p = 171–173 °C, FT–IR (KBr) cm⁻¹ υ: 3338 (NH), 3179 (NH₂), 2939 (CH), 1735 (C=O), 1632 (C=C_Ar), 1587 (C=N), 869 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 3.16 (br.s, 2H, CH₂), 3.38 (s, 2H, CH₂), 3.78 (br.s, 2H, CH₂), 7.36–8.18 (m, 4H, CH_Ar), 8.77 (br.s, 1H, CH=N_Ar) 8.78 (br.m, 2H, NH₂), 8.91 (br.m, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 39.52, 51.09, 53.52, 121.44, 125.65, 128.50, 128.84, 136.80, 140.75, 143.11, 149.08, 150.71, 175.15. EI–MS m/z (C₁₄H₁₇ClN₄O): calcd., 293; found: 293 [M]⁺, 295 [M + 2H]⁺.

N-(3-((6-chloronaphthalen-1-yl)amino)propyl)-2-hydrazineylacetamide (9c)

Off-white solid, yield (0.46 g, 70%), m.p = 185–187 °C, FT–IR (KBr) cm⁻¹ υ: 3411 (NH+NH₂), 2940 (CH), 1727 (C=O), 1662 (C=C_Ar), 1595 (C=N), 817 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.89 (t, 2H, CH₂, J = 8Hz), 3.48 (br.s, 4H, 2CH₂), 3.63 (t, 2H,CH₂, J = 8Hz), 4.16 (br.s, 2H, NH₂), 7.29–8.28 (m, 4H, CH_Ar), 8.45 (d, 1H, CH=N_Ar), 8.73 (br.s 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 22.68, 29.87, 36.59, 55.34, 116.51, 119.34, 121.51, 130.52, 131.08, 131.68, 132.14, 142.42, 172.47. EI–MS m/z (C₁₅H₁₉ClN₄O): calcd., 307; found: 307 [M]⁺, 309 [M+2H]⁺.

3.2.2. General Procedure for Synthesis of Conjugates of 10 and 11

To (1 mmol) 3a,b equimolar ratio of aminoquinoline derivatives 6a–c or 9a–c (1 mmol) was added in presence of excessive amount (3mmol) of triethyl amine and 3 mL of DMF under reflux for 4h to afford the target conjugates 10a–f and 11a–f after the consumption of the reactants. The reaction mixture was poured into ice water then filtered off and recrystallized from methanol.

1-(4-(2-(7-chloroquinolin-4-yl)hydrazineyl)butyl)indoline-2,3-dione (10a)

Brown solid, yield (0.26 g, 64%), m.p = 205–207 °C, FT–IR (KBr) cm⁻¹ υ: 3374 (NH), 2930 (CH), 1710 (C=O), 1675 (C=O), 1607 (C=C_Ar), 1562 (C=N), 1146 (C-C), 745 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.90 (br.m, 4H, 2CH₂), 3.79 (br.m, 4H, CH₂ + N-CH₂), 6.89–8.12 (m, 8H, CH_Ar), 8.54 (br.s, 1H, CH=N_Ar), 13.22 (s, 1H, NH), 13.56 (s, 1H, NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 31.65, 36.95, 41.65, 99.76, 103.84, 117.85, 124.31, 125.33, 125.97, 126.34, 137.52, 150.23, 153.10, 167.35, 183.20. EI–MS m/z (C₂₁H₁₉ClN₄O₂): calcd., 394; found: 394 [M]⁺, 396 [M+2H]⁺.

1-(4-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)butyl)indoline-2,3-dione (10b)

Reddish-orange solid, yield (0.27 g, 71%), m.p = 215–217 °C, FT–IR (KBr) cm⁻¹ υ: 3319 (NH), 2936 (CH), 1703 (C=O), 1665 (C=O), 1608 (C=C_Ar), 1577 (C=N), 1224 (C-C), 748 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.95 (br.m, 4H, 2CH₂), 3.47 (br.m, 4H, 2CH₂), 3.78 (br.m, 2H, CH₂), 3.88 (br.m, 2H, N-CH₂), 6.90–8.39 (m, 8H, CH_Ar), 8.77(br.s, 1H, CH=N_Ar), 13.80 (s, 1H, NH), 13.87 (s, 1H, NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.09, 25.90, 29.61, 34.79, 35.89, 44.81, 110.67, 117.37, 123.10, 124.42, 124.94, 126.87, 130.15, 136.24, 138.11, 142.79, 150.61, 158.17, 183.40. EI–MS m/z (C₂₃H₂₃ClN₄O₂): calcd., 422; found: 422 [M]⁺, 424 [M+2H]⁺.

