Discovery and druggability evaluation of pyrrolamide-type GyrB/ParE inhibitor against drug-resistant bacterial infection

Abstract

The bacterial ATP-competitive GyrB/ParE subunits of type II topoisomerase are important anti-bacterial targets to treat super drug-resistant bacterial infections. Herein we discovered novel pyrrolamide-type GyrB/ParE inhibitors based on the structural modifications of the candidate AZD5099 that was withdrawn from the clinical trials due to safety liabilities such as mitochondrial toxicity. The hydroxyisopropyl pyridazine compound 28 had a significant inhibitory effect on Gyrase (GyrB, IC₅₀ = 49 nmol/L) and a modest inhibitory effect on Topo IV (ParE, IC₅₀ = 1.513 μmol/L) of Staphylococcus aureus. It also had significant antibacterial activities on susceptible and resistant Gram-positive bacteria with a minimum inhibitory concentration (MIC) of less than 0.03 μg/mL, which showed a time-dependent bactericidal effect and low frequencies of spontaneous resistance against S. aureus. Compound 28 had better protective effects than the positive control drugs such as DS-2969 (5) and AZD5099 (6) in mouse models of sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) infection. It also showed better bactericidal activities than clinically used vancomycin in the mouse thigh MRSA infection models. Moreover, compound 28 has much lower mitochondrial toxicity than AZD5099 (6) as well as excellent therapeutic indexes and pharmacokinetic properties. At present, compound 28 has been evaluated as a pre-clinical drug candidate for the treatment of drug-resistant Gram-positive bacterial infection. On the other hand, compound 28 also has good inhibitory activities against stubborn Gram-negative bacteria such as Escherichia coli (MIC = 1 μg/mL), which is comparable with the most potent pyrrolamide-type GyrB/ParE inhibitors reported recently. In addition, the structure–activity relationships of the compounds were also studied.

Graphical abstract

A novel pyrrolamide-type GyrB/ParE inhibitor against drug-resistant bacterial infection with excellent pharmacological activity and druggability was discovered by the structural modifications of the AZD5099 which was withdrawn from clinical trials.

1. Introduction

Drug-resistant bacterial infections pose a serious threat to global public health and have become one of the leading causes of human death worldwide. In 2019, approximately 1.2 million deaths were associated with bacterial resistance, and the annual number of deaths resulting from multi-drug-resistant bacterial infections is predicted to reach 10 million by 2050^1,2. Therefore, in addition to limiting the abuse of antibiotics and advocating the rational use of antibiotics, it is necessary to develop novel antibiotics to fight the increasingly serious infections of drug-resistant bacteria^3,4.

Bacterial type IIA topoisomerase can inhibit double-stranded DNA from tangling during separation and from rotation when it changes the DNA topology so that DNA replication, transcription, and recombination can proceed normally. It plays a vital role in maintaining bacterial growth and reproduction^5,6. Except for a few bacteria such as Helicobacter pylori and Mycobacterium tuberculosis, most bacteria express two different type IIA topoisomerases, including DNA gyrase and topoisomerase IV (Topo IV)^7,8.

DNA gyrase was first discovered in Escherichia coli. It has a GyrA₂GyrB₂ heterotetrameric structure composed of two subunits, A and B, and is mainly involved in negative supercoiling during DNA replication⁹. The subsequently discovered Topo IV has structures similar to that of gyrases. They are ParC₂ParE₂ tetramers composed of C and E subunits, which are mainly involved in the separation of progeny DNA after DNA replication. The GyrA and ParC subunits can initiate double-stranded breaks in the DNA molecule, thereby releasing the torsional tension caused by supercoiling and subsequent reconnection of the two strands. GyrB and ParE contain ATP-binding sites, and ATP hydrolysis provides the energy required for the catalytic action of GyrA and ParC^10,11. Blocking of the catalytic or ATP-binding sites can result in antibacterial effects, and the inhibitors are safe because the type II topoisomerase structures of eukaryotes and bacteria are different¹².

Quinolone antibiotics commonly used in clinics nowadays are DNA GyrA/ParC inhibitors, but they have certain side effects and worrisome drug resistance issues^13,14. By contrast, novobiocin (1) is the only ATP-competitive GyrB/ParE inhibitor that has ever been on the market, which was withdrawn from the market by the FDA in 2011 due to efficacy and safety issues (Fig. 1)¹⁵. The novobiocin derivative L-C43 (2) developed by Bristol‒Myers Squibb once entered clinical trials, but its development was stopped due to side effects such as jaundice, rash, and congestive heart failure in healthy volunteers¹⁶.

Figure 1.

Representative GyrB/ParE inhibitor structure and key binding sites (in red color) for critical hydrogen bonding with GyrB/ParE subunit.

With the increasingly serious problem of bacterial drug resistance in recent years, the development of antibacterial drugs targeting ATP-competitive GyrB/ParE has become a hotspot again. Many pharmaceutical companies and scientific research institutions have begun to refocus the research and development of these targets. The rational drug design based on the X-ray structures of complexes also accelerated research progress¹⁷. At present, several GyrB/ParE inhibitors have been reported, including novobiocin derivatives, as well as synthetic pyrazoles, indazoles, arylaminopyrimidines, pyrrolopyrimidines, pyrrolamides, imidazolamides, ethylureas, aminotricyclics and other structural classes18, 19, 20, 21, 22. Like the novobiocin (1), they also have critical hydrogen bonding interactions with key amino acid Asp73 and water in the E. coli GyrB (Asp81 of S. aureus GyrB) with different groups as highlighted in red color in Fig. 1. Nevertheless, only two compounds are in clinical trials now, including SPR-720 (4) and DS-2969 (5). The ethylurea-type SPR-720 (4) developed by Spero Therapeutics was in phase II clinical trials for the treatment of non-tuberculous mycobacterial infections, which was the phosphate of the SPR-719 (3)^23,24. The imidazolamide-type inhibitor DS-2969 (5) developed by Daiichi Sankyo for the treatment of C. difficile infection, is currently in Phase I clinical research²⁵. It is worth noting that the above drug candidates have few inhibitory effects on most Gram-negative bacteria such as E. coli, similar to the vast majority of the GyrB/ParE inhibitors previously reported18, 19, 20, 21, 22.

Besides the novobiocin and the three drug candidates in clinical trials, the pyrrolamide-type GyrB/ParE inhibitors are also attractive. The AZD5099 (6) developed by AstraZeneca once entered phase I clinical trials, but it was withdrawn due to safety issues^18,26. Although plenty of pyrrole GyrB/ParE inhibitors have been discovered by different groups since then, there was no drug candidate developed in clinical trials. Nevertheless, the efforts to find and develop pyrrolamide-type GyrB/ParE inhibitors as antibacterial drugs are continued18, 19, 20, 21, 22. Herein, we reported the discovery of a novel pyrrolamide-type GyrB/ParE inhibitor with better antibacterial activities and druggability based on the chemical modifications of the AZD5099 (6).

2. Results and discussion

2.1. Design strategy

From the co-crystal complex structure with S. aureus GyrB (3TTZ), it can be found that the methyl dichloropyrrole carboxamide fragment of earlier AZD1279 (7) developed by AstraZeneca also has the critical hydrogen bonds with Asp81 (Asp73 in the E. coli GyrB) like other ATP-competitive GyrB/ParE inhibitors (Fig. 1)²⁷. It is noteworthy that the dichloropyrrole carboxamide moiety also existed in the natural GyrB/ParE inhibitor amycolamicin (8)²⁸. The substituent of a 3-S-methoxy group on the 4-R-amino-piperidine ring is also beneficial to the enzyme inhibitory activity. In addition, the thiazole interacts with Arg84 of S. aureus GyrB (Arg76 in E. coli GyrB) by cation–π stacking. Although the carboxyl group on thiazole can generate electrostatic interaction with arginine Arg144 in S. aureus GyrB (Arg136 in E. coli GyrB) to enhance the enzyme inhibitory activity, the AZD1279 showed moderate antibacterial activities because of the unsatisfactory cell membrane permeabilities. Moreover, the carboxyl group may lead to this compound being easily metabolized by bile anion transporters, so the in vivo clearance (CL) was high²⁶. After systematic structural optimization, the addition of the amide side chain on the thiazole ring can improve antibacterial activities and reduce the in vivo clearance, but the candidate AZD5099 (6) was still withdrawn from clinical trials because of mitochondrial toxicity in 2011. By contrast, the ethylurea SPR720 (4) and imidazolamide DS2969 (5) under clinical research had better safety and pharmacokinetic properties, suggesting that replacement of the thiazole and the carboxyl group may be a promising strategy to improve the druggability of the pyrrolamide-type GyrB/ParE inhibitors.

2.1.1. The replacement of the thiazole fragment

At first, we synthesized the methyl ester of thiazole compound 9, but the inhibitory effect against S. aureus gyrase was significantly reduced compared with novobiocin (1) and AZD5099 (6), possibly because of the loss of the original electrostatic effect between the carboxyl group and Arg144 (Table 1). Nevertheless, the thiadiazole ester 10 was more potent than compound 9 against S. aureus gyrase, but the antibacterial activity (MIC = 0.125 mg/mL) was not improved possibly because of the insufficient membrane permeability. The following replacements of pyridine 11, pyrazine 12 and pyrimidine 13 led to the reduction of the activities, but the pyridazinyl 14 showed a comparable S. aureus gyrase inhibitory effect as compound 10. To our delight, the pyridazine derivative 14 showed 8 folds stronger antibacterial activity against S. aureus strain (MIC = 0.015 μg/mL) than thiadiazole 10, with the same MIC value as AZD5099 (6), possibly because of the promotion of physicochemical properties by the pyridazine. Although the oxazole derivative 16 showed better inhibitory activity than the oxadiazole compound 15, it was less active than thiadiazole compound 10.

Table 1.

In vitro gyrase inhibitory and antibacterial activity of compounds.

Compd.	A	R1	Gyrase IC50 (μmol/L)		MIC (μg/mL)
			S. aureus	E. coli	S. aureus (ATCC29213)	E. coli (ATCC25922)
9		Me	1.45 ± 0.111	>10	0.125	＞128
10		Et	0.137 ± 0.013	6.87 ± 1.221	0.125	16
11		Me	3.35 ± 0.154	>10	1	＞128
12		Me	4.87 ± 0.020	>10	0.125	＞128
13		Et	>10	>10	＞128	>128
14		Me	0.136 ± 0.020	3.12 ± 0.923	0.015	1
15		Et	>10	>10	2	＞128
16		Et	2.34	>10	0.5	128
1	/	/	0.040 ± 0.010	0.649 ± 0.227	0.5	128
5	/	/	0.376 ± 0.231	>10	0.5	64
6	/	/	0.032 ± 0.014	0.760	0.015	32

Overall, the synthesized compounds have a significantly stronger inhibitory effect on S. aureus gyrase than E. coli gyrase, which is similar to Novobiocin (1) and AZD5099 (6). Except for thiadiazole 10 and pyridazine 14, the IC₅₀ values of the other compounds against E. coli gyrase were larger than 10 μmol/L. Although compound 14 showed a weaker E. coli gyrase inhibitory effect than novobiocin (1) and AZD5099 (6), its antibacterial activity against E. coli was significantly improved, which is comparable to the most potent pyrrolamide-type GyrB/ParE inhibitors reported recently, indicating the compound could also penetrate impenetrable outer cell membranes of Gram-negative bacteria^29,30.

