The Discovery and Development of Thienopyrimidines as Inhibitors of Helicobacter pylori that act through Inhibition of the Respiratory Complex I

Abstract

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, mode of action studies identified H. pylori’s respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices and a good overall pharmacokinetic profile, with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.

Graphical Abstract

Helicobacter pylori is a major causative agent of gastritis, peptic ulcers, and gastric cancer. According to the Centers for Disease Control and Prevention (CDC), more than 500,000 cases of H. pylori infections are diagnosed annually in the United States. A triple therapy, consisting of a proton pump inhibitor and the broad-spectrum antibiotics clarithromycin and amoxicillin, is the recommended treatment against H. pylori infection.¹ However, the rate of successful treatment with the use of the triple therapy regimen has dropped below 80% due to the rise of antibiotic resistance.^2-5 Additionally, the intensive use of the broad-spectrum antibiotics for the treatment of H. pylori infections perturbs the gut microbiome, leading to side effects such as diarrhea and drug resistance in commensal pathogens.¹ Given the rise of H. pylori resistance, new drugs are needed for the treatment of H. pylori infections.

In a previous effort to discover novel chemical matter selective against H. pylori, a high-throughput screening protocol was developed to address the microaerophilic environment required to grow H. pylori.⁶ Hits found in the high-throughput screening campaign were then screened against Staphylococcus aureus to remove promiscuous compounds and prioritize selective compounds. From this approach, we previously reported Hpi1 (Figure 1A), a selective anti-H. pylori compound with excellent in vitro potency (H. pylori minimum inhibitory concentration (MIC) of 0.002 μg/mL) compared to clarithromycin (0.008 μg/mL).⁶ This protocol was subsequently applied to a larger library of 219,197 compounds, which yielded 2,027 compounds as positive hits. The hit compounds were clustered and subsequently prioritized based on their solubility and their chemical tractability. The second screen identified two thienopyrimidine compounds, 1 and 2 (Figure 1B, C), with H. pylori IC₅₀ of 1.55 μM and 1.72 μM respectively. Their activities were confirmed through re-synthesis and testing.

Figure 1.

A) Structure of Hpi1, a selective anti-H.pylori compound that emerged as a positive hit from a high through-put screen against H. pylori.⁶ B, C) Structures of thienopyrimidines hits 1 and 2, that were identified using a similar screening approach with the H. pylori IC₅₀ of 1.55 μM and 1.72 μM respectively. D) Areas substituted in this study to improve potency, drug-like properties, and cytotoxicity.

Herein, we report the structure-activity relationship (SAR) developed from the modification of thienopyrimidines 1 and 2 against H. pylori, which explored substitutions around the thienopyrimidine core and merged optimized side chain elements from the two hits. Hit to lead development was based on H. pylori activity, cytotoxicity, in vivo, and ex vivo efficacy using a mouse model of infection. During these studies, NADH:ubiquinone oxidoreductase (complex I) subunit NuoD was identified as a putative target of thienopyrimidine series by analyzing resistant mutants using whole-genome sequencing and performing genetic transformation and complementation of resistance. To help rationalize the SAR, explore the mechanism of action, and guide further development of series, a homology model of the H. pylori NuoB-NuoD binding interface was generated. These studies produced tractable thienopyrimidine leads with a defined mechanism of action suitable for further optimization and validated the NuoB-NuoD interface as a target for the development of novel narrow spectrum agents against H. pylori.

Results and Discussion

Generation of optimized thienopyrimidines

Four series of new compounds were synthesized to develop the SAR around the two thienopyrimidine hits as outlined in Schemes 1-3.^7-10 Initial SAR efforts focused on examining the N-alkyl hydroxyl moiety of hit compound 1, series-1 (Table 1), and the phenethylamine motif of compound 2, series-2 (Table 2). Sidechains based on lead compounds from each series were then merged into compounds for series-3 (Table 3). Finally, series-4 explored the differing substitution patterns observed for compounds 1 and 2 at C5 and C6 of the thienopyrimidine ring, in combination with an optimized phenylglycinol 4-position side chain from series-3 (Table 4).

Scheme 1. Synthesis of Thienopyrimidine Derivatives.

Reagents and conditions: (i) Sulfur, Morpholine, Ethanol, 70˚C, Microwave, 20 min, 90%; (ii) Formamide, reflux, 18 h, 80%; (iii) POCl₃, N, N dimethylaniline, reflux, 14 h, 90%; (iv) Ethanol, 150˚C, Microwave, 1 h, 80-93%.

Scheme 3: Synthesis of thienopyrimidine derivatives with modifications at position 5 and 6.^a.

^aReagents and conditions: (i) Et₃N, 150˚C, Microwave, 1 h, 89-93%.

Table 1.

The structure-activity relationship of series-1.

Compound		H. pylori ATCC 43504 pIC50 (M)	H. pylori SS1 pIC50 (M)	H. pylori SS1 nuoD A402P pIC50 (M)	FaDu Cells Cytotoxicity pIC50 (M)
1		5.8 ± 0.10	5.8 ± 0.037	4.4 ± 0.27	3.9 ± 0.051
8		4.8 ± 0.27	5.2 ± 0.15	4.7 ± 0.23	4.0 ± 0.023
9		5.3 ± 0.071	5.7 ± 0.081	4.0 ± 0.039	4.0 ± 0.046
10		7.4 ± 0.15	7.1 ± 0.24	5.3 ± 0.034	4.0 ± 0.045
11		6.0 ± 0.27	6.4 ± 0.16	4.8 ± 0.12	4.2 ± 0.013
12		5.3 ± 0.032	5.6 ± 0.12	4.6 ± 0.26	3.6 ± 0.043
13		6.8 ± 0.035	7.5 ± 0.10	5.2 ± 0.16	4.5 ± 0.035
14		5.9 ± 0.20	6.4 ± 0.16	4.8 ± 0.14	4.5 ± 0.046
15		5.8 ± 0.11	6.5 ± 0.10	5.0 ± 0.17	3.9 ± 0.0072
16		7.0 ± 0.063	7.7 ± 0.17	5.5 ± 0.21	4.4 ± 0.0011
17		6.1 ± 0.27	6.6 ± 0.15	4.9 ± 0.16	4.4 ± 0.0010
18		5.5 ± 0.028	6.1 ± 0.048	4.5 ± 0.25	4.3 ± 0.077

All pIC₅₀ (−log IC₅₀) data are the mean of at least 3 measurements. Error measured in S.E.M.

Table 2.

The structure-activity relationship of series-2.

Compound		H. pylori ATCC 43504 pIC50 (M)	H. pylori SS1 pIC50 (M)	H. pylori SS1 nuoD A402P pIC50 (M)	FaDu Cells Cytotoxicity pIC50 (M)
19		6.8 ± 0.26	7.4 ± 0.035	6.3 ± 0.23	3.9 ± 0.25
20		7.1 ± 0.039	6.3 ± 0.20	5.3 ± 0.25	>4.8
21		5.6 ± 0.092	5.9 ± 0.17	5.7 ± 0.28	3.9 ± 0.25
22		5.2 ± 0.11	5.5 ± 0.088	5.0 ± 0.21	4.0 ± 0.0053
23		5.8 ± 0.036	6.3 ± 0.17	4.9 ± 0.13	4.6 ± 0.087

All pIC₅₀ (−log IC₅₀) data are the mean of at least 3 measurements. Error measured in S.E.M.

Table 3.

The structure-activity relationship of series-3.

Compound		H. pylori ATCC 43504 pIC50 (M)	H. pylori SS1 pIC50 (M)	H. pylori SS1 nuoD A402P pIC50 (M)	FaDu Cells Cytotoxicity pIC50 (M)
24		6.9 ± 0.15	6.6 ± 0.057	5.0 ± 0.072	4.8 ± 0.0054
25		7.7 ± 0.070	7.7 ± 0.044	6.9 ± 0.15	3.4 ± 0.050
26		8.0 ± 0.13	8.2 ± 0.093	7.1 ± 0.20	4.8 ± 0.00023
27		7.3 ± 0.032	7.0 ± 0.13	5.7 ± 0.14	4.3 ± 0.047
28		7.6 ± 0.17	8.0 ± 0.23	6.1 ± 0.15	>4.7
29		7.7 ± 0.086	7.9 ± 0.040	6.5 ± 0.18	4.5 ± 0.012
30		7.8 ± 0.073	7.9 ± 0.055	6.5 ± 0.17	4.5 ± 0.017
31		5.5 ± 0.061	6.0 ± 0.20	4.8 ± 0.071	4.3 ± 0.13

All pIC₅₀ (−log IC₅₀) data are the mean of at least 3 measurements. Error measured in S.E.M.

