Synthesis of 1,3,5‐Triazine‐Indole Conjugates as Broad‐Spectrum Coronavirus Entry Inhibitors

Shazia Asghar; Leentje Persoons; Yanicka Smolders; Niels Willems; Dirk Daelemans; Steven De Jonghe; Najim A Al‐Masoudi; Shahid Hameed

doi:10.1002/cmdc.70434

. 2026 Aug 12;21(15):e70434. doi: 10.1002/cmdc.70434

Synthesis of 1,3,5‐Triazine‐Indole Conjugates as Broad‐Spectrum Coronavirus Entry Inhibitors

Shazia Asghar ¹, Leentje Persoons ², Yanicka Smolders ², Niels Willems ², Dirk Daelemans ², Steven De Jonghe ², Najim A Al‐Masoudi ³, Shahid Hameed ^1,^✉

PMCID: PMC13469574 PMID: 42587342

Abstract

A series of hybrid molecules containing indole and 1,3,5‐triazine scaffolds was synthesized and evaluated for their antiviral activity against several human RNA and DNA viruses. The conjugates displayed overall low cytotoxicity (CC₅₀ > 50 µM) and potent antiviral activity across a coronavirus panel, with the most promising analogs (compounds 5d, 5g and 5o) showing sub‐ to low‐micromolar EC₅₀ values against HCoV‐OC43, HCoV‐229E, and SARS‐CoV‐2. Structure‐activity relationship studies highlighted that the substitution pattern on the anilino moiety allowed to modulate antiviral activity. Time‐of‐drug addition, adsorption, and fusion assays indicated inhibition of viral replication at an early viral entry step, consistent with inhibition of spike‐mediated membrane fusion. Overall, these data suggest that 1,3,5‐triazine‐indole conjugates are a promising scaffold to develop broad‐spectrum coronavirus entry inhibitors. ADMET profiling of lead compounds (5d, 5g, and 5o) revealed favorable pharmacokinetic properties, including high oral absorption, limited CNS penetration, and non‐mutagenic safety profiles.

Keywords: 1,3,5‐triazines; coronavirus entry inhibitors; indole‐2‐carbohydrazides; SARS‐CoV‐2

The synthesized conjugates displayed overall low cytotoxicity (CC₅₀ > 50 µM) and potent antiviral activity across a coronavirus panel, the most promising analogs (compounds 5d, 5g and 5o) exhibiting sub‑ to low‑micromolar EC₅₀ values against HCoV‑OC43, HCoV‑229E, and SARS‑CoV‑2. Time‑of‑drug addition, adsorption, and fusion assays indicate that the lead compounds effectively inhibit viral entry by interfering with spike protein‐mediated membrane fusion.

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1. Introduction

Emerging viral pathogens represent a major global health threat, causing widespread disease and substantial global economic losses. Viral infections can have catastrophic consequences, impacting human health, animal welfare, and ecological stability, while posing risks to biodiversity [1]. Coronaviruses (CoVs), members of the Coronaviridae within the order Nidovirales, are characterized by their distinctive crown‐like surface spikes (Latin: corona) [2, 3, 4]. To date, seven human coronaviruses have been identified: HCoV‐229E, HCoV‐OC43, HCoV‐NL63, HCoV‐HKU1, Severe Acute Respiratory Syndrome CoV (SARS‐CoV), Middle East Respiratory Syndrome CoV (MERS‐CoV), and SARS‐CoV‐2 [5]. The endemic strains HCoV‐229E, HCoV‐OC43, HCoV‐NL63, and HCoV‐HKU1 typically cause mild upper respiratory tract infections and are responsible for 15%–30% of common cold cases in adults. However, in vulnerable populations such as infants, the elderly, and immunocompromised individuals, these viruses can occasionally lead to severe and potentially life‐threatening lower respiratory tract illnesses. In contrast, SARS‐CoV (and MERS‐CoV) exhibit high pathogenicity and primarily target cells in the lower respiratory tract, leading to acute life‐threatening conditions. Notably, SARS‐CoV‐2 has significant mortality in vulnerable populations [6, 7]. The COVID‐19 pandemic, driven by SARS‐CoV‐2, emerged in December 2019 in Wuhan, China, and has since then resulted in unprecedented global impact. By April 6, 2025, the World Health Organization reported over 777 million confirmed cases and more than 7 million deaths worldwide [8]. SARS‐CoV‐2 possesses a ~30 kb positive‐sense single‐stranded RNA genome encoding structural, nonstructural, and accessory proteins essential for replication and pathogenesis. The four structural proteins, spike (S), membrane (M), envelope (E), and nucleocapsid (N), are essential for viral assembly and infection [9]. Among these, the spike glycoprotein is involved in viral entry by binding to the angiotensin‐converting enzyme 2 (ACE2) receptor on host cells, while the membrane (M) protein is crucial for viral assembly and budding. Additionally, the envelope (E) protein contributes to viral morphogenesis and pathogenesis, enhancing infectivity. Despite extensive research, only a limited number of approved antiviral agents specifically targeting SARS‐CoV‐2 are available, highlighting the need for novel therapeutic approaches [10].

Vaccines [11, 12, 13, 14, 15] and neutralizing antibodies [16, 17, 18] targeting SARS‐CoV‐2 spike glycoprotein remain central to COVID‐19 prevention strategies, yet the emergence of viral variants such as Delta and Omicron has markedly compromised vaccine efficacy. Moreover, vaccines and antibody therapies require intramuscular or intravenous administration and strict cold‐chain storage, presenting logistical challenges for large‐scale deployment. In contrast, orally active, small molecule, antiviral agents offer distinct advantages, including improved stability, ease of distribution, and patient self‐administration. Such therapies can alleviate healthcare burdens and potentially limit rapid viral transmission. Consequently, while efforts to enhance vaccine performance continue, there is a dire need to identify and develop novel small molecule broad‐spectrum antiviral agents.

The indole nucleus is widely recognized as a privileged scaffold in medicinal chemistry. Both natural and synthetic indole‐based pharmacophores are associated with a broad range of antimicrobial properties including antibacterial [19, 20, 21, 22], antifungal [23, 24, 25], antiviral [24, 25, 26, 27, 28, 29, 30], and antimycobacterial [31]. Among the indole‐containing antivirals, umifenovir [32, 33] (arbidol) 1 (Figure 1) is a broad‐spectrum agent active against RNA (e.g., Zikavirus, influenza virus, hepatitis C virus, Ebola, and West Nile) and DNA viruses (such as hepatitis B virus and several herpesviruses). It has been repurposed for COVID‐19 treatment and exhibits an IC₅₀ value of 4.11 μM against SARS‐CoV‐2. Lufotrelvir [34] (PF‐07304814) 2, a SARS‐CoV‐2 main protease (M^pro) inhibitor developed by Pfizer for intravenous administration, exhibits potent activity with an IC₅₀ value of 0.692 μM. Similarly, obatoclax [35] (GX15‐070) 3, originally an antitumor drug, has been repurposed for COVID‐19 due to its ability to interfere with ACE2‐mediated viral entry. Natural products, such as neoechinulin A 4 and echinulin [36] 5, both isolated from Aspergillus fumigatus MR2012, have also demonstrated strong inhibitory activity against SARS‐CoV‐2 M^pro, with IC₅₀ values of 0.47 and 3.90 μM, respectively.

Compounds based on a 1,3,5‐triazine scaffold received considerable attention as broad‐spectrum antiviral agents owing to their promising pharmacological properties and flexibility for chemical modification [37]. These heterocyclic compounds have been shown to be potent inhibitors of key viral enzymes, including the main protease (3CLpro) [38] and the RNA‐dependent RNA polymerase (RdRp) [38], making them compelling leads for antiviral drug development. Beyond antiviral activity, both natural and synthetic triazines have shown activity against diverse biological targets, including tubulin [39], metalloproteinases [40], histone deacetylases [41], urease, and tyrosinase [42]. This broad target spectrum underscores the versatility and utility of the triazine scaffold in drug discovery [43, 44, 45, 46].

