Anthranilamides with quinoline and β-carboline scaffolds: design, synthesis, and biological activity.

In the present study, we report the design and synthesis of novel amide-type hybrid molecules based on anthranilic acid and quinoline or β-carboline heterocyclic scaffolds. Three types of biological screenings were performed: (i) in vitro antiproliferative screening against a panel of solid tumor and leukemia cell lines, (ii) antiviral screening against several RNA viruses, and (iii) anti-quorum sensing screening using gram-negative Chromobacterium violaceum as the reporter strain. Antiproliferative screening revealed a high activity of several compounds. Anthranilamides 12 and 13 with chloroquine core and halogenated anthranilic acid were the most active agents toward diverse cancer cell lines such as glioblastoma, pancreatic adenocarcinoma, colorectal carcinoma, lung carcinoma, acute lymphoblastic, acute myeloid, chronic myeloid leukemia, and non-Hodgkin lymphoma, but also against noncancerous cell lines. Boc-protected analogs 2 and 3 showed moderate activities against the tested cancer cells without toxic effects against noncancerous cells. A nonhalogenated quinoline derivative 10 with N-benzylanthranilic acid residue was equally active as 12 and 13 and selective toward tumor cells. Chloroquine and quinoline anthranilamides 10-13 exerted pronounced antiviral effect against human coronaviruses 229E and OC43, whereas 12 and 13 against coronavirus OC43 (EC50 values in low micromolar range; selectivity indices from 4.6 to > 10.4). Anthranilamides 14 and 16 with PQ core inhibited HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively. Compound 13 displayed significant anti-quorum/biofilm effect against the quorum sensing reporter strain (IC50 of 3.7 μM) with no apparent bactericidal effect.

Introduction

Anthranilic acid (2-aminobenzoic acid) is an aromatic non-peptide-forming amino acid. It is a biosynthetic precursor of tryptophan and the initial building block in the biosynthesis of Pseudomonas quinolone signal PQS (2-heptyl-3-hydroxyquinolin-4(1H)-one) and its precursor HHQ (2-heptylquinolin-4(1H)-one) [1]. It is also a motive present in several drugs and insecticides (Fig. 1) [2, 3].

Fig. 1

Drugs a and insecticides b based on anthranilic acid

Tranilast was first approved for the therapy of bronchial asthma and later for the treatment of keloids and hypertrophic scars [4]. It suppresses the proliferation of fibroblasts and decreases the viability of various cancer cell lines, along with the promotion of apoptosis, inhibition of angiogenesis, and cell migration [5-9]. These findings prompted us to design novel anthranilic acid-based hybrids as potential anticancer agents. To design compounds with improved activity, we combined anthranilic acid motif with other two scaffolds known for their antiproliferative properties, namely quinoline and β-carboline. Considering the numerous preclinical and clinical trials confirming the usefulness of antimalarial drugs in cancer therapy [10-16] and based on our previous experience with primaquine (PQ) and chloroquine derivatives with pronounced antiproliferative activities [17, 18], we have chosen N1-(7-chloroquinolin-4-yl)butane-1,4-diamine (CQ), as a simplified chloroquine molecule, and PQ-base as amino components for the preparation of anthranilamides. On the other hand, β-carboline alkaloids (harmane, harmine, and harmaline) possess diverse biological activities, including antimalarial and antiproliferative activity [19-37]. Zhao et al. investigated the cytotoxic activity of β-carboline conjugates with amino acid esters and found aromatic (Phe) and basic amino acid derivatives (Lys and Arg) as the most active against several cancer cell lines [38]. This finding further corroborated the idea to combine β-carboline and anthranilic acid moieties in hybrid molecules.

Chloroquine is also known for its antiviral effects [39-41]. During the COVID-19 pandemic, its antiviral potential was investigated intensively. A number of scientific papers reported chloroquine efficacy and acceptable safety in the treatment of COVID-19 associated pneumonia [42-46]. Several clinical trials based on chloroquine started as well, although some of them were discontinued due to the lack of clinical efficacy [47]. Since the antiviral effects of harmala alkaloids were also reported [48-52], the aim of the present study was to design and prepare a series of anthranilamides with quinoline and β-carboline cores and explore their antiproliferative and antiviral potential. Some quinoline derivatives, including chloroquine-based fumardiamides prepared by our research group, were previously shown to prevent cell-to-cell communication (i.e., quorum sensing, QS), expression of virulence factors, and biofilm formation in gram-negative pathogens [53, 54]. Based on these findings, we additionally evaluated potential of the novel quinoline derivatives as QS inhibitors.

Results and discussion

Chemistry

A series of anthranilamides 11–19 with quinoline- or harmane-based amines and their N-protected precursors 1–10 were prepared. The synthetic routes leading to the title compounds are outlined in Scheme 1.

Scheme 1

Synthetic route for the preparation of anthranilamides 1–19

The first step included the coupling of N-protected anthranilic acid (N-benzylanthranilic acid, N-Boc-4-chloroanthranilic acid or N-Boc-5-bromoanthranilic acid) with corresponding amines: N1-(7-chloroquinolin-4-yl)butane-1,4-diamine (CQ), primaquine (PQ) or harmane-based amine (1-methyl-9H-pyrido[3,4-b]indol-3-yl)methanamine (HA). N-protected anthranilic acids are commercially available compounds, whereas the starting amines were prepared following the published procedures. N1-(7-chloroquinolin-4-yl)butane-1,4-diamine was obtained from 4,7-dichloroquinoline and 1,4-diaminobutane under microwave irradiation (300 W) at 95 °C [17] and PQ was prepared from PQ diphosphate. The preparation of the aminoharmane derivative HA was more complex. The harmane scaffold was built from tryptophan ester utilizing a microwave-assisted Pictet–Spengler reaction [38, 55, 56]. Briefly, methyl 3-(1H-indol-3-yl)-2-aminopropanoate, acetaldehyde dimethyl acetal (ADMA), and trifluoroacetic acid (TFA) were heated under microwave irradiation to afford a corresponding tetrahydro-β-carboline product, which was then aromatized using KMnO4 at room temperature. Further steps included: (i) reduction of the harmane ester to alcohol using LiAlH4, (ii) conversion of the alcohol to azide by means of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and finally, (iii) reduction of the azide to amine HA under a hydrogen atmosphere.

Compound 1 was prepared by coupling of N-benzylanthranilic acid with CQ utilizing propanephosphonic acid anhydride (T3P) and triethylamine (TEA), whereas N-protected anthranilamides 2–9 were obtained by coupling of the corresponding anthranilic acid derivative with CQ, PQ or HA using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIEA) coupling system.

The deprotection of compound 1 with ammonium formate/Pd was rather unexpected and afforded product 10, still bearing benzyl group, but without chlorine in the quinoline part of the molecule. On the other hand, hydrogenation of compound 1 with Pd catalyst gave deprotected quinoline (Q) derivative 11 without chlorine atom. The removal of the Boc-group from compounds 2, 3, 5, 6, 8, and 9 proceeded smoothly with HCl and so did the removal of the benzyl (Bn) group from 4 and 7 using H2/Pd and HCOONH4/Pd, respectively. Altogether, one Q-derivative, two CQ, three PQ, and three HA anthranilamides with free amino groups were prepared.

A common set of physicochemical parameters (molecular weight, partition coefficient, number of H-bond donors and acceptors, molecular refractivity, topological polar surface area, rotatable bonds count) was calculated using Chemicalize.org software and is presented in Table 1 [57]. Compounds 7–19 are fully in agreement with Lipinski’s and Gelovani’s rules for prospective small molecular drugs (MW ≤ 500, log P ≤ 5, number of H-bond donors ≤ 5, number of H-bond acceptors ≤ 10, TPSA < 140 Å2, MR within the range of 40 and 130 cm3/mol, the number of atoms 20–70), while 1–6 show slight deviations in MR, MW and/or log P.