However, compound 14 showed a weak protective effect at 10 mg/kg by intragastric administration (ig) in a mouse model of sepsis induced by the methicillin-resistant S. aureus (ATCC43300) infection (Fig. 4). The poor metabolic stabilities of the methyl ester in vivo may be a major reason. Nevertheless, the excellent gyrase inhibitory and antibacterial activities of pyridazine 14 gave us great confidence to continue the optimization of its structure.

Figure 4.

The protective effects in vivo of the compounds on a mouse model of sepsis by intragastric administration (n = 4 for each group).

2.1.2. The structural optimization of substituents on the pyridazine ring

When the methyl ester group (14) was replaced by fluorine (17), bromine (18), or trifluoromethyl (19), the S. aureus gyrase inhibitory activities of the compounds were almost maintained, but the compound 17 with low lipophilicity was less potent in the bacterial cell (Table 2). Although the inhibitory activities of the cyano compound 20 and acetyl compound 21 were reduced, the trifluoroacetyl compound 22 showed better gyrase inhibitory activities than compound 14. The compound 22 also showed better antibacterial activities than the other compounds, with a MIC value of 0.015 μg/mL. Compared with the oxime 23 and its methyl derivative 24, the carboxyl oxime ether 25 and 26 have stronger inhibitory activities against S. aureus gyrase, possibly because the carboxyl groups have additional electrostatic interactions with the Arg144 of S. aureus gyrase. However, the neutral compounds 23 and 24 showed better antibacterial activities than carboxyl compounds 25 and 26, indicating that they are easier to penetrate the cell membranes of bacteria and the oxime/methyl oxime ether may enhance the antibacterial activities in other ways.

Table 2.

In vitro gyrase inhibitory and antibacterial activity of the compounds.

Compd.	R2	Gyrase IC50 (μmol/L)		MIC (μg/mL)
		S. aureus	E. coli	S. aureus (ATCC29213)	E. coli (ATCC25922)
17		0.084 ± 0.037	8.21 ± 1.21	0.25	16
18		0.13 ± 0.017	7.42 ± 1.01	0.03	4
19		0.12 ± 0.047	9.31 ± 0.923	0.03	16
20		0.701 ± 0.248	>10	0.06	＞128
21		0.825 ± 0.303	>10	0.06	＞128
22		0.045 ± 0.009	0.788 ± 0.289	0.015	1
23		1.49 ± 0.234	2.56	0.008	1
24		2.07 ± 0.568	>10	0.03	＞128
25		0.172 ± 0.056	>10	0.25	＞128
26		0.052 ± 0.013	0.869 ± 0.313	0.125	＞128
27		0.094 ± 0.040	5.11 ± 0.545	0.03	4
28		0.049 ± 0.005	1.48 ± 0.441	0.008	1
29		0.397 ± 0.024	>10	0.25	8
30		0.696 ± 0.056	>10	0.125	16
31		0.576 ± 0.045	>10	0.125	＞128
32		>10	>10	32	64
33		1.09 ± 0.146	>10	0.25	32
34		>10	>10	32	＞128
35		>10	>10	32	＞128
36		>10	>10	0.5	16

The hydroxyalkyl compounds 27 and 28 showed slightly better gyrase inhibitory activities than compound 14, especially compound 28 showed the best gyrase inhibitory and antibacterial activities against the S. aureus (IC₅₀ = 49 nmol/L, MIC = 0.008 μg/mL). The amide compounds 29–31 and 33 have moderate S. aureus gyrase inhibitory and antibacterial activities, but the basic N-aminoethyl amide 32, aminomethyl 34, amidine 35, and the acidic tetrazolium 36 were significantly less potent.

For the E. coli gyrase, the trifluoroacetyl compound 22 and carboxyl oxime ether 26 showed the best inhibitory activities with IC₅₀ values less than 1 μmol/L. In addition, the neutral compounds 17–19 and 27–28 also showed moderate inhibitory activities with IC₅₀ values below 10 μmol/L. The compounds 22, 23 and 28 also showed the best antibacterial activities against the E. coli strain with MIC as 1 μg/mL, similar to compound 14. By contrast, the carboxyl compound 26 was inactive, indicating that it was hindered from penetrating the outer cell membrane of the E. coli bacteria.

The Topo IV inhibitory activities (ParE) of representative compounds 14, 22, 26 and 28 were assayed subsequently with novobiocin (1) (Table 3). Just like novobiocin (1), the compounds show relatively weaker inhibitory activities on Topo IV than gyrase, with the IC₅₀ values as 1.513–7.180 μmol/L on S. aureus Topo IV and more than 10 μmol/L on E. coli Topo IV in our ATPase assay.

Table 3.

In vitro gyrase inhibitory on Topo IV [IC₅₀ (μmol/L)].

Strains	1	14	22	26	28
S. aureus	0.383 ± 0.120	7.180 ± 2.809	2.128 ± 0.753	5.706 ± 2.137	1.513 ± 0.460
E. coli	∼10	>10	>10	>10	>10

2.2. Surface plasmon resonance (SPR) and molecule docking

2.2.1. SPR studies

To verify further the interaction of the compounds with the gyrase B subunit (GyrB24; residues 2–220), we performed the surface plasmon resonance (SPR) experiment with novobiocin (1) and AZD5099 (6) as positive controls (Fig. 2 and Table 4)¹⁸. As expected, compound 28 had a value for K_D in the range 10⁻⁸–10^–9 mol/L, just like the novobiocin (1) and AZD5099 (6), indicating they bind with GyrB with high affinities.

Figure 2.

The affinity of the compounds with GyrB by SPR.

Table 4.

The affinity of the compounds with GyrB by SPR [K_D (nmol/L)].

Strains	1	6	28
S. aureus GyrB24	69.6	83.8	52

2.2.2. Molecule docking

From the molecule docking with S. aureus GyrB (cocrystal with AZD1279, 3TTZ), it can be found that compound 28 has a similar interaction mode with AZD5099 (6) (Fig. 3). At first, both of the methyl dichloropyrrole carboxamide fragments of compound 28 and AZD5099 (6) formed the critical hydrogen bonds with Asp81 and water in nearly the same manner. Although the 3-methoxyl groups on the piperidine rings took different conformations, the pyridazine of compound 28 and the thiazole of AZD5099 (6) formed similar cation–π stacking interactions with Arg84. In addition, the nitrogen atom of the pyridazine and oxygen atom of the hydroxyisopropyl group of compound 28 formed the hydrogen-bonding interactions with Arg144, which also can be found in the sulfur atom of the thiazole and oxygen atom of the carboxyl group of the AZD5099 (6).

Figure 3.

Molecule docking of compound 28 and AZD5099 (6) with S. aureus GyrB.

2.3. The mouse model of sepsis induced by MRSA

Using a mouse model of sepsis induced by the methicillin-resistant S. aureus (ATCC43300) infection, the protective effects of compounds 22, 23 and 28 that showed comparable in vitro inhibitory activities against S. aureus (MIC≤0.015 μg/mL) were evaluated through intragastric administration (ig) at a dose of 10 mg/kg, with DS2969b (5) and AZD5099 (6) as controls (Fig. 4)³¹. The experimental results showed that compounds 22 and 23 show weak protective effects, just like the lead compound 14. Although the DS2969 (5) protected 50% of the mice from death on day 3, only 25% of the mice survived on Day 7. By contrast, all the mice in the groups treated with compound 28 and AZD5099 (6) survived. Further experiments proved that compound 28 has a lower median effective dose (ED₅₀) than AZD5099 (6) (2.5 mg/kg vs 7.5 mg/kg). It turns out that the hydroxyisopropyl group of pyridazine 28 has unique advantages compared with the other substitutions, which may result in better pharmacokinetic properties in vivo.

2.4. Antibacterial spectrum of compound 28

Then the antibacterial spectrum of compound 28 was assayed, with DS2969 (5) and AZD5099 (6) as the positive control drugs (Table 5). The quality control strains were purchased from ATCC. The other strains were clinically isolated pathogenic bacteria collected in Sichuan, Beijing, and Shanghai from 2015 to 2020. Compound 28 showed slightly better inhibitory effects than AZD5099 (6) against susceptible and resistant Gram-positive bacteria including S. aureus (MRSA, MSSA, VISA), S. epidermidis (MRSE, MSSE), E. faecalis and E. faecium strains, with the MIC values less than 0.03 μg/mL, which are potent than DS-2969 (5). Moreover, compound 28 also has much better inhibitory activity against Gram-negative bacteria strains, with the MIC value of P. aeruginosa (ATCC27853) as 4 μg/mL, A. baumannii (ATCC19606) and K. pneumoniae (KNP-15-1) as 8 μg/mL. By contrast, AZD5099 (6) and DS2969 (5) were almost inactive against the assayed strains.

Table 5.

Antibacterial spectrum of compound 28 (MIC μg/mL).

Strains	DS-2969 (5)	AZD5099 (6)	28
S. aureus ATCC43300	0.5	0.03	0.015
S. aureus MRSA15-1a	0.5	0.03	0.015
S. aureus MRSA15-2a	0.5	0.03	0.015
S. aureus MSSA15-1a	0.5	0.03	0.015
S. aureus MSSA15-2a	0.5	0.03	0.015
S. aureus VISA5836	0.5	0.015	0.015
S. aureus VISA5827	0.5	0.015	0.015
S. epidermidis MRSE15-1a	0.25	0.03	0.015
S. epidermidis MRSE15-2a	0.125	0.03	0.015
S. epidermidis MSSE15-3a	0.25	0.03	0.015
E. faecalis ATCC51575	0.5	0.03	0.03
E. faecalis ATCC51299	0.5	0.03	0.015
E. faecium EFM19-1a	0.5	0.03	0.015
P. aeruginosa ATCC27853	>64	>64	4
A. baumannii ATCC19606	>64	>64	8
K. pneumoniae KPN-15-1	>64	>64	8

^aClinically isolated pathogenic bacteria collected in Sichuan, Beijing, and Shanghai from 2015 to 2020.

2.5. Evaluation of spontaneous resistance

The frequencies of spontaneous resistance of compound 28 on S. aureus (ATCC29213) and methicillin-resistant S. aureus (ATCC43300) at 4 × MIC have been determined, with DS-2969 (5) and AZD5099 (6) as controls. Compound 28 displayed low spontaneous resistance frequencies of less than 8 × 10^–12 and 4.3 × 10^–12 on the two strains, similar to the control drugs²⁶.

2.6. Time-killing curves

A time–kill curve of compound 28 against the susceptible S. aureus (ATCC29213) was then plotted, with DS-2969 (5) and AZD5099 (6) as positive controls (Fig. 5). It showed a time-dependent bactericidal effect at 4, 8, and 16 × MIC concentrations, similar to DS2969 (5) and AZD5099 (6).

