Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine

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Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria Robert J Moreau, Colin K Skepper, Brent A. Appleton, Anke Blechschmidt, Carl J. Balibar, Bret M. Benton, Joseph E Drumm, Brian Y Feng, Mei Geng, Cindy Li, Mika K Lindvall, Andreas Lingel, Yipin Lu, Mulugeta Mamo, Wosenu Mergo, Valery Polyakov, Thomas M Smith, Kenneth Takeoka, Kyoko Uehara, Lisha Wang, Jun-Rong Wei, Andrew H. Weiss, Lili Xie, Wenjian Xu, Qiong Zhang, and Javier de Vicente J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01691 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria Robert J. Moreau,* Colin K. Skepper, Brent A. Appleton, Anke Blechschmidt, Carl J. Balibar, Bret M. Benton, Joseph E. Drumm, III, Brian Y. Feng, Mei Geng, Cindy Li, Mika K. Lindvall, Andreas Lingel, Yipin Lu, Mulugeta Mamo, Wosenu Mergo, Valery Polyakov, Thomas M. Smith, Kenneth Takeoka, Kyoko Uehara, Lisha Wang, Jun-Rong Wei, Andrew H. Weiss, Lili Xie, Wenjian Xu, Qiong Zhang, Javier de Vicente Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California 94608, United States KEYWORDS. FBS, FBDD, PPAT, phosphopantetheine, adenylyltransferase, coaD, Gramnegative, antibacterial, antibiotic.

ABSTRACT. The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria is a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria.

Utilizing a

ACS Paragon Plus Environment

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fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli ΔtolC mutant strain.

INTRODUCTION The discovery of safe and effective broad-spectrum antibiotics stands as one of the landmark scientific achievements of the 20th century. The golden age of antibiotics, beginning in the 1940s, was characterized by the development of drugs such as penicillin and vancomycin that transformed the practice of medicine, turning life-threatening infections into curable, short-lived illnesses. Antibiotics also enabled the development of many medical procedures that we now consider routine including complex surgeries, implantable devices and cancer chemotherapy. Over the past few decades, however, it has become increasingly evident that continued use of available antibiotics is threatened by the rise of resistance, a situation that has prompted the World Health Organization and others to warn of an impending “post-antibiotic era”.1 The CDC currently estimates that antibiotic-resistant infections are responsible for at least 2 million illnesses and 23,000 deaths each year in the United States, with direct societal costs as high as $20 billion.2 Of particular concern is the spread of multidrug-resistant and pandrug-resistant Gram-negative bacteria. The Gram-negative cell envelope presents a formidable barrier to entry for small-molecule drugs3 and additional, specific resistance mechanisms have evolved that dramatically decrease the efficacy of many known classes of antibiotics (e.g. β-lactams, fluoroquinolones, aminoglycosides, etc.).4 As a result, the toxic, polycationic peptide colistin


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(polymyxin E) has seen a resurgence in use and is now widely regarded as the antibiotic of last resort. Alarmingly, strains of E. coli were recently identified that harbored a transferable, plasmid-mediated mechanism of resistance to colistin (MCR-1), and the mcr-1 gene is spreading.5 Compounding the issue of antibiotic resistance is the fact that very few new drugs have been approved to treat Gram-negative infections in recent decades, and the majority of these have come from well-known classes of drugs discovered half a century ago.6 Indeed, since the introduction of carbapenems in the 1980s, no compound from a new structural class has been approved for the treatment of Gram-negative infections. This decline in antibiotic discovery is due in part to the withdrawal from the field by many large pharmaceutical companies as a result of the conjoined factors of unfavorable economics and the considerable scientific challenge posed by the impermeability of Gram-negative pathogens.7 One strategy to mitigate the risks associated with antibiotic development is to identify analogs of an existing structural class with reduced susceptibility to resistance and/or improved intrinsic activity.6 However, the inevitability of cross-resistance dictates that there is a clear, unmet need for agents that inhibit novel bacterial targets. One such target that has received attention recently is phosphopantetheine adenylyltransferase (PPAT, CoaD). PPAT is a hexameric enzyme that catalyzes the penultimate step in CoA biosynthesis. CoA is an essential cofactor found in all living organisms, functioning as a carrier for activated acyl groups in many vital metabolic processes.

For example, CoA plays a central role in lipid metabolism by providing

phosphopantetheine prosthetic groups that are post-translationally appended to acyl carrier proteins upon which fatty acids are biosynthesized. CoA also has roles in various other cellular processes, from the citric acid cycle to amino acid degradation.8 In bacteria, CoA is a critical


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cofactor in the biogenesis of membrane lipids, peptidoglycan, teichoic acids in Gram-positive organisms, and Lipid A in Gram-negative organisms.9,10 The biochemical pathway for the formation of CoA from pantothenic acid (vitamin B5) consists of five enzymatic steps that are conserved across all taxa (Figure 1). Biosynthesis begins with the phosphorylation of pantothenic acid to 4’-phosphopantothenate catalyzed by pantothenate kinase (PanK). 4’-Phosphopantothenate is subsequently condensed with cysteine by phosphopantothenoylcysteine synthetase (PPCS) to provide 4’-phosphopantothenoylcysteine, which is in turn decarboxylated by phosphopantothenoylcysteine decarboxylase (PPCDC) to form 4’-phosphopantetheine. An AMP unit is appended to 4’-phosphopantetheine by PPAT to give dephospho-CoA, the substrate for phosphorylation of the ribose 3’-hydroxyl by dephosphoCoA kinase (DPCK) to form CoA.11 O

O N H

HO

OH

PanK (CoaA)

O

O

-

N H

HO

OH

pantothenic acid (vitamin B5)

N

HS

N H

O

O

O N H

N

O P O P O OH

O

-

O

R = H, dephospho-CoA

DPCK (CoaE)

HO2C HS

OH

-

O

OH

-

N

OH

PPCDC (CoaC)

6

N1

OPO32

N H

N H

NH2

PPAT (CoaD)

O

R

O

O

4'-phosphopantothenoylcysteine

4'-phosphopantothenate

7

O

OPO32

PPCS (CoaB)

PPi

O

O

ATP + HS

-

N H

N H

OPO32 OH

4'-phosphopantetheine

R = PO32-, coenzyme A

Figure 1. Biosynthetic pathway for the conversion of pantothenic acid (vitamin B5) to CoA. PPAT (CoaD), which catalyzes the reaction between ATP and 4’-phosphopantetheine, is essential for bacterial growth and exhibits high sequence homology amongst bacterial isoforms.12 In the human CoA biosynthesis pathway, the role of CoaD is performed by a bifunctional PPAT/DPCK enzyme called CoA synthase (CoaSY), which does not share significant homology


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with bacterial PPAT, though it is similar to bacterial DPCK.13

As such, bacterial PPAT

represents an attractive target for development of a new class of antibacterial agents. This is supported by a report from AstraZeneca describing the discovery of a series of cycloalkylpyrimidine PPAT inhibitors using high-throughput screening.14 Optimization of this series provided compounds with potent inhibitory activity against Gram-positive species, but much weaker activity against Gram-negative isolates.

While small molecule inhibitors of

bacterial PPAT had been reported previously (1, Figure 2),15 the AstraZeneca compounds (2, 3) were the first to demonstrate inhibition of bacterial growth at the cellular level and in an animal infection model. However, several observed issues including toxicity, solubility, protein binding and high in vivo clearance appear to limit the future potential of this class of compounds. Below and in the accompanying manuscript, we describe our own work on the discovery and optimization of potent inhibitors of PPAT from Gram-negative bacteria using an alternative approach based on fragment-based screening. O N O N

N

NH N H

O

H N O

HN

CO2H

N H

CO2H

N

O N H H

NHFmoc

1

O N

N Cl

H

N

OH

O N H H

Cl

2

NH2

Cl Cl

3

Figure 2. Structures of known inhibitors of bacterial PPAT. RESULTS AND DISCUSSION The selection of a target enzyme for screening was guided by in silico analysis of PPAT from four Gram-negative organisms: E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii. Based on the high degree of conservation observed for residues in and around the enzyme active site, E. coli and P. aeruginosa PPAT were selected as representative enzymes for assay development. In


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the event, the greater stability of E. coli PPAT led to a larger suite of more robust biochemical and biophysical assays, including SPR, and this enzyme was chosen for assay miniaturization and compound screening. Validated inhibitors would be profiled against P. aeruginosa PPAT, and evaluated for selectivity against S. aureus PPAT and human CoaSY. Broad biochemical and cellular profiling was planned for later in the program once sufficient gains in potency had been achieved. In an effort to circumvent the well-described incompatibilities between HTS campaigns and antibacterial drug targets, we placed an emphasis on fragment-based lead discovery to escape the confines of our optimized compound library and discover smaller hits with lower lipophilicity.7,16

Primary fragment screening against E. coli PPAT was conducted using

orthogonal biochemical and biophysical techniques.