Table 4.

The structure-activity relationship of series-4, with substitution at the 5 and 6-positions of the thienopyrimidine core.

Compound			H. pylori ATCC 43504 pIC50 (M)	H. pylori SS1 pIC50 (M)	H. pylori SS1 nuoD A402P pIC50 (M)	Cytotoxicity pIC50 (M)
	R1	R2				FaDu Cells
37			5.2 ± 0.0084	6.2 ± 0.15	5.1 ± 0.17	>4.7
38			6.5 ± 0.015	7.3 ± 0.056	5.1 ± 0.10	4.7 ± 0.057
39			4.8 ± 0.067	5.1 ± 0.044	4.8 ± 0.20	3.9 ± 0.025
40			5.4 ± 0.044	6.3 ± 0.19	6.0 ± 0.18	4.5 ± 0.13

All pIC₅₀ (−log IC₅₀) data are the mean of a least 3 measurements. Error measured in S.E.M.

To generate compounds in series 1-3 (Scheme 1) phenylacetaldehyde 3 was reacted with ethyl 3-cyanopropanoate 4 to give phenylthiophene 5. Heating 5 in an excess formamide formed pyrimidone 6. Chlorination and dehydration of the pyrimidone with phosphoryl chloride afforded the chlorothienopyrimidine 7, which was coupled with a variety of primary amines and a secondary amine to give targeted 4-position substituted H. pylori inhibitors (8–30). Commercially available amine starting materials with hydroxyl and aromatic moieties, similar to the moieties identified within the hit compounds, were used to synthesize analogs. These amines were selected based on their structural resemblance to the amine substituents of the hit compounds and refined as data from synthesized compounds developed the SAR of each series.

Compounds in each series were evaluated for their activity against H. pylori, and cytotoxicity (Tables 1-4). Antimicrobial activity was measured using IC₅₀ assays against a H. pylori type strain ATCC 43504 and SS1, a mouse adapted strain used in the mouse model of infection. Activity was also monitored against a target based resistant mutant H. pylori SS1 nuoD A402P to ensure the developing anti-H. pylori SAR remained on target. Poor solubility of these 4 compound series sometimes resulted in large standard deviations, therefore comparison of the IC₅₀ between the two strains served to guide the development of the series, leading to successful identification of lead compounds 25 and 26 with a >69 fold increase in potency for both strains. The IC₅₀ data is presented as pIC₅₀, the negative log of the molar IC₅₀ value to better follow the emerging SAR.

In series-1, variations to the propyl alcohol motif of compound 1 were evaluated (Table 1). Compound 8 was generated first to investigate the effect of N-methylation of the amine group attached at the 4-position of the thienopyrimidine found in hit compound 1. Compound 8 did not improve the H. pylori pIC₅₀ values and increased cytotoxicity. Therefore the free secondary aromatic was retained for the remainder of series-1. To examine the length of the spacer contribution between the hydroxyl group and the amine moiety in 1, compound 9 was synthesized with a shorter ethyl motif as a spacer and compound 15 was synthesized with a longer spacer that included an additional methylene unit. Neither compound consistently improved the potency compared with 1.

To investigate the potential for branching between the amine moiety and the hydroxyl group of 1, methyl branched regio-isomers 10 and 11 were synthesized (Table 1). Compound 10 demonstrated 20-fold greater potency against H. pylori SS1 than 1, and compound 11 was 4-fold more potent than 1. The IC₅₀ in ATCC43504 confirmed this with a 37-fold increase in potency for compound 10 and only a negligible less than 2-fold increase for 11. This suggested there may be a preference for modification at the amine α-carbon over the β carbon. The methylation at this amine α-carbon creates a chiral center, therefore the contribution of each isomer in 10 was determined through the synthesis of enantiomers 12 (S) and 13 (R). The R enantiomer 13 was found to be the active isomer demonstrating 56-fold increased activity over 1 in SS1, while the S enantiomer 12 did not improve the activity over compound 1. To examine the contribution of the hydroxyl group in 10, the O-methylated analog 14 was synthesized. Overall, compound 14 demonstrated less potency when compared to 10. The activity of 14 against ATCC43504 was 29-fold less potent than 10. Therefore, the free hydroxyl group was retained in the remainder of this series. The importance of this hydroxyl group was later highlighted in the modeling studies of NuoD in complex with an active ligand (Figure 2).

Figure 2.

a. Homology model of the NuoD and NuoB subunits of complex I. b. Snapshot of lead compound 26 (gold) in the average conformation over a 250 ns molecular dynamic simulation in the wild type protein. The 250ns MD simulation shows that compound 26 is a hydrogen bond donor to the carbonyl oxygen at T395 and accepts a hydrogen bond from the hydroxyl side chain of T400 labeled with magenta dotted lines respectively. Compound 26 methoxybenzyl group pi-stacks with Y376 illustrated with blue dotted lines. c. A ligand interaction diagram between compound 26 and the protein. Pink lines show hydrogen bonds and the green lines show pi-pi interactions d. Analysis of compound 26 hydrogen bond interactions with respective residues over the entire 250 ns simulation.

To determine if further substitution could be tolerated on the α and β carbons of 10 and 11, regio-isomeric compounds 16 and 17 with an additional methylene unit were generated, respectively. The pIC₅₀ values of 16 and 17 were very similar to the parent compounds 10 and 11, in both SS1 and ATCC 43504, indicating branching can be accommodated at this position. However, compound 18, with a larger isopropyl substitution over the ethyl motif found in 16, showed decreased potency. Overall, within compound series-1, clear SAR was observed consistent with the analogs engaging a defined molecular target. However, increased potency did come with an increase in cytotoxicity for this series. Important to the SAR, and carried forward into the design of series-3 compounds, was a 2-carbon linker between the amine moiety and the hydroxyl group, branching at the amine α-carbon over the β-carbon and retention of the terminal hydroxyl group.

Series-2 was designed to evaluate substitution of the phenyl ring in the phenylethyl sidechain of compound 2. Four analogs with varying phenyl substitutions, 19, 20, 21, and 22 (Table 2), were synthesized on the thienopyridine core. Compound 19 displayed improved potency (11-fold against ATCC43504) over compound 2 with a pIC₅₀ of 6.8 ± 0.26 (IC₅₀ 0.16 μM), and improved cytotoxicity. 19 is the direct phenyl- thienopyrimidine hybrid of 2 with the 3,4-dimethoxyphenethyl side chain. Substitution of the dimethoxyphenyl motif with dimethylphenyl, compound 20, decreased comparative potency and increased cytotoxicity. 3 and 4 substituted pyridyl analogs 21 and 22 were designed to increase metabolic stability and solubility, but also showed substantially decreased potency.

The SAR progression revealed series leads 16 and 19 from hits 1 and 2, respectively. We hypothesized that merging both substituents at the 4-position of phenyl-thienopyrimidine could produce analogs with increased potency. Thus, we synthesized compound 24 (Table 3), which demonstrated activity similar to 16 and 19 against ATCC43504 but weaker activity against SS1 strain. Series-3 was designed to evaluate modifications of hybrid analogs based on 24 (Table 3). First compound 25, with a shorter spacer between the aryl motif and the amine was generated, which demonstrated high potency with pIC₅₀ 7.7 ± 0.070 (IC₅₀ 0.021 μM). To evaluate the contribution of each enantiomer, compounds 26 and 27 were synthesized. Although the difference in magnitude is less, like enantiomers 12 and 13, compound 26 pIC₅₀ ATCC43504 8.0 ± 0.13 and SS1 8.2 ± 0.093 (0.010 μM and 0.0068 μM respectively), the R enantiomer of 25, retains the activity of 25, while the S enantiomer 27 decreases activity consistently in both strains ATCC43504 7.3 ± 0.032 and SS1 7.0 ± 0.13 (0.053 μM and 0.11 μM respectively). Unexpectedly, the cytotoxicity of 25 was less than each individual isomer (26 and 27), which was reproducible upon re-synthesis and testing. The reason for this observation is unclear at this time. Unsubstituted 28 retained the reasonable potency of the series-3 compounds with an pIC₅₀ 7.6 ± 0.17 (IC₅₀ of 0.023 μM). Fluorinated compounds 29 and 30 were synthesized in efforts to increase metabolic stability. Fluorination in these compounds was tolerated as both retained similar IC₅₀ values to 26, which may be useful in future development of this series.