Figure 5.

Time-killing curves of compound 28.

2.7. Safety evaluation

For pyrrolamide-type GyrB/ParE inhibitors such as AZD5099 (6), the mitochondrial toxicity is a worrying deficiency and obstacle. Mitochondrial toxicity was tested in vitro using HepG2 cells as reported by Cotman and coworkers³⁰. Although AZD5099 (6) showed significant mitochondrial toxicity (IC₅₀ < 200 μmol/L) and resulted in complete damage at 400 μmol/L, compound 28 had much less toxicity at the 50–800 μmol/L, indicating it had much better safety and therapeutic indexes (Fig. 6). Compound 28 also has little inhibitory effect on hERG potassium current (IC₅₀ > 30 μmol/L), indicating a low risk of cardiac QT syndrome. Mini–Ames test proved that this compound did not show any mutagenic effects on Salmonella typhimurium TA98 and TA100 strains. It also showed excellent selectivities in human cells such as renal tubular epithelial cell HK2, normal hepatocyte LO2 and embryonic kidney cell HEK293 (IC₅₀ > 10 μmol/L). The mice median lethal doses (LD₅₀) of compound 28 by intragastric administration (ig), intraperitoneal injection (ip) and tail vein injection (iv) were over 800, 300 and 100 mg/kg respectively in acute toxicity tests. The following in vivo subacute toxicity studies in mice also showed great drug safety of compound 28 by repeated gavage administration at 150 and 300 mg/kg for one week (n = 10 for each group). During this process, no significant damage to the main organs of the mice was found, suggesting it has wide therapeutic indexes.

Figure 6.

The mitochondrial toxicity assay of the compounds.

2.8. Neutropenic murine thigh MRSA infection models

The in vivo antibacterial efficacy of compound 28 was evaluated in neutropenic murine thigh MRSA (ATCC43300) infection models with clinically used vancomycin as positive control through tail vein injection administration (iv) (Fig. 7)³². It can be found that compound 28 was efficacious against neutropenic mice thigh infections in a dose-dependent manner from 0.5 to 2.5 mg/kg, resulting in a 3–4-log₁₀ reduction of colony-forming unit (CFU) in the thigh at 2.5 mg/kg. By comparison, vancomycin required a higher effective dose of 110 mg/kg.

Figure 7.

Neutropenic murine thigh infection models (n = 4 for each group).

2.9. Pharmacokinetics study on beagle dogs

For antibacterial drugs, the pharmacokinetic properties are crucial to antibacterial activities in vivo. The pharmacokinetics studies on beagle dogs of compound 28 were performed, which showed fine pharmacokinetics after intravenous injection (iv) and oral administration (po) at 1 mg/kg, with the good absorption (AUC_0–48 = 56,432.101 vs 38,552.203 μg h) and metabolic stability and low clearance (t_1/2 = 11.285 vs 8.533 h, CL = 0.33 mL/min/kg), as well as high oral bioavailability (66.84%) (Fig. 8, Table 6).

Figure 8.

Drug time curve of compound 28 in Beagle dogs.

Table 6.

Pharmacokinetic parameters of compound 28 in Beagle dogs.

Parameter	Unit	iv (1 mg/kg)	po (1 mg/kg)
AUC0–48	μg·h	56,432.101	38,552.203
AUC0–∞	μg·h	58,890.621	39,362.651
MRT0–48	h	9.792	9.071
MRT0–∞	h	12.329	9.853
t1/2	h	11.285	8.533
Tmax	h	0.873	1.75
Cmax	μg/L	5510.488	3748.103
CL	mL/min/kg	0.33	/
F	–	/	66.84%

2.10. Chemistry

The starting material ethyl 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylate was hydrolyzed to carboxylic acid under alkaline conditions to obtain intermediate I-1 (Scheme 1). Then the chiral 1-tert-butoxycarbonyl-3-methoxyl-4-aminopiperidine was subjected to an amide condensation reaction with I-1 under the action of HATU and DIPEA to obtain intermediate I-2, followed by the removal of the protective group under the action of HCl/1,4-dioxane to obtain amine I-3. It reacted with halogenated heteroaryl formate through nucleophilic substitution reaction to obtain compounds 9–16 in 70%–90% yields.

Scheme 1.

Synthesis of compounds 9–16. Reagents and conditions: (a) i) 3 mol/L NaOH, EtOH.r.t.; ii) HCl (aq), r.t.85%; (b) HATU, DIPEA, DMSO, r.t. 87%; (c) HCl/1,4-dioxane, 50 °C, 91%; (d) DIPEA, DMF, 60 °C, 70%–90%.

Under the action of DIPEA, the intermediate I-3 reacted with a substituted pyridazine ring through a nucleophilic substitution reaction to give fluoro, bromo, trifluoromethyl and cyano compounds 17–20 (Scheme 2). Then the same intermediate I-3 underwent nucleophilic substitution reaction with 6-chloro-3-acetylpyridazine and 6-chloro-3-trifluoroacetylpyridazine under the action of DIPEA to obtain compounds 21 and 22. Compound 21 reacted with hydroxylamine hydrochloride or its derivatives to obtain compounds 23–26 (Scheme 2).

Scheme 2.

Synthesis of compounds 17–26. Reagents and conditions: (a) DIPEA, DMF, 60 °C, 70%–80%; (b) Hydroxylamine, MeOH, H₂O, 60 °C, 67%–81%.

The methyl ester 14 was reduced by sodium borohydride to give hydroxymethyl compound 27, and then compound 28 was obtained by Grignard reaction with methyl magnesium bromide (Scheme 3). The amide compounds 29–32 were obtained by aminolysis of ester 14, while the pyrazinamide compound 33 was obtained through amide condensation with acid.

Scheme 3.

Synthesis of compounds 27–33. Reagents and conditions: (a) NaBH₄, MeOH, 41%, r.t.; (b) CH₃BrMg/THF, −5 °C–r.t., 50%; (c) Amine, MeOH, r.t., 50%–80%; (d) i) 3 mol/L NaOH, MeOH, 70 °C, ii) HCl (aq), r.t. 75%; iii) HATU, DIPEA, r.t. 65%.

The cyano compound 20 was reduced under the action of palladium–carbon hydrogenation to obtain the primary amine 34 (Scheme 4). After the hydrolysis of compound 20, the amidino compound 35 was obtained by the following amination reaction. And then compound 20 underwent a cyclization reaction with sodium azide to obtain the tetrazole compound 36. The chemical structure of the target compounds was confirmed by ¹H NMR, ¹³C NMR and HR ESI MS, and the purity of the compounds was confirmed by UPLC–MS.

Scheme 4.

Synthesis of compounds 34–36. Reagents and conditions: (a) H₂ (0.4 MPa), Pd/C, MeOH, 41%; (b) i) NaOCH₃, MeOH, r.t.; ii) NH₄Cl, MeOH, 65 °C, 56%; (c) NaN₃, Py. HCl, 110 °C, 45%.

3. Conclusions

ATP-competitive GyrB/ParE are important targets for antibacterial drugs, but since the novobiocin (1) has been withdrawn from the market, there is no GyrB/ParE inhibitor yet approved in the clinic. Although a great number of GyrB/ParE inhibitors in different types with excellent antibacterial activities have been reported, there are only three drug candidates in clinical trials currently.

AZD5099, a representative pyrrolamide-type GyrB/ParE inhibitor, was withdrawn from clinical trials because of mitochondrial toxicity and other drug safety issues in 2011. Herein we discovered novel pyrrolamide-type GyrB/ParE inhibitors based on the structural modifications of the AZD5099 (6). After systematic replacement of the thiazole and the carboxyl group, the hydroxyisopropyl pyridazine compound 28 with better antibacterial activities and druggability was discovered. Compound 28 had a significant inhibitory effect on Gyrase (GyrB, IC₅₀ = 49 nmol/L) and a modest inhibitory effect on Topo IV (ParE, IC₅₀ = 1.513 μmol/L) of S. aureus. It also showed significant antibacterial effects against several susceptible or resistant Gram-positive bacteria strains with MIC values below 0.03 μg/mL. The frequencies of spontaneous resistance of this compound on the S. aureus bacteria were low. The killing curve showed that this compound had a time-dependent bactericidal effect at concentrations of 4, 8 and 16 × MIC. Compound 28 had better protective effects than DS-2969 (5) and AZD5099 (6) in mouse models of sepsis induced by MRSA. Moreover, the compound 28 had an obvious bactericidal effect at a dose of 2.5 mg/kg by tail vein injection in mouse thigh infection (MRSA) mode, resulting in 3–4-log₁₀ reductions in CFU in the thigh, and the effective doses were significantly lower than the positive drug vancomycin. What's more, the compound 28 also has much better safety and a higher therapeutic index than AZD5099 (6). It also showed good absorptions, metabolic stabilities as well as oral bioavailability in beagle dogs. At present, compound 28 has been evaluated as a pre-clinical drug candidate for the treatment of drug-resistant Gram-positive bacterial infections.

On the other hand, for Gram-negative bacterial strains, the outer membrane of lipopolysaccharide and the intracellular efflux pumps prevent most antibiotics from entering the cell, so they are less sensitive to antibiotics than Gram-positive bacteria^33,34. Therefore, although many compounds with strong inhibitory effects on E. coli gyrase and Topo IV have been reported in the literature before, their antibacterial effects on E. coli strains are relatively weak18, 19, 20, 21, 22. To our delight, compound 28 as well as 14, 22 and 23 with moderate E. coli gyrase inhibitory activities have comparable antibacterial activities against Gram-negative bacteria with the most potent pyrrolamide-type GyrB/ParE inhibitors reported recently^29,30. They can be further studied as the lead compounds against drug-resistant Gram-negative bacterial infections.

4. Experimental

4.1. Chemistry

All of the solvents and reagents used were obtained commercially and used without further purification unless noted otherwise. All the reactions were monitored by thin layer chromatography (TLC) on the silica gel plates GF254 and visualized with UV light or iodine vapor. Products were purified by flash chromatography on silica gel (200–300 mesh). ¹H and ¹³C NMR spectra were recorded on a Varian 400 or 500 NMR spectrometer using chloroform-d or DMSO-d₆ solutions with tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry was conducted by an Agilent Technologies LC/MSD TOF mass spectrometer in positive and negative polarity. All final compounds (compounds 9–36) were determined via analysis by Waters UPLC/SQD system using a reversed-phase C18 column with 5%–95% CH₃CN in water (0.1% HCOOH) for 10 min at a flow rate of 0.2 mL/min.

3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxylic acid (I-1). A mixture of 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylate (3 g, 13.6 mmol), NaOH (1.63 g, 41 mmol) in EtOH (9 mL) and water (15 mL) was heated at 60 °C for about 6 h. After the completion of the reaction determined by TLC, the excess EtOH was removed under reduced pressure. The reaction mixture was acidified with 1 mol/L HCl to pH 2–3 and the residue was extracted with DCM (50 mL × 3). The organic layer was washed with H₂O, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford 2.25 g of the I-1 as a pink solid in 86% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 12.78 (s, 1H), 12.17 (s, 1H), 2.18 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 160.2, 129.3, 116.1, 114.7, 109.1, 10.8.