Table 1

The Lipinski and Gelovani parameters of the anthranilamides 10–19 calculated with Chemicalize.org program [57]

Cmpd	Molecular formula	Number of atoms	MW	log P	HBD	HBA	MR (cm3 mol−1)	TPSA (Å2)	RBC
1	C27H27ClN4O	60	458.99	5.51	3	4	137.36	66.05	10
2	C25H28Cl2N4O3	62	503.42	5.18	3	5	137.65	92.35	10
3	C25H28BrClN4O3	62	547.88	5.34	3	5	140.47	92.35	10
4	C29H32N4O2	67	468.60	5.16	3	5	143.44	75.28	11
5	C27H33ClN4O4	69	513.04	4.83	3	6	143.73	101.58	11
6	C27H33BrN4O4	69	557.49	5.00	3	6	146.54	101.58	11
7	C27H24N4O	56	420.52	4.73	3	3	128.78	69.81	6
8	C25H25ClN4O3	58	464.95	4.40	3	4	129.07	96.11	6
9	C25H25BrN4O3	58	509.40	4.57	3	4	131.89	96.11	6
10	C27H28N4O	60	424.55	4.90	3	4	132.55	66.05	10
11	C20H22N4O	47	334.423	2.88	3	4	102.45	80.04	7
12	C20H20Cl2N4O	47	403.310	4.09	3	4	112.06	80.04	7
13	C20H20BrClN4O	47	447.760	4.25	3	4	114.88	80.04	7
14	C22H26N4O2	54	378.476	3.14	3	5	113.33	89.27	8
15	C22H25ClN4O2	54	412.920	3.74	3	5	118.13	89.27	8
16	C22H25BrN4O2	54	457.372	3.91	3	5	120.95	89.27	8
17	C20H18N4O	43	330.391	2.71	3	3	98.67	83.80	3
18	C20H17ClN4O	43	364.830	3.31	3	3	103.48	83.80	3
19	C20H17BrN4	43	409.287	3.48	3	3	106.30	83.80	3

MW, molecular weight; log P, partition coefficient; HBD, H-bond donor; HBA, H-bond acceptor; MR, molecular refractivity; TPSA, topological polar surface area; RBC, rotatable bond counts

Antiproliferative activity

Our first goal was to evaluate the anticancer activity of newly prepared quinoline- and harmane-based anthranilamides against a series of cancer cell lines. In vitro experiments on eight human solid tumor and leukemia cell lines (glioblastoma LN-229, pancreatic adenocarcinoma Capan-1, colorectal carcinoma HCT-116, lung carcinoma NCI-H460, acute lymphoblastic leukemia DND-41, acute myeloid leukemia HL-60, chronic myeloid leukemia K-562, and non-Hodgkin lymphoma Z-138) were performed. Normal human lung fibroblasts (HEL 299) were used as a model for noncancerous cells. The cells were treated with compounds at different concentrations and the IC50 values were determined (Table 2). Docetaxel (DTX) and staurosporine (STS) were included as positive controls. All anthranilamides with PQ core (4–6, 14–16) were inactive. In the CQ series, compounds with the free amino group were more active than their N-protected analogues pairs, suggesting the importance of amino moiety for activity. CQ derivatives 12 and 13 with two halogen atoms (chlorine in amine part and chlorine or bromine atoms in the carboxylic part of the molecules) and free amino group exerted the highest antiproliferative activity with IC50 between 2 and 10 µM, approximately ten times lower than the parent compound and the analogous compounds 1–3 with N-protected amino groups. However, these compounds exerted high antiproliferative activity against HEL 299 cells as well. On the other hand, Boc-protected analogues 2 and 3 showed moderate activities against tested cancer cells and no cytotoxicity against noncancerous cells. N-benzyl-protected quinoline derivative 10 without halogen substituents was equally active as compounds 12 and 13, whereas its analogue 11 with a free amino group had approximately 3–40 lower activity, depending on the cell type. Compound 10 showed favorable selectivity, ranging from 2 to 10 depending on the cell type. Comparison of N-protected CQ derivatives 1–3 with PQ derivatives 4–6 and HA derivatives 7–9 revealed the importance of the heterocycle part of the molecule. IC50 values of the most effective HA derivatives 8 and 9 were in the low micromolar range against four tested cancer cell types, but also against HEL 299. Within all tested anthranilamides, 2-(benzylamino)-N-[4-(quinolin-4-ylamino)butyl]benzamide (10) is the most promising compound with the best toxicity/activity ratio.

Table 2

Antiproliferative screening of anthranilamides 1–19 toward human cancer cell lines

Cmpd	Structural formula	IC50 (µM)a
		LN-229	Capan-1	HCT-116	NCI-H460	DND-41	HL-60	K-562	Z-138	HEL 299
1		53.5 ± 12.2	57.6 ± 14.6	> 100	> 100	> 100	> 100	55.7 ± 10.0	> 100	8.0 ± 4.4
2		30.1 ± 2.1	20.4 ± 9.0	32.4 ± 20.0	21.2 ± 10.0	46.2 ± 1.9	41.5 ± 17.0	54.5 ± 18.5	28.6 ± 14.6	> 100
3		48.4 ± 20.0	40.6 ± 4.2	66.1 ± 30.0	69.3 ± 30.7	51.3 ± 4.4	60.5 ± 17.0	51.2 ± 9.3	55.2 ± 9.2	> 100
4		> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100
5		> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100
6		> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100
7		35.8 ± 9.3	27.5 ± 8.1	34.6 ± 6.0	28.4 ± 4.0	53.5 ± 6.7	66.2 ± 18.3	90.7 ± 18.5	21.0 ± 10.1	50.9 ± 2.7
8		20.8 ± 13.8	6.5 ± 2.9	28.9 ± 24.1	8.3 ± 4.3	32.1 ± 20.8	6.7 ± 0.2	29.1 ± 21.3	8.2 ± 1.9	7.1 ± 1.8
9		8.3 ± 3.1	5.8 ± 3.0	32.9 ± 7.9	3.2 ± 1.8	12.5 ± 6.9	10.8 ± 3.3	63.1 ± 36.9	4.1 ± 1.8	7.5 ± 1.0
10		3.0 ± 2.0	4.2 ± 3.4	7.4 ± 1.5	2.2 ± 0.8	8.0 ± 3.2	14.9 ± 7.3	5.9 ± 3.5	4.6 ± 2.0	33.6 ± 15.6
11		39.7 ± 4.4	62.2 ± 9.7	45.4 ± 4.9	87.4 ± 12.6	59.6 ± 1.7	44.7 ± 11.0	18.7 ± 6.7	31.2 ± 1.1	41.6 ± 1.2
12		2.8 ± 1.2	6.3 ± 2.8	8.9 ± 3.0	7.3 ± 1.5	9.2 ± 2.2	9.8 ± 3.5	8.4 ± 3.2	7.2 ± 2.6	7.4 ± 1.2
13		3.1 ± 0.8	5.4 ± 1.8	7.0 ± 0.3	5.3 ± 0.4	7.8 ± 4.1	10.3 ± 3.2	7.7 ± 1.1	6.0 ± 2.6	7.8 ± 1.8
14		> 100	> 100	> 100	76.7 ± 25.0	> 100	> 100	> 100	> 100	> 100
15		> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100
16		> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100	> 100
17		41.0 ± 0.3	31.4 ± 1.9	48.4 ± 3.8	54.8 ± 5.9	61.5 ± 2.1	44.0 ± 8.4	52.0 ± 5.3	35.1 ± 4.5	32.8 ± 3.3
18		44.0 ± 7.6	56.3 ± 24.1	55.0 ± 17.7	43.8 ± 8.4	> 100	> 100	> 100	> 100	> 100
19		46.2 ± 7.5	38.9 ± 5.8	60.8 ± 31.0	55.0 ± 5.0	68.3 ± 12.2	66.3 ± 34.0	64.9 ± 4.9	55.5 ± 12.1	82.6 ± 17.4
CQ		8.9 ± 2.3	46.1 ± 1.3	65.5 ± 7.3	> 100	59.5 ± 3.5	> 100	50.6 ± 2.9	47.4 ± 5.3	84.7 ± 15.3
PQ		45.4 ± 1.2	45.6 ± 3.5	53.9 ± 4.2	> 100	50.2 ± 16.7	28.4 ± 1.6	40.5 ± 6.0	11.4 ± 1.1	> 100
HA		> 100	68.6 ± 3.4	> 100	85.1 ± 14.8	> 100	> 100	> 100	57.4 ± 32.6	> 100
DTXb		1.8 ± 3.0	10.0 ± 3.0	2.5 ± 3.0	27.1 ± 3.0	5.5 ± 3.0	13.7 ± 3.0	8.9 ± 3.0	2.5 ± 3.0	nt
STSb		51.1 ± 3.0	38.2 ± 3.0	68.7 ± 3.0	38.4 ± 3.0	40.7 ± 3.0	59.8 ± 3.0	73.9 ± 3.0	40.7 ± 3.0	nt

aIC50, the concentration required to decrease viability or cell growth by 50%; CQ, chloroquine, PQ, primaquine; HA, aminoharmane, DTX, docetaxel, STS, staurosporine

bConcentration in nM

Antiviral activity

In addition, the synthesized anthranilamides were evaluated against a broad variety of viruses including HIV-1 NL4.3 and HIV-2 ROD, influenza A/H1N1 A/Ned/378/05, influenza A/H3N2 A/HK/7/87, influenza B B/Ned/537/05, respiratory syncytial virus and human coronaviruses 229E and OC43 (in MT-4, MDCK, Hep-2 or HEL 299 cells; positive controls: PMPA, AMD3100, zanamivir, ribavirin, rimantadine, ribavirin, DS-10.000, remdesivir, and GS-441524, respectively). Chloroquine and quinoline anthranilamides 10–13 exerted pronounced antiviral effect against human coronavirus 229E and two of them, 12 and 13, against coronavirus OC43 as well, with EC50 values (50% effective concentrations) in the low micromolar range (< 0.8–7.7 µM), comparable with chloroquine’s EC50. Their selectivity indices (CC50 to EC50 ratio, SI) varied from 4.6 to > 10.4, while chloroquine SI was higher (approximately 30). PQ and HA derivatives were inactive against coronaviruses. Anthranilamide 14 and bromoanthranilamide 16 with PQ core were able to inhibit HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively, yielding a favorable selectivity index of 8.4 for 14. Primaquine itself showed no anti-HIV activity. Although chloroquine shows selective antiviral activity against both HIV-1 and HIV-2, none of the anthranilamides with CQ core showed antiretroviral activity. Also, influenza viruses and the respiratory syncytial virus did not respond to any of the anthranilamides tested. All compounds were tested, but only selected results are presented in Tables 3 and 4.

Table 3

Antiviral activity of selected anthranilamides against human coronaviruses 229E and OC43

Cmpd	Cytotoxicity (CC50)	Antiviral activity (EC50)
		229E	SI	OC43	SI
10	5.6 ± 3.0	< 0.8	> 7	> 100	–
11	46.3 ± 1.2	7.7 ± 0.3	6.0	> 100	–
12	8.3 ± 0.78	1.8 ± 1.2	4.6	< 0.8	> 10.4
13	7.5 ± 1.3	1.4 ± 0.1	5.4	1.5 ± 0.2	5.0
CQ	40.8 ± 2.2	1.4 ± 0.2	29.1	1.3 ± 1.2	31.4
PQ	> 100	> 100	–	> 100	–
HA	> 100	> 100	–	> 1002	–
Remdesivir	> 10	0.06 ± 0.02	> 160	0.04 ± 0.01	> 250
GS-441524	> 100	0.9 ± 0.1	> 110	1.3 ± 0.1	> 75

In HEL cell culture; CC50 (µM), 50% cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay; EC50 (µM), concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay; SI, selectivity index (CC50 to EC50 ratio); CQ, chloroquine, PQ, primaquine; HA, aminoharman; GS-441524, main plasma metabolite of remdesivir

Table 4

Antiviral activity of selected anthranilamides against HIV-1 and HIV-2

Cmpd	Cytotoxicity (CC50)	Antiviral activity (EC50)
		HIV-1 NL4.3	SI	HIV-2 ROD	SI
14	78.2 ± 9.3	9.3 ± 0.5	8.4	> 100	–
16	56.4 ± 1.6	14.1 ± 0.6	4.0	> 100	–
CQ	> 100	31.7 ± 4.7	> 3.2	3.8 ± 1.6	> 26.3
PQ	17.9 ± 8.0	> 100	–	> 100	–
HA	22.3 ± 4.9	> 100	–	> 100	–
PMPA	> 100	1.6	> 64	0.6	> 161

In MT-4 cell culture; CC50 (µM), 50% cytotoxic concentration; EC50 (µM), concentration producing 50% inhibition of virus-induced cytopathic effect; SI, selectivity index (CC50 to EC50 ratio); CQ, chloroquine, PQ, primaquine; HA, aminoharman; PMPA, 2-phosphonomethoxypropyl adenine [58]

Anti-QS and bactericidal effects on the Chromobacterium violaceum QS-reporter strain

Anthranilamides 1–19 were also tested for their ability to prevent the induction of QS and the following biofilm formation in gram-negative bacteria using C. violaceum ATCC31532 as the QS-indicator strain [59]. Three compounds, 11, 12, and 13, displayed significant anti-QS effect (> 34–63%) against the reporter strain with similar or minor (≤ 32%) bactericidal effect at 400 µM level under the screening conditions used (Fig. 2, SI Table 1).

Fig. 2

Three anthranilamide derivatives showed high anti-QS activity against the C. violaceum ATCC31532 QS-reporter strain. The selected compounds were tested at 400, 40 and 10 µM concentration levels and their effects on the violacein production (a) and viability (b) were recorded after an overnight incubation at 27 °C. CD, cells with 2% DMSO; A, azithromycin; Q, quercetin (both at 400 µM). Violacein production was monitored at 595 nm and the reduction of resazurin to resorufin with λ = 560/590 nm using the PerkinElmer Victor3 multilabel plate reader. The experiment was repeated three times. Error bars, ± SD (n = 3)

Compound 13 with IC50 of 3.7 µM (95% CI 0.4–10.3) was the most active: At 400 µM concentration, the compound reduced the QS-induced violacein production in C. violaceum by more than 60%, while the viability of the reporter strain was less affected (ca., 30% reduction) under the same conditions. This compound showed reasonably high anti-QS activity (> 40%) with no apparent bactericidal effect on the reporter also at 40 and 10 µM concentration levels. Interfering with the bacterial QS signaling is considered a method of choice to combat pathogenic biofilms and recalcitrant infections without increasing the risk of resistance development [60]. Thus, from all tested compounds, 13 shows promise as a potentially new anti-QS agent capable of preventing biofilm formation of gram-negative pathogens without posing selective pressure on the bacterial cell.

Conclusions

In conclusion, we have successfully prepared and characterized 19 novel anthranilamides with different heterocycle and anthranilic acid moieties and evaluated their potential as antiproliferative and antiviral agents. Of all tested anthranilamides, 2-(benzylamino)-N-[4-(quinolin-4-ylamino)butyl]benzamide (10) was the most promising compound with the best selectivity index. Several anthranilamides with CQ, Q and HA cores showed high antiproliferative activities against all or selected cancer cells, but also general toxicity, whereas two compounds exerted moderate, but selective activity toward cancer cell lines. Chloroquine and quinoline anthranilamides 10–13 exerted pronounced antiviral effect against human coronavirus 229E and two of them, 12 and 13, also against coronavirus OC43. Quinoline anthranilamide 13 displayed significant anti-QS/-biofilm activity in gram-negative QS-reporter strain, while primaquine anthranilamide 14 showed favorable selectivity against HIV-1. Taken together, obtained results could serve as a basis for the development of lead compounds with high and selective antitumor and/or antiviral/antibacterial properties.

Experimental

Materials and methods

Melting points were determined on the SMP3 apparatus (Barloworld Scientific, UK) in open capillaries and are uncorrected. CEM Discover microwave reactor was used for microwave reactions (CEM GmbH, Germany). IR spectra were recorded on Spectrum One (PerkinElmer, UK) and UV–VIS spectra on Lambda 20 double-beam spectrophotometers (PerkinElmer, UK). NMR 1H and 13C spectra were recorded at 25 °C on the NMR Avance 600 spectrometer (Bruker, Germany) at 300.13 or 600.13 and 75.47 or 150.9 MHz for 1H and 13C nuclei, respectively. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane in the 1H and the dimethyl sulfoxide (DMSO) residual peak as a reference in the 13C spectra (39.51 ppm). Coupling constants (J) are reported in hertz (Hz). Mass spectra were collected on an HPLC–MS/MS instrument (HPLC, Agilent Technologies 1200 Series; MS, Agilent Technologies 6410 Triple Quad) using electrospray ionization in positive mode. Elemental analyses were performed on a CHNS LECO analyzer (LECO Corporation, USA). All compounds were routinely checked by TLC with Merck silica gel 60F-254 glass plates using dichloromethane/methanol 9.5:0.5, 9:1, 8.5:1.5, cyclohexane/ethyl acetate 1:1 and cyclohexane/ethyl acetate/methanol 3:1:0.5, 1:1:0.5 as the solvent systems. Spots were visualized by short-wave UV light and iodine vapor. Column chromatography was performed on silica gel 0.063–0.200 mm (Sigma-Aldrich, USA) with the same eluents used in TLC.

All chemicals and solvents were of analytical grade and purchased from commercial sources. 4,7-Dichloroquinoline, butane-1,4-diamine, acetaldehyde dimethyl acetal (ADMA), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), lithium aluminum hydride, palladium on activated charcoal, propanephosphonic acid anhydride (T3P), ammonium formate, triethylamine (TEA), DIEA, and HATU were purchased from Sigma-Aldrich. N-Boc-4-chloroanthranilic acid and N-Boc-5-bromoanthranilic acid were purchased from Fluorochem. N-Benzylanthranilic acid, 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP) and L-tryptophan methyl ester hydrochloride were purchased from TCI. Potassium permanganate was purchased from Gram-Mol and hydrochloric acid from Carlo Erba. Anhydrous solvents were dried and redistilled prior to use. PQ was prepared from PQ diphosphate. All reactions with PQ were run protected from light.

Syntheses

To a mixture of N1-(7-chloroquinolin-4-yl)butane-1,4-diamine (0.067 g, 0.27 mmol), N-benzylanthranilic acid (0.067 g, 0.297 mmol) and TEA (0.055 g, 0.54 mmol) in N,N-dimethylformamide (DMF, 1 mL) was added dropwise a 50% solution of T3P in EtOAc (160 µL, 0.27 mmol) at 0 °C. The reaction mixture was slowly allowed to warm to r.t. and stirred for 19 h. The reaction mixture was diluted with EtOAc and extracted with a 5% solution of NaOH in water (3 × 30 mL) and washed with water (1 × 30 mL). The organic layer was dried through phase separator, filtrated, and evaporated under reduced pressure. After purification by column chromatography (mobile phase dichloromethane/methanol 9:1) and crystallization from diethyl ether, 0.082 g (66%) of white solid 1 was obtained; mp 102–104 °C; IR (KBr) (νmax/cm−1): 3284, 3032, 2934, 1623, 1578, 1535, 1449, 1426, 1368, 1352, 1328, 1279, 1245, 1202, 1159, 1136, 1064, 910, 871, 806, 747, 695, 499; 1H NMR (δ/ppm): 8.38–8.35 (m, 2H), 8.28 (d, 1H, J = 9.1 Hz), 8.23 (t, 1H, J = 5.7 Hz), 7.78 (d, 1H, J = 2.2 Hz), 7.54 (dd, 1H, J = 7.8, 1.3 Hz), 7.43 (dd, 1H, J = 9.0, 2.2 Hz), 7.35–7.28 (m, 5H), 7.26–7.16 (m, 2H), 6.62 (d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.4 Hz), 6.48 (d, 1H, J = 5.5 Hz), 4.36 (d, 2H, J = 5.8 Hz), 3.32–3.26 (m, 4H), 1.75–1.60 (m, 4H); 13C NMR (δ/ppm): 169.10, 151.90, 150.05, 149.12, 148.93, 139.57, 133.31, 132.24, 128.44, 128.23, 127.48, 127.10, 126.82, 124.09, 123.94, 117.45, 115.47, 114.38, 111.45, 98.64, 46.10, 42.14, 38.49, 26.75, 25.28; ESI–MS m/z = 459.4 [M + 1]+; Anal. Calcd. for C27H27ClN4O: C, 70.65; H, 5.93; N, 12.21, found: C, 70.58; H, 5.75; N, 12.38.

General procedure for the synthesis of -protected anthranilamides 2–9

A solution of the N-protected anthranilic acid or halogenoanthranilic acid (0.297 mmol), 0.068 g (0.54 mmol) DIEA and 0.103 g (0.27 mmol) HATU in DMF (1 mL) was stirred at room temperature. After 10 min, 0.27 mmol amino reagent was added to the mixture (0.060 g CQ, 0.096 g PQ or 0.057 g HA). The reaction mixture was stirred overnight at room temperature, diluted with EtOAc, extracted with a 5% solution of NaOH in water (3 × 30 mL) and washed with water (1 × 30 mL). The organic layer was dried through a phase separator, filtrated and evaporated under reduced pressure.

2 was obtained from the reaction of N-Boc-4-chloroanthranilic acid (0.081 g) and after crystallization from acetone as a white solid (0.101 g, 74%); mp 158–160 °C; IR (KBr) (νmax/cm−1): 3333, 3194, 2975, 2938, 2854, 1726, 1629, 1578, 1537, 1506, 1474, 1450, 1430, 1403, 1367, 1325, 1296, 1272, 1246, 1222, 1203, 1153, 1113, 1102, 1081, 1046, 8886, 868, 834, 800, 773, 690, 652, 599, 532, 496; 1H NMR (δ/ppm): 10.67 (s, 1H), 8.87 (t, 1H, J = 5.5 Hz), 8.38 (d, 1H, J = 5.4 Hz), 8.28 (d, 1H, J = 9.1 Hz), 8.22 (d, 1H, J = 9.0 Hz), 7.78 (t, 2H, J = 2.7 Hz), 7.53 (dd, 1H, J = 9.0, 2.5 Hz), 7.44 (dd, 1H, J = 9.0, 2.3 Hz), 7.31 (t, 1H, J = 5.3 Hz), 6.49 (d, 1H, J = 5.5 Hz), 3.33–3.25 (m, 4H), 1.76–1.62 (m, 4H), 1.45 (s, 9H); 13C NMR (δ/ppm): 166.93, 151.97, 151.97, 150.05, 149.10, 138.47, 133.32, 131.67, 127.64, 127.48, 125.17, 124.08, 123.95, 120.92, 120.26, 117.44, 98.64, 80.06, 42.05, 38.89, 27.88, 26.30, 25.24; ESI–MS m/z = 503.4 [M + 1]+; Anal. Calcd. for C25H28Cl2N4O3: C, 59.65; H, 5.61; N, 11.13, found: C, 59.79; H, 5.47; N, 10.98.

3 was obtained from the reaction of N-Boc-5-bromoanthranilic acid (0.094 g) and after purification by column chromatography (mobile-phase dichloromethane/methanol 9:1) and crystallization from diethyl ether as a white solid (0.117 g, 79%); mp 184–186 °C; IR (KBr) (νmax/cm−1) 3348, 3261, 2968, 2932, 1723, 1614, 1572, 1542, 1504, 1454, 1432, 1367, 1354, 1325, 1295, 1271, 1246, 1223, 1153, 1083, 1049, 1025, 965, 910, 884, 870, 833, 812, 761, 639, 615, 522, 508; 1H NMR (δ/ppm): 10.68 (s, 1H), 8.88 (t, 1H, J = 5.3 Hz), 8.39 (d, 1H, J = 5.4 Hz), 8.29 (d, 1H, J = 9.1 Hz), 8.17 (d, 1H, J = 9.0 Hz), 7.90 (d, 1H, J = 2.2 Hz), 7.78 (d, 1H, J = 2.1 Hz), 7.65 (dd, 1H, J = 9.0, 2.2 Hz), 7.45 (dd, 1H, J = 9.0, 2.1 Hz), 7.39 (t, 1H, J = 5.0 Hz), 6.50 (d, 1H, J = 5.5 Hz), 3.32 (dd, 4H, J = 11.7, 5.9 Hz), 1.79–1.61 (m, 4H), 1.45 (s, 9H); 13C NMR (δ/ppm): 166.86, 151.93, 151.54, 150.24, 148.70, 138.88, 134.56, 133.49, 130.42, 127.17, 124.14, 121.25, 120.56, 117.37, 113.00, 98.64, 80.08, 42.08, 38.89, 27.87, 26.29, 25.24; ESI–MS m/z = 549.3 [M + 3]+; Anal. Calcd. for C25H28BrClN4O3: C, 54.81; H, 5.15; N, 10.23, found: C, 54.85; H, 5.25; N, 10.11.

4 was obtained from the reaction of N-benzylanthranilic acid (0.067 g) and after purification by column chromatography (mobile-phase cyclohexane/ethyl acetate 1:1) as a brown oil (0.126 g, 80%); IR (film) (νmax/cm−1) 3352, 3061, 3029, 3002, 2960, 2933, 2862, 1710, 1630, 1615, 1594, 1514, 1451, 1422, 1386, 1362, 1334, 1273, 1219, 1200, 11,578, 1051, 1029, 900, 791, 747, 698, 679, 624, 529; 1H NMR (δ/ppm): 8.53 (dd, 1H, J = 4.2, 1.6 Hz), 8.35 (t, 1H, J = 5.5 Hz), 8.20 (t, 1H, J = 5.8 Hz), 8.07 (dd, 1H, J = 8.3, 1.6), 7.52 (dd, 1H, J = 7.9, 1.4 Hz), 7.42 (dd, 1H, J = 8.2, 4.2 Hz), 7.36–7.28 (m, 4H), 7.26–7.20 (m, 1H,), 7.19–7.15 (m, 1H), 6.60 (d, 1H, J = 7.9 Hz), 6.56–6.51 (m, 1H), 6.47 (d, 1H, J = 2.5 Hz), 6.28 (d, 1H, J = 2.5 Hz), 6.15 (d, 1H, J = 8.8 Hz), 4.35 (d, 2H, J = 5.8 Hz), 3.81 (s, 3H), 3.70–3.62 (m, 1H), 3.29–3.20 (m, 2H), 1.77–1.53 (m, 4H), 1.22 (d, 3H, J = 6.3 Hz); 13C NMR (δ/ppm): 169.03, 159.00, 148.89, 144.64, 144.64, 139.60, 134.79, 134.53, 132.01, 129.58, 128.43, 128.23, 127.09, 126.80, 122.09, 115.56, 114.38, 111.41, 96.11, 91.60, 54.97, 47.04, 46.07, 38.82, 33.50, 25.94, 20.20; ESI–MS m/z = 491.5 [M + 23]+; Anal. Calcd. for C29H32N4O2: C, 74.33; H, 6.88; N, 11.96, found: C, 74.66; H, 6.79; N, 12.04.

5 was obtained from the reaction of N-Boc-4-chloroanthranilic acid (0.081 g) and after purification by column chromatography (mobile-phase cyclohexane/ethyl acetate/methanol 3:1:0.5) as a yellow solid (0.107 g, 77%); mp 66–67 °C; IR (KBr) (νmax/cm−1) 3339, 2973, 2932, 2866, 1726, 1616, 1511, 1454, 1388, 1367, 1246, 1221, 1155, 1050, 1024, 902, 821, 790; 1H NMR (δ/ppm): 10.64 (s, 1H), 8.84 (t, 1H, J = 5.4 Hz), 8.53 (dd, 1H, J = 4.2, 1.6 Hz), 8.21 (d, 1H, J = 9.0 Hz), 8.07 (dd, 1H, J = 8.3, 1.6 Hz), 7.76 (d, 1H, J = 2.5 Hz), 7.52 (dd, 1H, J = 9.0, 2.5 Hz), 7.42 (dd, 1H, J = 8.2, 4.2 Hz), 6.46 (d, 1H, J = 2.5 Hz), 6.27 (d, 1H, J = 2.5 Hz), 6.16 (d, 1H, J = 8.8 Hz), 3.80 (d, 3H, J = 6.5 Hz), 3.70–3.63 (m, 1H), 3.28 (dd, 2H, J = 11.5, 5.6 Hz), 1.76–1.57 (m, 4H), 1.45 (s, 9H), 1.23 (d, 3H, J = 6.3 Hz); 13C NMR (δ/ppm): 166.87, 158.98, 151.97, 145.54, 144.62, 138.43, 134.78, 134.54, 131.60, 129.57, 127.64, 125.16, 122.07, 120.99, 120.22, 96.12, 91.60, 80.02, 54.94, 46.99, 38.88, 33.40, 27.88, 25.49, 20.19; ESI–MS m/z = 513.4 [M + 1]+; Anal. Calcd. for C27H33ClN4O4: C, 63.21; H, 6.48; N, 10.92, found: C, 62.70; H, 6.43; N, 11.09.

6 was obtained from the reaction of N-Boc-5-bromoanthranilic acid (0.094 g) and after purification by column chromatography (mobile-phase dichloromethane/methanol 9.5:0.5) and crystallization from ether as a yellow solid (0.081 g, 54%); mp 90–92 °C; IR (KBr) (νmax/cm−1) 3326, 2966, 2930, 2867, 1726, 1615, 1578, 1506, 1453, 1388, 1367, 1296, 1246, 1220, 1153, 1049, 1023, 901, 820, 790, 638; 1H NMR (δ/ppm): 10.65 (s, 1H), 8.85 (t, 1H, J = 5.2 Hz), 8.53 (dd, 1H, J = 4.0, 1.1 Hz), 8.16 (d, 1H, J = 9.0 Hz, 1H), 8.07 (d, 1H, J = 8.2 Hz), 7.88 (d, 1H, J = 2.1 Hz), 7.64 (dd, 1H, J = 9.0, 2.0 Hz), 7.42 (dd, 1H, J = 8.2, 4.2 Hz), 6.47 (d, 1H, J = 2.2 Hz), 6.27 (d, 1H, J = 2.1 Hz), 6.16 (d, 1H, J = 8.7 Hz), 3.81 (s, 17), 3.72–3.60 (m, 1H), 3.27 (d, 2H, J = 5.7 Hz), 1.77–1.56 (m, 4H), 1.45 (s, 9H), 1.23 (d, 3H, J = 6.2 Hz); 13C NMR (δ/ppm): 166.79, 158.98, 151.93, 144.62, 144.20, 138.84, 134.78, 134.54, 134.49, 130.46, 129.57, 122.07, 121.32, 120.52, 112.99, 96.12, 91.61, 80.04, 54.95, 46.99, 38.88, 33.38, 27.87, 25.50, 20.19; ESI–MS m/z = 559.4 [M + 3]+; Anal. Calcd. for C27H33BrN4O4: C, 58.17; H, 5.97; N, 10.05, found: C, 58.45; H, 5.90; N, 10.33.

7 was obtained from the reaction of N-benzylanthranilic acid (0.067 g,) and after purification by column chromatography (mobile-phase dichloromethane/methanol 8:1) and crystallization from cyclohexane/diethyl ether/petroleum ether as a white solid (0.061 g, 54%); mp 114–116 °C; IR (KBr) (νmax/cm−1) 3633, 3252, 3060, 3028, 2924, 2849, 1625, 1573, 1514, 1450, 1326, 1275, 1250, 1163, 746, 699; 1H NMR (δ/ppm): 11.49 (s, 1H), 8.98 (t, 1H, J = 5.9 Hz), 8.30 (t, 1H, J = 5.8 Hz), 8.15 (d, 1H, J = 7.9 Hz), 7.85 (s, 1H), 7.75 (dd, 1H, J = 7.9, 1.6 Hz), 7.57 (d, 1H, J = 8.1 Hz), 7.54–7.49 (m, 1H), 7.37–7.33 (m, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.26–7.21 (m, 2H), 7.21–7.17 (m, 1H), 6.65 (dd, 1H, J = 8.5, 1.1 Hz), 6.63–6.57 (m, 1H), 4.66 (d, 2H, J = 5.9 Hz), 4.38 (d, 2H, J = 5.8 Hz), 2.76 (s, 3H); 13C NMR (δ/ppm): 169.17, 149.09, 146.66, 141.12, 140.75, 139.61, 133.50, 132.25, 128.50, 128.45, 127.79, 127.74, 127.11, 126.82, 121.64, 120.99, 119.07, 115.31, 114.52, 111.90, 111.52, 109.42, 46.09, 44.58, 20.33; ESI–MS m/z = 421.2 [M + 1]+; Anal. Calcd. for C27H24N4O: C, 77.12; H, 5.75; N, 13.32, found: C, 76.96; H, 5.73; N, 13.67.

8 was obtained from the reaction of N-Boc-4-chloroanthranilic acid (0.081 g) and after purification by column chromatography (mobile-phase dichloromethane/methanol 9.5:0.5) and crystallization from cyclohexane as a white solid (0.049 g, 40%); mp 203–205 °C; IR (KBr) (νmax/cm−1) 3334, 3185, 3117, 3071, 2987, 2932, 2862, 1634, 1651, 1626, 1597, 1578, 1512, 1440, 1398, 1365, 1355, 1314, 1244, 1159, 1114, 1066, 1050, 1029, 967, 914, 844, 770, 751, 740, 653, 590, 530; 1H NMR (δ/ppm): 11.52 (s, 1H), 10.73 (s, 1H, 9.49 (t, 1H, J = 5.5 Hz), 8.26 (d, 1H, J = 9.0 Hz), 8.20 (d, 1H, J = 7.8 Hz), 7.98 (d, 1H, J = 2.3 Hz), 7.91 (s, 1H), 7.60–7.48 (m, 3H), 7.20 (t, 1H, J = 7.4 Hz), 4.68 (d, 2H, J = 5.6 Hz), 2.77 (s, 3H), 1.44 (s, 9H); 13C NMR (δ/ppm): 167.05, 152.02, 145.60, 141.29, 140.75, 138.64, 133.60, 131.79, 127.98, 127.82, 127.75, 125.28, 121.79, 121.01, 120.88, 120.29, 119.08, 111.90, 109.93, 80.09, 45.01, 27.86, 20.33; ESI–MS m/z = 465.1 [M + 1]+; Anal. Calcd. for C25H25ClN4O3: C, 64.58; H, 5.42; N, 12.05, found: C, 64.27; H, 5.19; N, 12.21.

9 was obtained from the reaction of N-Boc-5-bromoanthranilic acid (0.094 g) and after purification by column chromatography (mobile-phase dichloromethane/methanol 9.5:0.5) and crystallization from cyclohexane/diethyl ether/petroleum ether as a white solid (0.080 g, (58%); mp 134–136 °C; IR (KBr) (νmax/cm−1) 3339, 2978, 2926, 2850, 1726, 1696, 1627, 1572, 1505, 1437, 1392, 1367, 1314, 1246, 1154, 1099, 1049, 1024, 904, 828, 756, 736; 1H NMR (δ/ppm): 11.52 (s, 1H), 10.74 (s, 1H), 9.50 (s, 1H), 8.20 (d, 2H, J = 8.8 Hz), 8.09 (d, 1H, J = 2.2 Hz), 7.91 (s, 1H), 7.68 (dd, 1H, J = 9.0, 2.2 Hz), 7.58 (d, 1H, J = 8.2 Hz,), 7.53 (d, 1H, J = 7.6 Hz), 7.21 (d, 1H, J = 7.5 Hz), 4.67 (d, 2H, J = 5.6 Hz), 2.77 (s, 3H), 1.44 (s, 9H); 13C NMR (δ/ppm): 166.97, 151.98, 145.60, 141.30, 140.75, 139.06, 134.67, 133.60, 130.75, 127.83, 127.75, 121.79, 121.19, 121.00, 120.59, 119.08, 113.09, 111.90, 109.97, 80.12, 45.01, 27.86, 20.33; ESI–MS m/z = 511.1 [M + 3]+; Anal. Calcd. for C25H25BrN4O3: C, 58.95; H, 4.95; N, 11.00, found: C, 59.15; H, 4.74; N, 11.29.

General procedure for the synthesis of anthranilamides 10–19

Method A: To a solution of 0.1 mmol of N-Boc protected compound in MeOH (3 mL), 1 mmol of HCl in MeOH was added dropwise to the flask. The reaction mixture was stirred for 6–24 h at 60 °C. The solvent was removed under reduced pressure.

Method B: To a suspension of N-benzyl-protected compound (0.1 mmol) and 10% Pd/C (10 mg) in methanol (3 mL), ammonium formate (0.5 mmol) was added. The reaction mixture was stirred at 65 °C for 0.5–5 h under an inert atmosphere. The catalyst was filtered off, and the mother liquor was concentrated under reduced pressure.

Method C: A suspension of N-benzylanthranilic acid (0.067 g,) benzyl-protected compound (0.1 mmol) and 10% Pd/C (10 mg) in methanol (3 mL) was stirred at room temperature for 1–18 h under a hydrogen atmosphere. The catalyst was filtered off, and the mother liquor was concentrated under reduced pressure.

10 was obtained by method B (0.5 h, 65 °C), from the reaction of 0.046 g (0.1 mmol) 1 and after purification by column chromatography (mobile-phase dichloromethane/methanol 8.5:1.5) and crystallization from diethyl ether as a white solid (0.026 g; 64%); mp 117–119 °C; IR(ATR) (νmax/cm−1) 3396, 3296, 3252, 3074, 2932, 2849, 2806, 2723, 1751, 1623, 1594, 1575, 1548, 1512, 1449, 1358, 1335, 1278, 12,278, 1152, 1029, 963, 806, 766, 693, 662, 528; 1H NMR (δ/ppm): 8.67 (s, 1H), 8.49 (d, 1H, J = 7.3 Hz), 8.44 (d, 1H, J = 6.4 Hz), 8.42 (t, 1H, J = 5.7 Hz, 8.24 (t, 1H, J = 5.8 Hz), 7.91 (dd, 1H, J = 8.5, 1.3 Hz), 7.84–7.80 (m, 1H), 7.61–7.54 (m, 2H), 7.37–7.28 (m, 4H), 7.26–7.17 (m, 2H), 6.73 (d, 1H, J = 6.5 Hz), 6.62 (d, 1H, J = 7.5 Hz), 6.57–6.52 (m, 1H), 4.35 (d, 2H, J = 5.7 Hz), 3.49 (q, 2H, J = 6.8 Hz), 3.30 (q, 2H, J = 6.5 Hz), 1.78–1.71 (m, 2H), 1.69–1.62 (m, 2H); 13C NMR (δ/ppm): 169.13, 153.53, 148.95, 145.00, 141.32, 139.57, 132.11, 131.73, 128.46, 128.27, 127.11, 126.83, 125.47, 123.10, 122.79, 117.40, 115.39, 114.40, 111.46, 98.02, 46.10, 42.54, 38.41, 26.60, 25.22; ESI–MS m/z = 425.3 [M + 1]+; Anal. Calcd. for C27H28N4O: C, 76.39; H, 6.65; N, 13.20, found: C, 76.54; H, 6.78; N, 13.08.

11 was obtained by method C (5 h, r.t.), from the reaction of 0.046 g (0.1 mmol) 1 and after crystallization from diethyl ether/petroleum ether, as a white solid (0.025 g, 60%); mp 66–68 °C; IR(ATR) (νmax/cm−1) 3332, 3064, 2930, 2863, 1627, 1575, 1532, 1519, 1459, 1452, 1436, 1393, 1373, 1229, 1268, 1253, 1160, 1134, 1130, 1035, 844, 809, 748, 701, 656; 1H NMR (δ/ppm): 9.50 (t, 1H, J = 5.3 Hz), 8.67 (d, 1H, J = 8.4 Hz), 8.49 (d, 1H, J = 7.0 Hz), 8.29 (t, 1H, J = 5.5 Hz), 8.01 (d, 1H, J = 8.1 Hz), 7.93 (t, 1H, J = 7.5 Hz), 7.68 (t, 1H, J = 7.4 Hz), 7.48 (dd, 1H, J = 7.9, 1.0 Hz), 7.16 – 7.08 (m, 1H), 6.88 (d, 1H, J = 7.1 Hz), 6.69 (d, 1H, J = 7.7 Hz), 6.49 (t, 1H, J = 7.1 Hz), 6.39 (s, 2H), 3.58 (dd, 2H, J = 12.7, 6.5 Hz), 3.29 (dd, 2H, J = 12.4, 6.4 Hz), 1.81–1.70 (m, 2H), 1.69–1.59 (m, 2H); 13C NMR (δ/ppm): 168.87, 155.35, 149.56, 142.09, 137.81, 133.25, 131.51, 128.02, 126.35, 123.41, 120.10, 116.67, 116.67, 114.84, 114.49, 97.94, 42.72, 38.20, 26.55, 25.11; ESI–MS m/z = 335.4 [M + 1]+; Anal. Calcd. for C20H22N4O: C, 71.83; H, 6.63; N, 16.75, found: C, 71.75; H, 6.69; N, 16.70.

12 was obtained by method A (16 h, 60 °C), from the reaction of 0.050 g (0.1 mmol) 2 as a white solid (0.036 g, 89%); mp 264–266 °C; IR(ATR) (νmax/cm−1) 3235, 3087, 3042, 3037, 2945, 2920, 2858, 2830, 2818, 2802, 2695, 2642, 2623, 2520, 1994, 1923, 1636, 1632, 1592, 1565, 1550, 1485, 1454, 1432, 1385, 1355, 1332, 1322, 1302, 1281, 1231, 1211, 1169, 1151, 1106, 1030, 961, 913, 890, 874, 840, 791, 779, 763, 701, 677, 656; 1H NMR (δ/ppm): 9.75 (t, 1H, J = 5.5 Hz), 8.77 (d, 1H, J = 9.2 Hz), 8.61 (t, 1H, J = 5.2 Hz), 8.51 (d, 1H, J = 7.0 Hz), 8.12 (d, 1H, J = 2.1 Hz), 7.74 (dd, 1H, J = 9.1, 2.1 Hz), 7.64 (d, 1H, J = 2.4 Hz), 7.29 (dd, 1H, J = 8.7, 2.4 Hz), 6.92 (dd, 2H, J = 17.2, 8.0 Hz), 6.42 (s, 2H), 3.57 (q, 2H, J = 6.6 Hz), 3.29 (q, 2H, J = 6.3 Hz), 1.80–1.61 (m, 4H); 13C NMR (δ/ppm): 167.03, 155.30, 145.58, 142.55, 138.55, 137.83, 134.12, 130.32, 126.67, 125.99, 121.26, 119.95, 118.94, 115.46, 98.52, 42.83, 38.41, 26.30, 25.03; ESI–MS m/z = 403.3 [M + 1]+; Anal. Calcd. for C20H20Cl2N4O: C, 59.56; H, 5.00; N, 13.89, found: C, 59.39; H, 4.85; N, 13.97.

13 was obtained by method A (22 h, 60 °C), from the reaction of 0.055 g (0.1 mmol) 3 as a white solid (0.043 g, 95%); mp 255–257 °C; IR(ATR) (νmax/cm−1) 3243, 3085, 3058, 3032, 2918, 2858, 2822, 2801, 2691, 2639, 2619, 2516, 1995, 1986, 1632, 1616, 1592, 1563, 1547, 1483, 1453, 1433, 1386, 1355, 1333, 1322, 1309, 1230, 1211, 1169, 1152, 1139, 1102, 1096, 1028, 958, 915, 874, 871, 837, 794, 765, 762, 676, 656; 1H NMR (δ/ppm): 9.72 (t, 1H, J = 5.5 Hz), 8.76 (d, 1H, J = 9.2 Hz), 8.59–8.46 (m, 2H), 8.12 (d, 1H, J = 2.1 Hz), 7.74 (dd, 1H, J = 9.1, 2.1 Hz), 7.71 (d, 1H, J = 2.3 Hz), 7.36 (dd, 1H, J = 8.7, 2.3 Hz), 7.00 (s, 2H), 6.89 (d, 1H, J = 7.2 Hz), 6.83 (d, 1H, J = 8.7 Hz), 3.57 (q, 2H, J = 12.7, 6.6 Hz), 3.29 (q, 2H, J = 6.3 Hz), 1.83–1.58 (m, 4H); 13C NMR (δ/ppm): 167.03, 155.30, 145.58, 142.55, 138.55, 137.83, 134.12, 130.32, 126.67, 125.99, 120.63, 119.95, 118.94, 118.61, 115.45, 98.52, 42.83, 38.41, 26.30, 25.03; ESI–MS m/z = 449.2 [M + 3]+; Anal. Calcd. for C20H20BrClN4O: C, 53.65; H, 4.50; N, 12.51, found: C, 53.84; H, 4.81; N, 12.68.

14 was obtained by method C (18 h, r.t.), from the reaction of 0.047 g (0.1 mmol) 4 and after purification by column chromatography (mobile-phase cyclohexane/ethyl acetate/methanol 1:1:0.5) and crystallization from ether/petroleum ether as a yellow solid (0.016 g, 41%); mp 123–124 °C; IR(ATR) (νmax/cm−1) 3436, 3400, 3289, 3070, 3009, 2975, 2939, 2857, 1651, 1615, 1592, 1549, 1520, 1452, 1424, 1386, 1319, 1302, 1266, 1225, 1203, 1157, 1056, 1029, 903, 832, 818, 789, 738. 681, 627, 543; 1H NMR (δ/ppm): 8.53 (dd, 1H, J = 4.2, 1.6 Hz), 8.20 (t, 1H, J = 5.5 Hz), 8.07 (dd, 1H, J = 8.3, 1.6 Hz), 7.49–7.38 (m, 2H), 7.16–7.06 (m, 1H), 6.67 (d, 1H, J = 7.5 Hz), 6.52–6.45 (m, 2H), 6.35 (s, 2H), 6.28 (d, 1H, J = 2.4 Hz), 6.15 (d, 1H, J = 8.7 Hz), 3.82 (s, 3H), 3.71–3.60 (m, 1H), 3.27–3.19 (m, 2H), 1.78–1.53 (m, 4H), 1.22 (d, 3H, J = 6.3 Hz); 13C NMR (δ/ppm): 168.76, 159.00, 149.50, 144.64, 144.23, 134.79, 134.53, 131.44, 129.58, 128.98, 122.09, 116.23, 115.01, 114.49, 96.11, 91.60, 54.97, 47.03, 38.75, 33.51, 25.99, 20.20; ESI–MS m/z = 379.2 [M + 1]+; Anal. Calcd. for C22H26N4O2: C, 69.82; H, 6.92; N, 14.80, found: C, 69.99; H, 6.86; N, 15.15.

15 was obtained by method A (6 h, 60 °C), from the reaction of 0.051 g (0.1 mmol) 5 as an orange solid (0.037 g, 90%); mp 220–222 °C; IR(ATR) (νmax/cm−1) 3311, 3221, 3161, 3111, 3030, 3010, 2975, 2957, 2930, 2861, 2805, 2707, 2586, 1969, 1879, 1639, 1611, 1586, 1534, 1474, 1456, 1424, 1387, 1364, 1324, 1319, 1199, 1167, 1233, 1130, 1033, 898, 841, 811, 762, 672, 617, 542, 503; 1H NMR (δ/ppm): δ 8.75 (d, 1H, J = 4.1 Hz), 8.66 (d, 1H, J = 7.8 Hz), 8.56 (t, 1H, J = 5.1 Hz), 7.85–7.75 (m, 1H), 7.63 (d, 1H, J = 2.0 Hz), 7.31 (dd, 1H, J = 8.6, 1.8 Hz), 6.97 (d, 1H, J = 8.6 Hz), 6.86 (s, 1H), 6.64 (s, 1H), 3.88 (s, 3H), 3.82–3.72 (m, 1H), 3.25 (d, 2H, J = 5.1 Hz), 1.86–1.58 (m, 4H), 1.27 (d, 3H, J = 6.2 Hz); 13C NMR (δ/ppm): 166.67, 160.09, 148.50, 143.24, 141.92, 140.76, 131.35, 131.26, 127.65, 122.18, 121.92, 120.47, 119.55, 101.14, 94.01, 55.53, 48.37, 32.69, 25.49, 19.57; ESI–MS m/z = 413.3 [M + 1]+; Anal. Calcd. for C22H25ClN4O2: C, 63.99; H, 6.10; N, 13.57, found: C, 63.86; H, 5.99; N, 13.67.

16 was obtained by method A (19 h, 60 °C), from the reaction of 0.058 g (0.1 mmol) 6 as an orange solid (0.034 g, 74%); mp 141–142 °C; IR(ATR) (νmax/cm−1) 3331, 3227, 3090, 3069, 3025, 2967, 2938, 2866, 2735, 2655, 2615, 2544, 2462, 1998, 1899, 1815, 1637, 1608, 1591, 1538, 1466, 1389, 1356, 1334, 1305, 1278, 1238, 1221, 1202, 1178, 1168, 1147, 1131, 1059, 1028, 1006, 902, 833, 820, 777, 761, 678, 659, 624, 517; 1H NMR (δ/ppm): 8.75 (dd, 1H, J = 4.9, 1.5 Hz), 8.64 (d, 2H, J = 8.3 Hz), 8.52 (t, 1H, J = 5.2 Hz), 7.78 (dd, 1H, J = 8.4, 4.9 Hz), 7.71 (d, 1H, J = 2.3 Hz), 7.38 (dd, 1H, J = 8.7, 2.3 Hz), 6.86 (d, 2H, J = 8.8 Hz), 6.63 (s, 1H), 3.88 (s, 3H), 3.77 (dd, 1H, J = 12.2, 6.1 Hz), 3.24 (q, 2H, J = 6.2 Hz), 1.84–1.58 (m, 4H), 1.26 (d, 3H, J = 6.3 Hz); 13C NMR (δ/ppm): 166.79, 160.07, 144.73, 141.62, 141.00, 140.33, 138.34, 134.12, 131.23, 131.23, 122.19, 120.34, 119.29, 108.55, 93.96, 55.52, 48.37, 38.90, 32.75, 25.55, 19.61; ESI–MS m/z = 457.3 [M + 1]+; Anal. Calcd. for C22H25BrN4O2: C, 57.77; H, 5.51; N, 12.25, found: C, 57.97; H, 5.79; N, 12.51.

17 was obtained by method B (1 h, 65 °C), from the reaction of 0.042 g (0.1 mmol) 7 and after crystallization from diethyl ether as an off-white solid (0.022 g, 73%); mp 127–130 °C; IR(ATR) (νmax/cm−1) 3435, 3330, 3149, 3109, 3057, 2946, 2926, 2878, 2794, 2711, 1633, 1574, 1524, 1451, 1355, 1341, 1289, 1248, 1161, 1151, 1150, 1133, 1107, 1032, 962, 906, 840, 774, 751, 729, 529; 1H NMR (δ/ppm): 11.51 (s, 1H), 8.82 (t, 1H, J = 5.9 Hz), 8.21–8.13 (m, 2H), 7.84 (s, 1H), 7.66 (d, 1H, J = 7.6 Hz), 7.57 (d, 1H, J = 8.2 Hz), 7.51 (t, 1H, J = 7.6 Hz), 7.19 (t, 1H, J = 7.5 Hz), 7.16 (t, 1H, J = 7.6 Hz), 6.72 (d, 1H, J = 8.2 Hz), 6.45 (s, 2H), 4.65 (d, 2H, J = 5.8 Hz), 2.77 (s, 3H); 13C NMR (δ/ppm): 168.86, 149.71, 146.78, 141.09, 140.76, 133.49, 131.67, 128.21, 127.78, 127.74, 121.65, 120.99, 119.08, 116.35, 114.79, 114.65, 111.90, 109.43, 44.48, 20.32; ESI–MS m/z = 331.2 [M + 1]+; Anal. Calcd. for C20H18N4O: C, 72.71; H, 5.49; N, 16.96, found: C, 72.84; H, 5.71; N, 16.78.

18 was obtained by method A (24 h, 60 °C), from the reaction of 0.046 g (0.1 mmol) 8 as a white solid (0.028 g, 77%); mp 206–209 °C; IR(ATR) (νmax/cm−1) 3395, 3118, 3018, 2939, 2934, 2870, 2607, 2571, 1655, 1640, 1560, 1509, 1461, 1368, 1343, 1315, 1248, 1135, 1103, 985, 888, 863, 825, 752, 71; 1H NMR (δ/ppm): 13.04 (s, 1H), 9.33 (t, 1H, J = 5.6 Hz), 8.53 (s, 1H), 8.50 (d, 1H, J = 8.1 Hz), 7.84 (d, 1H, J = 2.5 Hz), 7.78–7.73 (m, 2H), 7.43–7.37 (m, 1H), 7.27 (dd, 1H, J = 8.8, 2.5 Hz), 6.86 (d, 1H, J = 8.8 Hz), 4.90 (d, 2H, J = 5.5 Hz), 4.77 (s, 2H), 3.13 (s, 3H); 13C NMR (δ/ppm): 167.73, 143.67, 140.19, 138.45, 133.12, 132.20, 132.03, 131.38, 127.86, 123.63, 121.17, 119.70, 119.17, 113.49, 112.78, 40.13, 15.84; ESI–MS m/z = 365.1 [M + 1]+; Anal. Calcd. for C20H17ClN4O: C, 65.84; H, 4.70; N, 15.36, found: C, 65.99; H, 4.86; N, 15.35.

19 was obtained by method A (24 h, 60 °C), from the reaction of 0.051 g (0.1 mmol) 9 as a white solid; mp 242–244 °C (decomp.) (0.037 g, 90%); IR(ATR) (νmax/cm−1) 3377, 3268, 3013, 2867, 2607, 2568, 1923, 1747, 1640, 1574, 1506, 1489, 1430, 1392, 1377, 1342, 1328, 1287, 1253, 1168, 1146, 1100, 988, 909, 881, 838, 826, 752, 712, 542, 502; 1H NMR (δ/ppm): 13.05 (s, 1H), 9.32 (t, 1H, J = 5.6 Hz), 8.53 (s, 1H), 8.49 (d, 1H, J = 8.1 Hz), 7.94 (d, 1H, J = 2.3 Hz), 7.79–7.73 (m, 2H), 7.42–7.35 (m, 2H), 6.80 (d, 1H, J = 8.8 Hz), 4.92–4.88 (m, 2H), 4.78 (s, 2H), 3.13 (s, 3H); 13C NMR (δ/ppm): 167.65, 143.67, 140.18, 138.43, 134.74, 133.11, 132.18, 131.37, 130.64, 123.62, 121.15, 119.68, 119.54, 113.49, 112.78, 40.13, 15.84; ESI–MS m/z = 409.0 [M + 1]+; Anal. Calcd. for C20H17BrN4O: C, 58.69; H, 4.19; N, 13.69, found: C, 58.99; H, 4.09; N, 13.35.

Antiproliferative activity assays

Adherent cell lines LN-229, Capan-1, HCT-116 and NCI-H460 cells were seeded at a density between 500 and 1500 cells per well, in 384-well tissue culture plates (Greiner). After overnight incubation, cells were treated with the test compounds. After incubation for 3 days at 37 °C, the formazan-based 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega) was performed to assess cell viability, and the spectrophotometric data were used to calculate the IC50 values [61-65].

Suspension cell lines DND-41, HL-60, K-562 and Z-138 were seeded at densities ranging from 2500 to 5500 cells per well in 384-well tissue culture plates containing the test compounds at the same concentration points. The plates were incubated at 37 °C and monitored for 72 h in an IncuCyte® device (Essen BioScience Inc.) for real-time imaging. Images were taken every 3 h, with one field imaged per well under 10 × magnification. Cell growth was then quantified based on the percent cellular confluence as analyzed by the IncuCyte® image analysis software and used to calculate IC50 values by linear interpolation.

Antiviral activity assays

The antiviral evaluation of anthranilamides 1–19 against influenza viruses A/H1N1 A/Ned/378/05, A/H3N2 A/HK/7/87, B/H1N1 and B/Ned/537/05, respiratory syncytial virus and human coronaviruses HCoV-229E and HCoV-OC43 was performed by seeding MDCK, Hep-2 or HEL 299 cells into 384-well dishes. The previously described procedure was applied [66, 67]. 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 and RSV) or 7 days post-infection (HCoV), the virus-induced cytopathogenic effect was measured colorimetrically by the formazan-based MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay), 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 or HEL 299 cells. The activities were compared with reference compounds such as zanamivir, ribavirin, rimantadine, ribavirin, DS-10.000, and remdesivir, respectively.

The antiretroviral assays in MT-4 cells have previously been described in detail [62]. Briefly, MT-4 cells (1 × 106 cells/mL) were pre-incubated for 30 min at 37 °C with the test compounds in a 96-well plate. Next, HIV-1 NL4.3 or HIV-2 ROD strains were added according to the CCID50 of the viral stock. The CPE was scored microscopically 5 days post-infection, and the EC50 values were determined using the MTS/PES method [68].

Anti-QS and bactericidal activity screening

The previously reported screening method using C. violaceum ATCC31532 (ATCC; Wesel, Germany) as the reporter was applied to test if the anthranilamides show anti-QS/-biofilm or bactericidal activities [54, 59, 69]. Shortly, the C. violaceum strain was cultured overnight at 27 °C on Luria-Berthani agar (Fischer Scientific, Leicestershire, UK) LBA to produce single colonies, which were suspended in PDYT (0.5% peptone, 0.3% D-glucose, 0.25% yeast extract, 0.05% L-tryptophan, m/v) to achieve OD600 = 0.02. The obtained cell suspension (200 µL at OD600 = 0.02) with 2% DMSO (control) or with the indicated compounds dissolved in DMSO and tested at varying concentrations (400, 200, 100, 40 and 10 μM) were added into the wells in two parallel 96-well plates (Tissue Culture Treated, polystyrene, flat-bottom, Becton Dickinson). In both 96-well plate, quercetin [70] and azithromycin (Sigma-Aldrich) at 400 μM (dissolved in DMSO as 20 mM stocks) were used as positive controls for QS inhibition and cell viability (bactericidal agent), respectively. The plates were incubated at 27 °C under aerobic conditions (200 r.p.m.) for 22 h. Resazurin, a redox‐sensitive dye that is reduced to fluorescent resorufin only by viable cells, was added at 200 μM per each well in the first 96-well plate to assess the bactericidal effects of the compounds [71, 72]. The 96-well plates, with/without the resazurin, were shaken for an additional 30 min (200 r.p.m.) in dark and then centrifuged (4000 rpm, for 20 min, 20 °C) to pellet insoluble violacein and cells. Resorufin containing supernatants (100 µL) were transferred into a new plate and the produced/remaining fluorescence was recorded with a PerkinElmer Victor3 multilabel microtiter plate reader using an excitation/emission wavelengths of 550/590 nm. Supernatants from the 96-well plate without the added resazurin were removed and the pelleted violacein was dissolved in 96% (v/v) ethanol. The supernatants with soluble violacein were cleared from cells by centrifugation (4000 rpm, for 20 min, 20 °C), the supernatants (100 µL) were transferred into new 96-well plate and changes in violacein yield were monitored at 595 nm using the PerkinElmer Victor3 reader. Both the anti-QS screening and bactericidal experiment was repeated three times with at least three technical replicates in each plate. Statistical parameters (Z´, S/N and S/B) [73, 74] for each assay were calculated throughout the screening process to monitor assay performance and confirm high quality of the obtained results. Potency (half inhibitory concentrations, IC50) calculation was conducted using the GraphPad Prism version 8 (GraphPad software Inc., San Diego, CA, USA).

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 3498 KB)