The final compound synthesized in in series-3 was compound 31, the primary amine analog of 30. It was generated to explore the potential of amine replacement, as primary amines have been shown to enhance uptake of drugs into Gram-negative bacteria.¹¹ Compound 31 was synthesized as described in Scheme 2, starting from amino alcohol intermediate 32, Boc-protection yielded alcohol 33.^12,13 Substitution of the hydroxyl group of 33 with a phthalimide group was carried out under Mitsunobu conditions to give the intermediate 34. Next, the removal of the Boc protecting group yielded the amine 35, which was coupled to chlorothienopyrimidine 7 to afford the intermediate 36. In the final step, the deprotection of the phthalimide group of 36 afforded the targeted thienopyrimidine 31. Substituting the hydroxyl group for a primary amine resulted in a large increase (>79-fold for both SS1 and ATCC 43504) in the H. pylori IC_50, indicating that the amine replacement was not tolerated and the hydroxyl group is required to maintain the low nM potency observed in the other compounds in series-3. This concurs with the modeling data for 26 (Figure 2), showing the hydroxyl group acting as a hydrogen bond acceptor to the T400 residue.

Scheme 2: Synthesis of Amine Derivative 31.^a.

^aReagents and conditions: (i) di-tert-butyl dicarbonate, Et₃N, DCM, RT, 2 h; (ii) phthalimide, DIAD, Ph₃P, DCM, RT, 4 h; (iii) 4 M HCl in dioxane, DCM, methanol, RT, 4 h; (iv) Et₃N, reflux, 6 h; (v) NH₂NH₂, methanol, RT, 6 h.

Series-3 confirmed our hypothesis that the lead side chains from series-1 and series-2 could be combined, producing leads with low nM anti-H. pylori activity. Like compounds 12 and 13 in Series-1, the R enantiomer was found to be more potent, but again led to an increase in cytotoxicity. Substitution of the phenyl ring was tolerated, which suggests modification of the phenyl ring may be possible for development of future compounds with improved metabolic stability. Unfortunately, replacement of the hydroxyl with the primary amine was not tolerated.

Compounds in series-4 explored the differing substitution patterns observed for compounds 1 and 2 at C5 and C6 of the thienopyrimidine ring, in combination with the phenylglycinol 4-position side chain from series-3 compound 28. Compounds 37–40 were synthesized as described in Scheme 3 and their activities were compared to compound 28 with the simplified phenylglycinol substitution (Table 4). Commercially available chlorothienopyrimidines (41-44) were used in the coupling reaction with the optimized phenylglycinol sidechain to yield final compounds (37-40). Moving the phenyl ring to the 5-position (39 and 40) substantially reduced anti-H. pylori potency. The dimethyl analog 37 with a similar substitution pattern of compound 2, was not as active as compound 28, instead having similar activity to compound 2. Compound 38, with a similar phenyl substitution pattern to series-3 compounds and an additional methyl substitution, demonstrated somewhat decreased activity compared to 28. These data suggested the phenyl ring at the 6-position is preferred, and the 5-position can be a hydrogen or methyl but not a larger phenyl group possibly due to steric clashes in the NuoB-NouD menaquinone binding tunnel targeted by these inhibitors.

Thienopyrimidines inhibit H. pylori respiratory Complex I

To determine the target of the thienopyrimidines, one resistant H. pylori mutant was successfully isolated by serial passaging (five times) on agar medium containing increasing concentrations of compound 1 and two additional spontaneous first-step mutants were obtained by plating on compound 28. Whole genome sequencing indicated mutations in NuoD with no other mutations present in all three strains compared to the isogenic wildtype. The serial passage mutant had multiple non-synonymous mutations in nuoD, while the two first-step resistant mutants had single nucleotide polymorphisms in nuoD causing an amino acid change - either A402P or T400I (Figure 3). Seven additional resistant mutants were generated using compound 28 all with a non-synonymous single-nucleotide polymorphism in nuoD, which resulted in amino acid mutations T400I or A402P. Further, the A402P mutation generated by compound 28 was transformed into a drug susceptible strain of H. pylori for site directed resistance studies to confirm that resistance was caused by non-synonymous mutations in nuoD. IC₅₀ studies with this strain demonstrated resistance was transferable with the A402P mutation alone.

Figure 3.

Sequence alignment of the wildtype nuoD and two H. pylori mutant NuoD. Resistant mutants were generated by exposing H. pylori to compound 1 or 28 on agar plates. Whole genome sequencing was performed, and single nucleotide polymorphism analysis identified two spontaneous mutants, which had non-synonymous point mutations in nuoD. The resulting amino acid changes were Threonine to Isoleucine (T400I) and Alanine to Proline (A402P). nuoD encodes for the subunit D of NADH:Quinone eductase also known as Complex I, a membrane protein that catalyzes the first step of oxidative phosphorylation. Complex I is the putative target of the thienopyrimidine.

To validate that all the compounds in the thienopyrimidine series were on target, each analog was tested against the mutant H. pylori strain SS1 NuoD A402P (Tables 1-4, Figure S1). All of the compounds tested demonstrated significantly decreased anti-H. pylori activity against the NuoD mutant strain, with the exception of compounds 8, 21, 22, 39, and 40 which did not demonstrate statistical significance, indicating that the mechanism of the action is likely consistent throughout the compound series (Supplementary data, Figure S1). The gene nuoD encodes for the NuoD subunit of NADH:quinone oxidoreductase, also known as respiratory complex I.^14-17 These results suggest that complex I, the protein that catalyzes the first step of oxidative phosphorylation, is the putative target of thienopyrimidines.^18,19 Interestingly, NuoD is also the putative target of a previously described series of benzimidazole anti-H. pylori agents, with resistance conferring mutations found at NuoD: G398S, F404S, and V407M (Figure S2).^20,21 This group further determined that while nuoD is essential in H. pylori, it is dispensable in other bacteria, suggesting that targeting NuoD may spare the microbiome. In accordance with this, we found that compounds 25, 26, 28, 29, 30, and 31 had limited to no activity (MIC ≥ 16 μg/ml) against a panel of representative Gram-negative and Gram-positive species, including commensals (supplementary data Table S3).

Recent advances in the structural characterization of complex I, one of the largest membrane protein complexes known, have now enabled the computational analysis of complex I inhibitors.²² The complete structure of NADH:ubiquinone oxidoreductase (complex I) from Thermus thermophilus was determined by Baradaran et al in which they modeled in decylubiquinone, showing key interactions with Y87 and H38, which correspond to Y85 and H36 in H. pylori, respectively.¹⁸ A protein sequence alignment showed that the NuoB and NuoD subunits of H. pylori’s complex I have a high degree of sequence similarity with T. thermophilus’ complex I subunits Nqo6 (63% identity) and Nqo4 (43% identity) (Figure S2). As discussed above, we have shown that thienopyrimidine resistance-conferring mutations occur at T400 and A402, positions which fall within the menaquinone-binding pocket (Figure 3, and Supplementary Figure S3).^23-25 The location of T400I and A402P mutations indicate that the thienopyrimidine series bind in the menaquinone active site. In comparing the published benzimidazole resistance mutations and the two mutations found above, we hypothesize that the menaquinone active site is a target for H. pylori therapy.²⁰

In order to further characterize interactions of the thienopyrimidine series of inhibitors with H. pylori’s complex I, a model of the wildtype protein (subunits NuoD and NuoB) was built using T. thermophilus’ complex I as a template. Inhibitor binding interactions were modeled by computationally docking 26 into the predicted active site, which showed hydrogen binding to T400. In order to determine stability of the docked pose of 26, a 250 ns molecular dynamic simulation was run using AMBERMD. Simulation trajectory analysis revealed key ligand-receptor hydrogen bonding interactions with T400 and T395 with pi-stacking interactions to Y376, all located within the menaquinone binding pocket (Figure 3b,c). Additional analysis revealed that the protein-ligand interaction stabilizes at 50ns and has a stable energy profile (Figure S4) concluding that the menaquinone pocket is the active site of 26.