Inhibition of enzymatic activity was

evaluated in the context of a PPAT/pyrophosphatase coupled-enzyme assay that relied on indirect measurement of pyrophosphate, the stoichiometric by-product of the PPAT-catalyzed reaction between 4’-phosphopantetheine and ATP.17 During the course of the assay, inorganic pyrophosphatase converted pyrophosphate into phosphate, which was subsequently detected using a molybdate/malachite green reagent.18 This coupled-enzyme assay is amenable to both absorbance- and fluorescence-based readouts.19 Two separate biochemical FBS campaigns were conducted.

With an absorbance-based readout in 384-well format at 500 µM compound

concentration, the Novartis 4th generation core fragment library of 1408 members,20 an expanded library of their fragment analogs, and other compounds solubilized at 50 mM in DMSO-d6 were screened in the coupled-enzyme assay. From a total of 25K compounds screened, 631 hits were identified that achieved ≥30% inhibition. In a complementary screen with a fluorescence-based readout in 1536-well format at 625 µM compound concentration, the core and expanded


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fragment libraries were screened. From a total of 16K compounds, 126 hits were identified using a similar cutoff. While it was reassuring to find a high degree of overlap between screening hits (>80%) despite the different assay formats and readouts, we corrected the low hit rate in the latter screen by rescuing ~300 weakly-active hits and carrying them forward through hit validation. We also obtained 26 fragment hits by screening an additional 4K fragments at 50 µM compound concentration as part of an HTS of the Novartis compound archive. In parallel, biophysical evaluation of the core fragment library from above20 in a set of 176 8fragment mixtures was performed by screening at 200 µM in three ligand-observed 1H NMR binding experiments: T1ρ, water-ligand observed by gradient spectroscopy (WaterLOGSY), and saturation transfer difference (STD).21–23 These NMR experiments produced 201 hits, which were combined with the biochemical hits from above. A limited hit expansion to include very close analogs gave 1963 fragments that were advanced to biochemical and biophysical hit validation, where fragments that were either ≥3-fold selective for PPAT over inorganic pyrophosphatase or showed >1 °C ΔTm by DSF were advanced. These criteria provided 488 validated fragment hits that displayed low overlap between validation methods, and 125 fragments were prioritized based on structural diversity, polarity, ligand efficiency and level of hit validation.

These fragments were examined using a robust E. coli PPAT X-ray

crystallography soaking system that typically produced crystals that diffracted at ≤2 Å, and 39 high-quality X-ray co-crystal structures were obtained. A diverse set of these co-crystallized fragments is shown in Figure 3.


7


N

N

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H N

N

OH

N

N N CF3

NH N

O F

N H

4 PPAT IC50 = 273 µM PPAT DSF DTµ = 2.3 °C LE = 0.31, cLogD7.4 = 2.0 N N

5 PPAT IC50 = 230 µM PPAT DSF DTµ = 8.1 °C LE = 0.36, cLogD7.4 = 2.1


O

N

N

N

Cl

N

t-Bu

NH2

O



N

N H

N

N

CF3

9 PPAT IC50 >2500 µM PPAT DSF DTµ = 2.5 °C cLogD7.4 = 2.0

O O O S NH

t-Bu N

N

N H

NH2

NH2

O

N N H

H 2N



12 PPAT IC50 = 1076 µM PPAT DSF DTµ = N/A LE = 0.32, cLogD7.4 = -0.38

O H N

N N

NH2

O

N H

O

O F

N HN

H N

O F


O


F

N


Figure 3. Selected fragments with available E. coli X-ray co-structure and associated biochemical, crystallography and DSF data are shown. Atoms colored in green highlight their interaction with the backbone NH of M74. For ligand efficiency, LE = (1.37/HA) × pIC50, where HA is the number of non-hydrogen (heavy) atoms.


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Interestingly, all fragment hits that crystallized with E. coli PPAT were found to bind at the pantetheine site of PPAT. This was somewhat unexpected considering the well-defined adenine site in the reported24a X-ray co-crystal structures of E. coli PPAT, though binding does not appear to involve the typical N1/N6 hydrogen bond acceptor/donor interactions that are well represented by fragments in our library. Instead, adenine enjoys hydrophobic interactions with several surrounding residues, a direct hydrogen bond between N6 and the carbonyl of W124, and a water-mediated hydrogen bond between N7 and the sidechain hydroxyl of T15. Consideration of the full ATP binding site reveals several polar interactions with the triphosphate that ensure proper orientation of the α-phosphate and stabilization of the pentavalent transition state during catalysis.24 Overall, this results in a more polar, shallow and solvent-exposed binding site relative to the phosphopantetheine site, which is deeper and has a good balance of hydrophobicity and polarity. Given that we screened at 10x the Km for phosphopantetheine and only 2x the Km for ATP, it is especially surprising that we enriched so strongly for phosphopantetheine site binders. This suggests that the phosphopantetheine site is primed for binding at lower concentrations, in line with each substrate’s Km and their expected relative intracellular concentrations. However, a recent report on P. aeruginosa PPAT nicely delineated the complex regulation and binding dynamics of PPAT, and especially the ATP pocket thereof, which could lead to alternate proposals that help rationalize our observations.24b A feature common to the binding of most of our fragments to E. coli PPAT is the ability to accept a hydrogen bond from the NH of M74, which is the same residue at the center of the pantetheine site that engages the cysteamide carbonyl of the natural substrate. Detailed analysis of our fragment-bound X-ray co-crystal structures, especially those with overlapping fragments, enabled us to map the phosphopantetheine binding site of PPAT and identify “hot spots”25 and


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polar interactions to help drive our structure-based drug discovery effort. This information would prove valuable in the subsequent optimization campaign.26 To advance our lead generation efforts, we performed fragment hit prioritization according to the following desirable properties: high ligand efficiency and polarity, attractive vectors for expansion, and high-quality interactions with residues conserved across multiple bacterial species. As a result of this analysis, fragments 4–6 were chosen as starting points for medicinal chemistry. Fragment optimization efforts for compound 4 began with the installation of a methyl group at C5 (16, Figure 4) in order to gain additional interactions with V135, M105 and L131 in a small, hydrophobic site in the binding pocket into which several other ligands were observed to project a small, lipophilic group (Figure 5A). To our delight, this modification yielded a 15-fold improvement in biochemical activity.27 Incorporation of the halogen found in closely-related fragment 7 produced m-chlorobenzyl analog 17, which possessed single-digit micromolar inhibitory activity.

Unfortunately, attempts to grow this fragment at the 2-position of the

azabenzimidazole for further optimization proved challenging due to the substituent at N1, so we deprioritized this fragment and focused instead on exploring a more efficient inhibitor, 4hydroxybenzimidazole 5, which possessed a slightly different binding mode from that of the azabenzimidazoles. 5

N

N

N

N

N

N

2

N

N

N Cl

4 PPAT IC50 = 273 µM LE = 0.31, cLogD7.4 = 2.0


17 PPAT IC50 = 4.3 µM LE = 0.41, cLogD7.4 = 3.1

Figure 4. Fragment hit optimization of E. coli PPAT inhibitors azabenzimidazoles 4, 16–17.