Based on the remarkable antiviral potential of indole‐based pharmacophores and the therapeutic efficacy of 1,3,5‐triazines against coronaviruses, a novel series of 1,3,5‐triazine‐indole conjugates was synthesized and evaluated for their in vitro antiviral activity against multiple human coronavirus strains. The current paper reports the synthesis of triazine‐indole hybrids as promising candidates for the development of treatments targeting current and future coronavirus outbreaks.

2. Results and Discussion

2.1. Chemistry

The synthesis of N′‐(2‐methoxy‐4‐((4‐morpholino‐6‐(arylamino)‐1,3,5‐triazin‐2‐yl)oxy)benzylidene)‐1H‐indole‐2‐carbohydrazides 5a–u is outlined in Scheme 1. 2‐(2‐Methoxy‐4‐formylphenoxy)‐4‐morpholino‐6‐arylamino‐1,3,5‐triazines 3a–u were synthesized from cyanuric chloride using a previously reported method [47]. To access intermediate 1H‐indole‐2‐carbohydrazide 4, 2‐(methoxycarbonyl)indole was treated with hydrazine hydrate [48]. The target molecules 5a–u were prepared via condensation of intermediates 3a–u with 1H‐indole‐2‐carbohydrazide 4. Structural proof of the identity of compounds 5a–u was obtained by recording their spectra. The N—H stretching vibrations of the indole ring appeared from 3463 to 3242 cm⁻¹, while the N—H stretching absorptions of hydrazide and aniline ring were observed in the range of 3310–3175 cm⁻¹. The peaks from 1646 to 1600 cm⁻¹ corresponded to the C=N stretch of azomethine and heteroaromatic 1,3,5‐triazines, whereas C=C stretching of aromatic rings appeared in the range from 1590 to 1473 cm⁻¹. Furthermore, the absence of characteristic C—H stretchings of the aldehyde group of vanillin also indicated the condensation of the aldehyde group with 1H‐indole‐2‐carbohydrazide (4). The ¹H NMR spectra of the target carbohydrazides 5a–u showed singlets in the range of 10.35–8.17 ppm arising from the imine moiety. The signal observed from 11.96 to 11.81 ppm was attributed to the proton of the indole nitrogen. The aromatic protons appeared in the region between 8.82 and 6.51 ppm. In ¹³C NMR spectra, signals observed in the range of δ 147.2–141.1 ppm were assigned to the azomethine carbon, further confirming the newly established imine linkage. Moreover, the presence of aromatic signals corresponding to both parts of the molecules (from 3a–u and 4) further confirmed the synthesis.

2.2. Broad Antiviral Evaluation

The activity of 1,3,5‐triazine–indole conjugates 5a–u against various human DNA and RNA viruses was investigated. This antiviral panel included influenza A virus (IAV H1N1, IAV H3N2), influenza B virus (IBV), respiratory syncytial virus (RSV), yellow fever virus (YFV), Zika virus (ZIKV), herpes simplex virus 1 (HSV‐1) and human coronavirus HCoV‐OC43. In parallel, cytotoxicity for all host cell lines (MDCK, Hep2, Hep3B, HEL 299) was tested to determine compound selectivity (Table 1).

TABLE 1.

Cytotoxicity and antiviral activity of 1,3,5‐triazine–indole conjugates 5a–u.

Compound	CC₅₀, µM^a				EC₅₀, µM^b
	MDCK	Hep2	Hep3B	HEL 299	IAV‐H1N1	IAV‐H3N2	IBV	RSV	YFV	ZIKV	HSV‐1	HCoV‐OC43
	MDCK	Hep2	Hep3B	HEL 299	MDCK	MDCK	MDCK	Hep2	Hep3B	Hep3B	HEL 299	HEL 299
5a	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	1.5 ± 0.4
5b	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5c	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	3.0 ± 1.6
5d	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	0.6 ± 0.07
5e	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5f	>50	>50	>50	>50	>50	>50	>50	4.0 ± 1.8	>50	>50	>50	1.5 ± 1.0
5g	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	0.8 ± 0.3
5h	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5i	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	3.6 ± 0.9
5j	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	≥23.8
5k	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	3.2 ± 2.6
5l	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	3.1 ± 2.5
5m	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5n	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	1.6 ± 0.4
5o	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	0.9 ± 0.01
5p	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5q	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
5r	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	1.2 ± 0.08
5s	19.9	>50	9.7	13.5	>50	>50	>50	>50	>50	>50	>50	>50
5t	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	5.5 ± 0.3
5u	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
Remdesivir	—	>100	>100	>100	—	—	—	0.007 ± 0.001	4.7	1.5	—	0.04 ± 0.03
Zanamivir	>100	—	—	—	0.01	2.1	0.1	—	—	—	—	—
Acyclovir	—	—	—	>100	—	—	—	—	—	—	0.1	—
DS‐10,000	—	>100	>100	>100	—	—	—	0.06 ± 0.03	>100	—	2.1	—

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Note: Values represent mean ± SD from two independent experiments.

CC₅₀ (µM): concentration causing 50% cytotoxicity in host cells.

EC₅₀ (µM): concentration required to reduce viral replication by 50%.

Overall, the compounds exhibited negligible cytotoxicity across all tested cell lines (CC₅₀ > 50 μM for most derivatives), indicating a favorable safety profile. Remarkably, several derivatives were endowed with potent and selective antiviral activity against HCoV‐OC43. The nature of the substitution pattern of the anilino moiety determined their antiviral potency. Analogs carrying either a halogen or an electron‐donating group at position 2 of the phenyl ring (e.g., compounds 5b, 5e, 5h, 5j, 5m, 5s, and 5u) are usually devoid of antiviral activity. The exception is the 2,4‐dimethylanilino derivative 5t that still maintains good activity against HCoV‐OC43 (EC₅₀ = 5.5 µM). The insertion of the strong electron‐withdrawing nitro group on the phenyl ring, such as in compounds 5p and 5q, gave inactive analogs. The most congeners that emerged from this series were the 4‐substituted analogs 5d (4‐methoxyanilino), 5g (4‐methylanilino) and 5o (4‐bromoanilino), with EC₅₀ values ranging from 0.6 to 0.9 μM. Other groups at position 4, such as a fluorine, a chlorine or trifluoromethyl (compounds 5i, 5l, and 5r, respectively) gave a slightly reduced potency against HCoV‐OC43, with EC₅₀ values in the 1–3 µM range. Similarly, the presence of various substituents at position 3 of the anilino group (e.g., a methoxy group in compound 5c, a methyl group in compound 5f, a chlorine in compound 5k and a bromine in compound 5n) gave antiviral potencies in the low micromolar range. In general, these 1,3,5‐triazine–indole conjugates lacked activity against other viruses, except for compound 5f being active against RSV (EC₅₀ = 4 µM).

2.3. Antiviral Evaluation Against Human Coronaviruses

To assess their potential as pan‐coronavirus antivirals, the 1,3,5‐triazine–indole conjugates were also evaluated against HCoV‐229E (an alphacoronavirus) and SARS‐CoV‐2 (similar to HCoV‐OC43, also a betacoronavirus). Cytotoxicity was assessed on HEL 299 and VeroE6 cells to confirm selectivity (Table 2).

TABLE 2.

Cytotoxicity and antiviral activity of 1,3,5‐triazine–indole conjugates 5a–u against human coronaviruses.