To determine the extent of potential selectivity, 26 was also docked into a model of the A402P mutated NuoD subunit using the same methods as the wildtype. A weaker docking score of −4.4, as opposed to −11.5 in the wildtype, was observed, suggesting a significantly reduced affinity of 26 for the mutated subunit.

Pharmacological properties of anti-H. pylori Thienopyrimidines

To examine if the lead thienopyrimidine 26 was suitable for advancement into animal testing, the pharmacological properties of its racemate 25 were determined for reasons of cost. The in vitro ADME and in vivo pharmacokinetic properties of 25 were determined.^26-28 The full results of these studies are provided in supplementary data Table S1 and S2, respectively, in the Supporting Information. Compound 25 showed good microsomal stability (t_1/2 = 3.8 hr) and permeability (Ave Pe = 239.5 x 10⁻⁶ cm/s) but has low solubility (1μM) and was highly bound to human plasma protein (HPPB % = 99). 25 showed good in vivo exposure (AUC = 31,907.71 hr*ng/mL, Table S2) at 50 mg/kg, intraperitoneal (IP) dose, with a lower exposure found upon oral (PO) dosing in mice. The total drug concentration of 25 in the plasma was above its IC₅₀ value for more than 8 hours by IP dosing. With good plasma exposure, which we believe is required to treat deep seated ulcerative H. pylori infections, we proceeded to test 25 in the H. pylori murine infection model.^29,30 Further details of this study are provided in the Supporting Information (Figure S5).³¹ Compared to the metronidazole control, 25 did not show efficacy at 50 mg/kg (PO), 50 mg/kg (IP) or 25 mg/kg (IP).³¹

An ex vivo H. pylori infection model was used to investigate the lack of efficacy of compound 25 in the in vivo infection model. Mice were infected with an H. pylori SS1 strain, as described previously, however, instead, the infected stomach tissues were dissected and placed in sterile growth media containing the active compound.^29,30 These experiments were designed to test the effectiveness of compound 25 independent of drug metabolism and excretion. Compound 25 was found to be highly effective in the ex vivo model with colony counts below the limit of detection for 6 of the 10 samples (Figure 4). These data suggest that insufficient levels of compound 25 are present at the site of infection in the in vivo model and optimization of the pharmacokinetic properties of the thienopyrimidines series is likely required to demonstrate in vivo efficacy.

Figure 4.

The effect of Compound 25 against H. pylori in an ex vivo infection model. Ten mice were infected with H. pylori strain SS1. After 3 days, stomachs were harvested and bisected laterally. One-half each stomach was placed in a 12-well plate containing either Brucella-FBS medium or Brucella-FBS medium with 0.6 μg/mL (1.6μM) of compound 25. Plates were incubated for 24 hours and tissues were washed, homogenized, diluted, and plated for colony counts.

In summary, we report the synthesis and evaluation of four series of thienopyridines, through which we establish: a clear anti-H. pylori SAR at the 4, 5 and 6 position of the thienopyrimidine core; identification of potent compounds, including lead compound 26 with the IC₅₀ of 0.0068 μM; mode of action studies identifying H. pylori’s respiratory complex I as the target of the thienopyrimidines; computational analysis utilizing a NuoD homology model that supported the SAR observed in microbiological studies; and efficacy in an ex vivo H. pylori model. The SAR of the thienopyridines occurred iteratively, prior to the development of the NuoD homology model. From these data, it was found that modified side chains from compounds 1 and 2 on the C4 position of the thienopyrimidine core could be combined to generate more potent compounds. Branching at the amine α-carbon was found to be preferred over the β-carbon with the R enantiomer having greater potency at this position (13 and 26). Retention of the hydroxyl group on the amine moiety from compound 1 was required to maintain potency (14 and 31), which was shown in modeling studies to form key hydrogen bonding with resistance residue T400 in the NuoD binding site. Fluorination of the phenyl ring in series-3 was tolerated (29 and 30), which may be helpful in future development to increase the metabolic stability of these compounds. Finally, substitution of the C5 and C6 position of the thienopyrimidine ring suggested the phenyl ring at C6 is preferred. This was supported in modeling studies indicating accommodation for larger groups at C6, but steric hinderance in the binding site for a large substitution at C5.

Utilizing compound 28, resistance generation, whole-genome sequencing, and resistance transfer studies identified NADH:ubiquinone oxidoreductase (complex I) subunit NuoD as the putative target of the thienopyrimidine series. Complex I is the first protein of the electron transport chain, is located on the inner membrane of H. pylori and catalyzes the first step of oxidative phosphorylation. Although complex I is conserved through prokaryotes and eukaryotes, it uniquely important in H. pylori as the electron transport chain is the only pathway to form ATP. Previous research efforts have indicated complex I and NuoD may be a good target for development of anti-H. pylori agents. Researchers at AstraZeneca identified an anti-H. pylori benzimidazole series derived from omeprazole (Supplementary data, Figure S6), that targets complex I of the electron transport chain at the same site as the theinopyrimidines.^20,34 In these studies knockout mutagenesis studies demonstrated that nuoD is an essential gene for H. pylori but not for the majority of other bacteria, indicating that H. pylori can be targeted selectively without perturbation of the host microbiota. Targeting oxidative phosphorylation has been done safely in the clinic, as exemplified by metformin, a drug used for the treatment of diabetes³² and in the treatment of certain cancers including relapsed/refractory AML and solid tumors.³³ These examples, taken together with the studies described herein, suggest that complex I is a worthwhile target in eradicating H. pylori, which can now be exploited using structure-based drug design. In this study, the computational model supported the SAR of the thienopyridines developed from the biological studies and can be used going forward in the further development of these agents. Although in vivo efficacy could not be obtained for the compounds reported here, 26 is a promising chemical probe that is worth further pharmacological optimization to decrease cytotoxicity and improve in vivo efficacy in the H. pylori infected mouse model. Towards these goals, ongoing efforts are focused around substituting the thienopyrmidine core with more hydrophilic ring systems.

Methods

General Chemistry methods

Starting materials were purchased from Sigma-Aldrich and Enamine. Chemical reactions were monitored by the thin-layer chromatography (TLC) and the Waters ACQUITY-UPLC-MS-UV system. Microwave-assisted chemical reactions were carried out in the Biotage Initiator⁺. The reaction mixtures were purified using the Biotage Flash column chromatography system, with silica cartridges acquired from Biotage Inc. The solvents for chromatography were purchased from Sigma-Aldrich. NMR spectra were obtained using the Bruker 400 MHz NMR spectrometer and the Bruker AVANCE 500 MHz NMR spectrometer. NMR analysis was carried out using the MestReNova software. The chemical shifts and the coupling constants (J) are reported in ppm and hertz (Hz) respectively. The purity of compounds was determined to be >95% by UPLC-MS-UV system and NMR. Optical rotation of the chiral compounds was determined using JASCO P-1010 Polarimeter.

Ethyl 2-amino-5-phenylthiophene-3-carboxylate (5).

In a microwave tube, ethyl 3-cyanopropanoate 4 (1.771 mL, 16.65 mmol) was added to a solution of 2-phenylacetaldehyde 3 (2 g, 16.65 mmol), sulfur (0.587 g, 18.315 mmol) and morpholine (1.579 mL, 18.315 mmol) in ethanol (20 mL). The resulting mixture was submitted to microwave heating for 30 min at 70˚C. After cooling, the mixture was filtered, and the filtrate was poured in water. The precipitate was collected, dried and purified by flash column chromatography (Isolera 1, Biotage, 25 g size, elution gradient 12-60% ethyl acetate in hexanes) to afford 5 as a yellow solid; ¹H NMR (DMSO, 500 MHZ): δ 7.48-7.44 (m, 4H), 7.33 (t, J = 7.7 Hz, 2H), 7.23 (s, 1H), 7.19 (t, J = 7.4 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H); MS (ESI): m/z 248.5 (M⁺+H).