Tert-butyl(3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-metho-xypiperidine-1-carboxylate (I-2). To a solution of intermediate I-1 (1 g, 5.2 mmol) in DMSO (6 mL) was added HATU (3 g, 7.7 mmol) and DIPEA (1.3 mL, 7.7 mmol) successively. After stirring at room temperature for 15 min, the chiral 1-tert-butoxycarbonyl-3-methoxyl-4-aminopiperidine (1.4 g, 6.2 mmol) was added and stirring was continued until completion determined by TLC. The reaction mixture was poured into 10 mL water. The resulting precipitate was filtered, washed with 0.1 mol/L HCl and saturated NaHCO₃, filtered, and dried to afford I-2 (1.8 g, 85%) as a light gray solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.12 (s, 1H), 7.12 (d, J = 8.31 Hz, 1H), 4.28–4.16 (m, 1H), 4.13–4.07 (m, 1H), 3.89 (dd, J = 48.60, 13.47 Hz, 1H), 3.32 (s, 3H), 2.97–2.72 (m, 2H), 2.18 (s, 3H), 1.63–1.52 (m, 2H), 1.40 (s, 9H).¹³C NMR (126 MHz, DMSO-d₆) δ 157.5, 154.3, 127.8, 118.6, 109.6, 108.1, 78.6, 75.3, 56.4, 48.2, 43.5, 41.5, 28.0, 25.9, 10.6. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₇H₂₅Cl₂N₃O₄Na 428.1114, found 428.1105.

3,4-Dichloro-N-((3S,4R)-3-methoxypiperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (I-3). The solution of the intermediate I-2 (1 g, 2.5 mmol) in 1,4-dioxane (12 mL) and 4 mol/L HCl/1,4-dioxane (4 mL) was heated at 50 °C for about 1 h. The precipitate formed was filtered and dried to afford I-3 (680 mg, 90%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.40 (s, 1H), 9.63–9.51 (m, 1H), 8.43 (d, J = 9.5 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 4.30–4.22 (m, 1H), 3.66–3.63 (m, 1H), 3.54 (d, J = 13.1 Hz, 1H), 3.39 (s, 3H), 3.17 (d, J = 13.6 Hz, 1H), 3.12–2.95 (m, 2H), 2.19 (s, 3H), 1.86–1.78 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.3, 128.2, 119.0, 111.0, 108.7, 73.7, 57.5, 46.4, 43.1, 42.0, 24.6, 11.1. HRMS (ESI):m/z [M+H]⁺ calcd for C₁₂H₁₈Cl₂N₃O₂ 306.0771, found 306.0973.

General procedure A for the synthesis of compounds9–16. A solution of intermediate I-3 (1 equiv), halogenated heteroarylformate (1.2 equiv) and DIPEA (2.1 equiv) in DMF was heated at 60 °C for about 2 h. After the completion of the reaction determined by TLC, the reaction mixture was poured into water. The precipitated solid was filtered and dried to afford compounds 9–16.

Methyl 2-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy piperidin-1-yl)thiazole-5-carboxylate (compound9). Compound 9 (120 mg, 82%) was prepared as general procedure A from methyl 2-bromothiazole-5-carboxylate as a light yellow solid. UPLC purity 97.75%. ¹H NMR (500 MHz, chloroform-d) δ 10.35 (s, 1H), 7.84 (s, 1H), 7.28 (d, J = 8.5 Hz, 1H), 4.53 (d, J = 13.9 Hz, 1H), 4.31 (tq, J = 8.0, 3.8 Hz, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.82 (s, 3H), 3.52 (s, 1H), 3.46 (s, 3H), 3.28–3.20 (m, 1H), 3.17 (d, J = 14.4 Hz, 1H), 2.28 (s, 3H), 2.00 (qd, J = 12.1, 4.5 Hz, 1H), 1.89 (dt, J = 13.4, 4.2 Hz, 1H). ¹³C NMR (101 MHz, chloroform-d) δ 174.7, 162.5, 159.0, 148.1, 128.2, 118.2, 116.2, 111.6, 110.4, 75.7, 57.6, 51.9, 48.7, 48.2, 47.6, 26.3, 11.2. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₇H₂₁Cl₂N₄O₄S 447.0655, found 447.0943.

Ethyl 5-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy-piperidin-1-yl)-1,3,4-thiadiazole-2-carboxylate (compound10). Compound 10 (735 mg, 81%) was prepared as general procedure A from ethyl 5-bromo-1,3,4-thiadiazole-2-carboxylate as a light yellow solid. UPLC purity 96.01%. ¹H NMR (500 MHz, chloroform-d) δ 10.71 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 4.63 (d, J = 14.4 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 4.31 (tt, J = 11.8, 3.9 Hz, 1H), 3.92 (d, J = 13.7 Hz, 1H), 3.53 (s, 1H), 3.45 (s, 3H), 3.37 (td, J = 13.2, 3.0 Hz, 1H), 3.25 (d, J = 14.3 Hz, 1H), 2.26 (s, 3H), 2.02 (tt, J = 12.4, 6.2 Hz, 1H), 1.94–1.86 (m, 1H), 1.39 (t, J = 7.1 Hz, 3H).¹³C NMR (101 MHz, chloroform-d) δ 174.7, 159.3, 159.1, 149.3, 128.6, 118.1, 111.6, 110.4, 75.64, 62.5, 57.7, 49.8, 49.4, 48.7, 26.3, 14.2, 11.2. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₇H₂₂Cl₂N₅O₄S 460.0619, found 460.0609.

Methyl 6-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy-piperidin-1-yl)nicotinate (compound11). Compound 11 (170 mg, 79%) was prepared as general procedure A from methyl 6-chloronicotinate (101 mg, 0.59 mmol) as a white solid. ¹H NMR (500 MHz, chloroform-d) δ 10.23 (s, 1H), 8.78 (d, J = 2.3 Hz, 1H), 8.01 (dd, J = 9.0, 2.3 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 9.1 Hz, 1H), 5.05 (d, J = 14.6Hz, 1H), 4.34 (qd, J = 8.6, 3.2 Hz, 1H), 4.31–4.23 (m, 1H), 3.87 (s, 3H), 3.51 (q, J = 1.8 Hz, 1H), 3.42 (s, 3H), 3.12 (ddd, J = 14.3, 8.5, 6.8 Hz, 1H), 3.07–2.99 (m, 1H), 2.28 (s, 3H), 1.90 (h, J = 4.3, 3.7 Hz, 2H).¹³C NMR (126 MHz, chloroform-d) δ 166.6, 159.0, 151,3, 138.8, 128.0, 118.6, 114.6, 111.5, 110.6, 104.9, 76.4, 57.6, 51.8, 49.3, 44.9, 43.7, 27.1, 11.4. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₉H₂₂Cl₂N₄O₄Na 463.0910, found 463.0895.

Ethyl 2-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy-piperidin-1-yl)pyrimidine-5-carboxylate (compound12). Compound 12 (180 mg, 79%) was prepared as general procedure A from ethyl 2-bromopyrimidine-5-carboxylate (139 mg, 0.60 mmol) as a light yellow solid. UPLC purity 96.53%. ¹H NMR (500 MHz, chloroform-d) δ 9.77 (s, 1H), 8.83 (s, 2H), 7.27 (s, 1H), 5.32 (m, J = 14.4, 2.8 Hz, 1H), 4.98–4.90 (m, 1H), 4.34 (q, J = 7.1 Hz, 3H), 3.51 (s, 1H), 3.42 (s, 3H), 3.10–2.98 (m, 2H), 2.28 (s, 3H), 1.84 (m, J = 29.2, 16.9, 6.5 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 165.0, 163.0, 159.9, 158.9, 127.9, 118.6, 112.8, 111.5, 110.6, 76.4, 60.7, 57.5, 49.4, 44.3, 42.9, 29.8, 27.3, 14.5, 11.4. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₉H₂₃Cl₂N₅O₄Na 478.1019, found 478.1029.

Methyl-5-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxypiperidin-1-yl)pyrazine-2-carboxylate (compound13). Compound 13 (173 mg, 78%) was prepared as general procedure A from methyl 5-bromopyrazine-2-carboxylate (130 mg, 0.60 mmol) as a white solid. UPLC purity 96.86%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (s, 1H), 8.63 (d, J = 1.5 Hz, 1H), 8.45 (d, J = 1.5 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 4.85 (d, J = 14.4 Hz, 1H), 4.50 (d, J = 12.2 Hz, 1H), 4.34–4.25 (m, 1H), 3.81 (s, 3H), 3.55 (t, J = 2.9 Hz, 1H), 3.32 (s, 3H), 3.26 (d, J = 13.7 Hz, 1H), 3.22–3.12 (m, 1H), 2.18 (s, 3H), 1.81–1.74 (m, 1H), 1.71–1.59 (m, 1H).¹³C NMR (101 MHz, DMSO-d₆) δ 165.0, 158.1, 155.8, 144.9, 130.7, 129.7, 128.3, 119.1, 110.1, 108.6, 76.5, 57.5, 52.1, 48.6, 44.8, 42.9, 27.1, 11.1. HR-MS(ESI): m/z [M+Na]⁺ calcd for C₁₈H₂₁Cl₂N₅O₄Na 464.0863, found 464.0868.

Methyl-6-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxypiperidin-1-yl)pyridazine-3-carboxylate (compound14). Compound 14 (850 mg, 72%) was prepared as general procedure A from methyl 6-bromopyridazine-3-carboxylate (700 mg, 3.20 mmol) as a white solid. UPLC purity 97.14%. ¹H NMR (500 MHz, chloroform-d) δ 9.54 (s, 1H), 7.88 (d, J = 9.7 Hz, 1H), 7.25 (s, 1H), 6.89 (d, J = 9.5 Hz, 1H), 5.38 (d, J = 13.9 Hz, 1H), 4.37 (s, 1H), 4.24 (d, J = 13.4 Hz, 1H), 4.00 (s, 3H), 3.56 (s, 1H), 3.43 (s, 3H), 3.25–3.17 (m, 1H), 3.10 (d, J = 14.9 Hz, 1H), 2.28 (s, 3H), 1.99–1.86 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 165.1, 160.1, 158.8, 142.6, 128.8, 128.0, 118.4, 111.5, 110.5, 109.9, 76.1, 57.6, 52.6, 49.1, 44.6, 43.7, 26.9, 11.3. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₈H₂₁Cl₂N₅O₄Na 464.0863, found 464.0873.