Compound	Cytotoxicity (CC₅₀/µM)		Antiviral activity (EC₅₀/µM)			S.I. (CC₅₀/EC₅₀)
	HEL 299	VeroE6	HCoV‐OC43	HCoV‐229E	SARS‐CoV‐2	HCoV‐OC43	HCoV‐229E	SARS‐CoV‐2
	HEL 299	VeroE6	HEL 299	HEL 299	VeroE6	HEL 299	HEL 299	VeroE6
5a	>250	>250	1.5 ± 0.4	1.2 ± 0.7	5.4 ± 0.6	≥166.7	≥208.3	≥46.3
5b	>50	—	>50	>50	—
5c	>250	>250	3.0 ± 1.6	0.7 ± 0.1	0.8 ± 0.2	≥83.3	≥357.1	≥312.5
5d	>250	>250	0.6 ± 0.07	0.8 ± 0.1	2.4 ± 2.8	≥416.7	≥312.5	≥104.2
5e	>50	—	>50	>50	—
5f	>250	>250	1.5 ± 1.0	1.0 ± 0.2	2.7 ± 2.5	≥166.7	≥250.0	≥92.6
5g	>250	>250	0.8 ± 0.3	0.5 ± 0.03	0.8 ± 0.0	≥312.5	≥500.0	≥312.5
5h	>50	—	>50	>50	—
5i	>250	>250	3.6 ± 0.9	1.3 ± 0.2	>50	≥69.4	≥192.3
5j	>50	—	≥23.8	>50	—
5k	>250	>250	3.2 ± 2.6	4.7 ± 4.1	7.2 ± 3.1	≥78.1	≥53.2	≥34.7
5l	>250	>250	3.1 ± 2.5	0.8 ± 0.05	2.6 ± 1.7	≥80.6	≥312.5	≥96.2
5m	>50	—	>50	>50	—
5n	>250	—	1.6 ± 0.4	39.4	—	≥156.3	≥6.3
5o	>250	>250	0.9 ± 0.01	1.1 ± 0.5	0.9 ± 0.1	≥277.8	≥227.3	≥277.8
5p	>50	—	>50	>50	—
5q	>50	—	>50	>50	—
5r	>250	—	1.2 ± 0.08	48.9	—	≥208.3	≥5.1
5s	13.5	—	>50	>50	—
5t	>250	—	5.5 ± 0.3	9.5 ± 7.9	—	≥45.5	≥26.3
5u	>50	—	>50	>50	—
Remdesivir	102.3 ± 19.4	≥239.0	0.04 ± 0.03	0.2 ± 0.007	6.3 ± 0.2	2557.5	511.5	≥37.9

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Note: CC₅₀ (µM): concentration causing 50% cytotoxicity in host cells; EC₅₀ (µM): concentration required to reduce viral replication by 50%. Values represent mean ± SD from at least two independent experiments.

In the first round of screening, compounds were evaluated for antiviral activity and cytotoxicity at a concentration of 50 µM. For the analogs that showed promising antiviral activity, the cytotoxicity assay was repeated using 250 µM as the highest tested concentration, allowing to calculate selectivity indexes (SI, ratio of CC₅₀/EC₅₀) more accurately. Overall, the compounds maintained low cytotoxicity (CC₅₀ > 250 μM for most derivatives). Compounds that lacked activity against HCoV‐OC43 (i.e., compounds 5b, 5e, 5h, 5j, 5m, 5p, 5q, 5s, and 5u) were also inactive against HCoV‐229E. Analogs that were endowed with activity against HCoV43 also displayed activity against HCoV‐229E (compounds 5a, 5c, 5d, 5f, 5g, 5i, 5k, 5l, 5o, and 5t) with, in general, a good correlation between both antiviral assays. Analogs that showed potent activity (EC₅₀ values less than 5 µM) against both HCoV‐OC43 and HCoV‐229E were also evaluated against SARS‐CoV‐2. Compounds 5g and 5o emerged as the most promising pan‐coronavirus antivirals, combining submicromolar potency against HCoV‐OC43 (0.6–0.9 μM) with excellent activity against HCoV‐229E (0.5–1.1 μM) and SARS‐CoV‐2 (0.8–0.9 μM). Moreover, other derivatives such as 5a, 5c, 5d, 5f, 5k, and 5l displayed low micromolar activity, suggesting a broad‐spectrum anticoronavirus profile of this compound series. Compound 5i was the exception in this series, as it is active against HCoV‐229E and HCoV‐OC43, but completely lacked antiviral activity against SARS‐CoV‐2. It seems that subtle structural modifications have a profound influence on the anticoronavirus profile of this compound class, although the exact reasons for similar or differential activities on SARS‐CoV‐2 versus the seasonal coronaviruses are currently unknown.

2.4. Mechanistic Insights Into Antiviral Action

To elucidate the mode of action of the most active compounds, we performed complementary mechanistic assays investigating different stages of the coronavirus life cycle. First, a time‐of‐drug‐addition (TOA) experiment on HEL299 cells infected with HCoV‐229E was conducted in which compounds were added at different time points (1h before the infection, 0 , 1, 2, 4 and 6 h post‐infection). Various reference compounds with an established mode of action were included. Remdesivir (tested at 5 µM and EC₅₀ ~ 0.1 µM) started to lose its antiviral activity, when added 2 h post infection, which is consistent with its action as a viral RNA polymerase inhibitor. Aloxistatin (also known as E64d, included at 30 µM and EC₅₀ ~0.3 µM), chloroquine (CQ, tested at 50 µM and EC₅₀ ~0.5 µM) and Hippeastrum hybrid agglutinin (HHA, tested at 100 µg/mL and EC₅₀ ~5 µg/mL) were included as they are known to block early steps in the viral replication cycle. vRNA levels were potently inhibited when the compounds were added at −1 or 0 h p.i. Compound 5o (tested at 100 µM and EC50 ~1 µM) shows a similar profile as E64d, CQ and HHA in this TOA assay, suggesting interference with processes preceding or coinciding with viral entry (Figure 2A).

Mechanistic analysis of the antiviral action of most potent triazine–indole conjugates. (A) Time‐of‐drug‐addition assay with HCoV‐229E. (B) Adsorption assay with HCoV‐229E for 5d, **5g,** and 5o at the indicated concentrations. (C) Cell–cell fusion assay in HEK293T cells co‐expressing SARS‐CoV‐2 spike and human ACE2 with 5d, **5g,** and 5o at the indicated concentrations.

Next, we assessed whether these compounds affect viral attachment through an adsorption assay using HEL299 cells and HCoV‐229E (Figure 2B). Compounds 5d, 5g, and 5o showed minimal or no impact on the initial adsorption of the virus to host cells at the concentration range tested (30–0.1 µM), excluding receptor binding as the primary mechanism.

Finally, a fusion assay was performed in HEK293T cells co‐expressing SARS‐CoV‐2 spike and human ACE2, using mNeonGreen as a reporter (Figure 2C). Treatment with 5d, 5g, and 5o resulted in a marked dose‐dependent reduction in the number of fused cells, strongly supporting that these compounds exert their antiviral effect by inhibiting the membrane fusion process required for viral entry.

2.5. In Silico ADMET Studies

The ADMET‐related parameters (Table 3) of the lead compounds (5d, 5g, and 5o) were predicted using the pkCSM computational tool [49]. A thorough understanding of a compound's pharmacokinetic behavior, from absorption to elimination, is essential for determining its suitability as a drug candidate. An ideal drug candidate should possess high oral bioavailability, demonstrate efficient absorption, exhibit favorable tissue distribution, undergo safe and balanced clearance, and maintain a non‐toxic profile to ensure both therapeutic efficacy and patient safety. Therefore, ADMET profiling remains a cornerstone of rational drug design [50].

TABLE 3.

ADMET properties of lead 1,3,5‐triazine–indole conjugates (5d, 5g, and 5o).

Compound	Absorption	Distribution		Metabolism (CYP)				Excretion	Toxicity
	Intestinal absorption (human) (% absorbed)	BBB permeability (log BBB)	CNS permeability (log PNS)	Substrate		Inhibitor		Total clearance numeric (log mL min⁻¹kg⁻¹)	AMES toxicity (yes/no)
	Intestinal absorption (human) (% absorbed)	BBB permeability (log BBB)	CNS permeability (log PNS)	2D6	3A4	2D6	3A4	Total clearance numeric (log mL min⁻¹kg⁻¹)	AMES toxicity (yes/no)
5d	87.613	−1.992	−3.639	No	Yes	No	Yes	−0.096	No
5g	91.741	−1.819	−3.408	No	Yes	No	Yes	−0.073	No
5o	92.185	−1.993	−3.382	No	Yes	No	Yes	0.101	No

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The ADMET evaluation of compounds 5d, 5g, and 5o revealed consistently favorable absorption characteristics, with all the three compounds showing high predicted human intestinal absorption (HIA) (>87%), suggesting favorable oral bioavailability potential. Among them, compound 5o demonstrated the highest absorption (92.18%), indicating it may achieve better systemic exposure compared to 5d and 5g. The negative logBB and logPS values indicate limited blood‐brain barrier penetration and low central nervous system exposure. This property could be advantageous for therapeutic applications where CNS activity is undesirable, reducing the risk of central side effects. None of the compounds were predicted to be CYP2D6 substrates, which is advantageous since genetic variability in this enzyme often leads to inconsistent drug responses. In contrast, all lead compounds (5d, 5g, and 5o) were identified as substrates and inhibitors of CYP3A4, highlighting the need for further evaluation of potential drug–drug interactions. Drug clearance, defined as the volume of plasma cleared of a drug per unit time, is a key pharmacokinetic parameter. Predicted total clearance values were relatively low for all compounds. Lower clearance prolongs systemic retention, potentially enhancing therapeutic efficacy. In addition, toxicity assessments through AMES testing indicated that none of the compounds are mutagenic, which is a positive outcome for their safety profile. Overall, these results suggest a broadly favorable predicted ADMET profile for further investigation.

3. Conclusion

In this study, we report the synthesis of a novel series of 1,3,5‐triazine–indole conjugates by combining two privileged scaffolds (i.e., indoles and triazines). The newly synthesized compounds exhibited negligible cytotoxicity across all tested cell lines, suggesting a favorable safety profile. Antiviral screening revealed potent and selective activity against HCoV‐OC43, with several derivatives (e.g., compounds 5d, 5g, and 5o) displaying submicromolar EC₅₀ values. Expanding the evaluation to other coronaviruses (i.e., HCoV‐229E and SARS‐CoV‐2) revealed that these compounds retained strong antiviral activity across multiple coronaviruses, suggesting broad‐spectrum anticoronavirus potential.

Time‐of‐drug addition experiments indicated that these compounds interfere with an early stage event of the viral life cycle. Adsorption assays confirmed that these compounds do not significantly affect initial virus–cell binding, while fusion assays demonstrated a marked reduction in spike‐mediated cell–cell fusion. This mechanistic profile highlighted the potential of these indole‐triazine hybrid to act as entry inhibitors for broad‐spectrum coronavirus therapy.

Further research will need to focus on the enhancement of antiviral potency, as well as on the evaluation and improvement of the pharmacokinetic properties, in order to obtain analogs that can be evaluated in preclinical animal models.

4. Experimental Section

4.1. Chemistry

4.1.1. Materials

Cyanuric chloride was purchased from Sigma Aldrich (Switzerland). Anhydrous potassium carbonate was the product of Uni‐chem (Mumbai, India). 2‐Fluoroaniline, 4‐fluoroaniline, 4‐(trifluoromethoxy)aniline, 4‐(trifluoromethyl)aniline, 2,4‐dimethylaniline, and 3‐chloro‐2‐methylaniline were procured from Sigma Aldrich (St. Louis, Missouri, United States), whereas methoxycarbonyl indole, hydrazine monohydrate (98%), and benzophenone were purchased from Fluka (Steinhein, Germany). Vanillin was supplied by Aldrich (Steinhein, Germany). o‐Anisidine, m‐anisidine, p‐anisidine, o‐toluidine, m‐toluidine, p‐toluidine, 3‐nitroaniline, and 4‐nitroaniline were purchased from Merck (Schuchardt, Germany). 2‐Chloroaniline, 3‐chloroaniline, 4‐chloroaniline, 2‐bromoaniline, 3‐bromoaniline, and 4‐bromoaniline were obtained from Alfa Aesar (Kandel, Germany). Trimethylamine was acquired from Acros Organics (New Jersey, USA). Absolute ethanol, chloroform, and 1,4‐dioxane were purchased from E. Merck (Darmstadt, Germany), while n‐hexane, acetone, methanol, and ethyl acetate were obtained from commercial sources and distilled before use.

4.1.2. General Experimental Methods

Melting points of the compounds were measured using capillary tube method on Gallenkamp melting point apparatus MP.D350.BM3.5 (UK) equipped with a digital thermometer and are reported uncorrected. IR spectra were obtained using Thermo Scientific Nicolet 6700 FTIR spectrophotometer using ATR mode. Bruker Avance 300 MHz NMR spectrophotometer (Switzerland) was used to record ¹H and ¹³C NMR spectra at 300 (¹H) and 75 MHz (¹³C), in CDCl₃ or DMSO‐d₆ with TMS as the reference compound. The progress of reactions was monitored using thin‐layer chromatography (TLC) on silica gel plates 60 F₂₅₄ coated on aluminum sheets (Merck, Germany). Chromatograms were developed using CHCl₃: MeOH (9:1) as eluent and visualized under UV light (254–366 nm) and in iodine vapor.

4.1.3. Procedure for the Synthesis of 1H‐Indole‐2‐Carbohydrazide (4)

2‐Methoxycarbonylindole (0.1 mol) and hydrazine hydrate (0.5 mol) were refluxed in 20 mL of dry ethanol for 5 h. Reaction progress was monitored using TLC. On the completion of reaction, the white crystals formed in the reaction mixture were filtered, washed with chilled methanol and air dried. Yield: 95%; m.p.: 242–244 °C (Lit. 243–245 °C) [48]; R _f: 0.65 [CHCl₃: MeOH (9:1)].

4.1.4. General Procedure for the Synthesis of N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(phenylamino)‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide [51] (5a–u)

1H‐Indole‐2‐carbohydrazide (4) (0.01 mol) was added to a solution of corresponding 2‐(2‐methoxy‐4‐formylphenoxy)‐4‐morpholino‐6‐arylamino‐1,3,5‐triazine (3a–u) (0.012 mol) in 20 mL of dry ethanol. The mixture was stirred along with catalytic amounts of sodium hydrogen sulfite for 15 min at room temperature and then heated under reflux for 6–12 h. After completion of the reaction as indicated by TLC, the solid formed in the reaction mixture was filtered, washed with ice‐cold ethanol, and recrystallized from a mixture of dimethylformamide and ethanol (1:2).

4.1.4.1. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(phenylamino)‐1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5a)

Yield: 80%; m.p.: 194–196 °C; R _f: 0.83 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3246, 3189 (2 × N—H stretch.), 3050 (C_sp2—H stretch.), 2996, 2957 (2 × C_sp3—H stretch.), 1638 (C=O stretch.), 1614 (C=N stretch.), 1587, 1509 (2 × C=C stretch.), 1276, 1208 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.82 (2H, 2 × s, NH), 8.50 (1H, s, NH), 8.50 (1H, s, H—C=N), 7.69 (1H, d, J = 7.8 Hz, ArH), 7.53–7.47 (4H, m, ArH), 7.37–7.17 (6H, m, ArH), 7.10–6.93 (2H, m, ArH), 3.83 (3H, s, OCH₃), 3.75–3.63 (8H, m, morpholine‐H); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 165.4, 166.1 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.9, 139.8, 137.3, 133.2, 130.4, 128.7, 127.4, 124.3, 123.8, 122.7, 122.2, 121.1, 120.4, 112.8, 110.3, 104.1 (18 × ArC), 66.3 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂).

4.1.4.2. N′‐(2‐Methoxy‐4‐((6‐((2‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazides (5b)

Yield: 78%; m.p.: 208–210 °C; R _f: 0.79 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3426, 3231 (2 × N—H stretch.), 3050 (C_sp2—H stretch.), 2960, 2861 (2 × C_sp3—H stretch.), 1652 (C=O stretch.), 1616 (C=N stretch.), 1562, 1508 (2 × C=C stretch.), 1269, 1210 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.82 (2H, 2 × s, NH), 8.49 (1H, s, NH), 8.17 (1H, s, H—C=N), 7.69 (2H, d, J = 7.8 Hz, ArH), 7.52–7.47 (2H, m, ArH), 7.34–7.21 (4H, m, ArH), 7.10–7.00 (3H, m, ArH), 6.78 (1H, s, ArH), 3.82 (3H, s, OCH₃), 3.80 (3H, s, OCH₃), 3.74–3.60 (8H, m, morpholine‐H); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.1 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.8, 137.3, 133.1, 130.4, 128.7, 127.6, 127.4, 124.3, 123.8, 122.7, 122.2, 121.1, 120.4, 112.6, 111.4, 110.3, 104.2 (19 × ArC), 66.2 (OCH₂), 56.3, 56.4 (2 × OCH₃), 43.8 (NCH₂).

4.1.4.3. N′‐(2‐Methoxy‐4‐((6‐((3‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5c)

Yield: 78%; m.p.: 208–210 °C; R _f: 0.79 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3426, 3231 (2 × N—H stretch.), 3050 (C_sp2—H stretch.), 2960, 2861 (2 × C_sp3—H stretch.), 1652 (C=O stretch.), 1616 (C=N stretch.), 1562, 1508 (2 × C=C stretch.), 1269, 1210 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.83 (2H, 2 × s, NH), 8.49 (1H, s, NH), 9.63 (1H, s, (H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.52–7.46 (2H, m, ArH), 7.34–7.20 (5H, m, ArH), 7.10–7.05 (3H, m, ArH), 6.51 (1H, s, ArH), 3.83 (6H, s, 2 × OCH₃), 3.63 (8H, bs, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.0, 165.4 (3 × C‐triazine), 147.2 (H—C=N), 159.7, 158.1, 152.0, 142.8, 141.0, 137.3, 133.1, 130.4, 129.5, 127.4, 124.3, 123.7, 122.2, 121.2, 120.4, 112.8, 112.6, 110.4, 106.1, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 55.3 (OCH₃), 43.8 (NCH₂).

4.1.4.4. N′‐(2‐Methoxy‐4‐((6‐((4‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5d)

Yield: 70%; m.p.: 228–230 °C; R _f: 0.81 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3426, 3231 (2 × N—H stretch.), 3050 (C_sp2—H stretch.), 2960, 2861 (2 × C_sp3—H stretch.), 1652 (C=O stretch.), 1616 (C=N stretch.), 1562, 1508 (2 × C=C stretch.), 1269, 1210 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.86 (2H, 2× s, NH), 8.50 (1H, s, NH), 9.56 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.52–7.47 (3H, m, ArH), 7.34–7.20 (5H, m, ArH), 7.10–6.88 (3H, m, ArH), 3.83 (6H, s, 2 × OCH₃), 3.72–3.62 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.0, 165.4 (3 × C‐triazine), 147.2 (H—C=N), 159.7, 158.1, 152.0, 142.8, 141.0, 137.3, 133.1, 130.4, 129.5, 127.4, 124.3, 123.7, 122.2, 121.2, 120.4, 112.8, 112.6, 110.4, 106.1, 104.1 (20 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 55.3 (OCH₃), 43.8 (NCH₂).

4.1.4.5. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(o‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5e)

Yield: 82%; m.p.: 185–187 °C; R _f: 0.84 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3443, 3277 (2 × N—H stretch.), 3050 (C_sp2—H stretch.), 2960, 2879 (2 × C_sp3—H stretch.), 1636 (C=O stretch.), 1602 (C=N stretch.), 1583, 1498 (2 × C=C stretch.), 1278, 1207 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.86 (2H, 2 × s, NH),8.50 (1H, s, NH), 8.98 (1H, s, H—C=N), 7.68 (1H, d, J = 8.1 Hz, ArH), 7.48 (2H, m, ArH), 7.33–7.03 (9H, m, ArH), 3.84–3.57 (11H, m, H‐morpholine, OCH₃), 2.18 (1H, s, CH₃), ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.4, 166.0 (3 × C‐triazine), 147.2 (H‐C=N), 158.1, 152.1, 142.8, 137.3, 137.1, 133.9, 130.5, 130.4, 127.4, 126.3, 126.0, 125.4, 124.3, 123.8, 122.2, 121.1, 120.4, 112.8, 110.3, 104.1 (20 × ArC), 66.2 (OCH₂), 56.2 (OCH₃), 43.7 (NCH₂), 18.5 (CH₃).

4.1.4.6. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(m‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5f)

Yield: 90%; m.p.: 292–294 °C; R _f: 0.87 (chloroform : methanol; 8:2); IR (ATR, ū, cm⁻¹): 3438, 3256 (2 × N—H stretch.), 3078 (C_sp2—H stretch.), 2951, 2840 (2 × C_sp3—H stretch.), 1643 (C=O stretch.), 1606 (C=N stretch.), 1571, 1507 (2 × C=C stretch.), 1281, 1210 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.97, 11.82 (2H, 2 × s, NH), 8.50 (1H, s, NH), 8.49 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.53–7.46 (2H, m, ArH), 7.37–7.20 (6H, m, ArH), 7.10–7.05 (2H, t, J = 7.5 Hz, ArH), 6.73 (1H, s, ArH), 3.83 (3H, s, OCH₃), 3.75–3.63 (8H, m, H‐morpholine), 2.54 (1H, s, CH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.4 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.9, 139.7, 137.9, 137.3, 133.2, 130.4, 128.5, 127.4, 124.3, 123.8, 123.4, 122.2, 121.2, 120.4, 117.4, 112.8, 110.4, 104.1 (20 × ArC), 66.3 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂), 21.7 (CH₃).

4.1.4.7. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(p‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5g)

Yield: 74%; m.p.: 213–216 °C; R _f: 0.88 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3463, 3267 (2 × N—H stretch.), 3024 (C_sp2—H stretch.), 2954, 2846 (2 × C_sp3—H stretch.), 1638 (2 × C=O stretch.), 1613 (C=N stretch.), 1583, 1496 (2 × C=C stretch.), 1238, 1205 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.82 (2H, 2 × s, NH), 8.50 (1H, s, NH), 9.55 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.53–7.49 (3H, m, ArH), 7.37–7.20 (5H, m, ArH), 7.10–6.92 (3H, m, ArH), 3.83 (3H, s, OCH₃), 3.73–3.62 (8H, m, H‐morpholine), 2.20 (1H, s, CH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.5 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.9, 139.7, 137.2, 133.1, 131.6, 130.4, 129.2, 127.4, 124.3, 123.8, 123.4, 122.2, 121.2, 120.4, 120.1, 112.8, 110.3, 104.1 (20 × ArC), 66.3 (OCH₂), 56.3 (OCH₃), 43.9 (NCH₂), 20.8 (CH₃).

4.1.4.8. N′‐(4‐((6‐((2‐Fluorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5h)

Yield: 79%; m.p.: 277–279 °C; R _f: 0.79 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3413, 3310 (2 × N—H stretch.), 3007 (C_sp2—H stretch.), 2939, 2848 (2 × C_sp3—H stretch.), 1650 (C=O stretch.), 1629 (C=N stretch.), 1590, 1507 (2 × C=C stretch.), 1270, 1209 (2 × C—O stretch.), 1165 (C—F stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.93, 11.81 (2H, 2 × s, NH), 9.27 (1H, s, NH), 8.47 (1H, s, H—C=N), 7.69 (1H, d, J = 7.8 Hz, ArH), 7.55–7.46 (3H, m, ArH), 7.34–7.05 (8H, m, ArH), 3.84 (3H, s, OCH₃), 3.57 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.1, 166.0 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 155.7 (d, ¹ J _C‐F = 245.5 Hz, 152.1, 142.8, 137.3, 133.1, 130.4, 127.4, 126.5 (d, ² J _C‐F = 23.2 Hz), 126.5, 126.2, 124.3, 123.8, 122.2, 121.2, 120.4, 115.9 (d, ² J _C‐F = 19.5 Hz), 112.8, 110.2, 104.1 (20 × ArC), 66.2 (OCH₂), 56.2 (OCH₃), 43.7 (NCH₂).

4.1.4.9. N′‐(4‐((6‐((4‐Fluorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5i)

Yield: 81%; m.p.: 283–285 °C; R _f: 0.77 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3303, 3215 (2 × N—H stretch.), 3005 (C_sp2—H stretch.), 2907, 2863 (2× C_sp3—H stretch.), 1633 (C=O stretch.), 1620 (C=N stretch.), 1561, 1499 (2 × C=C stretch.), 1276, 1205 (2 × C—O stretch.), 1158 (C—F stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.94 (2H, bs, NH), 8.50 (1H, s, NH), 9.69 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.60–6.89 (11H, m, ArH), 3.83 (3H, s, OCH₃), 3.72–3.62 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.1, 165.3 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 158.0 (d, ¹ J _C‐F = 240.9 Hz), 152.1,142.8, 137.3, 136.2, 133.2, 130.5, 127.4, 124.3, 123.8, 122.2, 121.1, 120.4, 115.3 (d, ² J _C‐F = 22.5 Hz), 112.8, 110.3, 104.1 (18 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂).

4.1.4.10. N′‐(4‐((6‐((2‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5j)

Yield: 72%; m.p.: 264–270 °C; R _f: 0.78 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3266, 3175 (2 × N—H stretch.), 3052 (C_sp2—H stretch.), 2990, 2854 (2 × C_sp3—H stretch.), 1670 (C=O stretch.), 1646 (C=N stretch.), 1575, 1473 (2 × C=C stretch.), 1249, 1200 (2 × C—O stretch.), 1023 (C—Cl stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.94, 11.81 (2H, 2 × s, NH), 8.47 (1H, s, NH), 9.06 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.58 (1H, dd, J = 8.1, 1.5 Hz, ArH), 7.50–7.43 (3H, m, ArH), 7.34–7.20 (5H, m, ArH), 7.17–7.05 (2H, m, ArH), 3.84 (3H, s, OCH₃), 3.56 (8H, bs, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.2, 166.0 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.0, 142.7, 137.3, 135.7, 133.1, 130.4, 129.8, 128.5, 127.7, 127.5, 127.4, 126.7, 124.3, 123.7, 122.2, 121.1, 120.4, 112.8, 110.3, 104.1 (21 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.7 (NCH₂).

4.1.4.11. N′‐(4‐((6‐((3‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5k)

Yield: 70%; m.p.: 240–244 °C; R _f: 0.75 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3461, 3245 (2 × N—H stretch.), 3070 (C_sp2—H stretch.), 2953, 2841 (2 × C_sp3—H stretch.), 1640 (C=O stretch.), 1603 (C=N stretch.), 1577, 1545 (2 × C=C stretch.), 1251, 1206 (2 × C—O stretch.), 1018 (C‐Cl stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.82 (2H, 2 × s, NH), 8.49 (1H, s, NH), 9.85 (1H, s, H—C=N), 7.69 (2H, m, ArH), 7.54–7.47 (3H, m, ArH), 7.35–7.20 (5H, m, ArH), 7.07 (1H, t, J = 7.5 Hz, ArH), 6.96 (1H, s, ArH), 3.83–3.65 (11H, m, H‐morpholine, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.3 (3 × C‐triazine), 147.2 (H—C=N), 158.1 151.9, 142.7, 137.3, 135.5, 133.3, 130.4, 130.3, 127.4, 124.3, 123.6, 122.2, 121.4, 120.4, 119.4, 118.4, 112.8, 110.5, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂).

4.1.4.12. N′‐(4‐((6‐((4‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5l)

Yield: 80%; m.p.: 318–321 °C; R _f: 0.73 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3376, 3203 (2 × N—H stretch.), 3011 (C_sp2—H stretch.), 2916, 2866 (2 × C_sp3—H stretch.), 1632 (C=O stretch.), 1600 (C=N stretch.), 1551, 1490 (2 × C=C stretch.), 1255, 1204 (2 × C—O stretch.), 1018 (C—Cl stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.81 (2H, 2 × s, NH), 8.47 (1H, s, NH), 9.78 (1H, s, H—C=N), 7.69 (1H, d, J = 7.8 Hz, ArH), 7.54–7.46 (3H, m, ArH), 7.37–7.20 (6H, m, ArH), 7.07 (2H, t, J = 7.5 Hz, ArH), 3.83 (3H, s, OCH₃), 3.74–3.62 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.1, 165.3 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.7, 138.8, 137.3, 133.2, 130.4, 128.6, 127.4, 126.3, 124.3, 123.8, 122.2, 121.8, 121.1, 120.4, 112.8, 110.4, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂).

4.1.4.13. N′‐(4‐((6‐((2‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5m)

Yield: 87%; m.p.: 173–176 °C; R _f: 0.71 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3265, 3178 (2× N—H stretch.), 3054 (C_sp2—H stretch.), 2994, 2861 (2 × C_sp3—H stretch.), 1668 (C=O stretch.), 1646 (C=N stretch.), 1575, 1497 (2 × C=C stretch.), 1248, 1200 (2 × C—O stretch.), 745 (C—Br stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.94, 11.81 (2H, 2 × s, NH), 8.47 (1H, s, NH), 9.83 (1H, s, H—C=N) 7.69 (1H, d, J = 7.8 Hz, ArH), 7.62 (1H, dd, J = 8.1, 1.2 Hz, ArH), 7.54–7.47 (3H, m, ArH), 7.34–7.20 (5H, m, ArH), 7.11–7.07 (2H, m, ArH), 3.84 (3H, s, OCH₃), 3.38 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.2, 166.0 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.0, 142.8, 137.3, 133.1, 132.9, 131.7, 130.4, 128.2, 128.1, 127.4, 127.1, 124.3, 123.7, 122.2, 121.1, 120.4, 119.6, 112.8, 110.3, 104.1 (21 × ArC) 66.2 (OCH₂), 56.3 (OCH₃), 43.7 (NCH₂).

4.1.4.14. N′‐(4‐((6‐((3‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5n)

Yield: 91%; m.p.: 279–284 °C; R _f: 0.73 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3242, 3180 (2 × N—H stretch.), 3067 (C_sp2—H stretch.), 2954, 2834 (2 × C_sp3—H stretch.), 1637 (C=O stretch.), 1603 (C=N stretch.), 1576, 1546 (2 × C=C stretch.), 1251, 1205 (2 × C—O stretch.), 744 (C—Br stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.82 (2H, 2 × s, NH), 8.49 (1H, s, NH), 9.85 (1H, s, H—C=N), 7.69 (2H, d, J = 8.1 Hz, ArH), 7.54–7.47 (3H, m, ArH), 7.35–7.20 (5H, m, ArH), 7.07 (1H, t, J = 7.5 Hz, Ar), 6.96 (1H, s, ArH), 3.83–3.65 (11H, m, H‐morpholine, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.3 (3 × C‐triazine), 147.3 (H—C=N) 158.1, 151.9, 142.7, 141.5, 137.3, 133.3, 130.7, 130.4, 127.4, 125.2, 124.3, 123.6, 122.2, 121.4, 120.4, 118.8, 112.8, 110.6, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 44.1 (NCH₂).

4.1.4.15. N′‐(4‐((6‐((4‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5o)

Yield: 73%; m.p.: 323–326 °C; R _f: 0.72 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3379, 3300 (2 × N—H stretch.), 3016 (C_sp2—H stretch.), 2956, 2863 (2 × C_sp3—H stretch.), 1633 (C=O stretch.), 1616 (C=N stretch.), 1581, 1507 (2× C=C stretch.), 1267, 1207 (2 × C—O stretch.), 739 (C—Br stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.82 (2H, 2 × s, ArH), 8.50 (1H, s, ArH), 9.79 (1H, s, H—C=N), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.54–7.20 (10H, m, ArH), 7.07 (1H, t, J = 7.2 Hz, ArH) 3.83 (3H, s, OCH₃), 3.74–3.62 (8H, m, H‐morpholine); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.1, 165.3 (3× C‐triazine), 147.2 (H—C=N), 158.1, 152.1, 142.8, 139.3, 137.3, 133.2, 131.5, 130.4, 127.4, 124.3, 123.8, 122.2, 121.8, 120.4, 114.3, 112.8, 110.4 104.1 (17 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.8 (NCH₂).

4.1.4.16. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((3‐Nitrophenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5p)

Yield: 69%; m.p.: 310–313 °C; R _f: 0.66 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3313, 3234 (2 × N—H stretch.), 3079 (C_sp2—H stretch.), 2971, 2854 (2 × C_sp3—H stretch.), 1649 (C=O stretch.), 1631 (C=N stretch.), 1590, 1523 (2 × C=C stretch.), 1253, 1208 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.82 (2H, 2 × s, NH, 8.49 (1H, s, NH), 10.16 (1H, s, H—C=N), 8.82 (1H, s, ArH), 7.89 (1H, d, J = 7.8 Hz, ArH), 7.80 (1H, d, J = 7.8 Hz, ArH), 7.69 (1H, d, J = 8.1 Hz, ArH), 7.52–7.46 (3H, m, ArH), 7.34–7.20 (4H, m, ArH), 7.07 (1H, t, J = 7.8 Hz, ArH), 3.84–3.56 (11H, m, H‐morpholine, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.0, 165.5 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.0, 148.3, 142.7, 141.2, 137.3, 133.3, 130.4, 130.1, 127.4, 126.0, 124.3, 123.6, 122.2, 121.2, 120.4, 117.1, 114.3, 112.8, 110.6, 104.1 (21 × ArC), 66.8 (OCH₂), 56.3 (OCH₃), 43.9 (NCH₂).

4.1.4.17. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((4‐Nitrophenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5q)

Yield: 77%; m.p.: 322–324 °C; R _f: 0.63 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3412, 3295 (2 × N—H stretch.), 3041 (C_sp2—H stretch.), 2964, 2859 (2 × C_sp3—H stretch.), 1632 (C=O stretch.), 1606 (C=N stretch.), 1591, 1540 (2 × C=C stretch.), 1274, 1202 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.96, 11.82 (2H, 2 × s, NH), 8.52 (1H, s, NH), 10.35 (1H, s, H—C=N), 8.06 (2H, m, ArH), 7.82–7.07 (10H, m, ArH), 3.84–3.56 (11H, m, H‐morpholine, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.0, 165.4 (3 × C‐triazine), 147.1 (H—C=N), 158.1, 152.0, 146.5, 142.7, 141.5, 137.3, 133.4, 130.4, 127.4, 125.0, 124.3, 123.8, 122.2, 121.2, 120.4, 119.4, 112.8, 110.4, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.9 (NCH₂).

4.1.4.18. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((2‐(trifluoromethyl)phenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5r)

Yield: 89%; m.p.: 293–296 °C; R _f: 0.70 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3397, 3290 (2 × N—H stretch.), 3078 (C_sp2—H stretch.), 2996, 2855 (2 × C_sp3—H stretch.), 1642 (C=O stretch.), 1618 (C=N stretch.), 1590, 1502 (2 × C=C stretch.), 1272, 1211 (2 × C—O stretch.), 1111 (C—F); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.97, 11.83 (2H, 2 × s, NH), 8.49 (1H, s, NH), 9.85 (1H, s, H—C=N), 7.69 (2H, d, J = 7.8 Hz, ArH), 7.53–7.46 (3H, m, ArH), 7.34–7.05 (7H, m, ArH), 3.75–3.56 8H, m, H‐morpholine), 3.83 (3H, s, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.5, 166.1, 165.3 (3 × C‐triazine), 147.1 (H—C=N), 158.1, 152.1, 143.3, 142.8, 137.3, 133.2, 130.4, 127.4, 124.3, 123.8, 122.2, 121.6, 121.5, 121.1, 120.4, 118.8, 112.8, 110.4, 104.1 (19 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.9 (NCH₂).

4.1.4.19. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((2‐(trifluoromethoxy)phenyl)‐Amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5s)

Yield: 87%; m.p.: 268–271 °C; R _f: 0.68 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3287, 3213 (2 × N—H stretch.), 3004 (C_sp2—H stretch.), 2916, 2863 (2 × C_sp3—H stretch.), 1659 (C=O stretch.), 1632 (C=N stretch.), 1573, 1499 (2 × C=C stretch.), 1254, 1202 (2 × C—O stretch.), 1158 (C—F stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.95, 11.81 (2H, 2 × s, NH), 8.49 (1H, s, NH), 9.84 (1H, s, H—C=N), 7.69 (2H, d, J = 7.8 Hz, ArH), 7.60–7.46 (3H, m, ArH), 7.35–7.20 (5H, m, ArH), 7.07 (2H, t, J = 7.5 Hz, ArH), 3.83–3.61 (11H, m, H‐morpholine, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.7, 166.1, 165.3 (3 × C‐triazine), (H—C=N), 158.1, 152.1, 147.1, 139.1, 142.8, 139.1, 137.3, 133.2, 130.4, 127.4, 124.3, 123.8, 122.2, 143.3, 118.9, 121.1, 120.4, 112.8, 110.3, 104.1 (20 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.9 (NCH₂).

4.1.4.20. N′‐(4‐((6‐((2,4‐Dimethylphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5t)

Yield: 90%; m.p.: 226–228 °C; R _f: 0.81 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3269, 3198 (2 × N—H stretch.), 3013 (C_sp2—H stretch.), 2914, 2869 (2 × C_sp3—H stretch.), 1631 (C=O stretch.), 1616 (C=N stretch.), 1559, 1497 (2 × C=C stretch.), 1253, 1206 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.94, 11.82 (2H, 2 × s, NH), 8.46 (1H, s, NH), 8.91 (1H, s, H—C=N), 7.69 (1H, d, J = 7.8 Hz, ArH), 7.48 (2H, d, J = 7.8 Hz, ArH), 7.34–7.15 (5H, m, ArH), 7.10–6.89 (3H, m, ArH), 3.83 (3H, s, OCH₃), 3.54 (8H, bs, H‐morpholine), 2.22 (3H, s, CH₃), 2.13 (3H, s, CH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.4, 166.0 (3 × C‐triazine), 147.1 (H—C=N), 158.1, 152.1, 142.8, 139.8, 137.3, 134.5, 133.4, 132.9, 130.4, 127.4, 126.6, 126.4, 124.3, 123.8, 122.2, 121.1, 120.4, 118.4, 112.8, 110.4, 104.1 (21 × ArC), 66.2 (OCH₂), 56.3 (OCH₃), 43.6 (NCH₂), 20.94 (CH₃), 18.45 (CH₃).

4.1.4.21. N′‐(4‐((6‐((3‐Chloro‐2‐Methylphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5u)

Yield: 96%; m.p.: 241–243 °C; R _f: 0.79 (chloroform:methanol; 8:2); IR (ATR, ū, cm⁻¹): 3442, 3279 (2 × N—H stretch.), 3040 (C_sp2—H stretch.), 2970, 2869 (2 × C_sp3—H stretch.), 1637 (C=O stretch.), 1617 (C=N stretch.), 1575, 1496 (2 × C=C stretch.), 1251, 1203 (2 × C—O stretch.); ¹H‐NMR (300 MHz, DMSO‐d₆): δ (ppm) 11.93, 11.80 (2H, 2 × s, NH), 8.46 (1H, s, NH), 9.25 (1H, s, H—C=N), 7.69 (1H, d, J = 7.8 Hz, ArH), 7.48 (2H, d, J = 7.2 Hz, ArH), 7.33–7.20 (6H, m, ArH), 7.15–7.05 (2H, m, H), 3.83 (1H, s, OCH₃), 3.56 (8H, s, ArH), 2.18 (3H, s, CH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ (ppm) 170.8, 166.5, 166.0 (3 × C‐triazine), 147.2 (H—C=N), 158.1, 152.0, 143.3, 142.8, 137.3, 133.9, 133.0, 131.7, 130.4, 127.4, 126.8, 126.2, 125.7, 124.3, 123.7, 122.2, 121.0, 120.4, 112.8, 110.3, 104.1 (21 × ArC), 66.2 (OCH₂), 56.2 (OCH₃), 43.7 (NCH₂), 15.8 (CH₃).

4.2. Biology

4.2.1. Antiviral Evaluation

The antiviral evaluation against influenza viruses A/H1N1 A/Ned/378/05, A/H3N2 A/HK/7/87, and B/Ned/537/05, respiratory syncytial virus (RSV), yellow fever virus (YFV), Zika virus, Herpes simplex virus 1 (HSV‐1), and human coronaviruses HCoV‐229E, HCoV‐OC43 and SARS‐CoV‐2 was performed by seeding MDCK (CVCL_0422), Hep‐2 (CVCL_1906), Hep3B (CVCL_0326), HEL 299 (CVCL_2480) or VeroE6 (CVCL_0574) cells into 384‐well dishes, respectively. Briefly, after 24 h at 37 °C, serial dilutions of the compounds were added to the cells prior to infection. At 4 days post infection (influenza, RSV, HSV‐1, HCoV‐229E and SARS‐CoV‐2) or 6 days post infection (YFV, Zika virus and HCoV‐OC43), the virus‐induced cytopathogenic effect was measured colorimetrically by the formazan‐based MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay from Promega), and the antiviral activity was expressed as the 50% effective concentration (EC₅₀). In parallel, the 50% cytotoxic concentration (CC₅₀) was derived from mock‐infected MDCK, Hep‐2 Hep3B, HEL 299 and VeroE6 cells. The activities were compared with reference compounds remdesivir, zanamivir, acyclovir, and dextran sulfate with a molecular weight of ±10,000 (DS‐10,000). Values represent mean ± SD from at least two independent experiments.

4.2.2. Time‐of‐Drug‐Addition Assay

HEL 299 cells were infected with HCoV‐229E under standard conditions, and test compounds were added at different time points relative to infection (−1 h, 0 h, 30 min, and up to 6 h post‐inoculation) to identify the stage of the viral life cycle affected. Compound 5o was tested at 100 µM (EC₅₀ ~ 1 µM), and remdesivir at 5 µM (EC₅₀ ~ 0.1 µM), aloxistatin (E64d) at 30 µM (EC₅₀ ~ 0.3 µM), chloroquine (CQ) at 50 µM (EC50 ~ 0.5 µM), hippeastrum hybrid agglutinin (HHA) at 100 µg/mL (EC₅₀ ~ 5 µg/mL) were included as reference compounds. After 1 h adsorption at 37 °C, inoculum was removed, and fresh medium was added. Plates were incubated at 37 °C until 24 h post‐infection, then lysed for viral RNA quantification by qPCR targeting the HCoV‐229E membrane gene. Actin was measured in parallel as an internal control. Datapoints are results from two independent experiments.

4.2.3. Adsorption Assay

HEL299 cells were pre‐chilled on ice before infection with HCoV‐229E. Test compounds were prepared in cold medium and added to cells under adsorption conditions at 4 °C, followed by virus inoculation and incubation on ice for 1 h to allow binding without internalization. After washing with cold PBS, cells were shifted to 37 °C in fresh medium. Two parallel conditions were included:

(1) Compounds not re‐added after the temperature shift, to specifically evaluate their impact on the adsorption process; (2) Compounds re‐added after the shift, to confirm antiviral activity under standard conditions. Viral replication was measured after 5 days using an MTS‐based viability assay. Datapoints are results from two independent experiments.

4.2.4. Cell‐Based Fusion Assay

HEK293T (CVCL_0063) were maintained in DMEM supplemented with 5% FBS. For transfection, cells were transfected using Turbofectin 8.0 with plasmids encoding ACE2, SARS‐CoV‐2 spike, and mNeonGreen. After overnight incubation, cells were reseeded in 96‐well plates and treated with test compounds immediately after seeding. Fusion was monitored using an Incucyte live‐cell imaging system in phase contrast and green fluorescence channels, and quantified by measuring mNeonGreen‐positive syncytia, defined by their size. Values represent mean ± SD from three independent experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

CMDC-21-e70434-s001.pdf^{(4.3MB, pdf)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material

CMDC-21-e70434-s001.pdf^{(4.3MB, pdf)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Synthesis of 1,3,5‐Triazine‐Indole Conjugates as Broad‐Spectrum Coronavirus Entry Inhibitors

Shazia Asghar

Leentje Persoons

Yanicka Smolders

Niels Willems

Dirk Daelemans

Steven De Jonghe

Najim A Al‐Masoudi

Shahid Hameed

Abstract

1. Introduction

FIGURE 1.

2. Results and Discussion

2.1. Chemistry

SCHEME 1.

2.2. Broad Antiviral Evaluation

TABLE 1.

2.3. Antiviral Evaluation Against Human Coronaviruses

TABLE 2.

2.4. Mechanistic Insights Into Antiviral Action

FIGURE 2.

2.5. In Silico ADMET Studies

TABLE 3.

3. Conclusion

4. Experimental Section

4.1. Chemistry

4.1.1. Materials

4.1.2. General Experimental Methods

4.1.3. Procedure for the Synthesis of 1H‐Indole‐2‐Carbohydrazide (4)

4.1.4. General Procedure for the Synthesis of N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(phenylamino)‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide [51] (5a–u)

4.1.4.1. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(phenylamino)‐1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5a)

4.1.4.2. N′‐(2‐Methoxy‐4‐((6‐((2‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazides (5b)

4.1.4.3. N′‐(2‐Methoxy‐4‐((6‐((3‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5c)

4.1.4.4. N′‐(2‐Methoxy‐4‐((6‐((4‐Methoxyphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5d)

4.1.4.5. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(o‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5e)

4.1.4.6. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(m‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)‐Benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5f)

4.1.4.7. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐(p‐Tolylamino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5g)

4.1.4.8. N′‐(4‐((6‐((2‐Fluorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5h)

4.1.4.9. N′‐(4‐((6‐((4‐Fluorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5i)

4.1.4.10. N′‐(4‐((6‐((2‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5j)

4.1.4.11. N′‐(4‐((6‐((3‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5k)

4.1.4.12. N′‐(4‐((6‐((4‐Chlorophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5l)

4.1.4.13. N′‐(4‐((6‐((2‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5m)

4.1.4.14. N′‐(4‐((6‐((3‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5n)

4.1.4.15. N′‐(4‐((6‐((4‐Bromophenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5o)

4.1.4.16. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((3‐Nitrophenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5p)

4.1.4.17. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((4‐Nitrophenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5q)

4.1.4.18. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((2‐(trifluoromethyl)phenyl)amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5r)

4.1.4.19. N′‐(2‐Methoxy‐4‐((4‐Morpholino‐6‐((2‐(trifluoromethoxy)phenyl)‐Amino)−1,3,5‐Triazin‐2‐yl)oxy)benzylidene)‐1H‐Indole‐2‐Carbohydrazide (5s)

4.1.4.20. N′‐(4‐((6‐((2,4‐Dimethylphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5t)

4.1.4.21. N′‐(4‐((6‐((3‐Chloro‐2‐Methylphenyl)amino)‐4‐Morpholino‐1,3,5‐Triazin‐2‐yl)oxy)‐2‐Methoxybenzylidene)‐1H‐Indole‐2‐Carbohydrazide (5u)

4.2. Biology

4.2.1. Antiviral Evaluation

4.2.2. Time‐of‐Drug‐Addition Assay

4.2.3. Adsorption Assay

4.2.4. Cell‐Based Fusion Assay

Conflicts of Interest

Supporting information

Data Availability Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

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