6-phenylthieno[2,3-d]pyrimidin-4(3H)-one (6).

Compound 5 (1.15 g, 4.65 mmol) was dissolved in excess formamide and the resulting mixture was heated for 16 h at 160˚C. The mixture was concentrated in vacuo and purified by flash column chromatography (Isolera 1, Biotage, 25 g size, elution gradient 12-60% ethyl acetate in hexanes) to afford 6 as a red solid. ¹H NMR (CDCl₃, 500 MHZ): δ 11.05 (s, 1H), 8.58 (s, 1H), 7.65-7.60 (m, 2H), 7.45 (s, 1H), 7.43-7.38 (m, 2H), 7.33-7.30 (m, 1H); MS (ESI): m/z 230.1 (M⁺+H).

4-chloro-6-phenylthieno[2,3-d]pyrimidine (7).

Compound 6 (1 g, 4.38 mmol) and N,N-dimethylaniline (0.043 mL, 0.336 mmol) were dissolved in phosphoryl trichloride (5.10 mL, 54.8 mmol) and the mixture was heated for 16 h to 95˚C. The mixture was concentrated in vacuo and purified by flash column chromatography (Isolera 1, Biotage, 25 g size, elution gradient of 12-60% ethyl acetate in hexanes) to afford 7 as a yellow solid. ¹H NMR (CDCl₃, 500 MHZ): δ 8.85 (s, 1H), 7.79-7.77 (m, 2H), 7.62 (s, 1H), 7.54-7.46 (m, 3H); MS (ESI): m/z 247.1 (M⁺+H).

General procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine.

In a microwave tube, compound 7 (1 equiv), amine (1.5 equiv) and triethylamine (3 equiv) were dissolved in ethanol and subjected to microwave heating for 1 h at 150˚C. The mixture was extracted with Ethyl Acetate/Water and the organic phase was washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by column chromatography (Isolera 1, Biotage, elution gradient of 20-85% ethyl acetate in hexanes) to afford the targeted compound.

3-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (1).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 1 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.48 (s, 1H), 7.69-7.67 (m, 1H), 7.67-7.66 (m, 1H), 7.48-7.43 (m, 2H), 7.41-7.37 (m, 1H), 7.33 (s, 1H), 5.70 (br, 1H), 3.85 (q, J = 6.5 Hz, 2H), 3.74 (t, J = 5.9 Hz, 2H), 1.93-1.87 (m, 2H); MS (ESI): m/z 286.1 (M⁺+H).

3-(methyl(6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (8).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 8 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.41 (s, 1H), 7.74 (s, 1H), 7.70-7.64 (m, 2H), 7.49-7.43 (m, 2H), 7.41-7.36 (m, 1H), 4.67 (br, 1H), 3.96 (dd, J = 6.8, 5.4 Hz, 2H), 3.61-3.60 (m, 2H), 3.51 (s, 3H), 1.96-1.90 (m, 2H); MS (ESI): m/z 301.1 (M⁺+H).

2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (9).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 9 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 8.45 (d, J = 2.7 Hz, 1H), 7.72-7.60 (m, 2H), 7.43 (ddt, J = 9.5, 7.8, 1.5 Hz, 2H), 7.40-7.30 (m, 2H), 5.70 (s, 1H), 3.93 (dt, J = 5.0, 3.3 Hz, 2H), 3.82 (ddt, J = 7.2, 5.4, 3.0 Hz, 2H); MS (ESI): m/z 273.1 (M⁺+H).

2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (10).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 10 was obtained as a white solid.¹H NMR (CDCl₃, 400 MHZ): δ 8.38 (s, 1H), 7.62-7.57 (m, 2H 1.32 (d, J = 6.8 Hz, 3H), 7.37 (ddd, J = 7.7, 6.6, 1.6 Hz, 2H), 7.31-7.25 (m, 2H), 5.24 (d, J = 6.7 Hz, 1H), 4.42-4.40 (m, 1H), 3.81 (dd, J = 10.9, 3.1 Hz, 1H),3.68-3.64 (m, 1H); MS (ESI): m/z 286.9 (M⁺+H).

1-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-2-ol (11).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 11 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.33 (s, 1H), 8.12 (s, 1H), 7.99 (t, J = 5.9 Hz, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.51 (t, J = 6.8 Hz, 2H), 7.42-7.38 (m, J = 7.5 Hz, 1H), 4.86 (d, J = 4.8 Hz, 1H), 3.94-3.89 (m, 1H), 3.54-3.38 (m, 2H), 1.13 (d, J = 6.2 Hz, 3H); MS (ESI): m/z 268.7 (M⁺+H).

(S)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (12).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 12 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.47 (s, 1H), 7.71-7.68 (m, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.41-7.39 (m, 1H), 7.35 (s, 1H), 5.31 (s, 1H), 4.51-4.49 (m, 1H), 3.91 (dd, J = 11.1, 3.02 Hz, 1H), 3.75 (dd, J = 11.1, 6.2 Hz, 1H), 1.41 (d, J = 6.9 Hz, 3H); MS (ESI): m/z 286.9 (M⁺+H).

(R)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (13).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 13 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.47 (s, 1H), 7.70-7.68 (m, 2H), 7.46 (t, J = 8.2 Hz, 2H), 7.41-7.35 (m, 2H), 5.33 (s, 1H), 4.52-4.48 (m, 1H), 3.93-3.88 (m, 1H), 3.78-3.73 (m, 1H), 1.41 (d, J = 6.4 Hz, 3H); MS (ESI): m/z 286.9 (M⁺+H).

N-(1-methoxypropan-2-yl)-6-phenylthieno[2,3-d]pyrimidin-4-amine (14).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 14 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.34 (s, 1H), 8.12 (s, 1H), 7.74-7.68 (m, 3H), 7.51 (t, 2H, J = 7.4 Hz), 7.43-7.38 (m, 1H), 4.60-4.53 (m, 1H), 3.52-3.48 (m, 1H), 3.40-3.37 (m, 1H), 3.30 (s, 3H), 1.24 (d, J = 6.7 Hz, 3H); MS (ESI): m/z 300.8 (M⁺+H).

4-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-1-ol (15).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 15 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.47 (s, 1H), 7.67-7.64 (m, 2H), 7.45-7.25 (m, 4H), 5.67 (s, 1H), 3.79 (s, 2H), 3.69 (s, 2H), 2.16 (s, 1H), 1.87-1.84 (m, 2H), 1.75-1.73 (m, 2H); MS (ESI): m/z 301.3 (M⁺+H).

2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-1-ol (16).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 16 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.45 (s, 1H), 7.70-7.67 (m, 2H), 7.48-7.43 (m, 2H), 7.39 (t, J = 7.48 Hz, 1H), 7.35 (s, 1H), 5.34 (d, J = 7.5 Hz, 1H), 4.33-4.26 (m, 1H), 3.93 (dd, J = 11.1, 3 Hz, 1H), 3.80 (dd, J = 11.1, 5.8 Hz, 1H), 1.88-1.70 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS (ESI): m/z 300.9 (M⁺+H).

1-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-2-ol (17).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 17 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.33 (s, 1H), 8.12 (s, 1H), 7.96 (t, J = 5.73 Hz, 1H), 7.70-7.67 (m, 2H), 7.51 (dd, J = 8.41, 7.01 Hz, 2H), 7.42-7.38 (m, 1H), 4.83 (d, J = 5.24 Hz, 1H), 3.70-3.62 (m, 1H), 3.61-3.55 (m, 1H), 3.44-3.37 (m, 1H), 1.58-1.48 (m, 1H), 1.42-1.31 (m, 1H), 0.93 (t, J = 7.4 Hz, 3H); MS (ESI): m/z 300.9 (M⁺+H).

3-methyl-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)butan-1-ol (18).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 18 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.35 (s, 1H), 7.62-7.59 (m, 2H), 7.37 (t, J = 7.2 Hz, 2H), 7.32-7.25 (m, 2H), 5.30 (d, J = 7.7 Hz, 1H), 4.11-4.05 (m, 1H), 3.87-3.75 (m, 2H), 3.48 (m, 1H), 2.10-2.01 (m, 1H), 1.20-0.98 (m, 6H); MS (ESI): m/z 315.01 (M⁺+H).

N-(3,4-dimethoxyphenethyl)-6-phenylthieno[2,3-d]pyrimidin-4-amine (19).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 19 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.54 (s, 1H), 7.65 (d, J = 7 Hz, 2H), 7.45 (t, J = 7.04 Hz, 2H), 7.40-7.36 (m, 1H), 7.20 (s, 1H), 6.88-6.78 (m, 3H), 5.28 (br, 1H), 3.95-3.90 (m, 5H), 3.87 (s, 3H), 2.99 (t, J = 7.04 Hz, 2H); MS (ESI): m/z 392.01 (M⁺+H).

N-(3,4-dimethylphenethyl)-6-phenylthieno[2,3-d]pyrimidin-4-amine (20).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 20 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.37 (s, 1H), 8.07 (t, J = 5.62 Hz, 1H), 8.00 (s, 1H), 7.67 (d, J = 7.52 Hz, 2H), 7.51 (t, J = 8.08 Hz, 2H), 7.40 (t, J = 7.12 Hz, 1H), 7.06-7.05 (m, 2H), 6.99-6.97 (m, 1H), 3.73-3.68 (m, 2H), 2.87 (t, J = 7.49 Hz, 2H), 2.19 (s, 3H), 2.17 (s, 3H); MS (ESI): m/z 361.21 (M⁺+H).

6-phenyl-N-(2-(pyridin-4-yl)ethyl)thieno[2,3-d]pyrimidin-4-amine (21).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 21 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.56-8.47 (m, 3H), 7.65-7.63 (m, 2H), 7.44-7.41 (m, 2H), 7.38-7.36 (m, 1H), 7.21-7.18 (m, 3H), 5.26 (s, 1H), 3.98-3.93 (m, 2H), 3.05 (t, 2H, J = 7.52 Hz); MS (ESI): m/z 333.1 (M⁺+H).

6-phenyl-N-(2-(pyridin-3-yl)ethyl)thieno[2,3-d]pyrimidin-4-amine (22).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 22 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.56-8.53 (m, 3H), 7.68-7.65 (m, J = 6.88 Hz, 2H), 7.61 (d, J = 7.91 Hz, 1H), 7.45 (dd, J = 8.45, 6.60 Hz, 2H), 7.40-7.37 (m, 1H), 7.24 (s, 1H), 5.26 (s, 1H) 3.96 (q, J = 6.73, 2H), 3.08 (t, J = 6.96 Hz, 2H); MS (ESI): m/z 333.1 (M⁺+H).

N-(2,3-dimethoxybenzyl)-6-phenylthieno[2,3-d]pyrimidin-4-amine (23).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 23 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 8.54 (s, 1H), 7.69-7.63 (m, 2H), 7.49-7.41 (m, 2H), 7.40-7.33 (m, 1H), 7.30 (d, J = 1.2 Hz, 1H), 7.11-7.00 (m, 2H), 6.94 (dd, J = 7.8, 1.8 Hz, 1H), 5.73 (s, 1H), 4.89 (d, J = 5.6 Hz, 2H), 3.97 (d, J = 0.9 Hz, 3H), 3.92 (d, J = 1.1 Hz, 3H); MS (ESI): m/z 378.02 (M⁺+H).

3-(4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)propan-1-ol (24).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 24 was obtained as a white solid. ¹H NMR (CDCl₃, 500 MHZ): δ 8.38 (s, 1H), 7.55 (d, J = 7.65 Hz, 2H), 7.36 (t, J = 7.54 Hz, 2H), 7.30 (t, J = 7.7 Hz, 1H), 7.14-7.11 (m, 3H), 6.80 (d, J = 8.2 Hz, 2H), 5.39 (d, J = 6.25 Hz, 1H), 4.45-4.39 (m, 1H), 3.82 (d, J = 10.8 Hz, 1H), 3.72-3.66 (m, 4H), 2.97-2.80 (m, 2H); MS (ESI): m/z 391.9 (M⁺+H).

2-(4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (25).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 25 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.28 (s, 1H), 8.21 (s, 1H), 8.01 (d, J = 8.08 Hz, 1H), 7.75 – 7.67 (m, 2H), 7.55 – 7.47 (m, 2H), 7.44 – 7.33 (m, 3H), 6.95 – 6.84 (m, 2H), 5.48 – 5.33 (m, 1H), 4.89 (t, J = 5.69 Hz, 1H), 3.91 – 3.63 (m, 5H); MS (ESI): m/z 378.1 (M⁺+H).

(R)-2-(4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (26).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 26 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.28 (s, 1H), 8.24 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.73-7.70 (m, 2H), 7.54-7.50 (m, J = 8.12 Hz, 2H), 7.43-7.39 (m, 1H), 7.37-7.34 (m, 2H), 6.89 (d, J = 8.6 Hz, 2H), 5.43-5.38 (m, 1H), 4.99 (t, J = 6.16 Hz, 1H), 3.79-3.69 (m, 5H); MS (ESI): m/z 378.1 (M⁺+H).

(S)-2-(4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (27).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 27 was obtained as a white solid. ¹H NMR (DMSO, 400 MHZ): δ 8.28 (s, 1H), 8.24 (s, 1H), 8.14 (d, J = 7.76 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.52 (t, J = 7.72 Hz, 2H), 7.43-7.35 (m, 3H), 6.89 (d, J = 9 Hz, 2H), 5.41 (q, J = 7.88 Hz, 1H), 4.99 (t, J = 6.04 Hz, 1H), 3.79-3.69 (m, 5H); MS (ESI): m/z 378.1 (M⁺+H).

(R)-2-phenyl-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (28).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 28 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.46 (s, 1H), 7.69 (d, J = 6.88 Hz, 2H), 7.47-7.34 (m, 9H), 5.85 (s, 1H), 5.50-5.47 (m, 1H), 4.17-4.09 (m, 2H); MS (ESI): m/z 348.9 (M⁺+H).

(R)-2-(2-fluoro-4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (29).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 29 was obtained as a yellow solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.45 (s, 1H), 7.67 (d, J = 6.16 Hz, 2H), 7.45-7.29 (m, 5H), 6.70 (s, 2H), 6.02 (s, 1H), 5.70 (s, 1H), 4.14-4.05 (m, 2H), 3.81 (s, 3H); MS (ESI): m/z 396.01 (M⁺+H).

(R)-2-(3-fluoro-4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (30).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 30 was obtained as a yellow solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.42 (s, 1H), 7.67-7.65 (m, 2H), 7.54 (s, 1H), 7.45-7.35 (m, 3H), 7.22-7.16 (m, 2H), 6.98 (t, J = 8.6 Hz, 1H), 6.31 (s, 1H), 5.45-5.41 (m, 1H), 4.14-4.06 (m, 2H), 3.90 (s, 3H); MS (ESI): m/z 397.3 (M⁺+H).

(R)-2-((5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)amino)-2-phenylethan-1-ol (37).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 37 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.35(s, 1H), 7.44-7.28 (m, 5H), 6.28 (s, 1H), 5.47-5.43 (m, 1H), 4.07 (d, J = 4.58 Hz, 2H), 2.51 (s, 3H), 2.46 (s, 3H); MS (ESI): m/z 301.00 (M⁺+H).

(R)-2-((5-methyl-6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-2-phenylethan-1-ol (38).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 38 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.48 (s, 1H), 7.48-7.36 (m, 9H), 7.11-7.07 (m, 1H), 5.89 (d, J = 6.84 Hz, 1H), 5.49 (q, J = 5.44 Hz, 1H), 4.17-4.09 (m, 2H), 1.28 (s, 3H); MS (ESI): m/z 361.9 (M⁺+H).

(R)-2-((6-methyl-5-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-2-phenylethan-1-ol (39).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 39 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.42 (s, 1H), 7.57 (t, J = 7.63 Hz, 1H), 7.45-7.41 (m, 2H), 7.27-7.21 (m, 5H), 6.90-6.88 (m, 2H), 5.33 (d, J = 5.6 Hz, 1H), 5.20-5.16 (m, 1H), 3.78-3.69 (m, 2H), 2.33 (s, 3H); MS (ESI): m/z 361.9 (M⁺+H).

(R)-2-phenyl-2-((5-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol (40).

Following the general procedure for the amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine, compound 40 was obtained as a white solid. ¹H NMR (CDCl₃, 400 MHZ): δ 8.45 (s, 1H), 7.69-7.67 (m, 2H), 7.47-7.35 (m, 9H), 5.96-5.94 (m, 1H), 5.51-5.47 (m, 1H), 4.17-4.10 (m, 2H); MS (ESI): m/z 348.1 (M⁺+H).

tert-butyl (R)-(1-(3-fluoro-4-methoxyphenyl)-2-hydroxyethyl)carbamate (33).

Di-tert-butyl dicarbonate (0.290 g, 1.329 mmol) was added to a solution of (R)-1-(3-fluoro-4-methoxyphenyl)-2-hydroxyethanaminium chloride 32 (0.267 g, 1.208 mmol) and triethylamine (0.506 mL, 3.63 mmol) in DCM (4 mL). The reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated and purified by flash column chromatography (Isolera 1, Biotage, 10 g size, elution gradient 12-80% ethyl acetate in hexanes). Pure fractions were evaporated to afford 33 as a white solid; ¹H NMR (CDCl₃, 500 MHZ): δ 7.08-7.03 (m, 2H), 6.96 (t, J = 8.33 Hz, 1H), 5.21 (d, J = 6.55 Hz, 1H), 4.73 (s, 1H), 3.90-3.81 (m, 5H), 2.11 (s, 1H), 1.46 (s, 9H); MS (ESI): m/z 284.3 (M⁺+H).

tert-butyl (R)-(2-(1,3-dioxoisoindolin-2-yl)-1-(3-fluoro-4-methoxyphenyl)ethyl)carbamate (34).

DIAD (0.282 mL, 1.458 mmol) was added dropwise at room temperature, to a solution of 33 ( 0.320 g, 1.122 mmol), phthalimide (0.215 g, 1.458 mmol) and Ph₃P (0.382 g, 1.458 mmol) in THF (3.8 mL). The resulting mixture was stirred for 16 h at room temperature. The mixture was extracted with Ethyl Acetate/Water and the organic phase was washed with brine, dried (Na₂SO₄) and evaporated in vacuo. The residue was purified by flash column chromatography (Isolera 1, Biotage, 10 g size, elution gradient 12-60% ethyl acetate in hexanes), to afford 34 as a yellow solid; ¹H NMR (CDCl₃, 500 MHZ): δ 7.80 (m, 2H), 7.66 (m, 2H), 7.04 (m, 2H), 6.87 (m, 1H), 5.28 (d, J = 8.1 Hz, 1H), 4.94-4.92 (m, 1H), 3.89-3.81 (m, 5H), 1.20 (s, 9H); MS (ESI): m/z 415.7 (M⁺+H).

(R)-2-(2-amino-2-(3-fluoro-4-methoxyphenyl)ethyl)isoindoline-1,3-dione (35).

HCl (4 M in 1,4-dioxane, 0.9 mL, 3.6 mmol) was added to a solution of 34 (0.372 g, 0.897 mmol) in a mixture of DCM (2.5 mL) and methanol (1 mL), and the resulting mixture was stirred for 16 h at room temperature. The mixture was subjected to acid-base extraction with Ethyl Acetate/Water and the organic phase was washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The resulting residue 35 (yellow oil) was used in the next step without further purification. ¹H NMR (MeOD, 500 MHZ): δ 7.88-7.78 (m, 4H), 7.20 (dd, J = 12.4, 2.20 Hz, 1H), 7.08 (dd, J = 8.65, 2.14 Hz, 1H), 7.00(t, J = 8.49 Hz, 1H), 4.25 (t, J = 7.27 Hz, 1H), 3.88-3.84 (m, 5H); MS (ESI): m/z 315.5 (M⁺+H).

(R)-2-(2-(3-fluoro-4-methoxyphenyl)-2-((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)ethyl)isoindoline-1,3-dione (36).

Compound 36 was synthesized following the general procedure for amination of 4-chloro-6-phenylthieno[2,3-d]pyrimidine; ¹H NMR (CDCl₃, 500 MHZ): δ 8.31 (s, 1H), 7.88-7.85 (m, 2H), 7.75-7.73 (m, 4H), 7.54 (s, 1H), 7.49 (t, J = 7.55, 1H), 7.42 (t, J = 7.35 Hz, 1H), 7.25-7.22 (m, 2H), 6.99-6.95 (m, 1H), 6.91 (s, 1H), 5.66-5.62 (m, 1H), 4.24-4.12 (m, 2H), 3.89 (s, 3H); MS (ESI): m/z 525.5 (M⁺+H).

(R)-1-(3-fluoro-4-methoxyphenyl)-N¹-(6-phenylthieno[2,3-d]pyrimidin-4-yl)ethane-1,2-diamine (31).

Compound 36 ( 0.203 g, 0.393 mmol) was dissolved in methanol and a 80 % hydrazine hydrate solution (0.073 mL,1.2 mmol) was added dropwise to the solution at 0˚C. The reaction mixture was stirred for 16 h at room temperature. The mixture was extracted with Ethyl Acetate/Water and the organic phase was washed with brine, dried (Na₂SO₄) and evaporated in vacuo. The residue was purified by flash column chromatography (Isolera 1, Biotage, 10 g size, elution gradient 2-20 % methanol in ethyl acetate) to afford 31 as a yellow solid.¹H NMR (DMSO, 500 MHZ): δ 8.27 (s, 1H), 8.15 (s, 1H), 7.75 (d, 2H, J = 7.6 Hz), 7.54 (t, 2H, J = 7.6 Hz), 7.44 (t, 1H, J = 7.6 Hz), 7.26 (dd, J = 12.5, 2 Hz, 1H), 7.20 (d, 1H, J = 7.6 Hz), 7.12 (t, J = 8.6 Hz, 1H), 5.28-5.25 (m, 1H), 3.80 (s, 3H), 3.00-2.91 (m, 2H), 1.25-1.18 (m, 2H); MS (ESI): m/z 396.4 (M⁺+H).

H. pylori IC₅₀ dose responses

H. pylori strains ATCC 43504, SS1, or SS1 NuoD A402P were grown overnight in brucella broth containing Skirrow’s selective medium supplement and 10% fetal bovine serum (FBS) or brain heart infusion broth containing Skirrow’s and 10% FBS. Cultures were grown in vented 25-cm² tissue culture flasks at 37°C, 10% CO₂ with rotation at 50 rpm. Two-fold serial dilutions of test and control compounds were performed in DMSO or water as appropriate and 1 uL was transferred to a 96 well plate. 99 uL of a 1:10 dilution of an overnight culture of H. pylori in Brucella/Skirrow’s/10% FBS was added to each well. Plates were incubated for 18-20 h, and 5 ul of 3mM resazurin was added to each well. Plates were incubated for an additional 3-5 h, and resazurin reduction was measured using a BioTek Synergy Mx (BioTek, Winooski, VT) with an excitation wavelength of 540nm and emission read at 590nm. Percent inhibition compared to growth controls was calculated and the 50% inhibitory concentration (IC₅₀) was determined using XLFit software (IDBS, London, United Kingdom). pIC₅₀, the negative log of the IC₅₀ value when converted to molar, was calculated based on individual IC₅₀ values along with the standard error of the mean (S.E.M.). All pIC₅₀ data with 4 or more independent replicates were analyzed for outliers using Grubbs’s test with alpha set to 0.05 (GraphPad Prism). A t-test was used to compare the replicate pIC50 values for SS1 wild-type and NuoD A402P mutant (GraphPad Prism). P values <0.05 were considered significant.