Ethyl-2-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methox-ypiperidin-1-yl)oxazole-5-carboxylate (compound15). Compound 15 (147 mg, 90%) was prepared as general procedure A from ethyl 2-bromooxazole-5-carboxylate (80 mg, 0.36 mmol) as a white solid. UPLC purity 98.08%. ¹H NMR (500 MHz, chloroform-d) δ 10.30 (s, 1H), 7.52 (s, 1H), 7.25 (d, J = 4.6 Hz, 1H), 4.55 (d, J = 14.4 Hz, 1H), 4.32–4.28 (m, 2H), 4.26 (d, J = 11.4 Hz, 2H), 3.48 (d, J = 1.4 Hz, 1H), 3.44 (s, 3H), 3.18–3.07 (m, 2H), 2.26 (s, 3H), 1.95 (qd, J = 12.4, 4.5 Hz, 1H), 1.85 (d, J = 10.4 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, chloroform-d) δ 163.1, 159.0, 158.1, 136.9, 136.6, 128.2, 118.4, 111.60, 110.6, 75.61, 60.7, 57.5, 48.7, 46.0, 44.6, 26.6, 14.5, 11.4. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₈H₂₂Cl₂N₄O₅Na 467.0859, found 467.0856.

Ethyl-5-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy piperidin-1-yl)-1,3,4-oxadiazole-2-carboxylate (compound16). Compound 16 (219 mg, 87%) was prepared as general procedure A from ethyl 5-bromo-1,3,4-oxadiazole-2-carboxylate (150 mg, 0.68 mmol) as a light yellow solid. UPLC purity 95.91%. ¹H NMR (500 MHz, chloroform-d) δ 9.71 (s, 1H), 7.22 (d, J = 8.8 Hz, 1H), 4.53–4.41 (m, 3H), 4.33–4.26 (m, 1H), 4.22 (d, J = 13.5 Hz, 1H), 3.51 (s, 1H), 3.45 (s, 3H), 3.27–3.17 (m, 2H), 2.27 (s, 3H), 1.97 (m, J = 12.4, 6.5 Hz, 1H), 1.91–1.85 (m, 1H), 1.42 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 165.2, 158.7, 154.5, 151.3, 128.0, 118.3, 111.5, 110.56, 75.4, 62.8, 57.5, 48.4, 46.6, 45.2, 26.3, 14.2, 11.3. HR-MS (ESI): m/z [M–H]^– calcd for C₁₇H₂₀Cl₂N₅O₅ 444.0847, found 444.0836.

3,4-Dichloro-N-((3S,4R)-1-(6-fluoropyridazin-3-yl)-3-methoxypiperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound17). Compound 17 (40 mg, 70%) was prepared as general procedure A for 3,6-difluoropyridazine (20 mg, 0.17 mmol) as a white solid. UPLC purity 97.24%. ¹H NMR (500 MHz, chloroform-d) δ 9.93 (s, 1H), 7.29 (s, 1H), 7.13–7.08 (m, 1H), 7.00 (d, J = 9.7 Hz, 1H), 4.85 (d, J = 14.5 Hz, 1H), 4.33 (d, J = 5.4 Hz, 1H), 4.11 (d, J = 14.0 Hz, 1H), 3.54 (s, 1H), 3.42 (d, J = 1.5 Hz, 3H), 3.10 (dd, J = 35.3, 14.3 Hz, 2H), 2.31–2.25 (m, 3H), 2.00–1.89 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 161.2(d, J_C-F = 235.3 Hz), 159.7, 158.8, 127.9, 118.4, 117.3, 116.9, 111.4, 110.4, 76.2, 57.5, 48.9, 45.8, 44.6, 26.7, 11.3. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₆H₁₈Cl₂FN₅O₂Na 424.0714, found 424.0726.

N-((3S,4R)-1-(6-Bromopyridazin-3-yl)-3-methoxypiperidin-4-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (compound18). Compound 18 (233 mg, 74%) was prepared as general procedure A from 3,6-dibromopyridazine (195 mg, 0.82 mmol) as a white solid. UPLC purity 95.90%.¹H NMR (500 MHz, chloroform-d) δ 10.06 (s, 1H), 7.32–7.26 (m, 2H), 6.82 (d, J = 9.6 Hz, 1H), 4.97 (d, J = 14.4 Hz, 1H), 4.41–4.30 (m, 1H), 4.12 (d, J = 13.9 Hz, 1H), 3.53 (s, 1H), 3.45–3.40 (m, 3H), 3.12 (ddd, J = 13.5, 9.8, 4.9 Hz, 1H), 3.05 (d, J = 14.6 Hz, 1H), 2.28 (s, 3H), 1.95–1.88 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 158.1, 136.4, 132.1, 128.4, 119.2, 116.7, 110.0, 108.6, 76.5, 57.3, 48.6, 45.4, 43.2, 26.7, 11.1. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₆H₁₈BrCl₂N₅O₂Na 483.9913, found 483.9925.

3,4-Dichloro-N-((3S,4R)-3-methoxy-1-(6-(trifluoromethyl)pyridazin-3-yl)piperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound19). Compound 19 (70 mg, 77%) was prepared as general procedure A from 3-chloro-6-(trifluoromethyl)pyridazine (44 mg, 0.24 mmol) as a white solid. UPLC purity 99.90%. ¹H NMR (500 MHz, chloroform-d) δ 9.99–9.89 (m, 1H), 7.47 (dd, J = 9.6, 4.2 Hz, 1H), 7.30 (d, J = 4.9 Hz, 1H), 6.95 (dd, J = 9.7, 4.4 Hz, 1H), 5.25 (d, J = 14.7 Hz, 1H), 4.44–4.33 (m, 1H), 4.31–4.21 (m, 1H), 3.57 (s, 1H), 3.44 (d, J = 4.1 Hz, 3H), 3.19 (t, J = 12.8 Hz, 1H), 3.11 (dd, J = 15.0, 4.4 Hz, 1H), 2.29 (d, J = 4.2 Hz, 3H), 2.00–1.87 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 160.5, 158.8, 142.5(q, J_C-CF3 = 34.0 Hz), 128.0, 124.7, 122.1 (d, J_C-F = 271.0 Hz), 118.4, 111.5, 110.8, 110.5, 76.2, 57.7, 49.0, 44.8, 43.7, 26.8, 11.3. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₇H₁₈Cl₂F₃N₅O₂Na 474.0682, found 474.0695.

3,4-Dichloro-N-((3S,4R)-1-(6-cyanopyridazin-3-yl)-3-methoxypiperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound20). Compound 20 (97 mg, 72%) was prepared as general procedure A from 6-chloropyridazine-3-carbonitrile (56 mg, 0.40 mmol) as a white solid. UPLC purity 97.96%. ¹H NMR (500 MHz, chloroform-d) δ 9.70 (s, 1H), 7.44 (d, J = 9.6 Hz, 1H), 7.24 (s, 1H), 6.87 (d, J = 9.6 Hz, 1H), 5.28 (d, J = 11.3 Hz, 1H), 4.43–4.34 (m, 1H), 4.32–4.21 (m, 1H), 3.57 (s, 1H), 3.42 (s, 3H), 3.21 (t, J = 12.0 Hz, 1H), 3.11 (d, J = 14.5 Hz, 1H), 2.28 (s, 3H), 2.00–1.83 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 159.0, 158.7, 130.64, 129.0, 127.9, 118.3, 116.8, 111.5, 110.6, 109.4, 76.2, 57.7, 48.9, 44.8, 43.7, 26.8, 11.3. HR-MS (ESI): m/z [M–H]^– calcd for C₁₇H₁₇Cl₂N₆O₂ 407.0796, found 407.0784.

N-((3S,4R)-1-(6-Acetylpyridazin-3-yl)-3-methoxypiperidin-4-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (compound21). A mixture of 1-(6-chloropyridazin-3-yl)ethan-1-one (40 mg, 0.26 mmol), I-3 (80 mg, 0.26 mmol) and DIPEA (106 μL, 0.64 mmol) was heated to 60 °C for about 2 h. After the completion of the reaction determined by TLC, the reaction mixture was poured into water. The precipitated solid was filtered to afford compound 21 (86 mg, 78%) as a white solid. UPLC purity 99.12%. ¹H NMR (500 MHz, chloroform-d) δ 10.05 (s, 1H), 7.89 (d, J = 9.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 9.7 Hz, 1H), 5.21 (d, J = 15.1 Hz, 1H), 4.44–4.33 (m, 2H), 3.58 (d, J = 3.4 Hz, 1H), 3.44 (s, 3H), 3.24–3.10 (m, 2H), 2.77 (s, 3H), 2.28 (s, 3H), 1.99–1.91 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 197.3, 160.7, 158.1, 148.7, 128.4, 125.9, 119.1, 112.0, 110.1, 108.6, 76.5, 57.5, 48.7, 45.1, 43.1, 27.0, 25.6, 11.1. HR-MS (ESI): m/z [M–H]^– calcd for C₁₇H₂₁Cl₂N₅O₃ 424.0949, found 424.0931.

3,4-Dichloro-N-((3S,4R)-3-methoxy-1-(6-(2,2,2-trifluoroacetyl)pyridazin-3-yl) piperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound22). A mixture of 1-(6-chloropyridazin-3-yl)-2,2,2-trifluoroethan-1-one (100 mg, 0.39 mmol), I-3 (119 mg, 0.39 mmol) and DIPEA (171 μL, 0.98 mmol) was heated to 60 °C for about 2 h. After the completion of the reaction determined by TLC, the mixture was extracted with EA (10 mL × 3). The organic phase was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by chromatography on silica gel to afford compound 22 (130 mg, 70%) as a yellow solid. UPLC purity 97.00%. ¹H NMR (400 MHz, chloroform-d) δ 10.01 (s, 1H), 7.92 (d, J = 9.8 Hz, 1H), 7.25 (s, 1H), 6.95 (d, J = 9.8 Hz, 1H), 4.44–4.35 (m, 1H), 3.62–3.58 (m, 1H), 3.44 (s, 3H), 3.28–3.16 (m, 2H), 2.95 (s, 1H), 2.88 (d, J = 0.6 Hz, 1H), 2.28 (s, 3H), 2.02–1.89 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 178.7(q, J_C-CF3 = 35.0 Hz), 159.6, 158.8, 144.3, 128.60, 128.1, 118.3, 116.9 (q, J_C-F3 = 288.0 Hz), 111.6, 110.6, 110.0, 76.3, 57.9, 48.9, 45.1, 43.9, 27.0, 11.3. HR-MS (ESI): m/z [M–H]^– calcd for C₁₈H₁₇Cl₂F₃N₅O₃ 478.0666, found 478.0658.