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A

B Figure 5. Fragment-bound X-ray crystal structures of E. coli PPAT. (A) Azabenzimidazole 4. (B) Hydroxybenzimidazole 5. Protein carbon atoms are shown in gray, ligand carbon atoms in orange, nitrogen in blue, oxygen in red, and fluorine in green. Water molecules are shown as red spheres, hydrogen bonds as cyan dotted lines. In contrast to the binding mode of fragment 4, where the azabenzimidazole core accepted hydrogen bonds from both M74 and a conserved water molecule (Figure 5A), the hydroxyl group of fragment 5 displaced the conserved water to form direct interactions with the sidechain carbonyl of N106 and the NH of A75 (Figure 5B). This resulted in deeper binding of the hydroxybenzimidazole relative to the azabenzimidazole and slightly altered the trajectory of the


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lone pair at N3 without disrupting its ability to accept a hydrogen bond from M74; however, the substituent vectors away from this pivot point were significantly affected. Despite the successful improvement in biochemical activity observed following the installation of a methyl group at C5 of azabenzimidazole 4, the deeper binding and altered vector at C5 of hydroxybenzimidazole 5 suggested that the addition of a methyl group would be unfavorable due to a clash with the N106 sidechain, with which the hydroxyl made a key hydrogen bonding interaction, as well as the sidechain of L109. We turned our attention to the opposite end of the molecule, where the now repositioned and unsubstituted N1 of fragment 5 participated in a hydrogen bond between the benzimidazole NH and the sidechain of E134. This glutamate sidechain extends from the short α-helix that forms the upper limit of the pantetheine pocket and it engages the cysteamide NH of the natural substrate.

To maintain this interaction and explore the new vector at C2, we

conducted a survey of our compound archive for analogs differentiated at the 2-position of the benzimidazole.

We were fortunate to discover hydroxymethyl analog 18, which displayed

improved activity and efficiency (Table 1). An X-ray co-crystal structure with E. coli PPAT confirmed the previously observed network of hydrogen bonds, and an overlay with azabenzimidazole 17 (not shown) suggested that we could use the hydroxyl as a handle to similarly position a phenyl group and improve the potency. This was indeed the case – mchlorophenyl ether 19 was 70-fold more potent than 18, and further gains were realized when we replaced the ether oxygen with a methylene (20). Gratifyingly, compound 20 demonstrated cellular activity against Gram-negative bacteria, albeit with an MIC of 32 µg/mL against the efflux-deficient E. coli ΔtolC mutant strain.

While our efforts to reinforce a favorable

arrangement of the two aromatic groups through the installation of branching methyl groups were only modestly successful, cyclic linkers proved to be appropriate biasing elements that also


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better filled the surrounding pocket.

One such compound, racemic trans-cyclopentane 21,

displayed a several-fold improvement in biochemical activity and cellular potency with an MIC of 8 µg/mL against E. coli ΔtolC. From here, we investigated several cyclopentane replacements to correct our tendency toward more lipophilic analogs and to explore new interactions with the enzyme. Exchange of the distal stereogenic methine of 21 for nitrogen gave racemic pyrrolidine 22, which was clearly less active. Lactams 23 and 24 were efficient inhibitors (LipE two units more favorable than the corresponding cyclopentane) that showed promising biochemical activity, but these analogs did not address the large shift in potency between E. coli and P. aeruginosa PPAT that was generally observed for this scaffold.

Further, our reliance on

structural information from E. coli PPAT caused us to produce compounds with improved potency against E. coli that tended to lose P. aeruginosa activity. The two enzymes are 76% identical within 8 Å of the binding pocket with 58% overall sequence identity, and the divergence in activity between E. coli and P. aeruginosa was rationalized by the presence of several residue differences in the vicinity of the inhibitor, namely M74L, V135I, and H138L (E. coli numbering used throughout this discussion). The alterations at positions 74 and 138 may be responsible for the shift in potency through steric clashing with the appended arene and the linker chain of the inhibitor, respectively. By removing the linker substituents and modifying the nature of the aromatic ring or removing it completely, we identified compounds with more balanced (benzisoxazole 25) or completely inverted (benzimidazole 26 and acetamide 27) selectivity. More detailed studies were performed early in the program in an effort to better understand the binding of peptidic inhibitor 1 to E. coli PPAT and rationalize its high specificity for E. coli PPAT over P. aeruginosa PPAT.28 Here, seven reciprocal residue swaps between E. coli and P. aeruginosa PPAT identified that the residue at position 74 was one of three residues


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most heavily involved in determining susceptibility to 1 – the single M74L point mutation resulted in a 25-fold loss in potency against E. coli PPAT.

Interestingly, single residue

alterations at positions 135 and 138 in E. coli PPAT conferred greater susceptibility to 1. A complimentary effort to target interactions with conserved, polar residues in the nearby phosphate binding region of the active site led to the preparation of m-carboxylate 28, which was equipotent to m-chloro analog 21 against E. coli PPAT but suffered a 16-fold loss in P. aeruginosa activity (Table 1).

The E. coli X-ray co-crystal structure confirmed that an

interaction with the R88 guanidine was achieved and encouraged further exploration (Figure 6). The phosphate binding region of the enzyme accommodates the terminal phosphate group of the natural substrate, 4’-phosphopantetheine, as well as the phosphate ester linkage of the enzymatic reaction

product,

dephospho-CoA.

Pantetheine

substrate

analogs

such

as

N-

pentylpantothenamide have been reported as CoA pathway inhibitors with the ability to inhibit the growth of certain bacterial strains.29 It has been proposed that N-pentylpantothenamide does not inhibit the CoA biosynthetic enzymes, but instead acts as an alternative substrate, forming the CoA analog ethyldethia-CoA, which may be responsible for the observed antibacterial activity by inhibition of the CoA- and acetyl-CoA-utilizing enzymes.30 We confirmed that Npentylpantothenamide lacked activity against E. coli PPAT up to 500 µM but displayed cellular potency with MICs of 16 µg/mL against E. coli ΔtolC and 64 µg/mL against wild-type E. coli. In an effort to explore a similar potential substrate analog bearing our fragment core, we synthesized hydroxybenzimidazole-modified pantetheine 29, which showed modest on-target potency. The corresponding X-ray co-crystal structure with E. coli PPAT confirmed excellent overlap with dephospho-CoA, including an interaction between the terminal hydroxyl and the R88 guanidine (Figure 7). In contrast to N-pentylpantothenamide, compound 29 did not possess


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any cellular activity. Clearly, 29 is too weak of an inhibitor to achieve cellular potency by direct inhibition of PPAT, and it is likely not accepted by the biosynthetic enzymes along the CoA pathway that would convert it to a toxic CoA analog or phosphorylated intermediate. Ultimately, the challenges of achieving broad spectrum activity and the relatively high lipophilicity of many compounds in this series prompted us to focus on an alternative series. Table 1. Biochemical data of 4-hydroxybenzimidazole compounds 5, 18–29. OH N R1 N H

Compound

R1

5

CF3

18

E. coli PPAT IC50 (µM)

P. aeruginosa PPAT IC50 (µM)

LEa

cLogD7.4

230

>500

0.36

2.1

116

108

0.45

0.37

1.7

14

0.42

3.5

0.25

4.6

0.48

4.1

0.051

1.6

0.45

5.0

0.91

34

0.38

3.7

OH

19 O Cl

20 Cl H

21

H Cl

22 N

Cl


15


23

O

H

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1.0

11

0.36

1.5

0.32

25

0.37

1.9

1.0

2.7

0.41

2.7

23

0.13

0.30

2.2

106

1.9

0.30

0.69

0.051

25

0.42

0.76

30

114

0.28

0.21

N H

24

O N

25 N O

26 N N

27

H N O

H

28

H O HO

29 HN O HO

OH

a = LE for E. coli PPAT data.


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Figure 6. X-ray crystal structure of E. coli PPAT with hydroxybenzimidazole analog 28.

Figure 7. Overlapping X-ray crystal structures of compound 29 (orange) and dephosphocoenzyme A (light purple) bound to E. coli PPAT. Phosphorus atoms are shown in mustard. In parallel with the hydroxybenzimidazole efforts, fragment 6 was selected for further optimization. An X-ray co-structure of closely-related fragment 30 (Figure 8) revealed that this triazolopyrimidinone bound in a manner similar to azabenzimidazole 4. In addition to the


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common interactions with the conserved water molecule and M74, the exocyclic NH of 30 provided a new hydrogen bond with the backbone carbonyl of D72. Returning to fragment 6, we hypothesized that the 6-methyl substituent of the bicyclic system and o-fluorine of the benzylamine were not significantly contributing to the activity of this fragment; removal of both of these groups while maintaining the key 5-methyl substituent on the triazolopyrimidinone led to compound 31 and only a 2.5-fold drop in potency (Figure 9). As before, installation of a chlorine atom at the meta-position of the phenyl ring (32) provided a 10-fold boost in activity. Based on our observations from other fragments, substitution of the benzylic position of the benzylamine was expected to be tolerated; however, the 30-fold improvement observed for Rmethyl analog 33 exceeded our expectations. We believe the stereogenic methyl serves to facilitate de-solvation of the adjacent NH, provide lipophilic interactions, and favor the bound conformation.

The latter point was supported by a Cambridge Structural Database (CSD)

analysis of the dihedral angle of similar molecules, which showed enrichment of the 32° torsion angle that is preferred for the binding of 33 (Figure 10).31 Further confirmation was obtained by synthesis – the S-methyl analog of 31 was a weaker inhibitor of E. coli PPAT (IC50 = 120 μM) than the parent methylene compound, while the R-methyl analog again led to improved activity (IC50 = 6.4 μM) (structures not shown).

H N

N N

NH N

O

Br



18

Page 19 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Inhibition data and X-ray crystal structure of compound 30 bound to E. coli PPAT. H N

H N

N N

6

N

NH N

O

H N

N NH

NH N

O

Cl


N

N

O

N


Cl


Figure 9. Fragment hit optimization of E. coli PPAT for triazolopyrimidinones 6, 31–33.

Figure 10. CSD torsion angle population distribution for substituted α-methyl benzylamines. With an optimized benzylamine moiety in place, we explored modifications to the core heterocycle. The binding pocket surrounding the C5 methyl group was found to be restrictive, as exemplified by the several-fold loss in potency for 34 and 35 (Table 2).

Interestingly,

triazolopyrimidine 36 was almost 20-fold less potent than 33, even though 36 would be expected to enjoy similar interactions with the enzyme. It is perhaps misleading to structurally represent the parent inhibitors as triazolopyrimidinones when, in fact, they engage PPAT in the tautomeric 7-hydroxytriazolopyrimidine form (or its ionized state) to satisfy the hydrogen bonding network. In this form, N4 accepts a hydrogen bond from the nearby structural water, which is anchored by interactions with the sidechain carbonyl of N106 and the NH of A75. As such, the hydrogen


19


Page 20 of 52

bond acceptor strength of both N3 and N4 is expected to strongly influence overall binding affinity. Calculation of the electrostatic potential (ESP) was performed on a set of prospective core analogs, which were then ranked in order of increasingly negative electrostatic potential at the N3 and N4 surface to evaluate potential hydrogen bonding interactions with the enzyme (Figure 11).32 From this set, triazolopyrimidine 36 was calculated to possess the least negative electrostatic potential at the N3 and N4 surface, and so it was predicted to form weaker interactions with PPAT than parent compound 33. This was borne out in the relatively weak biochemical potency observed for 36. Our hypothesis was further validated when we found that the introduction of an amine at C7 (37) recaptured most of the lost potency, which could be again ablated by the incorporation of another ring nitrogen to provide the more electron-deficient triazolotriazine 38. Among azolopyridines 39–41, the calculated electrostatic potential at the N3 and N4 surface increased along the series O < S < N, and 4-azabenzoxazole 39, 4azabenzothiazole 40 and 4-azabenzimidazole 41 were found to be progressively more active against PPAT. From our limited analysis set, 41 was calculated to possess the most negative electrostatic potential at N3 and N4 surface, and it also proved to be the most potent inhibitor. One could reasonably assume that an amine at C7 would further enhance the electron density at the N3 and N4 surface for these analogs; the results of our efforts along these lines can be found in the accompanying manuscript on our lead optimization campaign.26 Beyond the triazolopyrimidinone-inspired, evolutionary expansion of the core SAR, we also evaluated several distinct core modifications.

For example, installation of the core from

fragment 5 provided 4-hydroxybenzimidazole 42, which was 5–6-fold less active against E. coli PPAT than both 33 and 41. Compound 42 also did not compare favorably to the most potent hydroxybenzimidazoles (see Table 1), nor did it resolve the large shift in potency between E. coli


20

Page 21 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and P. aeruginosa PPAT observed for many compounds from that series. Compared to the parent hydroxybenzimidazole analogs, the additional hydrogen bond between the exocyclic NH of 42 and the backbone carbonyl of D72 appears to result in a slight rotation of the inhibitor in the binding pocket that may weaken the other interactions with the enzyme and lead to lower potency. We also found that bolder core alterations were not well tolerated. This included analogs such as pyridopyrazine 43 and “reverse” azabenzimidazole 44, both of which were essentially inactive. These data support a concept that may be either underappreciated or masked during multi-parameter optimization of cellularly-active inhibitors: stronger enzyme inhibition can be achieved when the shape and the charge distribution of the protein is better matched by the shape and the charge distribution of the ligand, presumably as a result of tighter binding.33 Table 2. Biochemical data of triazolopyrimidinones and heterocyclic core replacements 33–44. R2 NH

Cl

Compound 33

R2 H N

N N



0.25

0.97

0.90

2.8

1.1

1.7

N

O

H N

34

N N

N

O

35

H N

N N

N

O


21


N

36

N N

2.6

0.41

0.38

5.9

4.6

2.4

4.9

0.41

2.3

0.19

0.61

1.2

15

348

271

401

>500

N

N

37

4.7

Page 22 of 52

N N

N

NH2

N

38 N

N N

N

NH2

N

39

N O

N

40

N S

N

41

N N H

42

OH N N H

N

43

N N

N

44

N HN


22

Page 23 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. Electrostatic potential (ESP) maps of bicyclic core replacements. The color codes of the ESP maps represent more negative (red) to more positive (violet) potentials from –50 to +50 kcal/mol. For each compound, the minimum atomic ESP value (in kcal/mol) on the molecular surface for N3 and N4 were labelled as a reference. From the preceding fragment hit optimization, compounds 33 and 41 emerged as promising leads. We selected triazolopyrimidinone 33 for further optimization primarily due to its greater polarity (LogD7.4 1.1 vs. 3.3), which we anticipated would facilitate the discovery of advanced inhibitors with the physicochemical properties expected to be required for wild-type Gramnegative permeability.34 If this hypothesis turned out to be flawed, there would likely be some SAR learnings that could then be applied to the 4-azabenzimidazole scaffold. The X-ray cocrystal structure between 33 and E. coli PPAT suggested that there was an opportunity to explore additional polar interactions with amino acid residues surrounding the stereogenic methyl. Branching off of this methyl group with a number of polar substituents provided a series of optically pure analogs (45–48) that were well tolerated; in the racemic series, both β-amino acid 49 and amide 50 showed an improvement in biochemical potency relative to the parent racemate, rac-33 (Table 3). In the X-ray co-crystal structures of 46 and 49, a hydrogen bond was observed between the newly installed polar sidechains and the NH of S39 (not shown). Similar to the alcohol vs. ether matched pair (46 vs. 47), terminal methylation of acid 49 and amide 50 led to a >10-fold loss in potency (51–52), perhaps due to size constraints in the vicinity of S39. Fortunately, a breakthrough was identified in the form of nitrile analog 53, which engaged in the desired interaction with S39 without increasing the hydrogen bond donor count for the inhibitor (Figure 12). Not only did 53 display strong, equipotent activity against both E. coli and P. aeruginosa PPAT with excellent selectivity against S. aureus PPAT (IC50 >500 μM), but it was


23


Page 24 of 52

the first analog in the triazolopyrimidinone series to demonstrate cellular activity with an MIC of 16 µg/mL against E. coli ΔtolC (Figure 13).