1-(4-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)butyl)indoline-2,3-dione (10c)

Pale yellow solid, yield (0.25 g, 73%), m.p = 232–234 °C, FT–IR (KBr) cm⁻¹ υ: 3397 (NH), 2931 (CH), 1713 (C=O), 1654 (C=O), 1609 (C=C_Ar), 1559 (C=N), 1231 (C-C), 750 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.85 (br.m, 6H, 3CH₂), 3.24 (m, 4H, 2CH₂), 3.77 (m, 2H, CH₂), 4.28 (br.m, 2H, N-CH₂), 6.79–8.18 (m, 8H, CH_Ar), 8.55 (br.s, 1H, CH=N_Ar), 13.38 (s, 1H, NH), 13.72 (s, 1H, NH).¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.43, 29.69, 31.33, 36.47, 39.50, 49.80, 110.15,116.70, 119.18, 121.77, 123.93, 125.58, 127.10, 133.60, 138.57, 140.48, 155.49, 162.39 184.53. EI–MS m/z (C₂₄H₂₅ClN₄O₂): calcd., 436; found: 436[M]+, 438 [M+2]⁺.

5-bromo-1-(4-(2-(7-chloroquinolin-4-yl)hydrazineyl)butyl)indoline-2,3-dione (10d)

Brown solid, yield (0.35 g, 76%), m.p = 208–210 °C, FT–IR (KBr) cm⁻¹ υ: 3360 (NH), 2933 (CH), 1732 (C=O), 1655 (C=O), 1606 (C=C_Ar), 1567 (C=N), 1253 (C-C), 808 (C-Cl), 660 (C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.33 (br.m, 4H, 2CH₂), 3.26 (br.m, 2H, CH₂), 3.91 (br.m, 2H, N-CH₂), 7.42–8.07 (m, 7H, CH_Ar), 8.82 (br.s, 1H, CH=N_Ar), 13.58 (s, 1H, NH), 13.71 (s, 1H, NH). ¹³C– NMR (CDCl₃, 100 MHz) ppm δ: 29.68, 31.43, 36.47, 39.49, 116.75, 121.26, 121.38, 122.32, 123.92, 124.66, 125.55, 127.06, 127.65, 129.06, 129.30, 130.54, 131.08, 133.26, 138.58, 140.80, 158.24, 162.55, 183.29. EI–MS m/z (C₂₁H₁₈BrClN₄O₂): calcd., 473; found: 473[M]⁺, 475 [M+2]⁺.

5-bromo-1-(4-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)butyl)indoline-2,3-dione (10e)

Reddish-brown solid, yield (0.30 g, 72%), m.p = 212–214 °C, FT–IR (KBr) cm⁻¹ υ: 3312 (NH), 2949 (CH), 1731 (C=O), 1657 (C=O), 1604 (C=C_Ar), 1555 (C=N), 1269 (C-C), 815 (C-Cl), 577(C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.93 (br.m, 4H, 2CH₂), 3.35 (br.m, 4H, 2CH2), 3.45 (br.m, 2H, CH₂), 3.81 (br.m, 2H, N-CH₂), 6.75–8.21 (m,7H, CH_Ar), 8.81 (br.s, 1H, CH=N_Ar), 13.17 (s, 1H, NH), 13.48 (s, 1H, NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.26, 35.07, 39.52, 53.44,111.82, 116.88, 121.45,125.05, 125.64, 128.38, 128.69, 128.76, 136.54, 140.78, 142.72,149.46, 151.02, 167.17, 181.95. EI–MS m/z (C₂₃H₂₂BrClN₄O₂): calcd., 501; found: 501 [M]⁺, 503 [M+2H]⁺.

5-bromo-1-(4-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)butyl)indoline-2,3-dione (10f)

Violet solid, yield (0.30 g, 70%), m.p = 230–232 °C, FT–IR (KBr) cm⁻¹ υ: 3343 (NH), 2939 (CH), 1705(C=O), 1653 (C=O), 1605(C=CAr), 1587(C=N), 1161(C-C), 808 (C-Cl), 585(C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.82 (br.m, 6H, 3CH₂), 2.67 (m, 4H, 2CH₂), 3.75 (t, 2H, CH₂, J = 8 Hz), 3.89 (t, m, 2H, N-CH₂, J = 8 Hz), 7.06–8.41 (m, 7H, CH_Ar), 8.65 (d, 1H, CH=N_Ar, J = 8 Hz), 13.22 (s, 1H, NH), 13.87 (s, 1H, NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.42, 31.43, 36.48, 39.49, 45.86, 108.34, 110.17, 116.15, 121.76, 123.91, 125.53, 126.93, 127.64, 131.06, 131.66, 133.19, 138.58, 150.68, 158.41, 165.91, 183.40. EI–MS m/z (C₂₄H₂₄BrClN₄O₂): calcd., 515; found: 515 [M]⁺, 517 [M+2H]⁺.

2-(2-(7-chloroquinolin-4-yl)hydrazineyl)-N’-(4-(2,3-dioxoindolin-1-yl)butyl)acetohydrazide (11a)

Brown solid, yield (0.24 g, 71%), m.p = 231–233 °C, FT–IR (KBr) cm⁻¹ υ: 3405 (NH), 2935 (CH), 1728 (C=O), 1668 (C=O), 1605 (C=C_Ar), 1577 (C=N), 1221(C-C), 813(C-Cl).¹H–NMR (CDCl3,, 400 MHz) ppm δ: 1.78 (br.m, 4H, 2CH₂), 3.46 (br.m, 4H, 2CH₂), 3.79 (br.s, 2H, N-CH₂), 7.44–8.27 (m, 8H, CH_Ar), 8.79 (d, 1H, CH=N_Ar, J = 8 Hz), 13.39 (s, 2H, 2NH), 13.65 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 22.78, 29.34, 29.80, 32.02, 59.91, 106.39, 123.67, 124.86, 127.80, 128.74, 129.14, 136.57, 152.24, 152.83, 171.88, 184.32. EI–MS m/z (C₂₃H₂₃ClN₆O₃): calcd., 466; found: 466 [M]⁺, 468 [M+2H]⁺.

2-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)-N’-(4-(2,3-dioxoindolin-1-yl)butyl)acetohydrazide (11b)

Reddish-orange solid, yield (0.27 g, 75%), m.p =224–226 °C, FT–IR (KBr) cm⁻¹ υ: 3293 (NH), 2933 (CH), 1721(C=O), 1664 (C=O), 1605 (C=C_Ar), 1577 (C=N), 1159 (C-C), 814 (C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.84 (br.m, 4H, 2CH₂), 3.38 (br.m, 4H, 2CH₂), 3.42 (br.m, 4H, 2CH₂), 3.79 (br.m, 2H, N-CH₂), 7.48–8.12 (m, 8H, CH_Ar), 8.77 (s, 1H, CH=N_Ar), 13.70 (s, 2H, 2NH), 13.78 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.62, 24.99, 39.73, 53.50, 57.30, 102.79, 110.12, 120.49, 121.54, 123.94, 125.73, 127.14, 128.86, 129.15, 151.12, 162.62, 176.13, 184.31. EI–MS m/z (C₂₅H₂₇ClN₆O₃): calcd., 494; found: 494 [M]⁺, 496 [M+2H]⁺.

2-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)-N’-(4-(2,3-dioxoindolin-1-yl)butyl) acetohydrazide (11c)

Brown solid, yield (0.25 g, 71%), m.p = 221–223 °C, FT–IR (KBr) cm⁻¹ υ: 3343 (NH), 2933 (CH), 1705 (C=O), 1676 (C=O), 1609 (C=C_Ar), 1566 (C=N), 1146(C-C), 745(C-Cl). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.83 (br.m, 6H, 3CH₂), 2.11 (br.m, 2H, CH₂), 3.30 (br.m, 2H, CH₂), 3.63 (br.m, 2H, CH₂), 3.74 (m, 2H, CH₂), 3.86 (m, 2H, N-CH₂), 6.89–8.07 (m, 8H, CH_Ar), 8.78 (s, 1H, CH=N_Ar), 13.67 (s, 2H, 2NH), 13.73 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 25.68, 31.65, 35.41, 36.95, 43.50, 53.95, 57.61, 103.50, 103.83, 110.81, 121.36, 122.55, 122.93, 124.53, 125.40, 128.05, 128.15, 128.59, 128.91, 130.99, 131.79, 137.16, 138.30, 149.91, 152.50, 152.94, 170.29, 182.65. EI–MS m/z (C₂₆H₂₉ClN₆O₃): calcd., 509; found: 509 [M]⁺, 511 [M+2H]⁺.

N’-(4-(5-bromo-2,3-dioxoindolin-1-yl)butyl)-2-(2-(7-chloroquinolin-4yl)hydrazineylacetohydrazide (11d)

Brown solid, yield (0.29 g, 75%), m.p = 232–234 °C, FT–IR (KBr) cm⁻¹ υ: 3352 (NH), 2937 (CH), 1710 (C=O), 1657 (C=O), 1679 (C=CAr), 1564 (C=N), 1151(C-C), 807 (C-Cl), 645 (C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.85 (br.m, 4H, 2CH₂), 3.77 (br.m,4H, 2CH₂), 4.20 (br.s, 2H, N-CH₂), 6.82–7.84 (m, 7H, CH_Ar), 8.38 (s, 1H, CH=N_Ar), 13.63 (s, 2H, 2NH), 13.71 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.27, 25.95, 29.69, 39.54, 62.88, 111.85, 116.86, 118.70, 119.36, 121.48, 128.34, 130.21, 131.45, 133.39, 140.77, 149.22, 157.75, 160.91, 178.31, 181.96. EI–MS m/z (C₂₃H₂₂BrClN₆O₃): calcd., 436; found: 545[M]⁺, 547[M+2H]⁺.

N’-(4-(5-bromo-2,3-dioxoindolin-1-yl)butyl)-2-((2-((7-chloroquinolin-4-yl)amino)ethyl)amino)acetohydrazide (11e)

Reddish-brown solid, yield (0.30 g, 73%), m.p = 227–230 °C, FT–IR (KBr) cm⁻¹ υ: 3324 (NH), 2938 (CH), 1715 (C=O), 1654 (C=O), 1606 (C=C_Ar), 1556 (C=N), 1174 (C-C), 815 (C-Cl), 679 (C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.85 (br.m, 4H, 2CH₂), 3.17 (br.m, 2H, CH₂), 3.34 (m, 4H, 2CH₂), 3.49 (br.s, 2H, CH₂), 3.79 (br.m, 2H, N-CH₂), 7.46–8.18 (m, 7H, CH_Ar), 8.77 (s, 1H, CH=N_Ar), 13.52 (s, 2H, 2NH), 13.66 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.26, 25.78, 39.52, 51.09, 53.52, 111.79, 121.44, 125.07, 125.65, 128.50, 128.84, 136.80, 140.75, 143.11, 149.08, 150.71, 170.19, 184.52. EI–MS m/z (C₂₅H₂₆BrClN₆O₃): calcd., 573; found: 573 [M]⁺, 575[M+2H]⁺.

N’-(4-(5-bromo-2,3-dioxoindolin-1-yl)butyl)-2-((3-((7-chloroquinolin-4-yl)amino)propyl)amino) acetohydrazide (11f)

Violet solid, yield (0.26 g, 72%), m.p = 235–237 °C, FT–IR (KBr) cm⁻¹ υ: 3365 (NH), 2937 (CH), 1731(C=O), 1651 (C=O), 1607 (C=C_Ar), 1555 (C=N), 1291(C-C), 815 (C-Cl), 679(C-Br). ¹H–NMR (CDCl₃, 400 MHz) ppm δ: 1.92 (m, 6H, 3CH₂), 3.16 (m, 2H, CH₂), 3.48 (m, 4H, 2CH₂), 3.78 (br.m, 2H, CH₂), 4.31 (m, 2H, N-CH₂), 6.83–8.18 (m, 7H, CH_Ar), 8.77 (s, 1H, CH=N_Ar), 13.41 (s, 2H, 2NH), 13.74 (s, 2H, 2NH). ¹³C–NMR (CDCl₃, 100 MHz) ppm δ: 24.26, 25.86, 29.70, 39.52, 51.13, 53.58, 111.80, 121.45, 125.08, 125.66, 128.36, 128.92, 136.93, 140.75, 143.32, 148.87, 150.55, 157.75, 178.83, 183.90. EI–MS m/z (C₂₆H₂₈BrClN₆O₃): calcd., 587; found: 587 [M]⁺, 589 [M+2H]⁺.

3.3. Antibacterial Screening

In order to test the antibacterial activity of our conjugates, we performed preliminary screening for them by the agar-well diffusion method [44]. For all tested isolates, we began with 1.5 × 10⁸ CFU/mL (colony-forming units) (0.5 McFarland scale) inoculum. Under aseptic conditions, nutrient agar was poured into sterilized petri plates. Then, wells were made by sterile cork borer (6 mm in diameter) into agar plates after inoculation and 100 µL of each compound (40 mg/mL) was added to the wells. Finally, we incubated the plates at 37 °C for 20 h. We detected antibacterial activity by measuring the inhibition zone (including the well diameter) appearing after the incubation period. The DMSO was employed as a negative control. Positive control experiments were conducted to determine the sensitivity of the microorganisms: ampicillin (Sigma-Aldrich) and chloramphnicol (Sigma-Aldrich, St. Louis, MO, USA). The whole experiment was performed in triplicate.

3.3.1. Minimum Inhibitory Concentration (MIC)

In this assay, the microtitre broth dilution method was adopted for all compounds. The MIC of the tested conjugates was determined by serially diluting them according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [45]. To initiate, 1.5mL of compound stock solution in microcentrifuge tubes (Eppendorff) was prepared by dissolving powder to a final concentration of 20 mg/mL in dimethylsulphoxide (DMSO). Mueller-Hinton broth (Becton Dickinson, Sparks, MD, USA) was used to make serial dilutions from the stock solution ranging from 10 mg/mL to 0.004 mg/mL in 96-well microplates. A 24 h culture plate produced a bacterial suspension containing approximately 1.5108 CFU/mL (colony-forming units) (0.5 McFarland scale). One hundred liters of the prepared suspension was inoculated into each well. A sterility control well and a growth control well were investigated for each strain. The microtiter plates were incubated at 37 °C for 24 h. Then, after incubation at 37 °C, 40 μL of 0.4 mg/mL INT solution was added to each well as an indicator of microbial growth. After 30 min of incubating the plates at 37 °C, the MIC values were determined visually. The minimum inhibitory concentration was determined to be the lowest concentration of each extract that showed no visible growth. The MIC value was determined by taking the concentration that completely inhibited bacterial growth (the first clear well). To confirm activity, MIC values were determined in triplicate. Mueller-Hinton broth (Becton Dickinson, Sparks, MD, USA) was used in 96-well microplates to determine the final concentrations, ranging from 10 mg/mL (row A1) to 0.004 mg/mL (row A12). To ensure sterility, a negative control experiment was performed using only DMSO and MHB.

3.3.2. Minimum Bactericidal Concentration (MBC)

The microtitre broth dilution method was also chosen for MBC determination. It was the lowest compound concentration, killing most (99.9%) of the bacterial inocula after 24 h incubation at 37 °C. The calculation of MBC was carried out using the Ozturk and Ercisli method [46]. It was performed on all conjugates tested. Under aseptic conditions, we took ten microliters from the MIC experiment well (MIC value) and two wells above the MIC value well and spread them on MHA plates. After 18–24 h of incubation at 37 °C, the colony count was determined. The MBC value was influenced by the concentration of sample that produced ten colonies. Each experiment was performed in triplicate.

3.3.3. Time-Kill Assay

Only compounds with the lowest MBC/MIC ratio were considered [47]. It was carried out against MRSA clinical strain using the microplate method [48]. Samples and inoculums were prepared, 100 μL of sample in each well was filled with 100L of microbial broth at a concentration of 1.5108 CFU/mL (colony-forming units) (0.5 McFarland scale), grown in Mueller Hilton Broth (MHB). This resulted in a total volume of 200 μL of the culture in each well. The plates were then incubated at 37 °C for 18 h, with optical density monitored every 1 h at a wavelength of 600 nm. Turbidity was plotted against time on a graph. The obtained growth rate was investigated for bactericidal effects of the compounds.

3.4. Anti-Biofilm Assay

3.4.1. Bacterial Culture Preparation

Three to five pure colonies of MRSA ATCC43300 were inoculated from the culture plate into 15 mL TSB, then the bacteria were revived in the shaker incubator at 200 rpm and at 37 °C for 18 to 24 h prior to the experiment so that they were preferably in their log phase of growth. Sterile TSB was used by autoclaving TSB at 121 °C, 1.5 atm for 20 min.

3.4.2. Anti-Biofilm Assay

Bacterial suspension (1.5 × 10⁸ CFU/mL equivalent to UV absorbance reading of 0.1 with wavelength at 600 nm) was prepared in 15 mL from a 24 h bacterial culture. A 1:100 dilution was made in a separate centrifuge tube to obtain a 106 CFU/mL bacterial suspension. One hundred microliters of the diluted bacterial suspension was inoculated in TSB into the respective well of a new 96-well microplate then sterile distilled water was added to the 4 corners of the microplate to prevent evaporation of water from the test wells. Evaporation of water in test wells can interfere with the results. Alternatively, the microplate can be kept in a container with moist filter paper during the incubation. The plate was incubated at 37 °C in the incubator for 24 h. After incubation, the TSB broth was decanted completely from the microplate, the well was gently washed without disrupting the biomass formed, attaching on the bottom and wall of the wells with sterile phosphate buffer saline (PBS) 3 times. After that, 100 µL of freshly prepared sterile TSB broth (control well) was added in, and tested compounds suspended in TSB with test concentrations and TSB containing the vancomycin in test concentration. Vehicle control in DMSO is also needed to be aliquoted into appropriate wells as it is used as the diluent for the tested substances. The plate was covered with the lid and the plate was placed in incubator at 37 °C for 18 to 24 h. The 96-well plate was taken out of incubator and the TSB was slowly removed either by decanting or pipetting. The plate was rinsed with sterile double-distilled water and allowed to air-dry under the biosafety cabinet. The plates were turned upside down to speed up the process of drying. Dryness was ensured before moving on to the next step.

3.4.3. Staining the Biofilm with Crystal Violet

One hundred microliters of aqueous crystal violet (1% w/v) was dispensed into the test wells to stain the bacterial cell walls for 10 to 15 min. The crystal violet was decanted onto clean disposable tissues. The test wells were rinsed three times with sterile double-distilled water and allowed to dry under the biosafety cabinet. Alternatively, the plate can be bathed subsequently with 3 dishes of water. Thirty percent (v/v) glacial acetic acid was dispensed in water to solubilize crystal violet and it was left standing for 15 min. It was ensured that there was clear violet solution with no visible residue in each of the test wells. Finally, the UV absorbance of all the wells at 570 nm was read. The anti-adherence activity of test substance and vancomycin were calculated using the following formula:

Anti-adherence activity% = 100 × ((Absorbance of control-Absorbance of test sample)/Absorbance of control)

3.5. Molecular Assay by Transmission Electron Microscopy (TEM)

Observation of TEM microscopy samples of MRSA was done by preparing slices of selected MRSA isolate. Bacterial samples were fixed for 1 h with pH 7.4 at 4 °C, immersed in 2.5% of glutaraldehyde (GA) and 0.1 M of sodium cacodylate buffer, then washed in cacodylate buffer. All bacterial samples were double-fixed in cacodylate buffer containing 1% of osmium tetroxide (OsO₄) for 90 min at room temperature. Furthermore, we dehydrated the samples in acetone and the dehydrated cells were embedded in Epon-Araldite (502 kit, Pelco, CA, USA). After that, 500–1000 nm sections of bacterial samples were obtained using a Leica EM UC6 ultra-microtome (Wetzlar, Germany) mounted on glass slides and stained with 1% toluidine blue stain. All sample sections were scanned and investigated using a JEM 1011 (JEOL) electron microscope set to 80 kv [49].

3.6. Molecular Modeling

The targeted enzyme was downloaded from Protein Data Bank along with its bounded co-crystallized ligand (PDB:4DKI). Preparation of the downloaded protein was performed following the literature [50]

For optimization of the synthesized compounds, all structures were copied as smiles from chem-draw, bonds and restraints were enabled, calculating of partial charges was performed and compounds were saved upon energy minimization in mol2 format.

The protocol for docking was performed using MOE software [51] following the reported procedures [41]

4. Conclusions

New conjugates 10a–f and 11a–f bearing quinoline and isatin scaffolds were prepared in good yields. The structures of the conjugates were established based on spectroscopic data. The antibacterial activity for all synthesized conjugates was evaluated and displayed activity higher than the first line antibiotics ampicilin and chloramphnicol used in this study. In addition, the synthesized conjugates exhibited good to excellent bactericidal and anti-biofilm activity. Moreover, the TEM study proved their mechanism of action where a damage in both cell wall and membrane was observed with the appearance of damaged vacuoles in cell cytoplasm. Molecular docking studies were performed on the prepared compounds 10a–f and 11a–f, which showed promising binding affinity for the transpeptidase receptor active site in MRSA and the main interactions between the synthesized ligands and active site involved THR 600, HIS583, GLN521, SER462 and TYR446. The range of binding affinity was −8.098 to −6.501 and the RMSD values ranged from 1.184 to 2.134 A. Compounds 10a–f and 11a–f were subjected to in silico assessment for their pharmacokinetic properties; all the assessed compounds were considered as “drug-like” molecules with promising bioavailability.