3,4-Dichloro-N-((3S,4R)-1-(6-((E)-1-(hydroxyimino)ethyl)pyridazin-3-yl)-3-methoxypiperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound23). To a solution of compound 21 (60 mg, 0.14 mmol) in MeOH (2 mL) was added hydroxylamine hydrochloride solution (1.5 g dissolved in 2 mL water and 1 mL MeOH). The mixture was stirred at 60 °C. After the completion of the reaction determined by TLC, the reaction mixture was poured into water (8 mL), and extracted with EA (10 ml × 3). The combined organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by chromatography on silica gel to afford compound 23 (50 mg, 81%) as a white solid. UPLC purity 99.90%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.15 (s, 1H), 11.38 (s, 1H), 7.70 (d, J = 9.7 Hz, 1H), 7.33 (d, J = 9.9 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 4.69 (d, J = 14.7 Hz, 1H), 4.40 (d, J = 12.6 Hz, 1H), 4.26 (d, J = 2.8 Hz, 1H), 3.49 (s, 1H), 3.21 (d, J = 14.3 Hz, 1H), 3.16–3.07 (m, 1H), 2.48 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H), 1.76–1.65 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.9, 158.1, 153.1, 148.5, 128.3, 124.7, 119.2, 113.3, 110.1, 108.6, 76.5, 57.3, 48.7, 45.1, 43.0, 26.9, 11.1, 10.3. HR-MS (ESI): m/z [M–H]^– calcd for C₁₈H₂₁Cl₂N₆O₃ 439.1058, found 439.1042.

3,4-Dichloro-N-((3S,4R)-3-methoxy-1-(6-((E)-1-(methoxyimino)ethyl) pyridazin-3-yl) piperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound24). Compound 24 (32 mg, 78%) was prepared from compound 21 (40 mg, 0.09 mmol) and methoxyammonium chloride (0.75 g dissolved in 2 mL water and 1 mL MeOH) in the same manner as described for compound 23 as a white solid. ¹H NMR (500 MHz, chloroform-d) δ 10.40 (s, 1H), 7.90–7.82 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 9.7 Hz, 1H), 5.05–4.96 (m, 1H), 4.35 (m, 1H), 4.28 (d, J = 12.8 Hz, 1H), 4.00 (d, J = 1.6 Hz, 3H), 3.55 (d, J = 3.4 Hz, 1H), 3.42 (s, 3H), 3.18–3.07 (m, 2H), 2.38 (d, J = 1.6 Hz, 3H), 2.28 (s, 3H), 1.94 (m, 2H).¹³C NMR (101 MHz, DMSO-d₆) δ 159.5, 159.0, 154.1, 148.5, 128.1, 125.5, 118.5, 112.3, 111.5, 110.5, 76.3, 62.4, 57.7, 49.2, 45.2, 43.8, 27.0, 11.4, 10.5. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₉H₂₅Cl₂N₆O₃ 455.1360, found 455.1378.

2-((((E)-1-(6-((3S,4R)-4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxypiperidin-1-yl) pyridazine-3-yl)ethylidene)amino)oxy)acetic acid (compound25). Compound 25 (33 mg, 70%) was prepared from compound 21 (40 mg, 0.09 mmol) and 2-(aminooxy)acetic acid (200 mg dissolved in 1 mL water and 1 mL MeOH) in the same manner as described for compound 23 as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.37 (s, 1H), 7.71 (d, J = 9.7 Hz, 1H), 7.38 (d, J = 9.7 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 4.74 (d, J = 14.5 Hz, 1H), 4.57 (s, 2H), 4.47 (d, J = 12.9 Hz, 1H), 4.33 (d, J = 7.4 Hz, 1H), 3.55 (s, 2H), 3.31 (s, 1H), 3.28 (s, 1H), 3.21 (dd, J = 16.8, 6.8 Hz, 2H), 2.35 (s, 3H), 2.23 (s, 3H), 1.76 (dd, J = 14.1, 9.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.6, 160.1, 158.1, 154.8, 147.2, 128.3, 125.0, 119.2, 113.1, 110.2, 108.6, 76.5, 71.4, 57.3, 48.7, 45.1, 43.0, 39.4, 26.8, 11.1. HR-MS (ESI): m/z [M+H]⁺ calcd for C₂₀H₂₅Cl₂N₆O₅ 499.1258, found 499.1273.

2-((((E)-1-(6-((3S,4R)-4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy piperidin-1-yl)pyridazin-3-yl)ethylidene)amino)oxy)-2-methylpropanoic acid (compound26). Compound 26 (42 mg, 67%) was prepared from compound 21 (50 mg, 0.12 mmol) and 1-carboxy-1-methylethoxyammonium chloride (100 mg dissolved in 0.5 mL water and 1 mL MeOH) in the same manner as described for compound 23 as a white solid. UPLC purity 98.37%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.56 (s, 1H), 12.14 (s, 1H), 7.64 (d, J = 9.7 Hz, 1H), 7.38 (d, J = 9.8 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 4.76–4.66 (m, 1H), 4.44 (d, J = 12.7 Hz, 1H), 4.34–4.25 (m, 1H), 3.52 (s, 1H), 3.30 (s, 3H), 2.89 (s, 1H), 2.73 (s, 1H), 2.29 (s, 3H), 2.18 (s, 3H), 1.79–1.67 (m, 2H), 1.50 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.2, 162.8, 159.9, 158.1, 153.8, 147.6, 128.4, 125.1, 119.2, 110.0, 108.5, 81.8, 76.5, 57.4, 48.7, 45.2, 43.1, 36.3, 31.2, 26.9, 24.4, 10.9. HR-MS (ESI): m/z [M+H]⁺ calcd for C₂₂H₂₉Cl₂N₆O₅ 527.1571, found 527.1571.

3,4-Dichloro-N-((3S,4R)-1-(6-(hydroxymethyl)pyridazin-3-yl)-3-methoxy piperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound27). To a solution of compound 14 (60 mg, 0.14 mmol) in MeOH (2 mL) was added sodium borohydride (15 mg, 0.41 mmol) in portions at 0 °C and stirred at room temperature for 4 h. After the completion of the reaction determined by TLC, 8 mL water was added to the reaction solution and the mixture was extracted with DCM (10 mL × 3). The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel to obtain compound 27 (24 mg, 41%) as a white solid. UPLC purity 95.94%. ¹H NMR (500 MHz, chloroform-d) δ 10.02 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 9.4 Hz, 1H), 6.99 (d, J = 9.5 Hz, 1H), 4.95 (dt, J = 14.5, 3.2 Hz, 1H), 4.80 (s, 2H), 4.37–4.30 (m, 1H), 4.23–4.16 (m, 1H), 3.54 (s, 1H), 3.42 (s, 3H), 3.11 (dd, J = 26.4, 11.5 Hz, 2H), 2.28 (s, 3H), 1.95–1.89 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 156.0, 158.9, 151.8, 127.9, 126.6, 118.6, 113.6, 111.5, 76.3, 63.5, 57.6, 49.1, 45.3, 44.0, 29.9, 26.9, 11.4. HR-MS (ESI): m/z [M–H]^– calcd for C₁₇H₂₀Cl₂N₅O₃ 412.0949, found 412.0941.

3,4-Dichloro-N-((3S,4R)-1-(6-(2-hydroxypropan-2-yl)pyridazin-3-yl)-3-methoxypiperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound28). To a suspension of compound 14 (400 mg, 0.91 mmol) in anhydrous THF (5 mL) was added CH₃BrMg (1.70 mL, 4.60 mmol, 3 mol/L in diethyl ether solution) dropwise under argon at −5 °C. The reaction mixture was stirred at room temperature for 1.5 h, then quenched with saturated NH₄Cl and diluted with water, extracted 3 times with EA. The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH = 20/1) to afford compound 28 (184 mg, 46%) as a white solid. UPLC purity 98.87%. ¹H NMR (500 MHz, chloroform-d) δ 9.58 (s, 1H), 7.36 (d, J = 9.6 Hz, 1H), 7.25 (s, 1H), 6.99 (d, J = 9.6 Hz, 1H), 4.91 (dd, J = 15.0, 2.8 Hz, 1H), 4.35 (dq, J = 6.4, 3.1 Hz, 2H), 4.20 (d, J = 13.6 Hz, 1H), 3.60–3.54 (m, 1H), 3.44 (s, 3H), 3.18–3.08 (m, 2H), 2.28 (s, 3H), 2.00–1.90 (m, 2H), 1.57 (s, 6H). ¹³C NMR (101 MHz, chloroform-d) δ 159.3, 159.0, 158.2, 128.2, 124.6, 118.3, 113.8, 111.4, 110.3, 76.1, 71.6, 57.5, 49.0, 45.2, 43.8, 30.4, 29.7, 26.8, 11.2. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₉H₂₆Cl₂N₅O₃ 442.1407, found 442.1425.

6-((3S,4R)-4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxami-do)-3-methoxy piperidin-1-yl)pyridazine-3-carboxamide (compound29). To the solution of compound 14 (60 mg, 0.14 mmol) in MeOH (2 mL) was added the ammonia (1 mL, 7 mol/L in MeOH) and the mixture was stirred at room temperature. After the completion of the reaction determined by TLC, the mixture was concentrated under reduced pressure, and the residue was purified by chromatography on silica gel to afford compound 29 (39 mg, 66%) as a white solid. UPLC purity 99.32%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.21 (s, 1H), 8.12 (s, 1H), 7.75 (d, J = 9.5 Hz, 1H), 7.48 (s, 1H), 7.37 (d, J = 9.6 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 4.79 (d, J = 13.6 Hz, 1H), 4.42 (d, J = 12.3 Hz, 1H), 4.25 (s, 1H), 3.48 (s, 1H), 3.21 (s, 1H), 3.14 (t, J = 10.8 Hz, 1H), 2.13 (s, 3H), 1.95 (d, J = 8.4 Hz, 1H), 1.74–1.61 (m, 2H), 1.18 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.8, 160.9, 158.1, 144.8, 128.3, 126.7, 119.1, 112.6, 110.3, 108.6, 76.4, 57.3, 48.7, 45.0, 43.0, 26.9, 11.1. HR-MS (ESI): m/z [M+Na]⁺ calcd for C₁₇H₂₀Cl₂N₆O₃Na 449.0866, found 449.0863.

6-((3S,4R)-4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxy piperidin-1-yl)-N-methylpyridazine-3-carboxamide (compound30). Compound 30 (43 mg, 70%) was prepared from compound 14 (60 mg, 0.14 mmol) and methylamine (1 mL, 30% in alcohol) in the same manner as described for compound 29 as a white solid.¹H NMR (500 MHz, chloroform-d) δ 10.08 (s, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.87 (d, J = 4.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 9.6 Hz, 1H), 5.09 (d, J = 15.2 Hz, 1H), 4.41–4.29 (m, 2H), 3.60–3.53 (m, 1H), 3.42 (s, 3H), 3.21–3.09 (m, 2H), 3.03 (d, J = 5.1 Hz, 3H), 2.28 (s, 3H), 1.74 (s, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 164.0, 160.5, 158.9, 144.5, 128.0, 126.8, 118.4, 111.7, 111.44, 110.5, 76.2, 57.6, 49.0, 45.1, 43.6, 26.8, 26.1, 11.3. HR-MS (ESI): m/z [M–H]^– calcd for C₁₈H₂₁Cl₂N₆O₃ 439.1058, found 439.1053.

6-((3S,4R)-4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxypiperidin-1-yl)-N-ethylpyridazine-3-carboxamide (compound31). To a solution of compound 14 (100 mg, 0.22 mmol) in MeOH (2 mL) and DCM (1 mL) was added 3 mol/L NaOH (1 mL, 3 mmol) and heated to 70 °C for 3 h. The solvent was removed under reduced pressure, the residue was acidified with 1 mol/L HCl to pH 3, and the precipitate solids were collected to give the carboxylic acid (70 mg, 75%) as a pink solid.