Cytotoxicity assays

FaDu (ATCC HTB-43) and HepG2 (ATCC HB-8065) cell lines were routinely maintained according to the protocols provided by the American Type Culture Collection (ATCC). For cytotoxicity IC₅₀ determination, 100uL of cell suspension in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% FBS was seeded into black sided, tissue culture treated 96 well plates (Corning 3603) at a concentration of 3 x 10⁵ cells/ml. Plates were incubated for 24 hours in a 37°C, 5% CO₂ incubator to allow for cell adhesion and growth. Spent media was aspirated from the test plates and serial two-fold dilutions of test compounds mixed with fresh EMEM + 10% FBS were added to the plates at 100 μL per well. The plates were incubated for 72 hours. The media was aspirated and FBS-free EMEM and resazurin (final concentration 0.15 mM) was added to the plate. Plates were incubated for 3-5 hours and then resazurin reduction was measured using a BioTek Synergy Mx (BioTek, Winooski, VT) with an excitation wavelength of 540nm and emission read at 590nm. Percent inhibition compared to growth controls were calculated and the 50% inhibitory concentration (IC₅₀) curves were generated using XLFit software (IDBS, London, United Kingdom). IC₅₀ values were converted to pIC₅₀ values as described above and the average and S.E.M. were calculated.

Generation of resistant mutants, sequencing, and target identification

Mutants resistant to the thienopyrimidine series were generated in two separate ways. Strain HM-274 was chosen for these studies, since it had been previously sequenced and annotated. Spontaneous mutants of HM-274 were selected by concentrating and harvesting susceptible wildtype cultures and spreading them onto plates containing twice the minimum inhibitory concentration (MIC) of 28. Two stably resistant isolates were recovered which had MICs 8-fold higher than the wildtype. In the second method, H. pylori ATCC 43504 was serially passaged onto plates containing increasingly higher concentrations of 1 until the strain was able to grow at 8X the MIC of the wildtype. The DNA from this resistant mutant was then purified and transformed into strain HM-274 to confirm stable genetically transferable resistance prior to sequencing. Genomic DNA was purified from the three resistant mutants as well as HM-274 wildtype using ZR Fungal/Bacterial DNA MiniPrep (Zymo Research) according to the manufacturer’s instructions. Whole genome sequencing was performed by Tufts Medical Center. Genome annotation was accomplished using RAST and single nucleotide polymorphism (SNP) identification was performed with CLC Workbench. The two spontaneous resistant mutants had SNPs in nuoD which caused one amino acid change, either A402P or T400I. The serial passage mutant had multiple non-synonymous mutations in nuoD. There were no other mutations shared by all three strains and not present in the isogenic background strain.

Seven additional spontaneous resistant mutants were subsequently isolated using the same methodology. Genomic DNA was purified from each and nuoD was amplified by PCR. The PCR products were sequenced and compared to wildtype nuoD. All seven had mutations in nuoD resulting in an amino acid change, either A402P or T400I.

In order to ensure that no secondary mutations were causing thienopyrimidine resistance, a wild-type copy of nuoD was modified to contain the A402P mutation using the Q5 Site Directed Mutagenesis kit (New England Biolabs) according to the manufacturer’s instructions. The mutation was confirmed by PCR and then wild-type SS1 was transformed with this plasmid and selected for on plates containing 28. The A402P mutation in nuoD conferred resistance in strain SS1. Several colonies were chosen and the mutation was confirmed by PCR and sequencing. An SS1 isolate with the NuoD A402P mutation was used in further testing to confirm on target activity of the thienopyrimidine series (see above).

ex vivo efficacy study

This model was adapted from the standard mouse model of H. pylori infection using SS1. Briefly, 6 to 8-week-old female C57BL/6 (specific-pathogen-free) mice were obtained from Charles River Laboratories. Five mice were housed per cage and allowed to acclimate for at least 72 h. Animals were inoculated by oral gavage, three times within one week with 0.2 ml of an H. pylori SS1 suspension (~10⁹ CFU/dose). The infection was allowed to stabilize for one to two weeks after the final gavage. Mice were euthanized and the stomachs were excised, bisected laterally into equal halves along the long curvature, and washed three times with sterile PBS. Each half was added to the well of a 12 well plate containing Brucella broth supplemented with 10% FBS and Skirrow’s with or without 100X the MIC of Compound 25. Each half from individual stomachs were assigned to a control and treatment group. After 24 h, the stomach halves were washed, weighed, homogenized, serially diluted, and plated on selective medium. These plates were incubated for 7 days and then colonies were counted to determine the H. pylori burden. Data was log transformed and statistical significance was determined using a Mann-Whitney test with GraphPad Prism software. A P-value less than 0.05 was considered significant. The study was repeated to ensure reproducibility. Animal use complied with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare.

Computational studies of H. pylori’s Complex I

General Computational Methods.

The sequence analysis of T. thermophilus and H. pylori was done using the online Clustal Omega tool. For residue numbering, a multiple sequence alignment was performed with M. musculus, B. taurus, H. sapiens, A. fumagatis, H. pylori, E. coli and M. tuberculosis using CLC Genomic Workbench 11. Schrödinger’s Maestro was used to build three comparative models and for docking.³⁵ AMBERMD was used to run all simulations.³⁶

Homology Modeling.

A homology model of the subunits NuoD and NuoB of NADH:ubiquinone Oxidoreductase in Helicobacter pylori was created using Schrödinger’s Phase program.³⁷ The comparative model was created using a Thermus thermophilus protein sequence as a template (PDB:4HEA). An additional homology model was built with a A402P mutation in the NuoD subunit using the same template. Both models were built using energy-based methods. The comparative models were then prepared by AMBERMD’s Leap and then minimized using steepest decent for 10,000 steps using AMBERMD’s PMEMD implementation.³⁶

Molecular Docking.

A series of thienopyrimidines and piericidin A were docked using Schrödinger’s Induced Fit docking based upon the quinone binding channel, based on previous Thermus thermophilus and mitochondrial studies of ubiquinone binding (Y85, R40, V403, and N401), mutations found from the Mills et al group (G398, F404, and V407), and from a ligand in our thienopyrimidine series (T400 and A402).^{14,18,19,23,33} All residue numbers are based on the protein sequence of H. pylori. The default protocol was used with a box size that was extended to allow docking of ligands with up 30 Å in length and sampled each ligand conformation using an energy window of 2.5 kcal/mol for ring conformation sampling and an energy penalty for nonplanar amide bond conformations.³⁸ The ligands were prepared in Schrödinger using LigPrep.³⁹ The best poses were chosen using the induced-fit docking (IFD) score (a combination of Glide Score and Prime energy calculations) and visual inspection.³⁸ The glide scores are representative ranking of ligands based upon the specific molecular docking run based on the interactions between ligand and protein. The thienopyrimidines were docked using the same methods into the T400I model and A402P model. The ligands were then compared using the IFD score.

Molecular Simulations.

AMBERMD was used for molecular dynamic simulations. The chosen poses from the molecular docking were parameterized using Leap.³⁶ The protein-ligand complex was solvated using TIP3P with a 12.0 Å octahedron periodic box and neutralized with Na. The apo protein was subjected to the same solvation and neutralization methods as the protein-ligand complex. The parameterized structures were saved and the protein-ligand complex was used for simulations using GPU accelerated PMEMD.³⁶ The ligand-protein complex was minimized using steepest decent for 10,000 steps utilizing the SHAKE algorithm. The minimized protein-ligand complex was then heated using 2 fs time steps over 50,000 steps to reach 303 K. The heated structure was equilibrated for 20 ns and then further subjected to a 250 ns production run using 2 fs timesteps. After the simulation was completed, the AMBER MM-PBSA protocol was used to calculate binding energies and ptraj was used for analysis of protein-ligand interactions.³⁶ All images were created using UCSF Chimera.⁴⁰

Supplementary Material

supplementary data

Supplementary Table S1. In vitro ADME profile of Compound 25

Supplementary Table S2. Mouse pharmacokinetic profile of Compound 25

Supplementary Table S3. MIC values of select thienopyrimidines against a panel of representative Gram-negative and Gram-positive species.

Supplementary Figure S1. A comparison of pIC50 values between SS1 and SS1 A402P mutant

Supplementary Figure S2. Multiple species sequence alignment of respiratory complex I Thienopyrimidine binding site

Supplementary Figure S3. Docked conformations of known complex I ligands and inhibitors

Supplementary Figure S4. A complete energy profile of the protein ligand complex

Supplementary Figure S5. In vivo efficacy of compound 25

Supplementary Figure S6. Structures of the optimized benzimidazole and Omeprazole.