Metal-free stereoselective C(sp³)–H indolation of N-heterocycles to potent antimicrobial non-canonical tryp–pro hybrids

Abstract

Indolyl aliphatic N-heterocycles are widely present as key structural units in many natural products and unnatural bioactive molecules. The known synthetic methods for indolyl N-heterocycles rely on metallic reagents/catalysts, hazardous oxidants, and a multistep process, often generating toxic byproducts. Herein, an unprecedented example of a metal- and oxidant-free stereoselective C(sp³)–H indolation of aliphatic N-heterocycles is reported. The C–H indolation reaction, which relies on a three-component condensation reaction, proceeds under operationally simple conditions and avoids the use of metallic reagents, oxidants, and pre-functionalization/functional group protection steps. The indolation was highly stereoselective, providing a single isomer of the six possible isomeric indolyl N-heterocycles with excellent enantiopurity (>99% ee). Interestingly, synthesized non-canonical tryptophan–proline hybrids constitute a new class of potent antibacterial agents that specifically target Gram-positive bacteria, including multidrug-resistant clinical isolates. These compounds are relatively non-toxic (SI > 20) to normal cells, have a low MIC (2 µg mL⁻¹), and exhibit a very low propensity to induce resistance.

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Green foundation

1. This work reports an unprecedented metal and oxidant-free diastereoselective C(sp³)–H indolation of alicyclic amines via a three-component condensation reaction of commercially available and cost-effective starting materials under operationally simple conditions.

2. Direct C(sp³)–H indolations of alicyclic amines were achieved, avoiding the use of hazardous metallic reagents/catalysts, oxidants, and involvement of multiple steps for pre-functionalizations, protection/deprotection. Additionally, unlike the hazardous byproducts produced by known methods, this method produces water as its main byproduct. Compared with related methods, better green metrics are achieved with enhanced atom economy (up to 200%) and reduced E-factor (69–87%).

3. Further research to develop a more reactive fluorenone derivative and to carry out the reaction under solvent-free conditions with a catalytic amount of an acid would make this reaction more efficient and greener.

Introduction

S. aureus is a well-known high-priority bacterial pathogen associated with skin and soft tissue infections and bacteremia. It is one of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) that are becoming resistant to almost all available antibiotics. Kharat et al. showed that of 20 177 ESKAPE isolates collected, 16.3% (3286) were S. aureus.¹ The drug-resistant strains of S. aureus, specifically, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), are resistant to most of the β-lactam drugs, as well as nafcillin, oxacillin, and flucloxacillin, which are specifically marked as anti-S. aureus drugs.² A rapid increase in the emergence of multidrug-resistant S. aureus (MDR-SA) highlights the urgent need to develop novel antibiotics to target these bugs.³ The synthesis and evaluation of the antibacterial potential of new scaffolds are being actively pursued for the development of new and effective antibacterial agents.⁴

However, identifying a new class of effective antibacterial agents remains challenging for several reasons, including the multistep synthetic processes required for complex molecules, associated cytotoxicity, and the propensity to develop resistance. Indole and the associated amino acid, tryptophan, are key participants in various bacterial processes, including plasmid stability, biofilm formation, and drug resistance.⁵ Indole moiety in the tryptophan-based antimicrobial peptide plays a crucial role in facilitating bacterial cell selectivity and antibacterial activity due to its hydrophobic effect and its ability to form π–π interactions.⁶ As with tryptophan, proline also influences various cellular processes.⁷ Importantly, proline serves as a carbon source during the growth of S. aureus.⁸ In addition, some organisms metabolize proline to create natural antibacterial and antifungal agents.⁹ Therefore, the non-canonical structural hybrid of indole/tryptophan and proline can be considered as an important molecular platform for developing new antibacterial agents. Here, we explored indolyl N-heterocycles, an unprecedented non-canonical tryptophan–proline (tryp–pro) hybrid derivatives to selectively target MDR S. aureus (Scheme 1a).

Scheme 1 Design of tryp–pro hybrid, natural products containing indolyl N-heterocycles and the known diastereoselective C–H indolations.

Arylated aliphatic N-heterocycles are found widely as the key structural unit of many natural products and synthetic bioactive molecules (Scheme 1b).¹⁰ In particular, many alkaloids are built on N-heterocycles attached to indoles or indolines, which could be derived from corresponding indolyl N-heterocycles. For relevant examples, (−)-actinophyllic acid contains α-indolyl pyrrolidine as one of the key structural fragments.¹¹ Aspidosperma alkaloids (e.g., vincadifformine and jerantinine) are constructed over indolinyl piperidine derivatives.¹² The C₃-position of the indoline moiety is connected with the α-position of piperidines. In contrast, piperidine is connected to the C₂-position of indolenines in the akuammiline family of alkaloids (e.g., strictamine).¹³ Other than natural products, medicinally important synthetic molecules, such as a 5-HT6 antagonist, are built on indolyl pyrrolidine.¹⁴

The widespread presence of enantioenriched indolyl aliphatic N-heterocycles in both biology and chemistry demands the development of a new, efficient, and atom- and step-economic method for their synthesis with application potential. Direct indolation reactions at C₁ of tetrahydroisoquinolines (THIQs) under various conditions have been studied extensively.¹⁵ Most of these reactions relied on metallic reagents and oxidants (e.g., IBX, mCPBA, etc.), which generate hazardous waste. As compared to the indolation of THIQ, indolation of aliphatic N-heterocycles having unactivated C–H bonds necessitates the use of harsh and sensitive reaction conditions involving strong bases and metallic reagents.¹⁶ Transition-metal-catalyzed (e.g., Pd, Ir, Ru, Ni, etc.) reactions in the absence or presence of stoichiometric amounts of a strong base (BuLi) and transmetalating agents (ZnCl₂) have been used for indolation of N-protected pyrrolidine (or pyrrolidine bearing a directing group).¹⁷ Indolation reaction of pyrrolidine under metal and oxidant-free conditions provides a mixture of regioisomeric products.¹⁸ Although examples of indolation of aliphatic N-heterocycles are known, very few examples of the diastereoselective indolation reaction are reported. Directing group-assisted Ru-catalyzed coupling reaction of N-protected pyrrolidine derivative and N-protected indolyl boronic ester has been developed (Scheme 1c(i)).¹⁹ The method requires the installation of amidine, pyridine and pyrimidine as a directing group and provides moderate yield and poor diastereoselectivity. Similarly, Pd-catalyzed coupling reactions using picolinoyl amide (Scheme 1c(ii))²⁰ and aminoquinoline amide (AQ) (Scheme 1c(iii) and (iv)) have been used for the indolation of N-heterocycles. Pre-formed imines derived from N-heterocycles, either via oxidation or intermolecular hydride transfer (in the presence of an organolithium compound), were also used for the diastereoselective indolation reaction.²¹ Similarly, amine N-oxide has been used as the substrate for the Cu-catalyzed diastereoselective indolation reaction.²² Preformation of imines via chemical and enzymatic oxidation of amines in the presence of hazardous oxidants, such as IBX and NBS, etc., which produce toxic byproducts, was essential to achieve indolation under metal-free conditions.^21b,23 In addition, the multistep synthetic sequence has been used for the synthesis of indolyl N-heterocycles during the total synthesis of natural alkaloids.²⁴ Therefore, the known diastereoselective indolation reactions have the limitation related to the involvement of a multistep process (installation of suitable directing groups, protection of amines and indole derivative, pre-functionalization of indoles, etc.), metallic reagents, oxidants, and sensitive reaction conditions. However, the report on the direct diastereoselective C–H indolation of substituted N-heterocycles under metal and oxidant-free conditions is not known. Herein, we report an unprecedented metal and oxidant-free highly stereoselective C–H indolation reaction via a single-step three-component condensation of unprotected aliphatic amines, indoles, and 9-fluorenone (Scheme 1d).

Results and discussion

Classical condensation of chiral alicyclic amine 1 with fluorenone or its imine derivatives 2 produces iminium ion 3, which readily isomerizes (via zwitterion 4) to the corresponding regioisomeric iminium ion 5 (Scheme 2).²⁵ We anticipated that the preferential reaction of iminium ion 5 (over ion 3) with indole derivatives 6 would lead to the direct indolation of N-heterocycles under metal and oxidant-free conditions to provide indolyl N-heterocycles 7. Indole derivatives react readily with carbonyl compounds due to their high tendency toward electrophilic aromatic substitution,²⁶ which may lead to the undesired side products 8 and 9. The higher reactivity of indoles may promote their reaction with the initial iminium ion 3 before its isomerization to 5, which will create the possibility of forming a mixture of undesired product 10 with the desired indolyl N-heterocycle 7. Therefore, these challenges need to be addressed to achieve the desired indolation reaction. Optimal reaction conditions facilitating the isomerization of 3 without promoting the electrophilic aromatic substitution reaction that leads to undesired isomer 10 need to be established. The nature and position of substituent R in the amine were expected to direct the facial approach of indole to the iminium ion 5 for controlling the diastereoselectivity.²⁷

Scheme 2 Reaction design for metal and oxidant-free stereoselective indolation of substituted pyrrolidines.

Our investigation began with the reaction of enantiopure (S)-prolinol with 9-fluorenone (2a) and indole in refluxing toluene (Table 1). However, the desired indolyl prolinol 12a could not be detected in the reaction mixture. Instead, the side product bis-indole 9 and unreacted starting materials were isolated. A similar reaction in the presence of 9H-fluoren-9-imine (2b), which is more reactive than 9-fluorenone, also failed to yield the desired product (entry 2). We then hypothesized that the presence of a proton source may facilitate the condensation of amine and ketone/imine, and help isomerize the iminium ion via protonation of zwitterion 4. Therefore, reactions of (S)-prolinol and indole with 9-fluorenone or its imine were carried out in the presence of acetic acid. As expected, the desired arylated prolinol 12a was isolated with 21–30% (entries 3 and 4) as a single syn-isomer with excellent enantiopurity (>99% ee). The reactions performed in the presence of other solvents (e.g., THF, dioxane, MeOH) and other acids (TsOH, H₃PO₄, PhCOOH, etc.) did not yield the desired product. After screening various reaction conditions, an improved yield of 12a (40%) was obtained from the reaction carried out with excess 9-fluorenone and acetic acid in refluxing benzene (entry 8). The reaction under microwave heating gave lower yields (entry 9). A significant increase in the yield of the desired product 12a was observed when the reaction was carried out in the presence of increased relative stoichiometry of (S)-prolinol (entries 10, 11 and 13). No significant increase in yield was observed with increased amounts of 9-fluorenone, AcOH, or reaction time (entries 12–14). Reactions in other apolar solvents (e.g., xylene, mesitylene, anisole) gave 12a with diminished yields (entries 15–17). The reaction parameters were further screened to obtain greener reaction conditions (Table S1). The reaction can be performed under solvent-free conditions; however, it yields slightly lower (entry 18). Reaction can also be performed at a lower temperature (at 110 °C–120 °C). The reaction carried out in the presence of a small quantity of toluene or butanol gave the best yield (entries 19 and 20).

Table 1 Screening of the reaction conditions

Entry	Conditions	Yield (%)
All reactions are carried out with 0.2 mmol of indole and 1 mL of solvent. Reactions under reflux (entries 1 to 8) were done without maintaining an inert atmosphere under conventional heating. Reactions from entry numbers 9 to 17 are performed under closed-vessel microwave (145 W) irradiation.a No solvent, heated at 75 °C for 2 min.b 0.2 mL toluene was added.c 0.1 mL of ^nBuOH was added, the power was slowly increased (7–9 W) for 15 min.
1	11 (1.2), 2a (1.2), no acid, toluene, reflux, 24 h	—
2	11 (1.2), 2b (1.2), no acid, toluene, reflux, 24 h	Trace
3	11 (1.2), 2a (1.2), AcOH (1), toluene reflux	21
4	11 (1.2), 2b (1.2), AcOH(1), toluene, reflux, 24 h	30
5	11 (1.2), 2a (1.2), H3PO4 (1), toluene, reflux, 24 h	—
6	11 (1.2), 2a (1.2), PTSA, (1), toluene, reflux, 24 h	—
7	11 (1.2), 2b (1.2), PhCOOH (1), toluene, reflux, 24 h	Trace
8	11 (1.2), 2a (5), AcOH (2.5), benzene, reflux, 48 h	40
9	11 (1.2), 2a (1.2), AcOH (1), toluene, MW, 145 °C, 45 min	28
10	11 (2.0), 2a (1.2), AcOH (1), toluene, MW, 145 °C, 45 min	53
11	11 (2.5), 2a (1.2), AcOH (1), toluene, MW, 145 °C, 45 min	62
12	11 (2.5), 2a (1.2), AcOH (1.6), toluene, MW, 145 °C, 45 min	38
13	11 (2.5), 2a (2), AcOH (1), toluene, MW, 145 °C, 45 min	62
14	11 (2.5), 2a (1.2), AcOH (1), toluene, MW, 145 °C, 60 min	63
15	11 (2.5), 2a (1.2), AcOH (1), xylene, MW, 145 °C, 45 min	33
16	11 (2.5), 2a (1.2), AcOH (1), mesitylene, MW, 145 °C, 45 min	26
17	11 (2.5), 2a (1.2), AcOH (1), anisole, MW, 145 °C, 45 min	25
18^a	11 (2.5), 2a (1.2), AcOH (1), MW, 120 °C, 40 min	43
19^b	11 (2.5), 2a (1.2), AcOH (1), MW, 120 °C, 40 min	63
20^c	11 (2.5), 2a (1.2), AcOH (1), MW, to 120 °C, 100 min	60

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The best conditions were then used to explore the substrate scope of this metal-free C–H indolation reaction. Structurally diverse indoles 15 were reacted with 2-substituted pyrrolidines 13, including L-prolinol and 3,4-disubstituted pyrrolidine derivatives 14, to obtain corresponding α-indolyl N-heterocycles 12a–v and 16a–c with good yields and excellent regio-and stereo-selectivity (Scheme 3). Like unsubstituted indole, the reaction of indoles containing electron-donating Me and –OMe groups with L-prolinol provided the desired product 12b–c and 12d as a single stereoisomer with good yields and excellent enantiopurity. Interestingly, in the case of the reaction with 5-OH indole, a 1 : 1 separable mixture of regioisomeric products 12e and 12e′, which originated through the indolation at C₃ and C₄ positions, respectively, was isolated with a combined yield of 92%. More challenging substrates bearing C₂ substitutions (Me, Ph, –CH₂–OH) on the indoles also participated in the reaction, providing the corresponding enantiopure arylated prolinols 12f–h with moderate to good yields. Indoles having halogen-substituents, which generally interfere in transition metal-catalyzed coupling reactions, reacted smoothly, furnishing desired products 12i–m. The reactions of indoles bearing other electron-withdrawing groups, such as CN and CO₂Me, afforded the corresponding indolyl prolinols 12n–q in good yields. However, a slightly lower yield of 12r was obtained from the reaction of indole containing a strong electron-withdrawing –NO₂ group. The reaction of pyrrole instead of indole also afforded the expected arylated prolinol 12s in good yield (63%) with a high enantiomeric excess (>99%).

Scheme 3 Substrate scope of the metal-free stereoselective C–H indolation reaction.

As with L-prolinol, excellent regioselectivity and enantiospecificity were observed for the reaction of O-methylated L-prolinol and 2-methyl pyrrolidine with indole (12t, 12u). The C–H indolation of ethyl L-prolinate under standard conditions yielded the desired product 12v in 60% yield and with excellent regioselectivity. However, the single syn-isomer 12v was isolated with 12% ee. In all other cases, the products were isolated as a single syn-diastereoisomer. The indolation of pyrrolidine derivatives with 3,4-disubstitution also proceeded smoothly to provide corresponding indolyl N-heterocycles 16a–c. Interestingly, in contrast to syn-isomers from the reaction of 2-substituted pyrrolidine, a single anti-isomers were obtained from the reaction of 3,4-disubstituted pyrrolidine. The relative stereochemistry was unambiguously determined through single-crystal X-ray analysis (12e′, 12g, 12v, 16a, 16c) and/or NOE experiments, and the enantiopurity was measured through HPLC analyses.

In the case of 2-substituted pyrrolidine, the iminium ion 17a initially formed in situ from the acid-mediated reaction of 2a and 11 underwent isomerization to yield a less-substituted iminium ion, 17b, out of the two possible regioisomeric iminium ions (17b and 17c, Scheme 4). Preferential formation of less substituted iminium ion 17b over 17c is probably to avoid A^1,3-strain, which is present in the more substituted regioisomer 17c. This allows retention of stereochemistry, leading to regioselective formation of enantioenriched product 17. The fluorenyl group of the iminium ion prefers to remain at the opposite face of R (as shown in 17d) to avoid steric interaction. Indoles approached from the less hindered face of the iminium ion 17b to provide the observed syn-isomer.¹⁴ However, indoles reacted through the less sterically hindered convex face in the case of 3,4-disubstituted pyrrolidines to provide exclusively anti-products.

Scheme 4 Reaction mechanism with the origin of regio- and stereoselectivity, controlled experiments, and synthetic diversifications.

Then we wanted to investigate the observation of reduced enantiomeric excess for 12v. The high thermodynamic acidity of the α-proton of the ester group likely facilitated the racemization of ethyl L-prolinate, resulting in the poor enantiopurity of 12v. Racemization of proline ester may proceed through the more substituted iminium ion corresponding to 17c. In that case, there would be a possibility of the formation of regioisomeric indolyl-proline ester, which was not identified. Controlled experiments were performed to better understand the mechanism of racemization of the proline ester. A reaction of enantiopure ethyl-L-proline ester in the presence of fluorenone and acetic acid was carried out under standard conditions (Scheme 4). Interestingly, the specific rotation of Boc-protected ethyl-L-proline ester 18 was found to be reduced from +46.40 to −1.11. A similar reduction (to +2.86) of specific rotation was observed after the reaction of ethyl-L-proline ester only in the presence of AcOH under standard conditions. These results indicated that racemization of the ethyl-L-proline ester likely occurred before the indolation reaction (Scheme 4).

The reaction could be carried out on a gram scale to obtain the indolyl prolinol derivative 12a with slightly reduced yield (3.27 g, 50%). The primary hydroxyl group of 12a could be readily oxidized via Swern oxidation to afford the corresponding aldehyde 19 with excellent yield. Incorporation of the Boc-group at the indole nitrogen, followed by removal of fluorene under Pd/C-catalyzed hydrogenolysis conditions, gave the desired chiral amino alcohol 20. Alternatively, silylation of the primary alcohol proceeded smoothly to give the desired chiral secondary amine 21 with very good yield. The reaction between secondary amine 21 and an isothiocyanate afforded the corresponding chiral thiourea 22 in excellent yield. Chiral thioureas are an important class of compounds for their well-known organocatalytic properties, which are used in various asymmetric reactions. Then, the non-canonical tryptophan–proline hybrid could be coupled with other amino acids, such as proline and tryptophan, under standard amide coupling conditions to furnish dipeptides 23 and 24, respectively, containing unnatural amino acid analogues.

The green chemistry parameters and related factors of this reaction were evaluated and compared with those of the best existing reaction (Table 2), as the same reaction was not known. In contrast to known methods, direct CH-indolation is achieved in a single step using this method, without the aid of hazardous metallic reagents/catalysts or toxic oxidants. The crucial green chemistry parameters, such as atom economy (AE: 80%) and E-factors (1.4), of this method are found to be much superior to those (AE: 40%, E-factor: ∼5) of the known best methods.

Table 2 Comparison of key green chemistry parameters and other related factors

Parameters	This work	Ref. 19	Ref. 21b	Ref. 22
Steps	1	2	2	3
Yield (%)	63	59	58	56
dr	Single isomer	1 : 5	—	93 : 7
AE (%)	80	37	40	44
E-Factor	1.4	4.1	4.48	5
Hazardous reagent/catalyst	None	Ru3(CO)12	IBX	mCPBA, CuBr2

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Evaluation of antibacterial potential

Antimicrobial activity against Gram-positive pathogens in the human pathogen panel

Metal-free diastereoselective direct C–H indolation of prolinol allowed the synthesis of a library of non-canonical structures of tryp–pro hybrid. The synthesized compounds were screened against a panel of Gram-positive and Gram-negative human pathogenic bacteria, including E. coli ATCC 25922, S. aureus ATCC 29213, K. pneumoniae BAA-1705, A. baumannii BAA-1605, Enterococcus faecium NRS 31912, and P. aeruginosa ATCC 27853 (Table 3). While the majority of the compounds are selectively active against Gram-positive S. aureus ATCC 29213, 12j was found to be active against both S. aureus ATCC 29213 and Enterococcus faecium NRS 31912. The extent of activity of these compounds varies significantly with the hybrid's structural features. The incorporation of a methyl group at C₅ of the indole (in 12c, MIC: 8 µg mL⁻¹) ring of the parent hybrid (12a, MIC: 16 µg mL⁻¹) resulted in a two-fold decrease in the MIC. In contrast, MIC of 12b (MIC: 32 µg mL⁻¹) increased by twofold when the methyl group was placed at the C₆ position. Similar MIC values were observed for the compounds having other functional groups such as –OH, –OMe, –CN, –NO₂ and –CO₂Et.

Table 3 MIC (µg mL⁻¹) and selectivity index (SI) of compounds against the ESKAPE pathogen panel

Compounds	MIC (µg mL^−1)						CC50 (µg mL^−1)	SI (CC50/MIC)
	S. aureus ATCC 29213	E. faecium NR 31912	K. pneumoniaeBAA-1705	A. baumanniiBAA-1605	P. aeruginosa ATCC 27853	E. coli ATCC 25922
ND: Not determined; CC50 is against Vero cells.
12a	16	ND	>64	>64	>64	>64	ND	ND
12b	32	ND	>64	>64	>64	>64	ND	ND
12c	8	ND	>64	>64	>64	>64	ND	ND
12d	64	ND	>64	>64	>64	>64	ND	ND
12e	32	ND	>64	>64	>64	>64	ND	ND
12e′	32	ND	>64	>64	>64	>64	ND	ND
12f	>64	ND	>64	>64	>64	>64	ND	ND
12g	>64	ND	>64	>64	>64	>64	ND	ND
12h	16	ND	>64	>64	>64	>64	ND	ND
12i	4	ND	>64	>64	>64	>64	ND	ND
12j	2	16	>64	>64	>64	>64	40	20
12k	2	ND	>64	>64	>64	>64	20	10
12l	2	ND	>64	>64	>64	>64	ND	ND
12m	>64	ND	>64	>64	>64	>64	ND	ND
12n	64	ND	>64	>64	>64	>64	ND	ND
12p	>64	ND	>64	>64	>64	>64	ND	ND
12r	16	ND	>64	>64	>64	>64	ND	ND
12s	64	ND	>64	>64	>64	>64	ND	ND
12u	>64	ND	>64	>64	>64	>64	ND	ND
12v	>64	ND	>64	>64	>64	>64	ND	ND
16a	8	ND	>64	>64	>64	>64	ND	ND
16b	32	ND	>64	>64	>64	>64	ND	ND
11	>64	ND	>64	>64	>64	>64	ND	ND
2a	>64	ND	>64	>64	>64	>64	ND	ND
Indole	>64	ND	>64	>64	>64	>64	ND	ND
Levofloxacin	0.0625	>64	64	4	0.5	0.0156	ND	ND

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The MIC could not be improved through the incorporation of substituents at the five-membered heterocyclic ring of the indole moiety (in 12f–h). Halogenated tryptophans bearing a halogen substituent on the indole ring are well known to serve as substrates for various enzymes in plants, animals, and bacteria.²⁸ Therefore, with anticipation of obtaining better MIC, we have synthesized non-canonical tryp–pro hybrids containing halogenated indole rings, and their antibacterial activities were tested. Pleasingly, as anticipated, the halogenated hybrids 12i, 12j, 12k, and 12l (bearing F, Cl, or Br substitution on the indole ring) were found to be the most potent candidates against Gram-positive S. aureus and Enterococcus, with MICs 4, 2, 2, and 2 µg mL⁻¹, respectively (Table 3). Interestingly, 12s showed no antibacterial activity when pyrrole was introduced in place of the indole moiety. This indicates the importance of the indole moiety for the observed antibacterial activity. The hybrids 12u and 12v, which have methoxy and ester groups, respectively, instead of the hydroxyl group in 12a, did not exhibit any antibacterial activity. Limited activity was observed for 16a and 16b, in which indole was linked to fused heterocycles as an alternative to prolinol. Studies also showed that the individual components (11, 2a, and indole) of the antibacterial tryp–pro hybrid are inactive against the bacteria tested.

The outer membrane is not a factor for the inactivity of 12j against GNB pathogens

One of the most common reasons for a lack of activity against Gram-negative bacterial pathogens is the inability of the compounds to penetrate the outer membrane (OM). This well-recognized permeability barrier prevents the entry of antibiotics and other xenobiotics. In this context, we determined the activity of 12j in the presence of OM-permeabilizing PMBN, along with controls, and the data are shown in Table 4. As shown, the addition of PMBN increased permeability, and consequently, the MICs decreased for both rifampicin and vancomycin, while the MIC of levofloxacin remained unchanged as expected. These results confirm that the antimicrobial activity of 12j is limited to Gram-positive organisms, making it species-specific, which is a key characteristic that helps avoid eliminating beneficial Gram-negative commensals and prevents microbiome dysbiosis.

Table 4 MIC (µg mL⁻¹) of compounds against S. aureus in the presence of PMBN

Bacterial strain	MIC (μg ml^−1)
	PMBN (10 µg ml^−1)	12j		Rifampicin		Vancomycin		Levofloxacin
		PMBN		PMBN		PMBN		PMBN
		(−)	(+)	(−)	(+)	(−)	(+)	(−)	(+)
Escherichia coli ATCC 25922	>64	>64	>64	4	0.0625	512	64	0.0312	0.0156
A. baumannii BAA-1605	>64	>64	>64	4	0.0625	128	32	8	8

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12j displays a high selectivity index against Vero cells

The two most potent compounds, 12j and 12k, were further assessed for cytotoxicity against Vero cells (ATCC CCL-81) along with doxorubicin as a positive control; the results are shown in Table 3. As shown, both compounds displayed >50% cell proliferation even at 10× MIC concentrations, with 12j displaying better safety profiles (CC₅₀ 40 mg L⁻¹) and a selectivity index of 20, as compared to 12k (Table 3). Therefore, the 12j was chosen to be progressed ahead based on the superior selectivity index.

12j is equi-potently active against clinical, MDR strains of S. aureus

As the next step, the antimicrobial activity of 12j was explored against drug-resistant clinical isolates of S. aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). This panel includes strains resistant to a variety of clinically used antibiotics, including vancomycin and meropenem. The data are tabulated in Table 5. 12j displayed equipotent activity against drug-resistant clinical MRSA and VRSA, with MICs of 2 mg L⁻¹, irrespective of the strain's antibiotic resistance, site of isolation, or virulence markers (Table 5). The potent activity of 12j against the drug-resistant, clinical S. aureus is a positive indication for further development.

Table 5 MIC (µg mL⁻¹) of 12j against clinical, drug-resistant S. aureus

Strain		Antibiotics resistant to	Molecular details of strains	MIC (µg mL^−1) of 12j
MSSA	ATCC 29213	None	Type strain	2
MRSA	NRS100	Methicillin, tetracycline	Contains subtype I mec cassette & a large variety of virulence factors	2
	NRS119	Methicillin, gentamicin, linezolid, trimethoprim/sulfamethoxazole	Contains subtype IV mec cassette & G2576T mutation in domain V in one or more 23S rRNA genes	2
	NRS129	Chloramphinecol	mecA negative	2
	NRS186	Methicillin, levofloxacin, meropenem	USA 300 type CA-MRSA, PVL virulence factor positive & contains mec type IV cassette	2
	NRS191	Methicillin, levofloxacin, meropenem	USA 600 type CA-MRSA, PVL virulence factor negative & contains mec type II cassette	2
	NRS192	Methicillin, levofloxacin, meropenem, erythromycin	CA-MRSA, PVL virulence factor negative & contains mec type II cassette	2
	NRS193	Methicillin, levofloxacin, meropenem	CA-MRSA, PVL factor negative & contains the mec type II cassette	2
	NRS194	Methicillin, meropenem	CA-MRSA, PVL virulence factor positive & contains mec type V cassette	2
	NRS198	Methicillin, levofloxacin, meropenem	USA 100 type CA-MRSA, PVL virulence factor negative & contains mec type II cassette	2
VRSA	VRS 1	Methicillin, levofloxacin, meropenem, vancomycin, gentamicin, teicoplanin & spectinomycin	USA 100, contains mec subtype II cassette & vanA, negative for vanB, vanC1, vanC2, vanD, vanE, PVL & ACME	2
	VRS 4	Methicillin, levofloxacin, meropenem, vancomycin, gentamicin, teicoplanin & spectinomycin	USA 100, contains mec subtype II cassette & vanA, negative for vanB, vanC1, vanC2, vanD, vanE, PVL & ACME	2
	VRS 12	Methicillin, levofloxacin, meropenem, vancomycin, gentamicin, teicoplanin & spectinomycin	Data not available	2

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Time kill kinetics of 12j against S aureus ATCC 29213

The time-kill kinetics of 12j were determined against S. aureus ATCC 29213, along with vancomycin at multiple concentrations and time points as a control to determine whether it exhibited bacteriostatic or bactericidal activity. The data are plotted in Fig. 1. When tested at 1× and 10× MIC, 12j at 1× MIC exhibited static activity, whereas at 10× MIC, it led to a ∼4 log₁₀ CFU mL⁻¹ reduction compared to the initial count. This activity is comparable to that of vancomycin at 10× MIC, which reduced ∼5 log₁₀ CFU mL⁻¹ compared to the initial count. Thus, 12j exhibits concentration-dependent bactericidal activity against S. aureus ATCC 29213, which is comparable to that of vancomycin.

Fig. 1 Time kill kinetics of 12j and vancomycin as a comparator against S. aureus ATCC 29213 at various time points.

12j does not interact with any standard drugs against S. aureus ATCC 29213

Treatment of infections caused by bacterial pathogens with a combination of drugs is an important consideration for reducing the emergence of resistance and decreasing the treatment duration, thereby leading to faster infection clearance. In this context, the interaction of 12j with FDA-approved drugs, including daptomycin, ceftazidime, linezolid, gentamicin, levofloxacin, meropenem, minocycline, rifampicin, and vancomycin, which are utilized for the treatment of Staphylococcal infections was explored via chequerboard method and the data are tabulated in Table 6. 12j does not interact in any synergistic or antagonistic manner with any tested drugs, thus, enabling its utilization in combination for the treatment of staphylococcal infections.

Table 6 Drug interaction of 12j with FDA approved drugs utilized for the treatment of staphylococcal infections

Drug	MIC of the drug alone (µg mL^−1)	MIC of the drug in the presence of 12j (µg mL^−1)	MIC of 12j alone (µg mL^−1)	MIC of 12j in the presence of drug (µg mL^−1)	FIC index	Indication
Ceftazidime	16	16	2	2	2	No interaction
Daptomycin	1	1	2	2	2	No interaction
Gentamicin	0.25	0.125	2	1	1	No interaction
Levofloxacin	0.25	0.25	2	2	2	No interaction
Linezolid	2	2	2	2	2	No interaction
Meropenem	0.125	0.125	2	2	2	No interaction
Minocycline	0.125	0.125	2	2	2	No interaction
Rifampicin	0.0039	0.0039	2	2	2	No interaction
Vancomycin	1	1	2	2	2	No interaction

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12j does not induce resistance in S. aureus ATCC 29213

One of the important characteristics of any new compound under pre-clinical development is its ability to induce resistance, or lack thereof. To determine the potential of 12j to induce resistance, S. aureus was exposed to sub-inhibitory concentrations of 12j, along with levofloxacin as a control, for 40 days, and the results are plotted in Fig. 2. As seen, 12j displayed an extremely low probability of inducing resistance, with only a twofold increase in MIC even after 36 days of exposure. In contrast, S. aureus exposed to levofloxacin showed a drastic increase in MIC to >64 µg mL⁻¹ from an initial MIC of 0.25 µg mL⁻¹, representing a 256-fold increase.

Fig. 2 Resistance induction studies of 12j against S. aureus ATCC 29213.

Conclusions

An unprecedented direct C–H indolation of substituted aliphatic N-heterocycles via a highly atom-economic three-component condensation reaction of indoles, N-heterocycles, and fluorenone has been developed for the synthesis of the tryptophan–proline hybrid. The indolation was achieved by avoiding the use of hazardous metallic reagents/catalysts and oxidants, and by avoiding multiple steps for pre-functionalization and protection–deprotection. The indolation reaction proceeded with high control over diastereoselectivity, regioselectivity, and enantiospecificity, providing one of the six possible isomers. Structurally diverse tryptophan–proline hybrids were isolated as single syn-isomer with good yields and excellent enantiopurity. Of these molecular hybrids, 12j displayed a significant species-specific activity against Gram-positive S. aureus, including the drug-resistant isolates. Moreover, the compound demonstrated a broad safety window. The compound displayed a significant bactericidal activity with no interaction with FDA-approved drugs. Interestingly, 12j-treated S. aureus possesses a non-significant propensity to develop resistance against the compound. Taken together, 12j exhibits concentration-dependent, bactericidal activity against MRSA and VRSA, with an extremely low propensity to induce resistance, and can thus be further developed for the treatment of serious staphylococcal infections.

Conflicts of interest

A patent application has been filed on the synthesis of indolyl N-heterocycles.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5gc04622j.

CCDC 2420329, 2420321, 2420325, 2420327 and 2503126 contain the supplementary crystallographic data for this paper.^29a–e

Acknowledgements

CKJ acknowledges Anusandhan National Research Foundation (ANRF) (CRG/2023/000770) and SERB for financial support (STR/2022/000025) through the Science and Technology Award for Research (SERB-STAR). We acknowledge the Department of Chemistry (FIST:SR/FST/CS-II/2017/23C), IIT Guwahati, for infrastructural facilities. SS thanks Dr. Sandeep Kumar, Department of Chemistry, IITG, for assistance with X-ray crystallography. RM thanks DST-INSPIRE for his fellowship, while AA and DS thank UGC for their fellowship. Intra-mural funding by CSIR-CDRI is acknowledged. The CSIR-CDRI communication number allotted to this paper is 11023. The following reagents were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: NR 119, NR 100, NR 10129, NR 10198, NR 10192, NR 10191, NR 10193, NR 10186, NR 10194, VRS1, VRS4, and VRS12.

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