To a solution of the above intermediate (50 mg, 0.12 mmol) in DMSO (2 mL) was added HATU (68 mg, 0.18 mmol) and DIPEA (30 μL, 0.18 mmol) successively. After stirring at temperature for 15 min, ethylamine (70 μL, 0.14 mmol, 2 mol/L in THF) was added and stirring was continued until completion determined by TLC. The reaction mixture was poured into 2 mL water. The resulting precipitate was filtered, washed with 0.1 mol/L HCl and saturated NaHCO₃ to afford compound 31 (34 mg, 63%) as a light yellow solid .¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (s, 1H), 8.82 (t, J = 6.0 Hz, 1H), 7.79 (d, J = 9.5 Hz, 1H), 7.40 (d, J = 9.5 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 4.86 (d, J = 14.3 Hz, 1H), 4.45 (d, J = 12.7 Hz, 1H), 4.34–4.23 (m, 1H), 3.54 (s, 1H), 3.30 (s, 3H), 3.28–3.21 (m, 2H), 3.17 (d, J = 10.4 Hz, 1H), 2.69 (s, 1H), 2.18 (s, 3H), 1.81–1.64 (m, 2H), 1.12 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.3, 160.9, 158.1, 144.8, 128.37, 126.5, 119.2, 112.6, 110.1, 108.6, 76.5, 57.3, 48.7, 45.0, 43.1, 34.0, 26.9, 15.4, 11.1. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₉H₂₅Cl₂N₆O₃ 455.1360, found 455.1382.

N-(2-Aminoethyl)-6-((3S,4R)-4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-methoxypiperidin-1-yl)pyridazine-3-carboxamide (compound32). To a solution of compound 14 (60 mg, 0.14 mmol) in EtOH (2 mL) was added (2-aminoethyl) tert-butyl carbamate (110 mg, 0.68 mmol) and the mixture was stirred at room temperature. After the completion of the reaction determined by TLC, the reaction solution was concentrated under reduced pressure, and the residue was purified by chromatography on silica gel to afford 45 mg of the white solid in a 56% yield. The above intermediate (30 mg, 0.05 mmol) was heated at 50 °C for 1h in a solution of 1,4-dioxane (1 mL) and 4 mol/L HCl/1,4-dioxane (0.5 mL). The precipitate was filtered and dried to afford compound 32 (22 mg, 90%) as a white solid. UPLC purity 98.43%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.31 (s, 1H), 9.07 (s, 1H), 8.09 (s, 3H), 7.88 (d, J = 9.3 Hz, 1H), 7.56 (d, J = 9.7 Hz, 1H), 7.24 (d, J = 7.3 Hz, 1H), 4.87 (d, J = 15.5 Hz, 1H), 4.47 (d, J = 11.4 Hz, 1H), 4.31 (s, 1H), 3.28 (d, J = 18.2 Hz, 5H), 2.98 (s, 2H), 2.50 (s, 2H), 2.18 (s, 3H), 1.74 (d, J = 25.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.7, 159.6, 158.1, 144.2, 128.2, 127.7, 119.1, 114.8, 110.5, 108.6, 76.5, 66.8, 57.5, 48.5, 45.8, 43.7, 37.1, 27.0, 11.1. HR-MS (ESI): m/z [M–H]^– calcd for C₁₉H₂₄Cl₂N₇O₃ 468.1323, found 468.1312.

3,4-Dichloro-N-((3S,4R)-3-methoxy-1-(6-(piperazine-1-carbonyl)pyridazin-3-yl)piperidin-4-yl)-5-methyl-1H-pyrrole-2-carboxamide (compound33). Compound 33 (72 mg, 65%) was prepared from tert-butyl piperazine-1-carboxylate (55 mg, 0.28 mmol) in the same manner as described for compound 31 as a white solid. UPLC purity 97.11%. ¹H NMR (500 MHz, chloroform-d) δ 7.74–7.68 (m, 1H), 7.27 (d, J = 4.7 Hz, 2H), 7.02–6.96 (m, 1H), 5.05 (d, J = 15.3 Hz, 1H), 4.33 (d, J = 16.0 Hz, 2H), 4.14–4.04 (m, 2H), 3.99–3.88 (m, 2H), 3.55 (s, 1H), 3.42 (d, J = 5.3 Hz, 3H), 3.18–3.08 (m, 6H), 2.28 (d, J = 5.3 Hz, 3H), 1.97–1.88 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 165.2, 159.6, 158.8, 146.7, 129.8, 128.0, 118.4, 111.8, 111.4, 110.5, 76.2, 57.7, 48.9, 43.5, 36.0, 31.9, 27.2, 26.8, 22.7, 14.1, 11.3. HR-MS (ESI): m/z [M+H]⁺ calcd for C₂₁H₂₈Cl₂N₇O₃ 496.1625, found 496.1647.

N-((3S,4R)-1-(6-(Aminomethyl)pyridazin-3-yl)-3-methoxypiperidin-4-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (compound34). To a solution of compound 20 (250 mg, 0.61 mmol) in MeOH (4 mL) was added 24% palladium on carbon (60 mg) and acetic acid (0.5 mL). The reaction mixture was stirred for 30 h at room temperature under a hydrogen atmosphere at 0.4 MPa. The reaction mixture was filtered through celite and concentrated under reduced pressure. The crude residue was purified by chromatography (water/acetonitrile = 40%/60%) to afford compound 34 (50 mg, 20%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 7.47 (d, J = 9.5 Hz, 1H), 7.40 (d, J = 9.5 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 4.68 (dt, J = 14.8, 2.9 Hz, 1H), 4.43–4.36 (m, 1H), 4.30–4.25 (m, 1H), 4.08 (s, 2H), 3.50 (t, J = 2.3 Hz, 1H), 3.28 (s, 3H), 3.24–3.18 (m, 1H), 3.12 (ddd, J = 13.9, 10.6, 3.9 Hz, 1H), 2.18 (s, 3H), 1.71 (ddd, J = 15.3, 6.8, 3.6 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 159.7, 157.6, 147.8, 127.9, 127.3, 118.7, 113.1, 109.7, 108.1, 76.0, 56.8, 48.3, 44.7, 42.5, 42.0, 26.3, 10.6. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₇H₂₃Cl₂N₆O₂ 413.1254, found 413.1246.

N-((3S,4R)-1-(6-Carbamimidoylpyridazin-3-yl)-3-methoxypiperidin-4-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (compound35). To a solution of compound 20 (250 mg, 0.61 mmol) in MeOH (6 mL) was added sodium methanol (150 mg, 2.78 mmol) under argon and the reaction mixture was stirred overnight at room temperature. Then ammonium chloride (150 mg, 2.80 mmol) was added to the reaction solution and the mixture was heated at reflux for about 1 h. After cooling to room temperature, the solvent was removed to give compound 35 (250 mg, 96%) as an orange solid.

UPLC purity 97.75%.¹H NMR (500 MHz, DMSO-d₆) δ 9.75 (s, 3H), 8.74 (s, 1H), 8.48 (d, J = 9.8 Hz, 1H), 7.88 (d, J = 9.8 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 5.23 (s, 1H), 4.87 (s, 1H), 4.69–4.57 (m, 1H), 3.89 (d, J = 3.8 Hz, 1H), 3.67 (d, J = 14.5 Hz, 1H), 3.62 (s, 3H), 3.60–3.54 (m, 1H), 2.50 (s, 3H), 2.11 (dq, J = 11.4, 3.6 Hz, 1H), 2.02 (qd, J = 12.5, 12.0, 3.7 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 166.9, 161.2, 160.6, 157.7, 138.6, 127.8, 126.5, 118.7, 109.9, 108.1, 76.1, 57.0, 48.1, 42.7, 26.6, 14.1, 10.6. HR-MS (ESI): m/z [M+H]⁺ calcd for C₁₇H₂₂Cl₂N₇O₂ 426.1207, found 426.1195.

N-((3S,4R)-1-(6-(2H-Tetrazol-5-yl)pyridazin-3-yl)-3-methoxypiperidin-4-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (compound36). To a solution of compound 20 (35 mg, 0.086 mmol) in DMF 2 mL was added sodium azide (6.5 mg, 0.1 mmol) and pyridine hydrochloride (10 mg, 0.086 mmol). The reaction mixture was heated for 8 h at 110 °C until completion determined by TLC, and cooled to room temperature. Then the reaction mixture was slowly poured into 5 mol/L NaOH (2 mL). After stirring for additional 30 min, the mixture was concentrated under reduced pressure, diluted with water, and acidified with 3 mol/L HCl to pH 3. The white precipitate formed was filtered, washed with water and dried to afford compound 36 (17 mg, 45%) as a white solid. UPLC purity 97.75%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.42–12.33 (m, 1H), 7.90 (d, J = 9.5 Hz, 1H), 7.43 (d, J = 9.7 Hz, 1H), 7.32–7.24 (m, 1H), 4.67 (d, J = 12.8 Hz, 1H), 4.40 (d, J = 12.9 Hz, 1H), 4.32–4.25 (m, 1H), 3.52 (t, J = 4.4 Hz, 1H), 3.39 (d, J = 9.0 Hz, 3H), 3.28 (s, 1H), 3.17 (d, J = 4.8 Hz, 1H), 2.18 (s, 3H), 2.03–1.93 (m, 1H), 1.80–1.71 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.8, 158.1, 128.2, 126.9, 119.1, 113.6, 110.4, 108.6, 76.4, 57.3, 48.7, 45.2, 43.0, 29.5, 29.2, 26.9, 11.1. HR-MS (ESI): m/z [M–H]^– calcd for C₁₇H₁₈Cl₂N₉O₂ 450.0966, found 450.0950.