In addition, 53 also exhibited significantly

improved solubility relative to descyano analog 33 (>300 μg/mL vs. 68 μg/mL). To test our earlier hypothesis regarding the relative merits of the more polar triazolopyrimidinone core and the potential for translation of new SAR discoveries to other scaffolds, we synthesized a 4azabenzimidazole analog bearing the cyanomethyl benzylamine (54).

Consistent with the

observations from Table 2, the E. coli and P. aeruginosa PPAT biochemical potency for lead compounds 53 and 54 were comparable (54 was not tested against S. aureus PPAT); however, 54 showed improved cellular activity with an MIC of 4 µg/mL against E. coli ΔtolC. Neither compound was active against wild-type E. coli. Table 3. Biochemical data of triazolopyrimidinones 45–53. H N

N N

R1 N

O

Compound 33

R1



0.25

0.97

0.55

0.66

OH

0.24

2.0

O

2.4

11

0.25

3.9

H N Ar

45

H N

OH Ar

46

H N Ar

47

H N Ar

48

O

H N

O

NH2

Ar


24

Page 25 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


rac-33

H N

0.76

1.4

OH

0.20

1.1

NH2

0.45

0.83

O

2.5

0.49

H N

15

10

0.037

0.049

Ar

49

H N Ar

50

H N Ar

51

O

H N Ar

53

O

H N Ar

52

O

O

H N Ar

N

Ar = m-chlorophenyl.

Figure 12. X-ray crystal structure of compound 53 bound to E. coli PPAT.


25


H N

N N

N

H N

N

N N H

N

Page 26 of 52

H N N

O Cl

53 PPAT (Ec) IC50 = 0.037 µM PPAT (Pa) IC50 = 0.049 µM LE = 0.44, LipE = 6.89, LogD7.4 = 1.2 E. coli DtolC MIC = 16 µg/µL

Cl

54 PPAT (Ec) IC50 = 0.056 µM PPAT (Pa) IC50 = 0.064 µM LE = 0.45, LipE = 3.88, LogD7.4 = 3.3 E. coli DtolC MIC = 4 µg/µL

Figure 13. Comparison of lead compounds 53 and 54, including data for E. coli (Ec) and P. aeruginosa (Pa) PPAT, as well as lipophilic efficiency (LipE = pIC50 – cLogP). The ability of the triazolopyrimidinones to interact with the pantetheine site of E. coli PPAT and inhibit enzymatic activity prompted us to briefly explore the mechanism of inhibition. We expected the inhibitory activity of these compounds to be sensitive to an increase in the concentration of 4’-phosphopantetheine employed in the biochemical assay, and this was indeed the case. With a 10-fold increase in 4’-phosphopantetheine, a shift in potency was observed for all 15 triazolopyrimidinone analogs that were evaluated; the data for compounds disclosed here are shown in Table 4. While not a rigorous kinetic analysis of enzyme inhibition, these data support the characterization of the triazolopyrimidinones as inhibitors competitive with 4’phosphopantetheine. Table 4. E. coli PPAT biochemical data with 1x and 10x 4’-phosphopantetheine. Compound


E. coli 10x PhP PPAT IC50 (µM)

Fold shift

32

7.6

43

5.7

33

0.25

0.92

3.7

rac-33

0.76

3.2

4.2


26

Page 27 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


46

0.24

0.44

1.8

a PhP = 4’-phosphopantetheine As mentioned in the introduction, bacterial PPAT shares no significant sequence similarity to the PPAT domain of human CoaSY. Nonetheless, we developed a complimentary biochemical assay capable of measuring the PPAT activity of CoaSY to assess the selectivity of our inhibitors.

Of the hit-to-lead compounds presented here, only hydroxybenzimidazole 42

displayed measureable inhibition of the human enzyme (IC50 = 11 μM), though it was almost 9fold selective for the E. coli enzyme. All other analogs were either >200-fold selective or inactive over the compound concentration range tested in our assay; advanced inhibitor 53 did not show inhibition up to 500 μM.35 Although we lacked a direct biophysical measure of compound binding to CoaSY, the strong correlation observed between the E. coli biochemical IC50 and the SPR KD across a potency range of 0.00934-1.54 μM (R2 = 0.878, n = 64) suggested that, by extension, any binding to CoaSY should be weak. The SPR data for compounds disclosed here are shown below (Table 5).

These data provided confidence that further

optimization would lead to analogs capable of achieving selective bacterial growth inhibition due to specific activity on bacterial PPAT. Table 5. E. coli PPAT SPR and biochemical data. Compound

ka (x 105 M-1sec-1)

kd (s-1)

KD (µM)


21

1.96

0.0071

0.038

0.051

33

0.16

0.0028

0.17

0.25

53

23

0.021

0.0090

0.037

54

13

0.013

0.010

0.056


27


Page 28 of 52

CHEMISTRY Synthesis of the N1-substituted azabenzimidazole analogs 16 and 17 began with nitropyridine 55 (Scheme 1). Displacement of fluoride with ammonia, followed by N-formylation and nitro reduction36 provided 56, which underwent tandem reductive amination/cyclization with benzaldehyde or 3-chlorobenzaldehyde to furnish 16 and 17, respectively. Scheme 1. Synthesis of azabenzimidazoles 16 and 17. N N

F

a-c

H N

N

O NH2

NO2

55

H

N N

d R3

56 16 R = H 17 R = Cl

(a) ammonium hydroxide, MW, 130 °C, 30 min, 93%. (b) Ac2O, HCOOH, 60 °C, overnight, 100%. (c) H2, Raney-Nickel, THF, MeOH, rt, overnight, 86%. (d) benzaldehyde or 3chlorobenzaldehyde, BH3·Py, 2 h; 16: 28% and 17: 34%. Compounds 19–27 from the 4-hydroxybenzimidazole series were uniformly prepared by condensation of 2,3-diaminophenol 57 with the requisite carboxylic acid (Scheme 2). The carboxylic acid required for a synthesis of 21 was prepared as shown in Scheme 3. Enolization and triflate formation from β-keto ester 58 gave 59, which underwent smooth Suzuki-Miyaura coupling with 3-chlorophenylboronic acid to give 60. Hydrogenation of the tetrasubstituted olefin was achieved with Adams’ catalyst to give cis-1,2-disubstituted cyclopentane 61, along with some reduction of the chloro group. cis-Cyclopentane 61 was then equilibrated under basic conditions to trans-cyclopentane 62, which was directly condensed with 57 to give 21. Scheme 2. Synthesis of 4-hydroxybenzimidazole analogs 19–20 and 22–27.


28

Page 29 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O OH

OH

R1

HO

NH2

N R1

neat, 150 °C

NH2

N H

19 20, 22-27

57 O

O

O O

HO

O

HO

HO

H

HO

O

N

Cl

N H

Cl

Cl

19 overnight, 3%

20 1 h, 35%

22 overnight, 7%

O

O

23 overnight, 13%

O O

HO

HO

HO

N N

N

O

N

O N

O

24 overnight, 7%

H

HO

26 130 °C, 18 h, 3%

25 130 °C, 18 h, 5%

27 130 °C, 18 h, 14%

Scheme 3. Synthesis of trans-cyclopentane 21.

O

a

H3CO2C

OTf H3CO2C

Cl

c

b H3CO2C

59

58

60 OH R3

R3

d H3CO2C

H

H

N

H

e H3CO2C

N H

H

H

H

Cl

61 R3 = Cl, R3 = H (3:1)

62 R3 = Cl, R3 = H (3:1)

21

(a) NaH, Tf2O, Et2O, 0 °C, 1 h, 97% (b) Pd(OAc)2, PPh3, Na2CO3, EtOH-benzene (1:3), 140 °C, 30 min, MW, 86% (c) PtO2, H2 (1 atm), EtOH, 1.5 h, 72% (d) NaOMe, MeOH, reflux, 1 h. (e) 57, neat, 150 °C, overnight, 18%. Compound 28 was prepared in a similar manner, with vinyl triflate 59 undergoing SuzukiMiyaura coupling with 3-trimethylsilylphenlyboronic acid to give 63.

Hydrogenation with

Adams’ catalyst yielded cis-cyclopentane 64, which after iodo-desilylation with ICl provided


29


Page 30 of 52

65.37 Equilibration to trans-cyclopentane 66 preceded direct condensation with 57 to give 67. Finally, palladium-catalyzed carbonylation followed by hydrolysis of the resulting ester gave carboxylate 28. Scheme 4. Synthesis of trans-cyclopentane 28. TMS

OTf H3CO2C

I

TMS

a

c

b H3CO2C

H3CO2C

H3CO2C

H

H

H

H

59 64

63

65

OH

OH

I

H

N

N

e

d H3CO2C

H

f, g N H

H

N H

H

H I

66

H

HO2C

28

67

(a) Pd(OAc)2, PPh3, Na2CO3, EtOH-benzene (1:3), 140 °C, 30 min, MW, quant. (b) PtO2, H2 (1 atm), EtOAc-MeOH, rt, overnight, quant. (c) ICl, 1 h, 0 °C (d) NaOMe, MeOH, reflux, 1 h (e) 57, neat, 150 °C, overnight, 82% (f) Mo(CO)6, Pd(OAc)2, DPPF, i-Pr2NEt, dioxane-DMF, 160 °C, 30 min, 79% (g) NaOH, THF-H2O, rt, 1 h, 52%. The pantetheine substrate analog 29 was synthesized as shown in Scheme 5. Coupling of benzyl-protected 2,3-diaminophenol38 68 with N-Cbz-β-alanine, followed by cyclization to the benzimidazole and hydrogenolysis of the O-benzyl and N-Cbz groups gave amine 70. Nucleophilic ring-opening of D-(−)-pantolactone (71) with 70 gave the target analog 29 directly. Scheme 5. Synthesis of pantetheine substrate analog 29. OH

NH2

a, b, c

O

+ HO

NH2

68

N

OH

OBn

NHCbz

69

O

N N H

70

O

+ H2N

d

N H

HN

OH

O

71 HO

OH

29


30

Page 31 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a) HATU, Et3N, DCM, rt, 18 h. (b) 1,1,1,3,3,3-hexafluoropropan-2-ol, 100 °C, MW, 10 min. (c) Pd/C, H2 (1 atm), MeOH, rt, overnight. (d) Et3N, MeOH, 120 °C, MW, 40 min, 3%. Synthesis of compounds 33–38 is shown in Scheme 6. Sequential treatment of commercially available amine 72 with diphenyl cyanocarbonimidate and hydrazine gave key intermediate triazole 73. Triazolopyrimidinones 33–35 were accessed by heating 73 in acetic acid in the presence of the requisite β-keto ester. Further diversification of 73 to triazolopyrimidines 36 and 37 was achieved by treatment with 4,4-dimethoxybutan-2-one and freshly-prepared NaOEt in EtOH at room temperature or by heating under forcing conditions with 3-oxobutanenitrile in the presence of a catalytic amount of p-TsOH, respectively.39,40 A combination of 73 and ethyl N-cyanoacetimidate in hot DME provided triazolotriazine 38. Scheme 6. Synthesis of triazole-derived core systems 33–38. H 2N

H 2N

R4

N

a

N

NH N H

e

33 R4 = Me, R5 = H 34 R4 = Et, R5 = H 35 R4 = Me, R5 = Me

c d

N

N

N

NH N

NH2

N

N N

NH N

NH2

38

Cl

NH N

Cl

73

N

N

R5 O

72

N

N

b

Cl

Cl

H N

N N

NH N

Cl

37

Cl

36

(a) i) Diphenyl N-cyanocarbonimidate, 2-propanol, 60 °C, 2 h; ii) N2H4·H2O, rt, 2 days. (b) R4COCH(R5)CO2Et, AcOH, 165 °C, MW, 10–30 min; 33: 15%, 34: 6%, 35: 16%. (c) 4,4dimethoxybutan-2-one, NaOEt, EtOH, rt, 24 h, 32%. (d) 3-oxobutanenitrile, p-TsOH·H2O, mesitylene, 200 °C, MW, 30 min, 16%. (e) ethyl N-cyanoacetimidate, DME-NMP (4:1), 200 °C, 30 min, 10%. The series of azolopyridines 39-41 was quickly accessed from three distinct 2-amino-6methylpyridine building blocks (Scheme 7).

Beginning with 3-hydroxypyridine analog 74,


31


Page 32 of 52

azabenzoxazole 39 was obtained in three steps: formation and alkylation of the intermediate 5methyloxazolopyridine-2-thiol with potassium ethyl xanthogenate and ethyl iodide, respectively, followed by SNAr reaction with 72. A two-step protocol was used to obtain azabenzothiazole 40 from bromide 76. Here, sequential treatment of 76 with thiocarbonyldiimidazole and amine 72 gave

thiourea

77,

which

underwent

palladium-catalyzed

cyclization

to

give

40.

Azabenzimidazole derivative 41 was prepared from 2-chloro-5-methyl-4-azabenzimidazole (79) (available in three steps from 78)41 by direct SNAr with 72. For hydroxybenzimidazole 42, amine 72 was converted to isothiocyanate 80 and then joined with 57 to provide a mixture of intermediate thioureas.42 Without isolation, this mixture was treated with EDC and heated to induce cyclization to the desired hydroxybenzimidazole 42. Scheme 7. Synthesis of azolopyridines 39–41 and hydroxybenzimidazole 42.


32

Page 33 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N N

NH2

N

a

N NH

b

N

O

S O

OH

Cl

75

74

39

N

N

NH2

H N

N

H N

c

N NH

d Br

Br

77

76

S

S Cl

Cl

40 N N

NH2

N

e, f, g

N

NH

h

Cl

N H

N H

NO2

78

N

Cl

79 41 OH

H 2N

SCN

i

Cl

N NH2

+

80

j

NH N H

NH2

Cl

72

OH

57

Cl

42

(a) i) EtOCS2K, EtOH, 90 °C, 3 h; ii) EtI, K2CO3, DMF, rt, 5 min. (b) 72, MW, 200 °C, 30 min, 62%. (c) i) 1,1'-thiocarbonyldiimidazole, THF, rt, 3 h; ii) 72, overnight, rt. (d) Pd2dba3, DPPF, Cs2CO3, 1,4-dioxane, 85 °C, overnight, 18%. (e) H2 (1 atm), Pd/C, MeOH, 4 h, 100%; (f) CDI, THF, rt, overnight, 92%. (g) POCl3, 95 °C, overnight. (h) 72, EtOH, 150 °C, 20 min. (i) iPr2NEt, SCCl2, CH2Cl2, 0 °C → rt, 20 h, 80%. (j) i) 130 °C, MW, 10 min; ii) EDC, 100 °C, MW, 10 min, 5%. The final two core analogs, pyridopyrazine 43 and azabenzimidazole 44, were prepared in a straightforward manner beginning with an SNAr reaction between chloropyridine 81 and amine 72, followed by reduction of the nitro group in the same pot to provide common intermediate 82. Condensation of 82 with glyoxal or trimethylorthoformate gave 43 and 44, respectively. Scheme 8. Synthesis of pyridopyrazine 43 and azabenzimidazole 44.


33


Page 34 of 52

N

H N

N

N

H 2N

N

H 2N Cl

a

H 2N

H N

N

b

Cl

43

+ H 2N

O 2N

81

Cl

c Cl

72

82

N

H N

N

N H Cl

44

(a) i) EtOH, 100 °C, 3 h; ii) Zn, aq. NH4Cl, THF, rt, 20 min. (b) glyoxal, THF, rt, 1 h, 48%. (c) HC(OCH3)3, 100 °C, 18 h, 18%. In a manner consistent with that described in Scheme 6, triazolopyrimidinones 45–52 were synthesized from commercially available benzylamines 83, 84 and 85 (Scheme 9). This gave triazolopyrimidinones 45, 46 and 86 directly, and these compounds were then transformed into the remaining analogs. Alcohol 45 was carbamylated with potassium cyanate in the presence of TFA to provide primary carbamate 48, while alcohol 46 was converted to methyl ether 47 by displacement of the intermediate mesylate with sodium methoxide. Ethyl ester 86 provided a convenient precursor for the synthesis of acid 49 by hydrolysis with LiOH; amides 50 and 52 by direct amide formation with ammonia or methylamine, respectively; and methyl ester 51 by transesterification during the amide formation reactions. Cyanomethyl analogs 53 and 54 were synthesized from 87 by either triazole formation and cyclization to the triazolopyrimidinone or direct SNAr with 2-chloro-5-methyl-4-azabenzimidazole 79 (Scheme 10). Scheme 9. Synthesis of triazolopyrimidinone analogs 45–52.


34

Page 35 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H N

H 2N

OH

a, b, c 20% (3 steps)

Cl

N N

N

a, b, c

OH

N

O

H N

Cl

45

48

H N

H N OH

e

N N

N

H N

Cl

Cl

46

47 H N

N N

H N

CO2Et

a, b, c 48% (3 steps)

Cl

N N

H N

N

H N

N

R6

O

O H N

O

N

O

O

84

H 2N

NH2

O

N

Cl

N N

13% (3 steps)

N

d O

H N

OH

Cl

H N

O

83

H 2N

H N

Cl

or h CO2Et g

50 R6 = H 52 R6 = Me

O H N

Cl

85

86

N

f H N

N N

H N

N

N

H N

O

N O

O Cl CO2H

51

O Cl

49

(a) Diphenyl N-cyanocarbonimidate, 2-propanol, 60 °C, 0.75–2.5 h.; b) N2H4·H2O, 50 °C, 1–4 h. (c) ethyl acetoacetate, AcOH, reflux or 165 °C, MW, 10–20 min. (d) KOCN, TFA, CH2Cl2, rt, 19%. (e) i) MsCl, Et3N, THF, –40 °C→ rt, then rt, 3 h; ii) NaOMe, DMSO-MeOH (10:1), rt, 2 h, 13%. (f) LiOH, THF-EtOH-H2O (3:1:1), rt, overnight, 97%. (g) NH3, MeOH, rt → 50 °C; 50: 19%, 51: 23%. (h) MeNH2, MeOH, 50 °C; 52: 10%, 51: 20%. Scheme 10. Synthesis of nitrile analogs 53 and 54.


35


H N

N N

H N

N

N

H 2N CN

CN

a, b, c

d

11% (3 steps) Cl

O Cl

87

53

Page 36 of 52

N

H N

N H

CN

Cl

54

(a) Diphenyl N-cyanocarbonimidate, 2-propanol, 60 °C, 2 h. b) N2H4·H2O, 50 °C, 1 h. (c) ethyl acetoacetate, AcOH, 165 °C, MW, 10 min. (d) 79, KH2PO4, 2-butanol, 100 °C, 26 h, 4%. CONCLUSIONS By employing fragment-based screening, we discovered a number of unique scaffolds that were capable of binding to the pantetheine site of E. coli PPAT and inhibiting enzymatic activity. The wealth of structural information obtained by X-ray crystallography enabled structure-based optimization of three related fragment hits. This fragment-based lead discovery effort improved potency by three orders of magnitude and resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli ΔtolC mutant strain. These lead compounds, and the effort that led to their discovery, provided the framework for our subsequent lead optimization campaign directed at maintaining favorable properties while further improving ontarget potency to achieve growth inhibitory activity against wild-type Gram-negative bacteria.26 EXPERIMENTAL SECTION General. All synthesized compounds (16, 17, 19–29, 31–54) possessed a purity of at least 95% as assessed by analytical reversed phase HPLC (see Supporting Information for details). IC50 values obtained from the E. coli and P. aeruginosa PPAT biochemical assays are reported as the average at least two replicates except for compounds 31, rac-33, 36 and 43. For the biochemical assay used to measure the PPAT activity of human CoaSY and assess the selectivity of our inhibitors, compounds 5, 17, 18, 19, 20, R-31, S-31, 32, 37, 46 and 49 are an average of


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two or more replicates. For all other compounds, the IC50 values are from a single replicate. Reported DSF measurements are an average of two or more replicates. The reported SPR data for compound 21 is an average of two measurements, while the data for compounds 33, 53 and 54 are from a single measurement. For MIC data, the most frequently occurring value from three or more replicates is reported. In cases where two values occurred with identical frequency, the higher of the two values is reported. (R)-3-(3-Chlorophenyl)-3-((5-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-2yl)amino)propanenitrile (53). A suspension of diphenyl N-cyanocarbonimidate (0.263 g, 1.11 mmol, 1.2 equiv.) in 5 mL 2-propanol was treated with 87 (0.200 g, 0.921 mmol, 1.0 equiv.) and the mixture was heated at 60 °C for 2 h, then allowed to stand at rt overnight. The resulting mixture was concentrated to a yellow oil, which was dissolved in 5 mL MeOH and treated with hydrazine hydrate (0.029 mL, 0.921 mmol, 1.0 equiv.) dropwise over 1–2 min. The resulting mixture was heated at 50 °C for 1 h, then concentrated to give an oil that was dissolved in 1.6 mL AcOH and treated with ethyl acetoacetate (0.151 mL, 1.20 mmol, 1.3 equiv.). The mixture was heated in the microwave at 165 °C for 10 min, then more ethyl acetoacetate (0.100 mL, 0.793 mmol, 0.86 equiv.) was added and the mixture was heated in the microwave at 165 °C for an additional 5 min. The mixture was allowed to stand at rt overnight before it was concentrated to dryness. The residue was dissolved in DMSO, filtered and purified by RP-HPLC to provide 32 mg (11%) of 53.

1

H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H), 7.57–7.50 (m, 2H), 7.43

(dt, J = 7.6, 1.4 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.34 (dt, J = 7.8, 1.6 Hz, 1H), 5.65 (d, J = 0.8 Hz, 1H), 5.06 (td, J = 8.8, 6.0 Hz, 1H), 3.11–2.93 (m, 2H), 2.21 (d, J = 0.6 Hz, 3H). [α]D20 + 112.1 (c 0.25, MeOH). HRMS (ESI): m/z 329.0920 [M+H]+.


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(R)-3-(3-chlorophenyl)-3-((5-methyl-1H-imidazo[4,5-b]pyridin-2-yl)amino)propanenitrile (54). A mixture of 79 (120 mg, 0.716 mmol, 1.0 equiv.), 87 (311 mg, 1.43 mmol, 2.0 equiv.) and potassium dihydrogen phosphate (487 mg, 3.58 mmol, 5.0 equiv.) in 3.58 mL 2-butanol was heated at 100 °C using a heating block. After 26 h, a mixture of desired product, sec-butyl ester side product and remaining amine starting material was observed. The reaction mixture was filtered through a cotton plug and the volatiles were evaporated under reduced pressure. The residue was dissolved in DMSO, filtered and purified by RP-HPLC to give 12 mg (4%) of 54 as a colorless powder. 1H NMR (500 MHz, Methanol-d4) δ 7.81 (d, J = 7.8 Hz, 1H), 7.55 (s, 1H), 7.44 (dt, J = 7.6, 1.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.37 (dt, J = 7.4, 1.7 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H), 5.45 (t, J = 7.0 Hz, 1H), 3.25–3.17 (m, 2H), 2.62 (s, 3H). HRMS (ESI): m/z 312.1017 [M+H]+. 2-Chloro-5-methyl-1H-imidazo[4,5-b]pyridine (79). Step 1: A solution of 78 (15.0 g, 97.9 mmol, 1.0 equiv.) in 90 mL MeOH was flushed with nitrogen, then 10% Pd/C (0.750 g, 7.05 mmol, 0.07 equiv.) was added. The black reaction mixture was flushed with hydrogen, then the mixture was stirred at rt overnight under a balloon of hydrogen. LC-MS analysis only showed starting material, so the mixture was flushed with nitrogen and more 10% Pd/C (0.750 g, 7.05 mmol, 0.07 equiv.) was added. The mixture was flushed with hydrogen again and then stirred at rt under a hydrogen atmosphere for 4 h. LC-MS indicated the reaction was not complete, so it was left to stir overnight with the reaction flask wrapped with aluminum foil. LC-MS analysis revealed that the reaction was complete and clean. The reaction was filtered through Celite® and the filtrate was evaporated under reduced pressure to provide 12.7 g (quant.) of 6methylpyridine-2,3-diamine as a brown, sticky oil. The material became darker in color with time. Step 2: A mixture of 6-methylpyridine-2,3-diamine (12.1 g, 98.2 mmol, 1.0 equiv.) and


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1,1’-carbonyldiimidazole (18.5 g, 114 mmol, 1.16 equiv.) in 50 mL THF was stirred at rt overnight. The solid that precipitated was isolated by filtration, washed with THF, and dried under vacuum at 50 °C to provide 16.6 g (92%) of 5-methyl-1H-imidazo[4,5-b]pyridin-2(3H)one that was 81% pure (contaminated with imidazole). 1H NMR (400 MHz, DMSO-d6) δ 11.11 (br s, 1 H), 10.63 (s, 1 H), 7.08 (d, J = 7.78 Hz, 1H), 6.76 (d, J = 7.78 Hz, 1H), 2.34 (s, 3H). MS (ESI): m/z = 150.0 [M+H]+. Step 3: A mixture of 5-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one (2.13 g, 11.6 mmol, 81% purity, 1.0 equiv.) in POCl3 (10.8 mL, 116 mmol, 10.0 equiv.) was stirred at 95 °C overnight. The reaction solution was chilled in an ice bath, then diluted with ice water and cautiously neutralized with 6 N NaOH until pH ≈ 7. A sticky, yellow oil precipitated from the black aqueous solution. The aqueous mixture was allowed to warm to rt before it was extracted with several portions of EtOAc. The combined EtOAc extracts were washed with water and brine, dried over Na2SO4, filtered, and concentrated to afford 376 mg (19%) of 79. The balance of the desired product remained in the aqueous layer. MS (ESI): m/z = 168.0 [M+H]+. ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: Molecular Formula Strings (CSV) Experimental procedures describing in vitro assays and methods, procedures for generating protein reagents and genetic strains, single residue susceptibility testing protocol and data, computational methods, general experimental information, synthesis and characterization of compounds 16-52, crystallographic methods and references (PDF).


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X-ray crystallographic and structure refinement statistics (PDF) Accession Codes Structural coordinates have been deposited in the RCSB Protein Data Bank under the accession codes 6CCL (4), 6CCS (5), 6CCQ (20), 6CCO (28), 6CCN (29), 6CCM (30), 6CCK (53). Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding author * E-mail: [email protected]. Phone: +1 510 879 9454 ORCID Robert J. Moreau: 0000-0002-9056-7128 Notes The manuscript was written through contributions of all authors.

All authors have given

approval to the final version of the manuscript. The authors declare no competing interest. ACKNOWLEDGEMENTS This work was funded by Novartis AG. The authors thank Shengtian Yang for NMR structure elucidation support; Weiping Jia, Heidi Struble, Dazhi Tang, Alice Wan Wang, and Da Wang for analytical support. ABBREVIATIONS USED CoaSY, CoA synthase; DPCK, dephospho-CoA kinase; DSF, differential scanning fluorimetry; ESP, electrostatic potential; FBS, fragment-based screening; MW, microwave irradiation; PanK, pantothenate kinase; PDR, pandrug-resistant; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine


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31. Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small molecule conformational preferences derived from crystal structure data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008, 48, 1–24. 32. Kenny, P. W. Prediction of hydrogen bond basicity from computed molecular electrostatic properties: implications for comparative molecular field analysis. J. Chem. Soc., Perkin Trans. 2 1994, 199–202. 33. (a) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. The J. Phys. Chem. 1993, 97, 10269–10280. (b) Hunter, C. A. Quantifying intermolecular interactions: guidelines for the molecular recognition toolbox. Angew. Chem. Int. Ed. 2004, 43, 5310–5324. (c) Kenny, P. W. Hydrogen bonding, electrostatic potential, and molecular design. J. Chem. Inf. Model. 2009, 49, 1234–1244. (d) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med. Chem. 2010, 53, 5061–5084. (e) Klebe, G. Applying thermodynamic profiling in lead finding and optimization. Nat. Rev. Drug Discov. 2015, 14, 95–110. 34. O’Shea, R.; Moser, H. E. Physicochemical properties of antibacterial compounds: implications for drug discovery. J. Med. Chem. 2008, 51, 2871–2878. 35. Assay top concentration points varied, but 23 compounds were tested up to 500 μM, five up to 50 μM, and two up to 100 μM. Compound 49 was not tested. This analysis excluded the fragment hits shown in Figure 1.


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36. Khanna, I. K.; Weier, R. M.; Lentz, K. T.; Swenton, L.; Lankin, D. C. Facile, Regioselective syntheses of N-alkylated 2,3-diaminopyridines and imidazo[4,5-b]pyridines. J. Org. Chem. 1995, 60, 960–965. 37. Wong, W. W.; Jones, D. J.; Yan, C.; Watkins, S. E.; King, S.; Haque, S. A.; Wen, X.; Ghiggino, K. P.; Holmes, A. B. Synthesis, photophysical, and device properties of novel dendrimers based on a fluorene-hexabenzocoronene (FHBC) core. Org. Lett. 2009, 11, 975–978. 38. Spinks, D.; Ong, H. B.; Mpamhanga, C. P.; Shanks, E. J.; Robinson, D. A.; Collie, I. T.; Read, K. D.; Frearson, J. A.; Wyatt, P. G.; Brenk, R. Fairlamb, A. H.; Gilbert, I. H. Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1. ChemMedChem 2011, 6, 302–308. 39. (a) Chen, Q.; Zhu, X.-L.; Jiang, L.-L.; Liu, Z.-M.; Yang, G.-F. Synthesis, antifungal activity and CoMFA analysis of novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives. Eur. J. Med. Chem. 2008, 43, 595–603. (b) Monte, W. T.; Kleschick, W. A.; Meikle, R. W.; Snider, S. W.; Bordner, J. Methods for controlling the regioselection in the reaction of 3-amino-5-benzylthio1,2,4-triazole with acetylacetaldehyde dimethyl acetal. J. Heterocyclic Chem. 1989, 26, 1393– 1396. 40. Dietz, J.; Grote, T.; Huenger, U.; Lohmann, J. K.; Mueller, B.; Renner, J.; Ulmschneider, S.; Grammenos, W.; Rheinheimer, J. Fungicidal 5-Methyl-6-phenyltriazolopyrimidinyl Amines and Their Preparation. PCT Int. Appl. WO 2007012602 A1, 2007. 41. (a) Ognyanov, V. I.; Balan, C.; Bannon, A. W.; Bo, Y.; Dominguez, C.; Fotsch, C.; Gore, V. K.; Klionsky, L.; Ma, V. V.; Qian, Y.-X.; Tamir, R.; Wang, X.; Xi, N.; Xu, S.; Zhu, D.; Gavva, N. R.; Treanor, J. J.; Norman M. H. Design of potent, orally available antagonists of the


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Table of Contents graphic H N

H N

N N

N

Structure-based design

NH

N

N

N H N

N

N

N

H N

N H

N

O

O F

Cl

6 E. coli PPAT IC50 = 31 µM LE = 0.30

Fragµent screening

53 E. coli PPAT IC50 = 0.037 µM LE = 0.44, LogD7.4 = 1.2 E. coli DtolC MIC = 16 µg/µL

Cl

54 E. coli PPAT IC50 = 0.056 µM LE = 0.45, LogD7.4 = 3.3 E. coli DtolC MIC = 4 µg/µL

1,000-fold improvement in potency Cellular activity on mutant strain E. coli


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Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine

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