4.2. Gyrase and topo IV inhibitory assay

Gyrases and Topo IVs of S. aureus and E. coli bacteria were obtained from Inspiralis Ltd. (Norwich, UK). The gyrases were used at a final concentration of 7.5 nmol/L in a solution of 40 mmol/L HEPES-KOH (pH 7.6), 10 mmol/L magnesium acetate, 10 mmol/L dithiothreitol, 50 g/L BSA, 500 mmol/L potassium glutamate, 1% DMSO, 100 mmol/L ATP, and 10 nmol/L linear pBR322 DNA. Topo IVs were used at a final concentration of 8.5 nmol/L in a solution of 100 mmol/L Tris (pH 7.5), 2 mmol/L magnesium chloride, 1 mmol/L dithiothreitol, 50 g/L BSA, 200 mmol/L potassium glutamate, 1% DMSO, 300 mmol/L ATP, and 10 nmol/L pBR322 DNA. Reactions were conducted in a volume of 10 μL per well. Reactions were initiated with the addition of ATP and mixtures were incubated at 20 °C for 30 min. To quantify ADP concentration, 40 μL of ADP-Glo reagent was added and incubated for 40 min at room temperature after the enzymatic reaction. The luminescence signal was measured using a BioTek Synergy 2 microplate reader. The luminescence data were analyzed using the computer software, Graphpad Prism. Activity (%) was calculated as Eq. (1):

$$\text{Activity}(\%)=\left[(\text{Lu}–\text{Luc})/(\text{Lut}–\text{Luc})\right]\times 100$$ (1)

4.3. Surface plasmon resonance (SPR)

Dilute the GYRB to 50 μg/mL in immobilization buffer 10 mmol/L sodium acetate, pH 4.0. The activator is prepared by mixing 400 mmol/L EDC and 100 mmol/L NHS (GE) immediately before injection. The CM5 sensor chip is activated for 420 s with the mixture at a flow rate of 10 μL/min 50 μg/mL of GYRB in immobilization buffer 10 mmol/L sodium acetate (pH 4.0) is then injected into the Fc4 sample channel for 420 s at a flow rate of 10 μL/min, and typically resulting in immobilization levels of 16,000 RU, the reference channel (Fc3) does not need Ligand Capturing step. The chip is deactivated by 1 mol/L ethanolamine hydrochloride-NaOH (GE) at a flow rate of 10 μL/min for 420 s. Dilute analytes with the same running buffer to 6 concentrations (400, 200, 100, 50, 25 and 0 nmol/L). The analyte is injected into Fc3‒Fc4 of the channel at a flow rate of 20 μL/min for an association phase of 120 s, followed by 180 s dissociation. The association and dissociation processes are all handled in the running buffer. Repeat 6 cycles of an analyte according to analyte concentrations in ascending order. After each cycle of interaction analysis, the sensor chip surface should be regenerated completely with 10 mmol/L glycine/HCl, pH 1.5 as injection buffer at a flow rate of 30 μL/min for 30 s to remove the analyte, then next concentration cycle of the analyte demethylzeylasteral need to repeat analyte injection and regeneration steps.

4.4. Molecular docking

Redock: At first, the ligand 07N1 (AZD1279 (7)) is docked with the prepared protein by the CDOCKER module of Discovery Studio 2016. (A). Protein preparation: Retain the A chain, ligand (07N1), and HOH2 in the protein, remove heteroatoms and other chains from the protein, clean the protein, Prepare the Protein and Define Site operations on the protein, and cut the ligand to a new window. Save the prepared protein as 3TTZ _ prep _ site _ rec.ds. (B). Ligand preparation: Perform Clean Geometry, Prepare Ligands, and Minimize Ligands operations on the ligand. The prepared ligand is saved as 07N1 _ lig _ rec.sd (C). Docking: CDOCKER the prepared ligands and prepared proteins (D). Results: After docking, the conformations with similar interaction modes and the RMSD value less than 2 were selected.

Dock: (A). Protein Preparation: Use the prepared protein in Redock (B). Ligand (compound 28 and AZD5099 (6)) preparation: Perform Clean Geometry, Prepare Ligands, and Minimize Ligands operations on the molecule to be docked, saving the docked molecule as active _ rec.sd. (C). Docking: CDOCKER the prepared docking molecule and the prepared protein (D). Results: After the docking is completed, a conformation with a similar interaction mode with the ligand is selected.

4.5. Antibacterial assay

The antibacterial activity of all compounds was measured according to the broth dilution method prescribed by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacterial cells were cultured overnight at 37 °C 100 μL of each different concentration of the test sample solution was added to the sterilized 96-well polystyrene plate, and the final concentrations of the drug were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008 μg/mL, respectively. 100 μL of the bacterial suspension was added to each well (200 μL per well), and the final concentration of the solution was about 10⁵ CFU/mL. The plates were sealed and incubated in an incubator at 35–37 °C for 18–24 h to determine the results. The lowest drug concentration that completely inhibits the growth of bacteria is the Minimal Inhibitory Concentration (MIC). All tests were performed at least twice with biological replicates.

4.6. Killing kinetic studies

The killing kinetic assay on S. aureus (ATCC 29213) was performed in 96-well plates on DS2969 (5), compound 28 at four different concentrations (1 × MIC, 4 × MIC, 8 × MIC, 16 × MIC), and the concentration of the bacterial solution in each well was 10⁵ CFU/mL. The microplates were incubated at 37 °C. The viable cell count was assessed at 0, 1, 2, 4, 8 and 24 h.

4.7. Spontaneous drug resistance frequency

S. aureus (ATCC43300 and ATCC29213) were incubated in Mueller-Hinton (MH) broth overnight at 37 °C. The bacteria were collected by centrifugation at 3000 r/min, and the supernatant was removed and the concentration of the bacteria was adjusted to 10¹⁰ CFU/mL with sterile physiological saline. The above solution (100 μL) was applied to MH agar dishes containing drugs at the concentration of 4 × MIC, incubated at 37 °C for 48 h, and the number of colonies on the plates was recorded.

$$\text{Spontaneous}\text{resistance}\text{frequency}=\text{The}\text{number}\text{of}\text{colonies}\text{on}\text{the}\text{plate}/\text{The}\text{actual}\text{inoculum}\text{CFU}\text{of}\text{the}\text{plate}$$ (2)

4.8. Mitochondrial toxicity

The protocol was reported by Cotman and coworkers³⁰. HepG2 cells (50,000) were seeded in each well of two 96-well plates, and 100 μL of growth medium (glucose media) was added. Cells were allowed to adhere overnight in a cell incubator at 37 °C. The next day, the medium was removed, cells were washed with PBS, and 100 μL of medium (with glucose) containing positive control (Rotenone) was added, before the addition of the test compounds. The same procedure was repeated for plate 2 with medium replaced with galactose. Cells were again incubated at 37 °C for 24 h. CellTiter-Glo 2.0 Cell Viability (100 μL) (Promega) was then added to each well to measure the amount of ATP. The plate was placed in a shaker for 2 min and incubated for 15 min in the dark at 22 °C. Luminescence was read in a luminometer Tecan Infinite 200 pro. Luminescence from each well treated with the compound was compared to the negative control (medium only).

4.9. Cytotoxicity assay

The selected cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. Stocks of cells were cultured in 25 cm² tissue culture flasks and subcultured two to three times per week. Cytotoxicity testing was performed in a transparent 96-well microplate. Outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. The cells were incubated at 37 °C under 5% CO₂ until confluent and then diluted with culture medium to 4 × 10⁵ cells/mL. Dilutions of the stock solutions resulted in final concentrations of 10 μg/mL in a final volume of 100 μL. After incubation at 37 °C for 48 h, the medium was removed, and the monolayers were washed twice with 100 μL of warm Hanks’ balanced salt solution (HBSS). Warm medium (100 μL) and 10 μL of freshly made methylthiazolyldiphenyltetrazolium bromide (MTT) were added to each well, and then the plates were incubated for 4 h. The absorbance was determined at 570 nm.

4.10. Inhibition evaluation on hERG K⁺ channel

HEK293 cells were stably transfected with the human ether-a-go-go-related gene (hERG channel). The voltage-gated hERG potassium channel current was recorded at room temperature (25 °C) from randomly selected transfected cells under whole-cell manual patch clamp systems equipped with EPC10 amplifier (HEKA, Germany), while electrical data were digitalized by Digidata1440A with sampling frequency at 10 kHz using Patchmaster v2.0. hERG current inhibition at 10 and 30 μmol/L was tested. Quinine was also included as a positive control to ensure the accuracy and sensitivity of the test system.

4.11. Animal experimental protocols

The animal experimental protocols were conducted in accordance with the principles of the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Institute of Material medica, Chinese Academy of Medical Sciences. The male ICR mice (20–22 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The beagle dogs (10–12 kg) were obtained from Beijing Mars Biotechnology Co., Ltd.

4.12. Mouse model of sepsis

ICR mice (2 males and 2 females/group) were intraperitoneal injection with a minimum lethal dose (∼10⁸ CFU) of S. aureus strain (ATCC 43300). After 1 h, the compounds 14, 22, 23, 28, DS2969 (5), and AZD5099 (6) were administered by intragastric administration (ig) at a dose of 10 mg/kg. The survival of animals was recorded daily for 7 days.

4.13. Neutropenic murine thigh S. aureus infection models

The ICR mice were rendered neutropenic by twice intraperitoneal injections of cyclophamide for and on the 1st day and 4th day (150 and 100 mg/kg). After the S. aureus strain (ATCC43300, 10⁶ CFU) was injected into the thigh muscles on the 5th day, compound 28 was administered by tail vein injection at doses of 0.05, 0.5, and 2.5 mg/kg respectively. At the end of 24 h, the animals were sacrificed, and their thigh muscles were removed and homogenized in the iced sterile physiological saline. The CFU of S. aureus was evaluated by serial dilutions.

4.14. Mouse acute toxicity test

ICR mice were divided into blank, intragastric administration (200, 300, 600, 800 mg/kg), intraperitoneal injection (200, 300, 600 mg/kg), and tail vein injection (25, 50, 100, 150 mg/kg) group with different dosage (2 males and 2 females/group). After the single-dose administration of compound 28, the survival of animals was recorded daily for 7 days.

4.15. Mouse subacute toxicity test

ICR mice were divided into blank, low-dose groups (150 mg/kg) and high-dose groups (300 mg/kg) (5 males and 5 females/group). After the repeated gavage administrations of compound 28 for one week (7 days), the mice were euthanized and the main organ tissues (heart, liver, spleen, lungs, kidneys, adrenal glands, thyroid gland, uterus, ovaries, brain, bladder, gallbladder, etc.) were separated and observed.

4.16. Pharmacokinetics study on the beagle dogs

The beagle dogs were fasted for 12 h before the experiment. The pharmacokinetics study of compound 28 in beagle dogs (5 males and 5 females) after oral administration and fore limb intravenous injection at the dose of 1 mg/kg. The blood samples were collected at 0.08, 0.17, 0.25, 0.50, 0.75, 1, 2, 3, 5, 6, 8, 10, 12, 24, 36, and 48 h after the administration of compound 28. The concentrations in plasma were determined using LC–MS/MS. The pharmacokinetic data were analyzed.

Acknowledgments

The authors thank the financial support from the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-030, China) and the Science and Technology Commission Foundation of Beijing (Z221100007922045, China).

Author contributions

Wenxuan Zhang and Song Wu: conceptualization, methodology, supervision, writing reviewing, and editing. Xintong Zhao, Jing Feng, Jie Zhang: methodology, investigation, acquisition of data, writing-original draft. Zunsheng Han, Yuhua Hu, Hui–Hui Shao, Tianlei Li, Jie Xia, Kangfan Lei, Weiping Wang, Fangfang Lai, Yuan Lin, Bo Liu, Kun Zhang, Chi Zhang, Qingyun Yang, Xinyu Luo, Hanyilan Zhang, Chuang Li: investigation, acquisition of data, technical or material support. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.

Conflicts of interest

The authors declare no competing interests.

The authors declare no competing interests.

The following is/are the supplementary data to this article:

Appendix A. Supplementary data

The following is/are the supplementary data to this article: