RU2805298C2 - New inhibitors of bacterial glutaminyl cyclases for use in the treatment of periodontal and related diseases - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: compound of Formula I, a pharmaceutical composition containing it, and the use of this compound or pharmaceutical composition in the therapy and/or prevention of a bacterial infection. In Formula I, A is selected from the group consisting of optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, and optionally substituted alkenyl; R¹ is independently selected from the group consisting of H and optionally substituted alkyl; L is ; R^a and R^b are the same or different from each other and are independently selected from the group consisting of H, halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroalkyl and optionally substituted heteroaryl, and R^a and R^b may optionally be connected together to form a carbocyclic or heterocyclic ring; the remaining substituent values are as defined in the claims.

I

EFFECT: compound of Formula I with inhibitory activity against bacterial glutamyl cyclases (bacQC).

12 cl, 10 dwg, 5 tbl, 29 ex

Description

TECHNICAL FIELD

The present invention relates to new compounds that are particularly useful as inhibitors of bacterial glutaminyl cyclases (bacQC); pharmaceutical compositions containing such compounds; compounds and/or pharmaceutical compositions for use in methods of treatment, in particular for use in the treatment of periodontitis and related conditions. The present invention also provides crystals containing bacterial glutamyl cyclases and methods for identifying candidate compounds that may associate with the bacQC binding pocket and/or are bacQC inhibitors.

BACKGROUND ART

Periodontal disease is widespread, with approximately 30% of the world's population affected, has a significant impact on individuals and society, and requires expensive treatment. The cost of dental care ranks fourth among all diseases and consumes 5 to 10% of all health care resources (Batchelor, P. British Dental Journal 2014, 217, 405–409). Representative population studies show that periodontal disease is widespread and has been increasing in prevalence since 1997 (Micheelis, W. et al. Vierte Deutsche Mundgesundheitsstudie (DMS IV), Deutscher Ärzte-Verlag, Köln, 2006). Among the adult population of Germany, 52.7% suffer from moderate periodontitis and 20.5% from severe forms of periodontitis. Health insurance costs in Germany for direct treatment of periodontitis amounted to approximately 1.1 billion euros (Statistisches Bundesamt, 2008), excluding costs for secondary diseases.

Periodontitis is a general term describing an inflammatory condition of the periodontal system that is caused by multibacterial induction and has a strong association with various systemic diseases such as cardiovascular disease, rheumatoid arthritis, chronic obstructive pulmonary disease and Alzheimer's disease.

According to the recommendations of the German Society of Dental, Oral and Craniomandibular Sciences, currently used therapy for periodontitis is usually carried out by manual supragingival and subgingival debridement (removal of bacterial plaque) with the use of antiseptics (daily disinfection with mouthwash), which destroy all biofilm of the cavity mouth and allow recolonization (inoculation) of a potential pathogen. In addition, in advanced forms of the disease, adjuvant systemic broad-spectrum antibacterial therapy is used. The latter also leads to non-selective biofilm destruction and must be administered in high doses and over a long period of time to achieve sufficient therapeutic levels at the specific site of action, i.e. the periodontal pocket. Standard adjuvant therapy for periodontitis includes, for example, systemic doxycycline (oral) 1 × 200 mg/day for 1 day and 2 × 100 mg/day for the next 18 days (Wissenschaftliche Stellungnahme: Adjuvante Antibiotika in der Parodontitistherapie, Deutsche Gesellschaft für Zahn - Mund- und Kieferheilkunde, DZZ 2003). As a result, there is the development of resistance in oral pathogens. Additionally, the microbiome in the patient's gut is disrupted, resulting in loss of metabolic support, immune modulation, and allowing potential pathogen recolonization. Specialized and targeted therapy along with preservation of remaining biofilm will represent a significant improvement in the treatment of periodontitis and related conditions.

The presence of periodontopathogenic bacteria varies among patients with periodontitis. However, the presence of certain types of bacteria in subgingival plaques has been found to be closely associated with the cause of periodontal disease (Socransky et al., Journal of Clinical Periodontology, 1998, 25, 134–144).

Thus, there is a great need to develop new treatments for periodontitis and related conditions that are capable of selectively targeting pathogens that cause periodontal disease while being substantially inactive toward homologous human protein targets and preferably being substantially inactive. preserving the rest of the natural biofilm. Such treatments could significantly improve the outcomes of patients and healthcare systems.

TECHNICAL PROBLEM SOLVED BY THE INVENTION

In view of the above, the present invention is directed to the identification and creation of compounds and/or pharmaceutical compositions useful for the treatment of periodontal diseases and related diseases. Said compounds and/or pharmaceutical compositions should preferably be capable of selectively targeting pathogens that cause periodontal disease. More preferably, the remainder of the natural biofilm is largely preserved; homologous human target proteins should not be significantly suppressed. Even more preferably, the compounds and/or pharmaceutical compositions exhibit high in vivo activity against the target pathogens.

Another goal is to obtain a crystal and/or cocrystal of therapeutic target protein(s) that can be used to identify inhibitors capable of targeting periodontal disease pathogens, for example, through a structure-supported drug design.

Another objective is to provide a method for identifying a candidate compound that may associate with the binding pocket of a specified therapeutic agent.

Another object of the present invention is to provide an inhibitor of said therapeutic target protein(s) and a pharmaceutical composition containing such inhibitor. Said inhibitor should preferably be a selective inhibitor, i.e. selectively killing or selectively inhibiting the growth of bacterial pathogen(s) while being substantially inactive against other bacterial and/or human protein targets.

It is yet another object of the present invention to provide a method of treating a human or animal body and/or a compound or pharmaceutical composition for use in such a method.

Another object of the present invention is a method of treating or preventing a bacterial infection and/or a compound or pharmaceutical composition for use in such a method, preferably by selectively killing or selectively inhibiting the growth of pathogenic bacterial species.

It is yet another object of the present invention to provide a method for the treatment or prevention of acute, chronic or recurrent periodontal disease and/or a compound or compound of a pharmaceutical composition for use in such a method.

In the treatment methods for the above purposes, the administration method should preferably be local or systemic administration, and the methods are preferably non-surgical methods.

DISCLOSURE OF INVENTION

As a solution to the problems formulated above, the present description proposes a compound according to one of the following formulas I or II,

its individual enantiomers, its individual diastereoisomers, its hydrates, its solvates, its crystalline forms, its individual tautomers or a pharmaceutically acceptable salt thereof, wherein L, A and R ¹ are defined in accordance with the attached formula.

The present specification further provides a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable excipient.

The present description further provides the above-described compound and/or the above-described pharmaceutical composition for use in a method of treating a human or animal body; for use in a method of therapy and/or prevention of bacterial infection; and for use in a method of therapy and/or prevention of acute, chronic or recurrent periodontal disease.

The present description further provides a crystal containing a bacterial glutamyl cyclase (bacQC) selected from PgQC and TfQC, and a co-crystal further containing a compound of interest, preferably a bacQC inhibitor; as well as methods for obtaining said crystal and/or co-crystal.

The present disclosure further provides methods for identifying a compound of interest that can bind to the bacQC binding pocket, preferably a bacQC inhibitor.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the amino acid sequence alignment of human QC (hQC, SEQ ID NO: 4) and putative bacterial glutamyl cyclases (QCs, i.e. glutamine cyclotransferases) from Porphyromonas gingivalis (PgQC, SEQ ID NO: 1) (A); amino acid sequence alignment of additional putative QCs from Prevotella intermedia (PiQC, SEQ ID NO: 3), P. gingivalis (PgQC, SEQ ID NO: 1) and Tannerella forsythia (TfQC, SEQ ID NO: 2)) (B); amino acid numbering of the full-length sequence (SEQ ID NO: 1) of glutamyl cyclase (ie, glutamine cyclotransferase) from P. gingivalis (PgQC), amino acids 1-333 (C); and the amino acid numbering of the full-length sequence (SEQ ID NO: 2) of T. forsythia glutamyl cyclase (ie, glutamine cyclotransferase) (TfQC), amino acids 1-333 (D).

In FIG. Figure 2 shows SDS-PAGE of purified recombinant putative bacterial QCs expressed in E. coli Rosetta(DE3)pLysS.

In FIG. Figure 3 shows Lineweaver-Burk plots for the cyclization of H-Gln-AMC catalyzed by PgQC (A), PiQC (B), and TfQC (C).

Figure 4 shows typical v/S characteristics, Lineweaver-Burk and Eady-Hofstee plots for the PgQC-catalyzed cyclization of H-Gln-AMC in the presence of the reference compound MWT-S-00431.

Figure 5 shows PhoA activity in permeabilized E. coli CC118 pGP1-2 cells expressing seqPgQC-`PhoA fusion proteins.

In FIG. Figure 6 shows a ribbon image for three glutamyl cyclases: PgQC (A); TfQC (B); hQC(C); and trim (D).

In FIG. Figure 7 shows the surfaces of molecules of three glutamyl cyclases in a crystal: PgQC (A); TfQC (B); and hQC (PDB code 3SI0) (C). The surface is colored by lipophilicity (green: more lipophilic, purple: more hydrophilic); the centered hole shows the active site.

Figure 8 shows the molecular surfaces for PgQC (A); TfQC (B); and hQC (C) in the crystal, where the hydrophilic regions are highlighted in darker gray (top pictures).

Figure 9 shows the molecular surfaces for PgQC (A); TfQC (B); and hQC (C) in the crystal, where the lipophilic regions are highlighted in darker gray (lower pictures).

In FIG. Figure 10 shows a comparison of the active sites and molecular surfaces of PgQC and hQC.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic targeted proteins (bacQC)

Socransky et al. (Journal of Clinical Periodontology, 1998, 25 134–144) described that the appearance of a so-called “red complex” in subgingival plaques, consisting of a closely related group of Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola, was significantly associated with clinical indicators of periodontitis , and in particular the depth of the pocket and bleeding during probing.

Another related complex (the so-called “orange complex”) includes representatives of the subspecies Fusobacterium nucleatum/periodonticum, Prevotella intermedia, Prevotella nigrescens and Peptostreptococcus micros. Colonization of healthy periodontal sites by members of the "orange complex" has been found to correlate with the occurrence of gingivitis. Orange complex bacteria also promote colonization by red complex bacteria, which in turn cause deep pockets and chronic periodontitis.

Red complex and orange complex bacteria secrete a variety of virulence factors. For example, P. gingivalis has been found to secrete cysteine proteases such as gingipain, P. intermedia to secrete salivary IgA proteases, and T. forsythia to secrete glycosidases.

The present inventors unexpectedly discovered that about 80% of the secreted signal peptide-carrying proteins in the secretions of the oral pathogens P. gingivalis, T. forsythia and P. intermedia are cleaved at the Xaa-Gln peptide bond by signal peptidase. The N-terminal ends of the released proteins contain a pGlu residue. This implies the existence of glutaminyl cyclases, which appear to be required for the translocation of growth proteins across the outer membrane and growth of these periodontal pathogens.

Glutaminyl cyclases (QC) (EC 2.3.2.5), also called glutamine cyclotransferases, are acyltransferases that catalyze the cyclization of N-terminal glutaminyl residues of proteins into pyroglutamate (pGlu) upon release of NH ₃ , thereby modifying the N-terminus of peptides:

To date, two types have been identified (Type I and Type II). Type I QCs have been found in plants as well as several pathogenic bacteria and human parasites (Huang et al., J. Mol. Biol. 2010, 401, 374–388). Papaya QC (pQC) is the best known type I QC. This enzyme was first discovered in the latex of the tropical plant Carica papaya (Messer, M. Nature 1963, 197, 1299). The enzyme exhibits catalytic activity over a wide pH range (pH 3.5–11). X-ray diffraction analysis revealed that pQC is a hatbox-shaped molecule consisting of a five-bladed β-propeller traversed by a central channel (Wintjens, R., Belrhali, H., Clantin, B., Azarkan, M., Bompard, C., Baeyens-Volant, D., Looze, Y., and Villeret, V. J. Mol. Biol. 2006, 357, 457–470). pQC contains a zinc ion but is not at all inhibited by heterocyclic chelators (Zerhouni et al. Biochim. Biophys. Acta 1998, 1387, 275–290), and therefore zinc is assumed to have only a structural and stabilizing function. pQC is highly resistant to proteolytic, chemical and thermal denaturation (Wintjens et al., Zerhouni et al.).

Type II QCs have been primarily identified in mammalian neuroendocrine tissues. Among type II QCs, the most studied is human QC (hQC), which is known to play an important role in the maturation of numerous neuropeptides and cytokines in their secretory pathways. Unlike type I QCs, hQC is quite sensitive to chemical and thermal denaturation. Schilling et al., 2003 (J. Biol. Chem. 2003, 278, 49773–49779) showed that hQC was significantly unstable at pH above 8.5 and below 6.0. hQC adopts an α/β topology (Huang et al. Proc. Natl Acad. Sci. USA, 2005, 102, 13117–13122) and has been identified as a metalloenzyme, as demonstrated by time-dependent inhibition by heterocyclic chelators. The inactivated enzyme can be completely restored by the addition of Zn ²⁺ in the presence of equimolar concentrations of EDTA (Schilling et al., 2003). Thus, type II QCs are metal-dependent transferases, suggesting that the metal bound to the active site (Zn ²⁺ ) is required for catalytic activity, in contrast to type I QCs in which zinc performs only structural and stabilizing functions. function.

Thus, type I QCs are commonly found in plants, some bacteria, and protozoa; they have a β-propeller structure; high resistance to proteolysis, heat and acid; optimal catalytic activity - at pH 3.5-11; and have a structural ion Ca ²⁺ /Zn ²⁺ .

In contrast, type II QCs are mainly found in vertebrates; they exhibit α/β topologies; low resistance to proteolysis, heat and acid; optimal catalytic activity - at pH 6.0-8.0; and there is a catalytically necessary Zn ²⁺ ion. Thus, type II glutamyl cyclases are zinc-dependent acyltransferases.

The present inventors have surprisingly discovered that type II QCs are expressed in the oral pathogens P. gingivalis, T. forsythia and P. intermedia.

The primary structure of the QC protein from P. gingivalis (PgQC, SEQ ID NO: 1) is 25% identical to human QC (hQC, SEQ ID NO: 4). Additionally, QCs from P. intermedia (PiQC, SEQ ID NO: 3) and T. forsythia (TfQC, SEQ ID NO: 2) were identified, which share 42% and 49% identity with PgQC, respectively. Further experimental evidence shows that these enzymes do indeed belong to the type II QC family, as evidenced by, among other things, the pH and ionic strength dependence of bacQC activity, inhibition of QC activity by metal chelators, folding patterns of all three proteins suggesting an α/β topology, as shown by spectroscopic analysis of CDs, and their thermal stability.

The present inventors have discovered that type II bacterial QCs are expressed and required for the growth of 2 of the 3 periodontitis-causing "red complex" bacterial species as well as at least one "orange complex" bacterial species, and are therefore critical as a target for development a therapeutic inhibitor with antibiotic properties for the treatment of periodontitis diseases and conditions.

Thus, in the context of the present invention, the therapeutic target proteins are bacterial glutaminyl cyclases (bacQCs), where bacQC is defined as a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (also referred to as PgQC), SEQ ID NO: 2 (also referred to as TfQC), SEQ ID NO: 3 (also referred to as PiQC) and an amino acid sequence having 90% or more sequence identity with any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 .

bacQCs can be identified, isolated and purified and used to identify inhibitors capable of selectively targeting pathogens that cause periodontitis, as further described in the examples in this disclosure. For the purpose of comparing two or more amino acid sequences, the degree of identity between two amino acid sequences (percentage of "sequence identity") can be determined by conventional methods, for example, using standard sequence alignment algorithms known in the art, such as, for example, BLAST ( Altschul SF et al J Mol Biol 1990, 215 (3), 403-10).

Connections

Using the methods described here for identifying candidate compounds and/or inhibitors, the present inventors have identified a new group as a leading structural motif for the design and production of potent and therapeutically effective inhibitors of bacterial glutamyl cyclases. The specified structural motif is an imidazo[4,5-b]pyridine group, as shown below (including ring atom numbering):

This structural motif represents a suitable metal binding group capable of reversibly binding to the Zn ²⁺ ion located in the binding pocket of bacQCs. Preferably, when the imidazo[4,5-b]pyridine group is substituted at position 6 of the pyridine ring, particularly high bacQC inhibitory activity and selectivity can be achieved.

More preferably, the group attached to the imidazo[4,5-b]pyridine group is either a benzylamine or an aromatic sulfonamide, each of which is bonded via an N atom to the pyridine ring. In contrast, it has been found that using aromatic amides instead provides weak activity and is therefore less preferred. In other words, R ^a and R ^b are preferably not represented together with =O.

In a further preferred embodiment, the compounds contain a moiety represented by the following structure E:

The presence of this moiety increases the uptake of the inhibitor compounds of the present invention into bacterial cells expressing type II glutaminyl cyclases (such as P. gingivalis, T. forsythia and P. intermedia), which recognize, actively uptake and capture the porphyrin moiety via a metal-independent system (eg, the HA2 domain, which is a receptor for both heme and hemoglobin) and/or a metal-dependent system (eg, HusA, which is a hemophore-like porphyrin-binding protein capable of recognizing the above group). Thus, coupling an inhibitor of general formula I or II to a moiety represented by structure E provides enhanced targeted delivery to pathogenic cells, improved selectivity for target oral pathogens, and/or improved in vivo antibiotic activity against these pathogens. These effects are particularly pronounced when the moiety represented by structure E is attached to group A of the inhibitor via a linker represented by structure E, as defined in the appended claims.

In particular, the present disclosure provides connections for any of the following aspects <1> - <19>.

<1> A compound according to one of the following formulas I or II,

its individual enantiomers, its individual diastereoisomers, its hydrates, its solvates, its crystalline forms, its individual tautomers or a pharmaceutically acceptable salt thereof, wherein:

A is selected from the group consisting of optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl and optionally substituted alkenyl;

R ¹ is independently selected from the group consisting of H and optionally substituted alkyl;

L is selected from And .

R ^a and R ^b are the same or different from each other and are independently selected from the group consisting of H, halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroalkyl and optionally substituted heteroaryl, and wherein R ^a and R ^b may optionally be connected together with the formation of a carbocyclic or heterocyclic ring.

<2> Connection according to aspect <1>, where:

said aryl independently represents a C _6-10 , preferably a C ₆ aryl group;

said heterocyclyl is independently a monocyclic or bicyclic C _1-11 , preferably a C _2-8 , more preferably a C _4-5 heterocyclic group containing 1 to 4 ring heteroatoms selected from N, S and O;

said alkyl is independently a straight or branched, open or cyclic C _1-6 , preferably a C _1-4 , more preferably a C _1-3 , even more preferably a C _1-2 alkyl group;

said cycloalkyl independently represents a cyclic C _3-6 , preferably a C _4-6 , more preferably a C _5-6 alkyl group;

said alkenyl is independently a linear or branched, open or cyclic C _2-6 group, preferably a C _2-4 group, more preferably a C ₂ group containing at least one C=C bond;

said heteroaryl is independently an aromatic, monocyclic or bicyclic C _1-11 , preferably C _2-8 , more preferably C _4-5 heterocyclic group containing from 1 to 4 ring heteroatoms selected from N, S and O; And

said heteroalkyl is independently a linear or branched, open or cyclic C _1-5 , preferably C _1-3 , more preferably C _1-2 heteroalkyl group containing from 1 to 4 heteroatoms selected from N, S and O.

<3> A compound according to any one of aspects <1> to <2>, which is represented by one of the following formulas Ia, IIa, Ib and IIb, preferably Ia:

where A is selected from optionally substituted aryl and optionally substituted heteroaryl; And

R ¹ , R ^a and R ^b are H.

<4> A connection according to any one of aspects <1> to <3>, wherein R ^a and R ^b are independently selected from ¹ H and D.

<5> A connection according to any one of aspects <1> to <4>, wherein either one of R ^a and R ^b is ¹ H and the other is D; or each of them represents ¹ H; or each of them D.

<6> A connection according to any of aspects <1> to <5>, where:

A is represented by one of the following structures A-1 to A-7;

where Y is selected from O and S; And

R ² to R ⁶ are each independently selected from the group consisting of H, fluorine, chlorine, bromine, iodine, alkyl, aryl, heteroaryl, alkoxy, aryloxy, (aminoalkyl)oxy, (heteroaryl)oxy, amino, (dialkyl)amino , (monoalkyl)amino, (alkyl)(heteroalkyl)amino, (diheteroalkyl)amino, [(alkyl)oxy]carbonyl and haloalkyl, each of which may be optionally further substituted.

<7> Connection according to aspect <6>, where either:

(a) R ⁴ , or

(b) R ³ , or

(c) R ³ and R ⁴

each independently selected from the group consisting of fluorine, chlorine, bromine, iodine, alkyl, aryl, heteroaryl, alkoxy, aryloxy, (aminoalkyl)oxy, (heteroaryl)oxy, amino, (dialkyl)amino, (monoalkyl)amino, (alkyl) )(heteroalkyl)amino, (diheteroalkyl)amino, [(alkyl)oxy]carbonyl and haloalkyl, each of which may optionally be further substituted;

and the rest from R ² to R ⁶ are H.

<8> A connection according to any one of aspects <6> to <7>, where

at least one of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ is independently represented by the following structure B:

B

where n = 1, 2, 3, 4, 5 or 6;

R ⁷ and R ⁸ are independently selected from alkyl, aryl, heteroalkyl, heteroaryl, carbamidoyl, formyl, alkylacyl and arylacyl, each of which may be further substituted, and wherein R ⁷ and R ⁸ may optionally be joined together to form a carbocyclic or heterocyclic ring .

<9> A connection according to any of aspects <6> to <8>, where:

R ¹ , R ^a and R ^b represent H; And

A is represented by the structure A-1.

<10> A compound according to any one of aspects <1> to <7>, wherein A is replaced by at least one substituent represented by the following structure B:

B

where n = 1, 2, 3, 4, 5 or 6; And

R ⁷ and R ⁸ are independently selected from alkyl, aryl, heteroalkyl, heteroaryl, carbamidoyl, formyl, alkylacyl and arylacyl, each of which may optionally be further substituted and wherein R ⁷ and R ⁸ may optionally be joined together to form a carbocyclic or heterocyclic ring .

<11> A connection according to any one of aspects <8> to <10>, wherein R ⁷ is represented by the following E structure:

where M is selected from Fe ²⁺ and Fe ³⁺ or is absent;

R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH; And

X is selected from covalent bond, alkylene, aralkylene, heteroalkylene, carbocyclene, heterocyclene, heteroarylene, heteroaralkylene, aminoalkylene, alkylamino and carbonylalkylamino groups; where each of them may have up to 12 carbon atoms and up to 11 heteroatoms in the main chain and may be replaced by one or more C _1-6 alkyl group(s), C _1-6 heteroalkyl group(s), C _1-6 carbocyclyl group (s), C _1-5 heterocyclyl group(s), C _1-5 heteroaryl group(s), halogen atom(s), hydroxyl group(s), cyano group(s), primary, secondary or tertiary amino group(s) , carboxyl groups and side chains derived from proteinogenic or non-proteinogenic amino acids.

<12> Connection according to aspect <11>, where X is represented by the following structure:

where m = 0, 1, 2 or 3, and

R ^e is a side chain derived from a proteinogenic amino acid.

<13> A compound according to any of aspects <1> to <12>, wherein A is replaced by a substituent represented by the following structure F:

where M is selected from Fe ²⁺ and Fe ³⁺ or is absent; And

R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH.

<14> A compound according to aspect <13>, where A has the meaning defined according to aspect <6>, and where at least one of R ² , R ³ , R ⁴ , R ⁵ and R ⁶ is represented by structure F.

<15> A compound according to any of aspects <1> to <14>, wherein A is replaced by at least one substituent represented by the following structure E1:

where M is selected from Fe ²⁺ and Fe ³⁺ or is absent;

R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH; And

X is selected from covalent bond, O, NH, alkylene group, aralkylene, heteroalkylene, carbocyclene, heterocyclene, heteroarylene, heteroaralkylene, aminoalkylene, alkylamino and carbonylalkylamino; where each of them may have up to 12 carbon atoms and up to 11 heteroatoms in the main chain and may be replaced by one or more C _1-6 alkyl group(s), C _1-6 heteroalkyl group(s), C _1-6 carbocyclyl group (s), C _1-5 heterocyclyl group(s), C _1-5 heteroaryl group(s), halogen atom(s), hydroxyl group(s), cyano group(s), primary, secondary or tertiary amino group(s) , carboxyl groups and side chains derived from proteinogenic or non-proteinogenic amino acids.

<16> A connection according to any one of aspects <1> to <15>, where

A is selected from the group consisting of alkoxyaryl, (alkoxy)(halogen)aryl, (alkoxy)heteroaryl, alkylaryl, alkylheteroaryl, aminoaryl, aminoheteroaryl, arylaryl, arylheteroaryl, (aryloxy)aryl, (aryloxy)heteroaryl, haloaryl, haloheteroaryl, (heteroalkyl) )aryl, (haloalkyl)aryl, (heteroaryl)aryl, [(heteroaryl)oxy]aryl, [(aminoalkyl)oxy]aryl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl, each of which may optionally be substituted.

<17> A connection according to any one of aspects <1> to <16>, in which:

A is selected from the group consisting of 2-alkoxyphenyl, 3-alkoxyphenyl, 4-alkoxyphenyl, 2,3-dialkoxyphenyl, 2,4-dialkoxyphenyl, 2,5-dialkoxyphenyl, 2,6-dialkoxyphenyl, 3,4-dialkoxyphenyl, 3 ,5-dialkoxyphenyl, 3,4,5-trialkoxyphenyl, 2,6-dihalo-4-alkoxyphenyl, 2-halo-4-alkoxyphenyl, 3-halogen-5-alkoxyphenyl, 4-halo-3-alkoxyphenyl, 2-alkylphenyl , 3-alkylphenyl, 4-alkylphenyl, 3-[(alkyl)(heteroalkyl)amino]phenyl, 3-[(dialkyl)amino]phenyl, 3-[(diheteroalkyl)amino]phenyl, 3-[(monoalkyl)amino] phenyl, 4-[(alkyl)(heteroalkyl)amino]phenyl, 4-[(dialkyl)amino]phenyl, 4-[(diheteroalkyl)amino]phenyl, 4-[(heteroaryl)oxy]phenyl, 4-[(monoalkyl )amino]phenyl, arylphenyl, 2-(aryloxy)phenyl, 3-(aryloxy)phenyl, 4-(aryloxy)phenyl, 2-halophenyl, 3-halophenyl, 4-halophenyl, 2,3-dihalophenyl, 2,4 - dihalophenyl, 2,5-dihalophenyl, 2,6-dihalophenyl, 3,4-dihalophenyl, 3,5-dihalophenyl, 2,3,4-trihalophenyl, 2,3,5-trihalophenyl, 3,4,5-trihalophenyl, 2,4,5-trihalophenyl, 2,4,6-trihalophenyl, 3-(haloalkyl)phenyl, 4-(haloalkyl)phenyl, 3-(C ₅ monoheteroaryl)phenyl, 4-(C ₅ monoheteroaryl)phenyl, 3- (C ₄ monoheteroaryl)phenyl, 4-(C ₄ monoheteroaryl)phenyl, 3-[(heteroaryl)oxy]phenyl, 4-{[amino(alkyl)]oxy}phenyl, 3-{[amino (alkyl)]oxy} phenyl, naphthyl, phenyl, C ₅ -monoheteroaryl, C ₄ -monoheteroaryl, morpholinyl and piperidinyl, each of which may optionally be further substituted.

<18> A connection according to any one of aspects <1> to <17>, where:

A is selected from the group consisting of 2-methoxyphenyl, 3-methoxyphenyl, 4-propoxyphenyl, 4-methoxyphenyl, 4-isopropoxyphenyl, 4-benzyloxyphenyl, 3,4-dimethoxyphenyl, 2,3-dihydro-1,4-benzodioxin-6 -yl, 2,2-difluoro-1,3-benzodioxol-5-yl, 7-methoxy-1,3-benzodioxol-5-yl, 2,6-difluoro-4-methoxyphenyl, 3-(1-piperidyl) phenyl, 4-morpholinophenyl, [1,1'-biphenyl]-3-yl, [1,1'-biphenyl]-4-yl, 3-phenoxyphenyl, 4-phenoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4- chlorophenyl, 3,4-dichlorophenyl, 3,4,5-trifluorophenyl, 4-(2-pyridyl)phenyl, 4-(2-carbamimidamidoethyloxy)phenyl, 4-(2-aminoethoxy)phenyl, 4-(2-morpholinoethoxy) phenyl, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl and 3-thienyl.

<19> Connection selected from the group consisting of:

The expression "alkyl" as used herein, unless specifically limited, means a C _1-12 alkyl group, suitably a C _1-8 alkyl group, eg a C _1-6 alkyl group, eg a C _1-4 alkyl group. Alkyl groups can be straight or branched chain. Suitable alkyl groups include, for example, methyl, ethyl, propyl (for example, n-propyl and isopropyl), butyl (for example, n-butyl, isobutyl, sec-butyl and t-butyl), pentyl (for example, n-pentyl), hexyl (eg n-hexyl), heptyl (eg n-heptyl) and octyl (eg n-octyl). The term "alkyl" also includes cycloalkyl groups. The expression "cycloalkyl", unless specifically limited, means a C _3-10 cycloalkyl group (ie, 3 to 10 ring carbon atoms), more preferably a C _3-8 cycloalkyl group, eg a C _3-6 cycloalkyl group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The most suitable number of carbon atoms in the ring is between three and six.

The expression "heteroalkyl", unless specifically limited, refers to an alkyl group in which one or more carbon atoms, preferably 1, 2 or 3, are replaced by heteroatoms selected from N, S and O.

The expressions "carbocyclyl" and "carbocyclic", unless specifically limited, mean any ring system in which all ring atoms are carbon, and which contains from three to twelve ring carbon atoms, more suitably from three to ten carbon atoms and more suitably from three to eight carbon atoms. Carbocyclyl groups may be saturated or partially unsaturated, but do not include aromatic rings. Examples of carbocyclic groups include monocyclic, bicyclic and tricyclic ring systems, in particular monocyclic and bicyclic ring systems. Other carbocyl groups include internally bridged ring systems (eg, bicyclo[2.2.1]heptenyl). A specific example of a carbocyclyl group is a cycloalkyl group. Another example of a carbocyclyl group is a cycloalkenyl group.

The expression "aryl", unless specifically limited, means a C _6-12 aryl group, a suitable C _6-10 aryl group, a more suitable C _6-8 aryl group. Aryl groups will contain at least one aromatic ring (eg one, two or three rings). An example of a typical aryl group with one aromatic ring is phenyl. An example of a typical aryl group with two aromatic rings is naphthyl.

The expressions “heterocyclyl” and “heterocyclic,” unless specifically limited, refer to a carbocyclyl group in which one or more (e.g., 1, 2, or 3) ring atoms are replaced by heteroatoms selected from N, S, and O. Specific Example of a Heterocyclic Group is a cycloalkyl group (e.g. cyclopentyl or more particularly cyclohexyl) in which one or more (e.g. 1, 2 or 3, especially 1 or 2, especially 1) ring atoms are replaced by heteroatoms selected from N, S or O Exemplary heterocyclic groups containing one heteroatom include pyrrolidine, tetrahydrofuran and piperidine, and exemplary heterocyclic groups containing two heteroatoms include morpholine and piperazine. Another specific example of a heterocyclic group is a cycloalkenyl group (e.g., a cyclohexenyl group) in which one or more (e.g., 1, 2, or 3, especially 1 or 2, especially 1) ring atoms are replaced by heteroatoms selected from N, S, and O An example of such a group is dihydropyranyl (for example, 3,4-dihydro-2H-pyran-2-yl-).

The expression "heteroaryl", unless specifically limited, means an aryl moiety where one or more (e.g., 1, 2, 3 or 4, suitably 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O or 5 -membered aromatic ring containing one or more (e.g. 1, 2, 3 or 4, suitably 1, 2 or 3) ring atoms selected from N, S and O. Heteroaryl groups are a specific subtype in the general class of "heterocyclic" or "heterocyclic" groups. Examples of monocyclic heteroaryl groups containing one heteroatom include: five-membered rings (e.g., pyrrole, furan, thiophene) and six-membered rings (e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl) . Examples of monocyclic heteroaryl groups containing two heteroatoms include: five-membered rings (eg, pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-1-yl, imidazol-2-yl imidazol-4-yl); six-membered rings (for example, pyridazine, pyrimidine, pyrazine). Examples of monocyclic heteroaryl groups containing three heteroatoms include 1,2,3-triazole and 1,2,4-triazole. Examples of monocyclic heteroaryl groups containing four heteroatoms include tetrazole. Examples of bicyclic heteroaryl groups include: indole (eg, indol-6-yl), benzofuran, benzothiophene, quinoline, isoquinoline, indazole, benzimidazole, benzothiazole, quinazoline and purine.

The expressions "alkoxyaryl", "carboxyaryl", "cyanoaryl", "haloaryl", "hydroxyaryl" and "heteroaryl", unless specifically limited, denote an aryl moiety that is substituted by at least one alkoxy, carboxy, cyano, halogen, hydroxy and heteroaryl group, respectively.

The expressions “alkoxyheteroaryl,” “carboxyheteroaryl,” “cyanoheteroaryl,” “haloheteroaryl,” and “hydroxyheteroaryl,” unless specifically limited, denote a heteroaryl moiety that is substituted with at least one alkoxyl, carboxyl, cyano, halogen, and hydroxyl group, respectively.

The expression "alk", for example in the expressions "alkoxy", "haloalkyl" should be interpreted in accordance with the definition of "alkyl". Examples of alkoxy groups include methoxy, ethoxy, propoxy (eg n-propoxy), butoxy (eg n-butoxy), pentoxy (eg n-pentoxy), hexoxy (eg n-hexoxy), heptoxy (eg n-heptoxy ) and octoxy (for example n-octoxy). Examples of haloalkyl groups include fluoroalkyl, for example CF ₃ ; exemplary haloalkoxy groups include fluoroalkyl, for example OCF ₃ .

The term "halogen" or "halogeno" includes fluorine (F), chlorine (CI), bromine (Br) and iodine (I).

The terms "hydrogen" or "H" as used herein cover all isotopes of hydrogen, particularly protium ( ^1H ) and deuterium ( ^2H , also referred to as D).

The term "optionally substituted" refers to optional substitution with one or more groups independently selected from: C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 carbocyclyl, C _1-5 heterocyclyl and C _1-5 heteroaryl group, each of which may be substituted with one or more halogen atoms and/or hydroxyl groups; a halogen atom; cyano group; a primary, secondary or tertiary amino group; hydroxyl group; and a carboxyl group.

Stereoisomers

All possible stereoisomers of the claimed compounds are included in the present invention.

If the compounds of the present invention have at least one chiral center, they may suitably exist as enantiomers. If compounds have two or more chiral centers, they may additionally exist as diastereomers. It should be understood that all such isomers and mixtures thereof are included within the scope of the present invention.

If the methods for preparing the compounds according to the invention lead to a mixture of stereoisomers, these isomers can be separated by conventional methods such as preparative chromatography. The compounds can be obtained in racemic form, or the individual enantiomers can be obtained either by enantiospecific synthesis or by resolution. The compounds can, for example, be separated into their enantiomer components by standard methods, such as the formation of diastereomeric pairs by salt formation with an optically active acid such as (-)-di-p-toluoyl-d-tartaric acid and/or (+) - di-p-toluoyl-1-tartaric acid followed by fractional crystallization and regeneration of the free base or formation of a salt with an optically active base such as quinine, quinidine, quinotoxin, zincotoxin, (S)-phenylethylamine, (1R, 2S)-ephedrine , (R)-phenylglycine, (S)-2-aminobutanol, followed by fractional crystallization and regeneration of the free acid. Compounds can also be separated by formation of diastereomeric esters or amides followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, compounds can be separated using a chiral high performance liquid chromatography (HPLC) column.

Polymorphic crystalline forms, solvates, hydrates

In addition, some of the individual crystalline forms of the compounds may exist as polymorphs and, as such, are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (ie, hydrates) or common organic solvents, and such solvates are intended to be included within the scope of the present invention. The compounds, including their salts, may also be obtained in the form of their hydrates or may include other solvents used for their crystallization. In view of the close relationship between free compounds and compounds in the form of their salts, hydrates or solvates, whenever a compound is mentioned in this context, the corresponding salt, solvate or polymorph is also intended, provided that this is possible or appropriate in the circumstances .

Tautomers

The term "tautomer" as used here refers to the migration of protons between adjacent single and double bonds. The tautomerization process is reversible. The compounds described herein can undergo any possible tautomerization that is within the physical characteristics of the compound.

Pharmaceutically acceptable salts

As used herein, the term "pharmaceutically acceptable" covers both human and veterinary use. For example, the term “pharmaceutically acceptable” includes a compound that is veterinarily acceptable or a compound that is acceptable in medical or health care applications.

Salts, hydrates and solvates of the compounds of formula I and their physiologically functional derivatives, which are suitable for use in medicine, are salts in which the counterion or associated solvent is pharmaceutically acceptable. However, salts, hydrates and solvates containing pharmaceutically unacceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds and their pharmaceutically acceptable salts, hydrates and solvates.

Suitable salts according to the invention include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable salts obtained by acid addition include salts formed from hydrochloric, hydrobromic, sulfuric, nitric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, triphenylacetic, sulfamic, sulfanilic, succinic, oxalic, fumaric, maleic, malic, almond, glutamic, aspartic, oxaloacetic, methanesulfonic, ethanesulfonic, arylsulfonic (e.g. p-toluenesulfonic, benzenesulfonic, naphthalene sulfonic or naphthalene disulfonic), salicylic, glutaric, gluconic, tricarballyl, cinnamic, substituted cinnamic (eg phenyl, methyl, methoxy or halogen substituted cinnamic acid, including 4-methyl and 4-methoxycinnamic acid), ascorbic acid, oleic acid, naphthoic acid, hydroxynaphthoic acid (e.g. 1-or 3-hydroxy-2-naphthoic acid), naphthalene acrylic acid (e.g. naphthalene acrylic acid), benzoic acid, 4- methoxybenzoic, 2- or 4-hydroxybenzoic, 4-chlorobenzoic, 4-phenylbenzoic, benzolacrylic (eg 1,4-benzenediacrylic), isethionic acid, perchloric, propionic, glycolic, hydroxyethanesulfonic, pamic, cyclohexanesulfamic, salicylic, saccharic and trifluoroacetic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine.

All pharmaceutically acceptable forms of the acid addition salt compounds of the present invention are intended to be included within the scope of the present invention.

Pharmaceutical compositions

The pharmaceutical composition according to the present invention includes a compound as described above and a pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutical composition" is intended to cover a product containing the claimed compounds in therapeutically effective amounts, as well as any product that is the direct or indirect result of combinations of the claimed compounds. As used herein, the term "excipient" refers to a carrier, binder, disintegrant and/or other suitable additive for galenic formulations, for example, for oral liquid preparations such as suspensions, elixirs and solutions; and/or for solid preparations for oral administration, such as, for example, powders, capsules, gelatin capsules and tablets. Carriers that may be added to the mixture include necessary and inert pharmaceutical excipients, including, but not limited to, suitable suspending agents, lubricants, flavoring agents, sweeteners, preservatives, coatings, granulating agents, colorants and pigments.

Therapeutic Use

Disclosed herein is a compound, for example, a compound according to any of the above aspects <1> to <19>, and/or a pharmaceutical composition as described above, for use in a method of treating a human or animal body. Also provided herein is a method of treating a human or animal body, wherein the method includes administering a therapeutically effective amount of said compound or composition to a subject in need thereof.

The present disclosure further provides a compound and/or pharmaceutical composition as described above for use in a method of treating or preventing a bacterial infection. The present disclosure also provides a method of treating or preventing a bacterial infection, wherein the method includes administering a therapeutically effective amount of said compound or composition to a subject in need thereof. The bacterial infection is preferably caused by a bacterium expressing bacterial glutamyl cyclase type II (bacQC). More preferably, the bacterial infection is caused by a bacterium selected from the group consisting of Porphyromonas, Prevotella and Tannerella species, preferably selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia species.

The bacQC inhibitor compound or pharmaceutical composition used in the methods of the present disclosure preferably selectively kills or selectively inhibits the growth of a bacterium selected from the group consisting of Porphyromonas, Prevotella and Tannerella species, preferably selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia within the biofilm, while the remaining bacteria in the biofilm preferably remain largely unaffected (ie, die or are significantly less inhibited in growth). Said biofilm is preferably a complex biofilm, more preferably a natural biofilm and even more preferably a natural oral biofilm.

The present disclosure further provides a bacQC inhibitor compound or pharmaceutical composition for use in a method of treating or preventing an acute, chronic or recurrent periodontal disease or condition. The main categories of periodontal diseases and conditions are divided into the groups of plaque-induced gum disease, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal disease, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections. As used herein, acute, chronic or recurrent periodontal disease is preferably selected from the group consisting of plaque-induced gum disease, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal disease, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections. Also provided herein is a method for the treatment or prevention of acute, chronic or recurrent periodontal disease, which is preferably selected from the group consisting of plaque-induced gum disease, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic diseases, necrotizing periodontal disease, periodontal abscesses , periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections, the method comprising administering a therapeutically effective amount of a bacQC inhibitor compound or a pharmaceutical composition according to the present disclosure to the subject in need thereof. Other acute, chronic or recurrent periodontal diseases are described, for example, in Armitage, A. Ann Periodontol 1999, 4, 1-6.

In one embodiment of the present disclosure, a compound or pharmaceutical composition according to the present disclosure is preferably used in any of the methods described above, where the method of administration is topical administration and/or where the method is a non-surgical method. In another embodiment, the inhibitor compound or pharmaceutical composition according to the present disclosure is preferably used in any of the methods described above, where the method of administration is systemic administration and/or where the method is a non-surgical method.

As used herein, the term “subject” refers to an animal, preferably a mammal, most preferably a human, who is or has been the subject of treatment, therapy, prophylaxis, observation or experiment.

The term "therapeutically effective amount" as used herein means that amount of an active compound or pharmaceutical agent that produces a biological or drug response in a tissue system, animal, or human being sought by a researcher, veterinarian, physician, or other health care professional, which includes alleviation of disease symptoms. or disorder being treated.

Protein crystals

Presented herein are crystals of bacterial glutamyl cyclases (bacQC).

The atomic coordinates in the crystal structure of PgQC are shown in Table 1 on pages 21–42 of European Patent Application EP18158343, filed with the European Patent Office on 23 February 2018, the priority of which is claimed by this application and to which reference is made; or alternatively, in the RCSB Protein Database (www.rcsb.org) under PDB identification code 6QQL, registered on February 19, 2019. In one embodiment, the bacterial glutamyl cyclase (bacQC) containing crystal is a PgQC containing crystal, where PgQC is a polypeptide containing the amino acid sequence of SEQ ID NO. 1, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 1, and preferably having:

- atomic coordinates according to Table 1 of EP18158343 or 6QQL as above, or where the standard deviation from the backbone atoms of the polypeptide chain according to Table 1 of EP18158343 or 6QQL as above is 2 Å or less, and/or

- binding pocket defined by Gln133 residues; Asp149; Glu182; Asp183; Tyr187; Gly188; Asp189; Asp190; Trp193; Cys194; Asp218; Met219; Phe232; Gly263; Ala264; Leu265; Thr266; Asp267; Val270; Ile284; Tyr286; Asn290; Glu291; His292; Gly293; Phe294; Trp298; His299 from SEQ ID NO: 1 and/or

- space group P 3 ₁ 2 1 and unit cell dimensions a = 89.9 Å, b = 89.9 Å, c = 164.7 Å, α = 90°, β = 90° and γ = 120°.

In addition, preferably the PgQC crystal diffracts x-rays to determine the atomic coordinates of the crystal with a resolution of 2.81 to 44.95 Å.

The atomic coordinates in the crystal structure of TfQC are shown in Table 2 on pages 43 to 91 of European Patent Application EP18158343, filed with the European Patent Office on February 23, 2018, the priority of which is claimed by this application and to which reference is made; or alternatively in the RCSB Protein Database (www.rcsb.org) under PDB ID code 6QRO, registered February 19, 2019.

In another embodiment, the bacterial glutamyl cyclase (bacQC)-containing crystal may be a TfQC-containing crystal, wherein TfQC is a polypeptide containing the amino acid sequence of SEQ ID NO. 2, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO. 2 and preferably having:

- atomic coordinates according to Table 2 of EP18158343 or 6QQL as above, or where the standard deviation from the backbone atoms of the polypeptide chain according to Table 2 of EP18158343 or 6QQL as above is 3 Å or less, and/or

- binding pocket defined by His126 residues; Arg130; His135; Glu183; Asp184; Gly186; Thr187; Glu189; Lys195; Pro196; Asp197; Trp199; Asp224; Met225; Gly269; Ala270; Ile271; Val272; Gln276; Tyr277; Ile290; Tyr292; Gln297; Ser298; Gly299; Phe300; Trp304; His305 from SEQ ID NO: 2 and/or

- space group P 1 and unit cell dimensions a = 56.1 Å, b = 79.2 Å, c = 83.1 Å, α = 89.9°, β = 90.0° and γ = 71.9° .

In addition, preferably the TfQC crystal diffracts x-rays to determine the atomic coordinates of the crystal with a resolution of 2.10 to 38.28 Å.

The standard deviation from the backbone atoms of a polypeptide chain can be defined as the positional standard deviation from the structural coordinates of the equivalent backbone atoms C _α after structural alignment of the structure, which will be compared with the structure of the protein in accordance with the present invention and using the DALI program (L. Holm and C. Sander, Science, 1996, vol. 273, 595-602). The term "binding pocket" as used herein includes references to a specific region (or atom) in a molecular entity capable of entering into a stabilizing interaction with another molecular entity. In some embodiments, the term also refers to the reactive portions of a macromolecule that are directly involved in its particular combination with another molecule. In an alternative embodiment, the binding region comprises or is defined by the three-dimensional arrangement of one or more amino acid residues in the folded polypeptide. Most preferably, the bacQC binding pocket described herein is defined, respectively, by the atoms of the residues listed for each of the bacQCs above.

In a specific embodiment of the invention, the bacterial glutaminyl cyclase (bacQC)-containing crystal is a crystal containing a polypeptide fragment containing a truncated amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 90% or more sequence identity with the truncated amino acid sequence of SEQ ID NO. 1. The specified truncated amino acid sequence is SEQ ID NO. 1 is obtained by deleting from 1 to 132, preferably from 1 to 20, more preferably from 2 to 10 consecutive amino acids, starting from the N-terminus of the amino acid sequence SEQ ID NO. 1, and/or by deletion of 1 to 34, preferably 1 to 10, more preferably 2 to 5 consecutive amino acids starting from the C-terminus of SEQ ID NO. 1.

In another specific embodiment of the invention, the bacterial glutamyl cyclase (bacQC)-containing crystal is a crystal containing a polypeptide fragment containing a truncated amino acid sequence of SEQ ID NO. 2, or an amino acid sequence having 90% or more identity with the truncated amino acid sequence of SEQ ID NO. 2. Said truncated amino acid sequence is SEQ ID NO. 2 is obtained by deleting from 1 to 125, preferably from 1 to 21, more preferably from 2 to 10 consecutive amino acids, starting from the N-terminus of the amino acid sequence SEQ ID NO. 2, and/or by deletion of 1 to 29, preferably 1 to 10, more preferably 2 to 5 consecutive amino acids starting from the C-terminus of SEQ ID NO. 2.

In yet another embodiment, a cocrystal containing bacQC as described above is provided along with a candidate compound. The candidate compound is preferably bound to the bacQC binding pocket. The candidate compound is further preferably a bacQC inhibitor. Most preferably, the candidate compound is a compound according to one of formulas I or II, as specified in any of aspects <1> to <19>.

The crystals and cocrystals of the present invention are preferably of sufficient quality and size to permit determination of the three-dimensional X-ray diffraction structure of bacterial glutamyl cyclase with a resolution of about 1.9 Å and about 2.8 Å.

Both crystal structures have an α-β hydrolase fold, which is defined by an 8-stranded β-sheet surrounded by 7 α-helices. In both cases, the Zn ion is tetrahedrally coordinated to 2 aspartates and 1 histidine, respectively. The fourth coordination site is occupied by a water molecule, substrate, or metal-binding group as a substructural part of the inhibitor. These small group metal–Zn ion interactions are important for the development of an active compound used as an antibacterial agent against the pathogens mentioned here. Moreover, the binding sites are formed by hydrophobic and hydrophilic regions, which are different from human enzymes and are responsible for creating selectivity for bacterial enzymes (see Fig. 7–10).

Crystals and co-crystals in accordance with the present invention can be obtained using batch methods, liquid bridge methods, dialysis, vapor diffusion, such as pendant or sessile drop vapor diffusion, as further described below.

One preferred method for preparing a bacQC crystal, as defined above, involves the steps of:

(a) providing a solution containing bacQC as defined above, preferably in the presence of 150 mM NaCl, in a suitable buffer such as 50 mM Tris-HCl pH 8.0;

(b) mixing said solution with an equal amount of crystallization solution containing (i) 1 M HEPES pH 8.0, 2 M ammonium sulfate, 3% (v/v) PEG400 in the case of PgQC; or (ii) 0.1 M sodium acetate, pH 4.6, 2 M ammonium sulfate in the case of TfQC; And

(c) incubating the resulting mixture under conditions conducive to vapor diffusion of the hanging or sessile drop for a time sufficient to obtain a bacQC crystal.

One method for preparing a bacQC cocrystal with a candidate compound involves the steps of:

(a) providing a solution containing bacQC as defined above, preferably PgQC, and the candidate compound dissolved in 100 mM HCl, in a molar ratio of bacQC: compound = 1: 2.3;

(b) mixing said solution with an equal amount of crystallization solution containing 1 M HEPES pH 8.0, 2 M ammonium sulfate, 3% (v/v) PEG400; And

(c) incubating the resulting mixture under conditions conducive to vapor diffusion of the hanging or sessile drop for a time sufficient to obtain a cocrystal of bacQC and the candidate compound.

Another method for preparing a bacQC co-crystal with a candidate compound involves the steps:

(d) providing a solution as a seed of collected and crushed bacQC, preferably TfQC crystals without the candidate compound obtained as defined above;

(e) providing a solution containing bacQC as defined above and the candidate compound dissolved in 100 mM HCl, in a molar ratio of bacQC: compound = 1: 1.2;

(f) mixing said solution with an equal amount of crystallization solution containing 1M potassium granules pH 6.6 or pH 6.8, 32.5% (v/v) PEG 600, 0.1 M NaCl and 15% stock solution seeds;

(g) incubating the resulting mixture under conditions conducive to vapor diffusion of the hanging or sessile drop for a time sufficient to obtain a cocrystal of bacQC and the candidate compound.

Methods for identifying candidate compounds and/or inhibitors

The present invention provides a method for identifying a candidate compound that can bind to the bacQC binding pocket, comprising the following steps:

(a) creating a 3D model of the corresponding bacQC using the structural coordinates described in Table 1 of EP18158343 or 6QQL as above, or 2,

(b) analysis of the binding pocket provided by Gln133 residues; Asp149; Glu182; Asp183; Tyr187; Gly188; Asp189; Asp190; Trp193; Cys194; Asp218; Met219; Phe232; Gly263; Ala264; Leu265; Thr266; Asp267; Val270; Ile284; Tyr286; Asn290; Glu291; His292; Gly293; Phe294; Trp298; His299 from SEQ ID NO: 1 according to the coordinates of Table 1 from application EP18158343 or 6QQL as above, or analysis of the binding pocket provided by His126 residues; Arg130; His135; Glu183; Asp184; Gly186; Thr187; Glu189; Lys195; Pro196; Asp197; Trp199; Asp224; Met225; Gly269; Ala270; Ile271; Val272; Gln276; Tyr277; Ile290; Tyr292; Gln297; Ser298; Gly299; Phe300; Trp304; His305 from SEQ ID NO: 2 according to the coordinates of table 2 of application EP18158343 or 6QQL as above;

(c) performing a computer simulation analysis to identify a candidate compound that may associate with the binding pocket of the corresponding bacQC.

Preferably the method further includes:

(d) testing in vitro whether the candidate compound reduces the catalytic activity of bacQC, thereby classifying the candidate compound that reduces the catalytic activity of bacQC as a bacQC inhibitor.

Testing whether a candidate compound reduces the catalytic activity of bacQC can be performed, for example, using a bacQC inhibitor identification method comprising the following steps (d1) to (d5):

(d1) providing a composition containing a bacQC substrate and bacQC;

(d2) providing the candidate compound specified in (c);

(d3) contacting the candidate compound with the composition;

(d4) monitoring the catalytic activity of bacQC;

(d5) classifying the candidate compound as a bacQC inhibitor based on the effect of the candidate compound on the catalytic activity of bacQC, where the candidate compound that reduces the catalytic activity of bacQC is classified as a bacQC inhibitor.

Said composition (in step d1) may be an aqueous solution, preferably containing a suitable buffer and/or salt component. Said substrate is preferably a peptide or peptide derivative containing a glutamine residue at its N-terminus. Said substrate is preferably labeled, more preferably isotopically labeled, most preferably a fluorogenic substrate. The fluorogenic substrate preferentially undergoes a change in fluorescence intensity after conversion through the bacQC-catalyzed reaction.

A suitable fluorogenic substrate for monitoring bacQC activity is H-Gln-AMC, as described in Schilling, S., Hoffmann, T., Wermann, M., Heiser, U., Wasternack, C., and Demuth, H.-U. Anal. Biochem. 2002, 303, 49-56 (Schilling et al., 2002), as shown in the following diagram:

The candidate compound may be contacted before, simultaneously with, or after the addition of the remaining components of the composition in step (d1). The candidate compound is preferably in solution, more preferably as a dimethyl sulfoxide (DMSO) solution.

Substrate conversion is monitored over time, for example by monitoring the emission of a fluorophore produced upon cleavage of a fluorogenic substrate. bacQC activity can be determined from the AMC standard curve under assay conditions.

The catalytic activity of bacQC can be determined using different concentrations of substrate, bacQC and/or candidate compound. Suitable indicators of the catalytic activity of bacQC are, for example, inhibition constants ( _Ki ), 50% residual activity (RA) in the presence of a given concentration of a candidate compound and/or IC ₅₀ value.

The present invention also provides a method for identifying a candidate compound that binds to the bacQC binding pocket, the method comprising the following steps:

(a) creating a 3D model of a cocrystal containing bacQC as described above along with a candidate compound;

(b) analysis of the distances between the atoms of the candidate compound and the atoms of the binding pocket provided by the Gln133 residues; Asp149; Glu182; Asp183; Tyr187; Gly188; Asp189; Asp190; Trp193; Cys194; Asp218; Met219; Phe232; Gly263; Ala264; Leu265; Thr266; Asp267; Val270; Ile284; Tyr286; Asn290; Glu291; His292; Gly293; Phe294; Trp298; His299 from SEQ ID NO: 1, or the atoms of the binding pocket atoms at residues XXXX of His126; Arg130; His135; Glu183; Asp184; Gly186; Thr187; Glu189; Lys195; Pro196; Asp197; Trp199; Asp224; Met225; Gly269; Ala270; Ile271; Val272; Gln276; Tyr277; Ile290; Tyr292; Gln297; Ser 298; Gly299; Phe300; Trp304; His305 from SEQ ID NO: 2 to identify a candidate compound that binds to the bacQC binding pocket.

Preferably the method further includes:

(c) testing in vitro whether the candidate compound reduces the catalytic activity of bacQC, thereby classifying the candidate compound that reduces the catalytic activity of bacQC as a bacQC inhibitor.

Testing whether a candidate compound reduces the catalytic activity of bacQC can be performed, for example, using a bacQC inhibitor identification method comprising the following steps (c1) to (c5):

(c1) providing a composition containing a bacQC substrate and bacQC;

(c2) providing the candidate compound referred to in (b);

(c3) contacting the candidate compound with the composition;

(c4) monitoring the catalytic activity of bacQC;

(c5) classifying the candidate compound as a bacQC inhibitor based on the effect of the candidate compound on the catalytic activity of bacQC, where the candidate compound that reduces the catalytic activity of bacQC is classified as a bacQC inhibitor.

EXAMPLES

I. Identification and production of putative bacterial glutamyl cyclases (bacQCs)

a) Identification

Bioinformatics analysis of the secretome of oral pathogens P. gingivalis, T. forsythia and P. intermedia (http://www.oralgen.lanl.gov/) showed that about 80% of secreted proteins carrying a signal peptide are cleaved at the Xaa- peptide bond Gln signal peptidase. The N-terminal ends of the released proteins contain a pGlu residue. This implies the existence of glutamyl cyclases, which appear to be required for the translocation of growth proteins across the outer membrane and the growth of periodontal pathogens.

Figure 1 shows an amino acid sequence alignment of human QC (hQC, SEQ ID NO: 4) and a putative bacterial QC from P. gingivalis (PgQC, SEQ ID NO: 1) (A), as well as an amino acid sequence alignment of additional putative QC Ps. intermedia (PiQC, SEQ ID NO: 3) and T. forsythia (TfQC, SEQ ID NO: 2) and P. gingivalis (PgQC, SEQ ID NO: 1) (B). The estimated PgQC is 25% identical to human QC. In addition, the putative TfQC is 49% identical to PgQC, and QCs from P. intermedia share 42% identity with PgQC. The gray underlined cysteine residues reflect disulfide bridges in hQC that are absent in PgQC. Sequences in bold in human QC reflect highly conserved type II QC residues. Gray sequences describe the putative signal sequences of PgQC and hQC. In addition, the typical Asp-Glu-His metal-binding motif is presented in bold with underlining. A putative metal binding motif was also identified in TfQC and PiQC (B, bold letters). Alignments were prepared using the Clustal Omega program in EMBL-EBInet; (*) indicates positions that have one completely conserved residue, (:) indicates conservation between groups of highly similar properties, (.) indicates conservation between groups of weakly similar properties (http://www.ebi.ac.uk/Tools /msa/clustalo/).

BLAST analysis identified an open reading frame (ORF) encoding a putative QC protein in P. gingivalis (WP_005874301). The primary structure of this putative QC protein is 25% identical to human QC. In addition, putative QC proteins were also identified in the genome of the oral pathogens P. intermedia (WP_014709208) and T. forsythia (WP_014225037), which are 42% or 49% identical to putative QCs from P. gingivalis (Fig. 1). As shown by amino acid alignments, conserved human QC residues appear to differ in bacterial QCs from those conserved cysteine residues that form the disulfide linked in human, and other type II QCs do not appear to be present in all three putative bacterial QCs . However, the highly conserved metal-binding motif Asp144, Glu184 (Asp), and His322 type II QC ( Wintjens et al., 2006 ) appears to be present in a putative bacterial QC that may represent the catalytic center. The primary structures of these proteins may indicate that these proteins are indeed QCs.

b) Preparation

In fig. 2 shows protein polyacrylamide gel electrophoresis (SDS-PAGE) of purified recombinant putative bacterial QCs expressed in pLysS E. coli Rosetta (DE3) and purified as detailed below. Therefore, 30 μg of purified protein was loaded onto 12% SDS-PAGE and visualized by Coomassie staining, lane 1, unstained PageRuler Broad Range (Thermofisher Scientific), lane 2, HisPgQC, lane 3, HisPiQC, and lane 5, HisTfQC. All three putative bacterial QCs have a theoretical molecular mass of ≈37 kDa.

aa) Acceptor strains and environment

E. coli strain DH5α or XL-1 blue (Stratagene) was used for cloning procedures. E. coli Rosetta(DE3)pLysS (Novagene) was used for protein expression. Alkaline phosphatase activity assay was performed in strain CC118 pGP1-2 (Tabor, S. and Richardson, CC Proc. Natl. Acad. Sci. USA 1985, 82, 1074-1078; Manoil, C., J. J. Mekalanos and Beckwith, J. J. Bacteriol. 1990, 172 (2), 515-518). All E. coli strains were grown in Luria-Bertani medium as indicated at 20°C, 30°C, or 37°C. Antibiotics (ampicillin [50 to 125 mg/liter], chloramphenicol [15 to 30 mg/liter], and kanamycin [25 mg/liter]) were added as needed. To prepare solid media, 1.5% agar (Roth) was added to the appropriate broth.

bb) Molecular cloning of plasmid vectors encoding bacterial QCs.

All cloning procedures were performed using standard molecular biology techniques. The open reading frames (ORFs) used for protein expression are PgQC (SEQ ID NO: 1, putative QC from P. gingivalis), PiQC (SEQ ID NO: 3 putative QC from P. intermedia), and TfQC (SEQ ID NO: 2, putative QC from T. forsythia) were amplified using synthesized DNA sequences purchased from Eurofins Genomics as templates in the pilimerase chain reaction (PCR) to introduce the NheI/NdeI restriction site required for direct cloning into the pET28a(+) vector (Novagen) . To generate the PgQC-´PhoA fusion protein, the putative pgQC with the predicted signal sequence was subcloned into pET26b(+) through the NheI/XhoI restriction site using the primer pair seqpgQC NdeI (forward) and seqpgQC XhoI (reverse). In addition, pgQC with a native signal sequence including the ribosome binding sites of the pET26b(+) vector was amplified using the primer pair seqpgQC RBS NotI (forward) and seqpgQC XbaI (reverse) for cloning into the phoA expression vector pECD637. All cloning primers were purchased from Metabion and are described in Table 3.

Table 1. Oligonucleotides used for cloning bacQC constructs

SEQ ID NO: Primer Sequence (5’→ 3’) 5 pgQC NdeI (direct) AAA CAT ATG AAC GGC AAT AAC ACA AGT GAA 6 pgQC NheI (reverse) TTT GCT AGC TCA GTG TGA AGC GGC TTT 7 piQC NdeI (direct) TTT CAT ATG AAA GGA AAA TCG TCT AAC 8 piQC NheI (reverse) AT G CTA GC T TAC ATG CTG TAA AGC AC 9 tfQC NdeI (direct) TCA CAT ATG GGT CAG AAA AAT ACG ACA 10 tfQC NheI (reverse) AT G CTA GC T TAT TTC TCA TTA TAA ATC AC eleven seqpgQC NdeI (direct) AAA CAT ATG AAA AGA CTG ATA ACA ACA GGA GCA GCC TTT CTA CTG GCT GCT ACA CTC TCT GCC TGC AAC GGC AAT AAC ACA AGT GAA ACG 12 seqpgQC XhoI (reverse) TTT CTC GAG GTG TGA AGC GGC TTT CAC 13 seqpgQC RBS NotI (direct) TG G CGG CCG C TA AGA AGG AGA 14 seqpgQC XbaI (reverse) TTT TCT AGA GTG TGA AGC GGC TTT CAC

cc) Expression of bacterial QC as a His Tag fusion protein or a ´PhoA fusion protein

The expression vector pET28a (+):: pgQC was transformed into E. coli Rosetta(DE3)pLysS. Bacteria were grown in Luria-Bertani medium containing kanamycin (25 μg/ml) and chloramphenicol (15 μg/ml) at 37 °C until the cell density reached OD 600 ~ 0.6. Cultures were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and supplemented with 2% (v/v) ethanol, followed by incubation for 16 h at 20°C. Cultures were harvested by centrifugation at 4 °C and 3900 g for 30 min, and the cell pellet was stored at –20 °.

To express the seqPgQC-´PhoA fusion protein, the recombinant vector pECD637::seqpgQC was transformed into CC118 (without, for example, the phoA gene) together with the helper plasmid pGP1-2. Cultures were inoculated at 30°C overnight. Additionally, overnight cultures were diluted in fresh Luria-Bertani medium to a final optical density (OD600) of ~0.4, followed by incubation at 42°C for 20 min to induce expression of seqPgQC-´PhoA fusion proteins. The cultures were then inoculated for another 2 h at 30°C. Finally, OD600 was determined using a spectrophotometer (BioRad) and 200 μl of cultures were collected by centrifugation at 4 °C and 16,000 g for 15 min to determine PhoA activity.

dd) Purification of bacterial QCs

Cell pellets from a 500-mL culture were resuspended in 20 mL of buffer consisting of 50 mM Tris-HCl, 150 mM NaCl pH 8.0, 10 μg/mL DNase, and protease inhibitor cocktail mixture (full mini-tablets, EDTA-free, Roche). Cells were disrupted by passing through a French press (Thermo Scientific, Waltman, MA, USA) 3–4 times. Cellular debris was removed by centrifugation at 4 °C and 30,000 g for 30 min. The supernatant was diluted 1:2 with equilibration buffer and applied to a 5 mL HisTrap column (GE Healthcare). The column was equilibrated with 50 mM Tris-HCl containing 150 mM NaCl, pH 8.0. After washing with several column volumes of equilibration buffer and at least 20 mM imidazole protein were eluted using multi-step gradients, reaching a final concentration of 250 mM imidazole, while most of the pure protein had already been eluted with approximately 100 mM imidazole. All fractions containing bacQC were pooled, concentrated using Vivaspin 20 (Sartorius AG) and applied to a HiPrep 26/10 desalting column (GE, Healthcare), which was equilibrated with 50 mM Tris-HCl, 150 mM NaCl, pH 8.0. Purified proteins were analyzed by SDS-PAGE and protein content was determined by absorbance at 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Science) or by the methods of Bradford or Gill and von Hippel (Bradford, MM 1976 Anal Biochem 72, 248-254; Gill , SC and von Hippel, PH 1989 Anal Biochem 182, 319-326). Finally, purified recombinant bacQC fusion proteins were frozen in liquid nitrogen and stored at −80°C or glycerol was added to a final concentration of 50% and stored at −20°C. Its N-terminal fusion protein tag was removed using 1 unit of thrombin per 1 mg of fusion protein (Thrombin Cleavage Capture Kit, Novagen) in the presence of 20 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl ₂ at pH 8.0 followed by incubation for 16 hours at 4 °C. Thrombin was removed by coupling to steptavidin agarose (Thrombin Cleavage Capture Kit, Novagen), and bacQC proteins without His Tag proteins were isolated by centrifugation. After an additional desalting step using HiPrep (26/10), bacQC fractions were pooled and concentrated using Vivaspin 20 (Sartorius AG). Finally, recombinant bacQC in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 was shock frozen in liquid nitrogen and stored at −80 °C or glycerol was added to a final concentration of 50% and stored at −20 °C. PiQC was expressed and purified in the same way as PgQC. TfQC was expressed in the same way as PgQC and purified using a HiTrap Talon column (GE, Healthcare).

Conclusions: All putative bacterial QCs were expressed in E. coli Rosetta(DE3)pLysS without the signal sequence as N-terminal His Tag fusion proteins and purified to homogeneity by affinity chromatography. 16-40 mg of recombinant pure His-Tag fusion proteins were isolated from approximately 500 ml of induced E. coli cultures. Subsequently, the His-Tag fusion proteins were digested with thrombin, followed by enzymatic characterization of these putative bacterial QCs.

II. Protein crystallization

a) Crystallization of PgQC.

Crystallographic quality crystals were obtained from PgQC expressed and purified as described above. PgQC was crystallized at a concentration of 90 μM (2.3 mg/mL) for 40 days at 15 °C using the hanging drop vapor diffusion method. In a drop, 1 μl of protein solution was mixed with an equal volume of crystallization buffer consisting of 0.1 M HEPES pH 8.0, 3% (v/v) PEG 400 and 2 M (NH ₄ ) ₂ SO ₄ . For the crystallization process, a droplet containing the protein was steam equilibrated with 500 μL of crystallization buffer located in the reservoir.

Before X-ray measurements, the crystals were cryoprotected by quick soaking in a crystallization buffer containing 18% (v/v) glycerol and then flash frozen at –180 °C. In-house data from the diffracting single crystal were collected using a CCD detector (SATURN 944+, Rigaku Europe) mounted on a rotating anode copper source (RA Micro 007, Rigaku Europe). Subsequently, the collected diffraction data were processed, scaled and combined using XDSGUI. The measured PgQC crystals belong to the trigonal space group P 3 ₁ 21 with cell constants a = 89.9 Å, b = 89.9 Å, c = 164.7 Å, α = 90°, β = 90°, γ = 120°. They contain two PgQC molecules in an asymmetric unit and diffract to 2.8 Å.

Initial phases for the PgQC structure were obtained by molecular replacement using the PHASER MR program from the CCP4 crystallographic suite and a PgQC search model with PQ 50, which underwent some rounds of manual recovery with the program COOT and maximum likelihood refinement with REFMAC5. The model was refined to an R _work value of 0.2428 and an R _free value of 0.30404. To obtain the final PgQC model, it is sufficient to repeat cycles of manual restructuring and refinement.

b) Crystallization of TfQC.

Crystallographic quality crystals were obtained from TfQC expressed and purified as described above. The protein could be crystallized at a concentration of 9.91 mg/mL (279 μM) for 91 days at 15 °C using the sessile drop vapor diffusion method. Therefore, 200 nl of protein was mixed with an equal volume of crystallization buffer consisting of 2M (NH ₄ ) ₂ SO ₄ and 0.1 M sodium acetate, pH 4.6, in a droplet remaining in contact by vapor diffusion with a reservoir containing 55 μl of crystallization buffer .

Before X-ray measurement, the crystals were cryoprotected by quick soaking in crystallization buffer containing 25% (v/v) ethylene glycol and then flash frozen at -180°C. The following data from a diffracting single crystal was collected in-house using a CCD detector (SATURN 944+, Rigaku Europe) mounted on a rotating anode copper source (RA Micro 007, Rigaku Europe).

The obtained diffraction data were processed, scaled and combined using the XDSGUI program. TfQC crystals belong to the triclinic space group P1 with cell constants a = 56.1 Å, b = 79.2 Å, c = 83.1 Å, α = 89.9°, β = 90°, γ = 71.9° . They contain four TfQC molecules in an asymmetric unit and diffract to 2.1 Å.

Initial phases of the proposed TfQC structure were obtained by molecular replacement using PHASER MR from the CCP4 crystallography package. For this decision strategy, a suitable search model must be generated since no TfQC model existed at this point in time. This model was prepared from the original PgQC model with PQ 50 by side chain trimming using the SCULPTOR program from the PHENIX kit. The final TfQC model was obtained after repeated rounds of manual rebuilding using the COOT program and maximum likelihood refinement using PHENIX.REFINE. It was refined to an R _work value of 0.1938 and an R _free value of 0.2404.

III. Fluorometric assay for glutaminyl cyclase activity

QC activity was assessed using H-Gln-AMC as substrate (as previously described in Schilling et al., 2002).

The assay consists of various concentrations of fluorogenic substrate and 0.5 units. Pyroglutaminylaminopeptidase in 50 mM Tris-HCl, 50 mM NaCl at pH 8.0. After 10 minutes of incubation at 30°C, the reaction was initiated by adding bacQC to a final volume of 125 μl of the reaction mixture. The excitation/emission wavelength was 380/460 nm. bacQC activity was determined from the AMC fluorophore standard curve under assay conditions. All determinations were performed in 96-well microtiter plates (Fisher Scientific) at 30°C using FluoStar Optima (BMG Labtech). Kinetic data were assessed using GraFit software (version 7, Erithacus software Ltd., Horley, UK).

In FIG. Figure 3 shows Lineweaver-Burk plots for the cyclization of H-Gln-AMC catalyzed by PgQC (A), PiQC (B), and TfQC (C). The inset shows a secondary plot of the resulting Lineweaver-Burk estimator slopes. Measurement of bacQC activity was carried out in 50 mM Tris-HCl, pH 8.0 and 50 mM Tris-HCl at 30°C. Kinetic data were assessed using GraFit software (version 7, Erithacus software Ltd., Horley, UK). Enzymatic parameters of bacterial QC have been determined (Table 4), which differ from those of hQC (Schilling, S., Manhart, S., Hoffmann, T., Ludwig, H.-H., Wasternack, C., and Demuth, H. - U. Biol. Chem. 2003, 384, 1583-1592).

Table 2. Kinetic parameters of H-Gln-AMC conversion using bacterial quality control. Determination of kinetic parameters was carried out in 50 mM Tris-HCl pH 8 and 50 mM NaCl at 30°C.

Enzyme Km [µM] kcat [With^-1] kcat/Km [mm^-1 With^-1] PgQC 510.94 ± 13.3 4.71 ± 0.22 9.24 ± 0.28 PiQC 645.33 ± 7.4 8.51 ± 0.37 13.18 ± 0.05 TfQC 1091.67 ± 34.36 4.49 ± 0.22 4.1 ± 0.14

Conclusions. Purified recombinant proteins were tested for enzymatic activity using a fluorometric assay with H-Gln-AMC as substrate. H-Gln-AMC as a substrate was transferred by these bacterial proteins, resulting in an increase in RFU and hence an increase in the reaction rate (Figure 3). The affinity for H-Gln AMC as a substrate in the case of PgQC and PiQC is approximately ten times lower than that of hQC for this substrate. The K _m value for TfQC is actually 20 times higher than the K _m value for hQC. In addition, the efficiency of bacterial proteins appears to be similar to that of hQC, with PiQC having twice the turnover compared to PgQC or TfQC.

IV. Bacterial glutaminyl cyclase activity inhibitor assay

For inhibitor testing, sample composition was the same as described above except for the addition of the putative inhibitory compound. This resulted in the presence of 1% or 2% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture. Inhibition constants were determined using different concentrations of H-Gln-AMC, ranging from 1/4 K _m to 2 K _m . The final bacterial QC concentration ranged from 25 nM to 50 nM. The inhibition constant was estimated by fitting a set of process curves to a general competitive inhibition equation using GraFit software (version 7, Erithacus software Ltd., Horley, UK).

In FIG. Figure 4 shows typical v/S characteristics, Lineweaver-Burk and Eadie-Hofstee plots for the PgQC-catalyzed cyclization of H-Gln-AMC (A) in the presence of (●) 1 µM, ( ) 0.5 µM, ( ) 0.25 µM, (△) 0.125 µM and ( ) 0.063 µM reference compound MWT-S-00431 ((3,5-dichlorophenyl-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methanone). (○) represents a reaction without inhibitor. Determinations were carried out in 50 mM Tris-HCl, 50 mM NaCl pH 8.0 and 1% (v/v) DMSO at 30°C. The kinetic parameters shown in Table 5 were obtained.

Table 3. Kinetic parameters of inhibition of H-Gln-AMC conversion by PgQC in the presence of the reference compound MWT-S-00431.

Parameter Meaning Standard error Vmax [µM min-1] 4.41161 1.0569 10-1 Km [M] 6.45485 10-4 3.14087 10-5 Ki[M] 1.03698 10-7 3.31239 10-9

V. Inhibition of glutaminyl cyclase by compounds

The following compounds were synthesized as described in the synthesis methods below. The average K _i values for inhibition of PgQC (isolated and purified as described above), TfQC, PiQC and hQC were measured using the inhibitor assay above and are shown. K _i denotes the average values of K _i measured as described above, and Std(K _i ) refers to the standard deviation of K _i .

Table 4. Inhibition of PgQC, TfQC, PiQC and hQC by bacQC inhibitors.

Structure of matter Example PgQC TfQC PiQC hQC No. Ki [nM] Ki [nM] Ki [nM] Ki [nM] 1 1945 ± 318 8255 ± 361 9400±325 8800 ± 127 2 149 ± 4 1400 ± 156 1945 ± 148 1280 ± 71 3 157 ± 6 no data no data no data 4 53 ± 1 no data no data 655 ± 27 5 106 ± 13 no data no data 769 ± 153 6 77 ± 2 no data no data 599 ± 40 7 83 ± 3 no data no data no data 8 185 ± 11 no data no data 1870 ± 78 9 442 ± 71 no data no data no data 10 395 ± 5 no data no data no data eleven 139 ± 4 no data no data no data 12 44 ± 1 680 ± 45 728 ± 35 543 ± 20 13 133 ± 6 n.d. n.d. 556 ± 28 14 53 ± 2 358 ± 64 542 ± 44 431 ± 19 15 34 ± 2 no data no data no data 16 30 ± 2 317 ± 52 815 ± 4 1067 ± 203 17 14 ± 0.1 123 ± 16 349 ± 17 455 ± 14 18 86 ± 5 no data no data no data 19 15 ± 0.1 no data no data no data 20 42 ± 4 no data no data no data 21 19 ± 1 no data no data no data 22 30 ± 1 no data no data no data 23 65 ± 13 no data no data no data 24 67 ± 1 no data no data no data 25 38 ± 3 no data no data no data 26 33 ± 1 no data no data no data 27 60 ± 2 no data no data no data

Conclusion : All compounds according to the present invention exhibit inhibitory activity against bacterial glutamyl cyclases. The above results indicate that the compounds and/or pharmaceutical compositions are useful for the treatment of periodontal disease and related diseases. In particular, the compounds and/or pharmaceutical compositions are capable of selectively targeting pathogens that cause periodontal disease (P. gingivalis, T. forsythia and P. intermedia) while being substantially inert towards the homologous human enzyme.

In addition, it was found that the remaining natural biofilm should be largely preserved. Even more preferably, the compounds and/or pharmaceutical compositions exhibit high in vivo activity against the target pathogens.

In addition, it has been discovered that when these pathogens are embedded in a biofilm (eg, complex biofilm, natural biofilm, or natural oral biofilm), the remaining bacteria in the biofilm preferably remain substantially unaffected by the compounds of the present invention (i.e., killed or their growth is suppressed to a much lesser extent).

As can be seen from the experimental data above, particularly high inhibitory activity can be achieved when the imidazo[4,5-b]pyridine ring is substituted at position 6 (i.e. in accordance with formula I, as in example 2), in different from the substitution in the 5-position (i.e. according to Fromula II, as in Example 1). However, in both cases there was significant inhibitory activity of bacQC and selectivity for PgQC compared to the human enzyme hQC (Table 7).

Table 5. Comparison of inhibition parameters of compounds from examples 1 and 2

Target
protein Ki [µM] Relative inhibitory activity
[Ki(Example 1)]/[Ki(Example 2)] Example 1 Example 2 PgQC 1.95 0.15 13 PiQC 9.4 1.95 4.8 TfQC 8.3 1.4 5.9 hQC 8.8 1.28 6.9

Finally, due to the relatively high degree of homology between PiQC and the other two bacQCs (as shown in Fig. 1), compounds identified as inhibitors of PgQC and/or TfQC using the methods of the present invention can also be expected to exhibit significant PiQC activity (as evidenced by the experimental data in Table 6 above).

VI. Determination of seqQC-´PhoA activity

Figure 5 shows PhoA activity in permeabilized E. coli CC118 pGP1-2 cells expressing seqPgQC-`PhoA fusion proteins. PgQC with the native putative signal sequence was cloned into pECD637 to generate the seqPgQC-`PhoA fusion protein. The resulting vector was transformed into E. coli strain CC118 pGP1-2. Expression of the fusion protein was initiated by incubating the culture at 42°C for 20 min. The PhoA activity of the induced culture was determined as described previously (Pribyl, T., Topologie des CzcCBA-Efflux-Komplexes aus Ralstonia Metallidurans CH34. Thesis, 2001, Martin Luther University Halle-Wittenberg). Black shading represents PhoA activity of E. coli cells expressing seqPgQC-`PhoA, striped shading represents PhoA activity of cells expressing `BlaM-`PhoA and served as a positive control, and cells expressing the vector without insert served as a negative control. (without filling).

PhoA activity was determined in cultures grown as described above using the chromogenic substrate p-nitrophenyl phosphate (PNPP) (Pribyl, 2001). Therefore, 200 μl of cell suspension with known OD600 was collected at 13,000 rpm at 4°C for 10 minutes. The cell pellet was washed with 0.5 ml of 10 mM Tris-HCl pH 8.0, 10 mM MgSO ₄ and 1 mM iodoacetamide. The resulting cell pellet was resuspended in 1 ml of 1M Tris-HCl pH 8.0, 0.1 mM ZnCl ₂ and 1 mM iodoacetamide. Then, 50 μL of 0.1% (w/v) sodium dodecyl sulfate (SDS) and 50 μL of chloroform were added to the mixture, followed by incubation for 5 minutes at 37°C. The reaction was then initiated by adding 100 μl of substrate solution (1 M Tris-HCl pH 8.0, 0.4% (w/v) PNPP). The reaction mixture was further incubated at 37°C until the solution turned yellow. The reaction was then stopped by adding 120 μl of 1 M KH ₂ PO ₄ , 0.5 M EDTA, pH 8.0. The time required for the yellow color to develop determined the specific enzymatic activity of PhoA. Cellular debris was removed by centrifugation (13,000 rpm, 20 min) and the optical density of the supernatant was determined at 420 nm. PhoA activity was determined according to the Lambert-Beer law. Cultures of E. coli CC118 pGP1-2 carrying the empty vector pECD637 served as negative controls, whereas E. coli CC118 pGP1-2 with pECD619 ( Pribyl, 2001 ) served as positive controls. This plasmid encodes a short form of β-lactamase (´blaM), including a signal sequence downstream of the 'phoA domain.

Conclusion: In silico analysis of the PgQC open reading frame revealed a putative signal sequence. This indicates that PgQC is localized outside the cytoplasm of P. gingivalis. C-terminal fusions of PgQC, including native signal sequences, with alkaline phosphatase (PhoA) were constructed to investigate the “nature” of PgQC localization. Fusions with alkaline phosphatases are active exclusively when localized in the periplasm. Expression of seqPgQC-`PhoA resulted in a high phosphatase activity of 638.5 U/L ± 50.9 (Fig. 5). This indicates that PgQC is localized in the periplasm.

VII. Description of synthetic methods

a) Synthesis of 5-substituted imidazo[4,5-b]pyridinamines

5-Substituted imidazo[4,5-b]pyridinamines were prepared by a general method comprising: treating 6-chloropyridine-2,3-diamine with triethylorthoformate at elevated temperature to give 5-chloroimidazo[4,5-b]pyridine (Step A1); followed by cross-coupling of the product from Step A1 with the appropriate amine in the presence of a suitable Pd catalyst and base to give the corresponding 5-substituted imidazo[4,5-b]pyridinamine (Step A2).

The compound of Example 1 was prepared as described above, as shown in the following Scheme 1.

Scheme 1: Synthetic scheme for obtaining Example 1.

Example 1: N-Benzylimidazo[4,5-b]pyridin-5-amine.

Step A1: 5-Chloroimidazo[4,5-b]pyridine

A solution of 6-chloropyridine-2,3-diamine (718 mg, 5 mmol) in methanol (10 ml) and triethyl orthoformate (10 ml) was refluxed for 3 hours. The volatile components were evaporated and the residue was purified by flash chromatography on silica using a gradient of CHCl ₃ -MeOH. Yield: 540 mg (58%); ESI-MS mass/charge 151.1 [M+H] ⁺ .

Step A2: N-benzylimidazo[4,5-b]pyridin-5-amine

5-Chloro[4,5-b]pyridine (207 mg, 1.1 mmol, 1 eq), DavePhos (10 mg, 0.0264 mmol, 0.25 eq) and Pd ₂ (dba) ₃ (9 mg, 0.011 mmol, 0.1 eq.) were dissolved in dry THF (15 ml) under argon. LiN(TMS) ₂ (1M in THF, 2.4 ml, 2.4 mmol, 2.2 eq.) and benzylamine (144 µl, 1.3 mmol, 1.2 eq.) were added and the mixture was stirred at 65° C for 18 hours. After cooling to room temperature, the mixture was acidified with aqueous HCl (5 N) and stirring was continued for another 10 minutes. The reaction mixture was poured into saturated aqueous NaHCO ₃ and extracted with EtOAc (3×25 ml). The combined organic layers were dried over MgCO ₃ and evaporated to dryness. The residue was purified by semipreparative liquid column chromatography (HPLC). Yield: 90 mg (24%). MS mass/charge: 225.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.93 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.55 (s, 2H); 6.80 (d, 1H, ³ J = 8.8 Hz); 7.22-7.27 (m, 1H); 7.30-7.37 (m, 4H); 7.83-7.91 (m, 2H); 9.08(s, 1H).

b) Synthesis of 6-substituted imidazo[4,5-b]pyridinamines.

6-Substituted imidazo[4,5-b]pyridinamines were prepared by a general process comprising the steps of: treating 2-chloro-3,5-dinitropyridine with ammonia in a suitable solvent or mixture of solvents to obtain 3,5-dinitropyridine-2-amine (Step B1 ); treating the product obtained in Step B1 with (NH ₄ ) ₂ S to obtain 5-nitropyridine-2,3-diamine (Step B2); treating the product obtained in Step B2 with triethyl orthoformate to obtain 6-nitroimidazo[4,5-b]pyridine (Step B3); treating the product obtained in step B3 with a suitable reducing agent to obtain imidazo[4,5-b]pyridin-6-amine (step B4); followed by treatment of the product obtained in step B4 with the appropriate aldehyde in the presence of a suitable reducing agent such as NaBH ₄ to obtain the corresponding N-aliklimidazo[4,5-b]pyridin-6-amine (step B5).

The synthetic route according to the above general method is illustrated in Scheme 2 below.

Scheme 2: Synthetic scheme for the preparation of 6-substituted imidazo[4,5-b]pyridinamines

aa) Synthesis of imidazo[4,5-b]pyridin-6-amine

Step B1: 3,5-dinitropyridine-2-amine

A solution of 2-chloro-3,5-dinitropyridine (5 g, 24.6 mmol, 1 eq.) in ethanol (150 ml) was treated dropwise with aqueous NH ₃ (7 ml, 123 mmol, 5 eq.) at 0°C . After addition was complete, the mixture was stirred at ambient temperature for 1 hour. The solid product was collected by filtration and dried. The compound was used without further purification. Yield: 4.17 g (92%).

Step B2: 5-nitropyridine-2,3-diamine

A solution of 3,5-dinitropyridin-2-amine (3.9 g, 21.2 mmol, 1 eq.) in methanol (80 ml) was treated with 20% aqueous (NH ₄ ) ₂ S (36.1 ml, 106 mmol, 5 eq.) and heated to boiling under reflux for 1 hour. After cooling, the solid was collected by filtration and dried. The product was used without further purification. Yield: 3.2 g (98%); ESI-MS mass/charge: 155.0 [M+H] ⁺ ; HPLC (gradient A): room temperature 4.08 min, 100%; ¹ H-NMR (DMSO-d6) δ: 5.31 (br s, 2H), 6.98 (br s, 2H), 7.36 (d, 1H, ⁴ J = 2.2 Hz), 8. 28 (d, 1H, ⁴ J = 2.6 Hz).

Step 3: 6-nitroimidazo[4,5-b]pyridine

A solution of 5-nitropyridine-2,3-diamine (2.7 g, 17.5 mmol) in methanol (14 ml) and triethyl orthoformate (14 ml) was stirred at 150°C in a microwave oven for 10 minutes. The solvents were evaporated, and the residue was used without further purification. Yield: 2.64 g (92%); ESI-MS mass/charge: 165.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 6.21 min, 100%.

Step 4: Imidazo[4,5-b]pyridin-6-amine

A solution of 6-nitroimidazo[4,5-b]pyridine (1240 mg, 7.5 mmol, 1 eq.) in aqueous hydrochloric acid (10%) was treated with SnCl ₂ (5076 mg, 22.5 mmol, 3 eq.) and heated in a microwave oven to 100°C for 30 minutes. After cooling to room temperature, the mixture was made alkaline with aqueous NaOH (1M) and evaporated. The residue was suspended in MeOH and filtered. The filtrate was evaporated and the residue was purified by flash chromatography (silica, gradient CHCl ₃ /MeOH with 0.5% NH ₃ ). Yield: 993 mg (98%). ESI-MS mass/charge: 135.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 1.47 min, 100%.

bb) General procedure for the synthesis of N-Aliclimidazo[4,5-b]pyridin-6-amines

Step 5: N-Aliclimidazo[4,5-b]pyridin-6-amines

Imidazo[4,5-b]pyridin-6-amine (1 eq.) and the corresponding aldehyde (1 eq.) were dissolved in EtOH (5 ml) and stirred at room temperature for 4 hours. NaBH ₄ (1.5 eq.) was added and stirring was continued overnight. The reaction was quenched with water and extracted with EtOAc (3×20 ml). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified by flash chromatography (silica, gradient CHCl ₃ /MeOH).

The following examples of 6-substituted imidazo[4,5-b]pyridinamines were prepared using the methods described above.

Example 2: N-Benzylimidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and benzaldehyde (38 μl, 0.4 mmol) as described above. Yield: 30 mg (36%); MS mass/charge: 225.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.45 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.33 (s, 2H); 6.35 (br s, 1H); 6.93(s, 1H); 7.21-7.26 (m, 1H); 7.31-7.36 (m, 2H); 7.39-7.43 (m, 2H); 7.94 (d, 1H, ⁴ J = 2.6 Hz); 8.07(s, 1H); 12.20 (br s, 1H).

Example 3: N-(3,4,5-Trifluorobenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 3,4,5-trifluorobenzaldehyde (80 mg, 0.5 mmol) as described above. Yield: 74 mg (53%). MS mass/charge: 279.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 10.13 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.34 (d, 2H, ³ J = 5.7 Hz); 6.44 (br s, 1H); 6.94(s, 1H); 7.31-7.39 (m, 2H); 7.92 (d, 1H, ⁴ J = 2.6 Hz); 8.10(s, 1H); 12.16 (br s, 1H).

Example 4: N-(4-Chlorobenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 4-chlorobenzaldehyde (52 mg, 0.4 mmol) as described above. Yield: 48 mg (50%); MS mass/charge: 259.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 9.97 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.33 (s, 2H); 6.39 (br s, 1H); 6.91(s, 1H); 7.36-7.45 (m, 4H); 7.93 (d, 1H, ⁴ J = 2.5 Hz); 8.08(s, 1H); 12.22 (br s, 1H).

Example 5: N-(3-Chlorobenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-chlorobenzaldehyde (42 μl, 0.4 mmol) as described above. Yield: 70 mg (73%); MS mass/charge: 259.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 9.84 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.36 (d, 2H, ³ J = 5.9 Hz); 6.42(s, 1H); 6.93(s, 1H); 7.27-7.31 (m, 1H); 7.35-7.39 (m, 2H); 7.45-7.47 (m, 1H); 7.93 (d, 1H, ⁴ J = 2.3 Hz); 8.08(s, 1H); 12.09 (br s, 1H).

Example 6: N-(3-Methoxybenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-methoxybenzaldehyde (46 μl, 0.4 mmol) as described above. Yield: 40 mg (42%). MS mass/charge: 255.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.51 min, 97%; ¹ H-NMR (DMSO-d6) δ: 3.73 (s, 3H); 4.28-4.32 (m, 2H); 6.34 (br s, 1H); 6.80 (dd, 1H, ³ J = 8.1 Hz, ⁴ J = 1.9 Hz); 6.92(s, 1H); 6.96-7.00 (m, 2H); 7.22-7.27 (m, 1H); 7.93 (d, 1H, ⁴ J = 2.5 Hz); 8.07(s, 1H); 12.18 (br s, 1H).

Example 7: N-(4-Methoxybenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-methoxybenzaldehyde (46 μl, 0.4 mmol) as described above. Yield: 25 mg (26%). MS mass/charge: 255.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.43 min, 97%; ¹ H-NMR (DMSO-d6) δ: 3.72 (s, 3H); 4.24(s, 2H); 6.25(br s, 1H); 6.85-6.97 (m, 3H); 7.29-7.35 (m, 2H); 7.92 (d, 1H, ⁴ J = 2.4 Hz); 8.06(s, 1H); 12.13 (br s, 1H).

Example 8: N-(3-thienylmethyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and thiophene-3-carbaldehyde (33 μl, 0.4 mmol) as described above. Yield: 60 mg (70%). MS mass/charge: 231.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 7.63 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.31 (s, 2H); 7.03 (d, 1H, ⁴ J = 2.5 Hz); 7.13 (dd, 1H, ³ J = 4.9 Hz, ⁴ J = 1.1 Hz); 7.37-7.40 (m, 1H); 7.49 (dd, 1H, ³ J = 4.9 Hz, ⁴ J = 3.0 Hz); 7.96 (d, 1H, ⁴ J = 2.5 Hz).

Example 9: N-(3-(1-piperidyl)benzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-(1-piperidyl)benzaldehyde (71 mg, 0.4 mmol) as described above. Yield: 56 mg (49%). MS mass/charge: 308.3 [M+H] ⁺ ; HPLC (gradient A): rt (double peak) 5.17/5.49 min, 100%; ¹ H-NMR (DMSO-d6) δ: 1.48-1.63 (m, 6H); 3.07-3.14 (m, 4H); 4.25(s, 2H); 6.76-6.81 (m, 2H); 6.95 (d, ⁴ J = 2.5 Hz); 6.99(s, 1H); 7.14 (t, 1H, ³ J = 7.8 Hz); 7.96 (d, 1H, ⁴ J = 2.5 Hz); 8.15(s, 1H).

Example 10: N-(2-Pyridylmethyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and pyridine-2-carbaldehyde (35 μl, 0.4 mmol) as described above. Yield: 6 mg (7%). MS mass/charge: 226.2 [M+H] ⁺ ; HPLC (gradient B): room temperature 4.96 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.43 (s, 2H); 6.97 (d, 1H, ⁴ J = 2.5 Hz); 7.25-7.30 (m, 1H); 7.42 (d, 1H, ³ J = 7.8 Hz); 7.72-7.78 (m, 1H); 8.01 (d, 1H, ⁴ J = 2.5 Hz); 8.27(s, 1H); 8.55 (d, 1H, ³ J = 4.5 Hz).

Example 11: N-(3-Pyridylmethyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and pyridine-3-carbaldehyde (35 μl, 0.4 mmol) as described above. Yield: 15 mg (18%). MS mass/charge: 226.2 [M+H] ⁺ ; HPLC (gradient B): room temperature 4.64 min, 94%; ¹ H-NMR (DMSO-d6) δ: 4.38 (s, 2H); 7.01 (d, 1H, ⁴ J = 2.5 Hz); 7.34-7.39 (m, 1H); 7.78-7.83 (m, 1H); 7.97 (d, 1H, ⁴ J = 2.5 Hz); 8.21(s, 1H); 8.46 (dd, 1H, ³ J = 4.7 Hz, ⁴ J = 1.4 Hz); 8.64 (d, 1H, ⁴ J = 1.6 Hz).

Example 12: N-(2,3-Dihydro-1,4-benzodioxin-6-yl-methyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 1,4-benzodioxane-6-carbaldehyde (61 mg, 0.4 mmol) as described above . Yield: 21 mg (20%). MS mass/charge: 283.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.59 min, 96%; ¹ H-NMR (DMSO-d6) δ: 4.16-4.23 (m, 6H); 6.27(br s, 1H); 6.77-6.94 (m, 4H); 7.91 (d, 1H, ⁴ J = 2.5 Hz); 8.08(s, 1H); 12.25 (br s, 1H).

Example 13: N-(3,4-Dimethoxybenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (134 mg, 1 mmol) and 3,4-dimethoxybenzaldehyde (166 mg, 1 mmol) as described above. Yield: 90 mg (32%); MS mass/charge: 285.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 7.79 min, 100%; ¹ H-NMR (DMSO-d6) δ: 3.70-3.76 (m, 6H); 4.24(s, 2H); 6.24(br s, 1H); 6.87-6.97 (m, 3H); 7.01-7.04 (m, 1H); 7.93 (d, 1H, ⁴ J = 2.3 Hz); 8.07(s, 1H); 12.16 (br s, 1H).

Example 14: N-(3,4-Dichlorobenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3,4-dichlorobenzaldehyde (65 mg, 0.4 mmol) as described above. Yield: 75 mg (69%); MS mass/charge: 293.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 11.09 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.36 (s, 2H); 6.45 (br s, 1H); 6.94 (d, 1H, ⁴ J = 1.6 Hz); 7.39 (dd, 1H, ³ J = 8.3 Hz, ⁴ J = 1.7 Hz); 7.59 (d, 1H, ³ J = 8.3 Hz); 7.66 (d, 1H, ⁴ J = 1.6 Hz); 7.93 (d, 1H, ⁴ J = 2.5 Hz); 8.09(s, 1H); 12.25 (br s, 1H).

Example 15: N-(4-propoxybenzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (134 mg, 1 mmol) and 4-propoxybenzaldehyde (158 μl, 1 mmol) as described above. Yield: 110 mg (39%); MS mass/charge: 283.3 [M+H] ⁺ ; HPLC (gradient A): room temperature 11.07 min, 100%; ¹ H-NMR (DMSO-d6) δ: 0.96 (t, 3H, ³ J = 7.4 Hz); 1.70 (sext, 2H, ³ J = 7.0 Hz); 3.88 (t, 2H, ³ J = 6.5 Hz); 4.24(s, 2H); 6.84-6.97 (m, 3H); 7.27-7.34 (m, 2H); 7.93 (d, 1H, ⁴ J = 1.4 Hz); 8.07(s, 1H); 12.18 (br s, 1H).

Example 16: N-(4-(2-aminoethoxy)benzyl)imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (268 mg, 2 mmol) and N-Boc-(4-(2-aminoethoxy)benzaldehyde (531 mg, 2 mmol) as described above followed by Boc deprotection using tetraacetic acid/dichloromethane (TFA/DCM) (1:1.2 ml) and TIS (100 µl), and purification by semipreparative HPLC. Yield: 53 mg (9%); MS wt/charge : 284.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 5.12 min, 100%; ¹ H-NMR (DMSO-d6) δ: 3.18-3.25 (m, 2H) ; 4.13 (t, 2H, ³ J = 5.0 Hz); 4.31 (s, 2H); 6.94-6.99 (m, 2H); 7.07 (d, 1H, ⁴ J = 2.4 Hz); 7.33-7.38 (m, 2H); 7.91-8.01 (m, 3H); 8.09 (d, 1H, ⁴ J = 2.4 Hz); 8.85(s, 1H).

Example 17: 1-(2-(4-((Imidazo[4,5-b]pyridin-6-ylamino)methyl)phenoxy)ethyl)guanidine.

A solution of N-(4-(2-aminoethoxy)benzyl)imidazo[4,5-b]pyridin-6-amine*TFA (41 mg, 0.08 mmol, 1 equiv.) in DMF was treated with N, N'-Bis (tert-butoxycarbonyl)-S-methylisothiourea (23 mg, 0.08 mmol, 1 eq.), 4-dimethylaminopyridine (DMAP) (1 mg, 0.008 mmol, 0.1 eq.) and triethylamine (TEA) (33 µl , 0.24 mmol, 3 equiv.). The mixture was stirred for 16 hours at room temperature, quenched with water and extracted with EtOAc (3×20 ml). The combined organic layers were dried over MgCO ₃ and evaporated. Boc deprotection was carried out using TFA/DCM (1: 1.2 ml) and TIS (100 μl). The volatile components were evaporated, and the residue was purified by semipreparative HPLC. Yield: 18 mg (41%); MS mass/charge: 326.1 [M+H] ⁺ ; HPLC (gradient A): room temperature 5.95 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.04 (t, 2H, ³ J = 5.3 Hz); 4.30(s, 2H); 6.91-6.95 (m, 2H); 7.07 (d, 1H, ⁴ J = 2.5 Hz); 7.32-7.36 (m, 2H); 7.64 (t, 1H, ³ J = 5.8 Hz); 8.09 (d, 1H, ⁴ J = 2.4 Hz); 8.86(s, 1H).

Example 18: N-[(4-Isopropoxyphenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 4-isopropoxybenzaldehyde (78 μl, 0.5 mmol) as described above. Yield: 6 mg (4%); MS mass/charge: 283.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 10.69 min, 89.7%; ¹ H-NMR (DMSO-d6) δ: 1.23 (d, 6H, ³ J = 5.9 Hz), 4.20-4.24 (m, 2H), 4.50-4.60 (m , 1H), 6.83-6.89 (m, 3H), 7.26. -7.32 (m, 2H), 7.92 (br s, 1H), 8.05 (br s, 1H), 12.02 (br s, 1H)

Example 19: N-[(3-Phenoxyphenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-phenoxybenzaldehyde (64 μl, 0.4 mmol) as described above. Yield: 19 mg (16%); MS mass/charge: 317.3 [M+H] ⁺ ; HPLC (gradient A): room temperature 12.13 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.30-4.36 (m, 2H), 6.41 (br s, 1H), 6.80-6.97 (m, 4H), 7.02 -7.20 (m, 3H), 7.28-7.36 (m, 3H), 7.82-8.16 (m, 2H), 12.03 (br s, 1H)

Example 20: N-[(4-Benzyloxyphenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 3-benzyloxybenzaldehyde (79 mg, 0.4 mmol) as described above. Yield: 47 mg (38%); MS mass/charge: 331.2 [[M+H] ⁺ ; HPLC (gradient A): room temperature 12.59 min, 97.8%; ¹ H-NMR (DMSO-d6) δ: 4.22-4.26 (m, 2H), 5.06 (s, 2H), 6.24 (br s, 1H), 6.86-7.00 (m, 3H), 7.28-7.45 (m, 7H), 7.90-7.93 (m, 1H), 8.05 (s, 1H), 12.05 (br s, 1H)

Example 21: N-[(4-Biphenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (50 mg, 0.4 mmol) and 4-biphenylcarboxaldehyde (68 mg, 0.4 mmol) as described above. Yield: 30 mg (27%); MS mass/charge: 301.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 12.37 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.37 (d, 2H, ³ J = 5.4 Hz), 6.40 (br s, 1H), 6.94 (br s, 1H), 7. 31-7.37 (m, 1H), 7.41-7.51 (m, 4H), 7.60-7.67 (m, 4H), 7.95 (s, 1H), 8.06 ( s, 1H), 12.04 (br s, 1H)

Example 22: N-[(2,6-Difluoro-4-methoxyphenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 2,6-difluoro-4-methoxybenzaldehyde (86 mg, 0.4 mmol) as described above . Yield: 77 mg (53%). MS mass/charge: 291.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 9.60 min, 100%; ¹ H-NMR (DMSO-d6) δ: 3.76 (s, 3H), 4.22 (d, 2H, ³ J = 5.9 Hz), 5.97 (br s, 1H), 6.71 -6.79 (m, 2H), 7.10 (br s, 1H), 7.92 (s, 1H), 8.09 (s, 1H), 12.16 (br s, 1H)

Example 23: N-[(7-Methoxy-1,3-benzodioxol-5-yl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (60 mg, 0.45 mmol) and 5-methoxypiperonal (81 mg, 0.45 mmol) as described above. Yield: 44 mg (33%); MS mass/charge: 299.2 [M+H] ⁺ ; HPLC (gradient A): room temperature 8.91 min, 100%; ¹ H-NMR (DMSO-d6) δ: 3.80 (s, 3H), 4.21 (s, 2H), 5.93 (s, 2H), 6.27 (br s, 1H), 6. 63 (s, 1H), 6.72 (s, 1H), 6.94 (s, 1H), 7.91 (s, 1H), 8.07 (s, 1H), 12.23 (s, 1H )

Example 24: N-[(2,2-Difluoro-1,3-benzodioxol-5-yl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 2,2-difluoro-1,3-benzodioxole-5-carboxaldehyde (93 mg, 0.5 mmol) as described above. Yield: 60 mg (40%); MS mass/charge: 305.3 [M+H] ⁺ ; HPLC (gradient A): room temperature 11.07 min, 100%; ^1H -NMR (DMSO-d6) δ: 4.34 (d, 2H, 3J = 6.4 Hz), 6.45/6.26 (br s, 1H, rotamer), 6.85/7.08 (br s, 1H, rotamer), 7.22-7.31 (m, 1H), 7.33-7.38 (m, 1H), 7.43 (s, 1H), 7.85-8, 15 (m, 2H), 12.02/12.59 (br s, 1H)

Example 25: N-[[4-(2-Pyridyl)phenyl]methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 4-(2-pyridyl)benzaldehyde (92 mg, 0.5 mmol) as described above. Yield: 25 mg (17%); MS mass/charge: 302.4 [M+H] ⁺ ; HPLC (gradient A): room temperature 6.00 min, 100%; ¹ H-NMR (DMSO-d6) δ: 4.39 (d, 2H, ³ J = 5.9 Hz), 6.45 (br s, 1H), 7.29-7.35 (m, 1H) , 7.51 (d, 2H, ³ J = 8.3 Hz), 7.82-7.88 (m, 1H), 7.91-7.98 (m, 2H), 8.02-8, 09 (m, 3H), 8.64 (d, 2H, ³ J = 4.9 Hz), 12.02 (br s, 1H)

Example 26: N-[[4-(2-morpholinoethoxy)phenyl]methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 4-(2-morpholinoethoxy)benzaldehyde (118 mg, 0.5 mmol) as described above. Yield: 21 mg (12%); MS mass/charge: 177.6 [M+2H] ²⁺ , 354.4 [M+H] ⁺ ; HPLC (gradient A): room temperature 5.76 min, 100%; ¹ H-NMR (MeOH-d4) δ: 2.55-2.61 (m, 4H), 2.78 (t, 2H, ³ J = 5.4 Hz), 3.67-3.72 (m , 4H), 4.11 (t, 2H, ³ J = 5.4 Hz), 4.30 (s, 2H), 6.87-6.93 (m, 2H), 7.02 (s, 1H ), 7.29-7.35 (m, 2H), 7.94 (s, 1H), 8.05 (s, 1H)

Example 27: N-[(4-morpholinophenyl)methyl]-3H-imidazo[4,5-b]pyridin-6-amine.

The compound was synthesized starting from imidazo[4,5-b]pyridin-6-amine (67 mg, 0.5 mmol) and 4-(4-morpholinyl)benzaldehyde (96 mg, 0.5 mmol) as described above. Yield: 42 mg (26%); MS mass/charge: 310.4 [M+H] ⁺ ; HPLC (gradient A): room temperature 6.91 min, 93.9%; ¹ H-NMR (MeOH-d4) δ: 3.07-3.11 (m, 4H), 3.79-3.82 (m, 4H), 4.29 (s, 2H), 6.91- 6.96 (m, 2H), 7.01 (br s, 1H), 7.27-7.32 (m, 2H), 7.95 (br s, 1H), 8.04 (br s, 1H )

c) General procedure for the synthesis of N-benzylimidazo[4,5-b]pyridin-6-amine-porphyrin conjugates.

Stage C1:

Imidazo[4,5-b]pyridin-6-amine (1 equiv) was dissolved in MeOH or EtOH (c=0.05–0.08M). The appropriate aldehyde was added and the mixture was stirred at room temperature until thin layer chromatography (TLC) indicated complete turnover of the starting materials. NaBH ₄ (1.5 eq.) (For R ^a/b = 1H) or NaBD ₄ (1.7 eq.) (For R ^a/b = D) was carefully added and stirring was continued overnight. The volatile components were evaporated and the residue was purified by flash chromatography (Al ₂ O ₃ gradient CHCl ₃ /MeOH).

The purified product was dissolved in MeOH (c=0.06–0.12M) and treated with freshly prepared 1M HCl in MeOH (16 eq.). After stirring at room temperature for 3 hours, the volatiles were evaporated and the residue was used without further purification.

Scheme 3: Approximate synthetic scheme for the synthesis of N-benzylimidazo[4,5-b]pyridin-6-amine-porphyrin conjugates

Stage C2:

N-[[4-(2-aminoethoxy)phenyl]-methyl]-1H-imidazo[4,5-b]pyridin-6-amine or N-[[4-(2-aminoethoxy)phenyl]dideuteriomethyl]-1H -Imidazo[4,5-b]pyridin-6-amine (1 equiv.) obtained in step C1 was dissolved in dimethylformamide (c = 0.1–0.13 M). Fmoc-Lys (Boc)-OH (1.2 eq), TBTU (1.2 eq) and DIPEA (4 eq) were added and the mixture was stirred at room temperature for 4-5 hours. Water was added and extracted with EtOAc (3×100 ml). The combined organic layers were dried over Na ₂ SO ₄ and evaporated. The residue was purified by flash chromatography (Al ₂ O ₃ gradient, CHCl ₃ /MeOH).

Stage C3:

The corresponding product obtained from step C2 (1 eq.) was dissolved in dimethylformamide (c=0.03 M), treated with DBU (1 eq.) and stirred at room temperature for 30-40 minutes. Suitable porphyrin (1.2 eq.), HOBt (1.2 eq.), TBTU (1 eq.) and DIPEA (4 eq.) were dissolved in dimethylformamide, pre-stirred for 50 minutes at room temperature and added to the solution. The mixture was stirred at room temperature overnight. The reaction was carefully quenched by adding TFA/DCM (1:1 v/v, 10 ml) and stirring was continued for 45 minutes. The volatile components were evaporated and the crude mixture was directly subjected to semipreparative HPLC.

Example 28: 3-[(1Z, 4Z, 9Z, 15Z)-18-[3-[[(1S)-5-amino-1-[2-[4-[(3H-imidazo[4,5-b ]]pyridin-6-ylamino)methyl]phenoxy]ethylcarbamoyl]pentyl]amino]-3-oxopropyl]-3,8,13,17-tetramethyl-21,23-dihydroporphyrin-2-yl]propanoic acid

Yield (last stage): 15 mg (6%); MS mass/charge: 453.1 [M+2H] ²⁺ ; 904.8 [M+H] ⁺ HPLC (gradient A): room temperature 12.43 min, 94%; ¹ H-NMR (DMSO-d6) δ: 0.96-1.53 (m, 8H), 2.40-2.48 (m, 3H), 3.02-3.26 (m, 5H), 3.59-3.80 (m, 12H), 4.20-4.26 (m, 2H), 4.26-4.33 (m, 2H), 4.33-4.45 (m, 2H ), 6.70 (d, 2H, J = 8.7 Hz), 7.04-7.08 (m, 1H), 7.17-7.24 (m, 2H), 7.42-7, 51 (m, 3H), 7.87 (t, 1H, ³ J = 5.4 Hz), 8.05-8.12 (m, 2H), 8.97 (br s, 1H), 9.34 (d, 1H, J = 24.0 Hz), 9.35 (s, 1H), 10.19 (d, 1H, J = 8.6 Hz), 10.32 (s, 1H), 10.33 (d, 2H, J = 9.8 Hz) 12.34 (br s, 1H).

Example 29: 3 - [(4Z, 10Z, 15Z, 19Z) -18- [3 - [[(1S)-5-amino-1- [2- [4- [dideuterio-(1H-imidazo [4,5 -b]pyridin-6-ylamino)methyl]phenoxy]ethylcarbamoyl]pentyl]amino]-3-oxopropyl]-3,8,13, 17 -tetramethyl-22,24-dihydroporphyrin-2-yl]propanoic acid

Yield (last stage): 37 mg (26%); MS mass/charge: 453.9 [M+2H] ²⁺ ; 906.4 [M+H] ⁺ HPLC (gradient A): room temperature 12.45 min, >99%; ¹ H-NMR (DMSO-d6) δ: 0.96-1.53 (m, 6H), 2.40-2.48 (m, 2H), 3.02 -3.26 (m, 6H), 3.59-3.80 (m, 14H), 4.26-4.45 (m, 5H), 6.70 (d, 2H, J=8.4 Hz), 7.04-7.08 ( m, 1H), 7.17-7.24 (m, 2H), 7.42-7.51 (m, 3H), 7.87 (m, 1H), 8.05-8.12 (m, 2H), 8.98 (br s, 1H), 9.34 (d, 1H, J = 23.7 Hz), 9.35 (s, 1H), 10.28 (d, 1H, J = 8, 4 Hz), 10.31 (s, 1H), 10.33 (d, 2H, J = 10.0 Hz).

c) Analytical methods

HPLC: The HPLC analytical system consisted of a Merck-Hitachi device (LaChrom model) using LUNA RP 18 (5 µm), an analytical column (length: 125 mm, diameter: 4 mm) and a diode array detector (DAD) with λ = 214 nm as the report wavelength. Compounds were analyzed using a gradient at a flow rate of 1 ml/min; wherein eluent (A) was acetonitrile, eluent (B) was water, both containing 0.04% (v/v) trifluoroacetic acid using one of the following gradients:

Gradient A: 0 min - 5% (A), 15 min - 60% (A), 20 min - 95% (A), 30 min - 95% (A), 31 min - 5% (A), 35 min - 5% (A)

Gradient B: 0 min - 1% (A), 5 min - 1% (A), 20 min - 20% (A), 30 min - 95% (A), 34 min - 95% (A), 35 min - 1% (A), 37 min - 1% (A)

The purity of all these compounds was determined by the percentage of peak area at 214 nm.

Mass spectrometry: ESI and APCI mass spectra were obtained using a SCIEX API 1200 spectrometer (Perkin Elmer) or Express CMS (Advion).

NMR spectroscopy. 1H NMR spectra were recorded on an Agilent DD2 400 MHz spectrometer. Chemical shifts are expressed in parts per million (ppm) at a lower field compared to tetramethylsilane. The splitting patterns were designated as follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), and br (broad signal).

INDUSTRIAL APPLICABILITY

The crystals and co-crystals of the present invention are crystals of therapeutic target proteins (bacterial glutamyl cyclases, bacQC) and can be specifically and substantially used to identify inhibitors capable of selectively targeting periodontitis-causing pathogens using candidate compound identification techniques that can bind to the bacQC binding pocket, and/or are bacQC inhibitors described herein. Thus, the crystals are suitable for the treatment of a specific disease, such as periodontitis, and are therefore susceptible to industrial application.

Methods for identifying candidate compounds that can bind to the bacQC binding pocket and/or are bacQC inhibitors in accordance with the present invention are industrially applicable in view of the pharmaceutical importance of bacQC enzymes for the occurrence of periodontitis and their importance for the growth of periodontal pathogens demonstrated here.

The compounds of the present invention, including compounds identified by methods for identifying candidate compounds that can bind to the bacQC binding pocket and/or are bacQC inhibitors, and the pharmaceutical composition of the present invention can also be used industrially as they can be used in methods of treatment human or animal body, in particular in a method for treating or preventing a bacterial infection, for example a bacterial infection caused by a bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, and/or in a method for treating or preventing acute, chronic or recurrent periodontal disease, such as periodontitis.

The research leading to these results was supported by the European Union.

Claims (54)

1. A compound according to the following Formula I, Where: A is selected from the group consisting of optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl and optionally substituted alkenyl; R ¹ is independently selected from the group consisting of H and optionally substituted alkyl; L is ; R ^a and R ^b are the same or different from each other and are independently selected from the group consisting of H, halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroalkyl and optionally substituted heteroaryl, and wherein R ^a and R ^b may optionally be connected together with the formation of a carbocyclic or heterocyclic ring; wherein "optionally substituted" refers to optional substitution with one to three groups independently selected from: C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 carbocyclyl, C _1-5 heterocyclyl and a C _1-5 heteroaryl group, each of which may be replaced by one or two halogen atoms and/or hydroxyl groups; halogen atom; cyano groups; primary, secondary or tertiary amino group; hydroxyl group and carboxyl group; or wherein A is optionally substituted by at least one substituent represented by the following structure B: where n = 1, 2, 3, 4, 5 or 6; and R ⁷ and R ⁸ are independently selected from alkyl, aryl, heteroalkyl, heteroaryl, carbamidoyl, formyl, alkylacyl and arylacyl, each of which may optionally be further substituted, and wherein R ⁷ and R ⁸ may optionally be combined together to form a carbocyclic or heterocyclic ring and/or R ⁷ is represented by the following structure E: where M is selected from Fe ²⁺ and Fe ³⁺ or is absent; R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH; And X is selected from covalent bond, alkylene, aralkylene, heteroalkylene, carbocyclene, heterocyclene, heteroarylene, heteroaralkylene, aminoalkylene, alkylamino and carbonylalkylamino groups; where each of them may have up to 12 carbon atoms and up to 11 heteroatoms in the main chain and may be replaced by one or more C _1-6 alkyl group(s), C _1-6 heteroalkyl group(s), C _1-6 carbocyclyl group (s), C _1-5 heterocyclyl group(s), C _1-5 heteroaryl group(s), halogen atom(s), hydroxyl group(s), cyano group(s), primary, secondary or tertiary amino group(s) , carboxyl group(s) and side chain(s) derived from proteinogenic or non-proteinogenic amino acids; said aryl independently represents a C _6-10 aryl group; said heterocyclyl independently represents a monocyclic or bicyclic C _1-11 heterocyclic group containing from 1 to 4 ring heteroatoms selected from N, S and O; said alkyl independently represents a linear or branched, open C _1-6 alkyl group; said cycloalkyl independently represents a cyclic C _3-6 alkyl group; said alkenyl independently represents a linear or branched, open C _2-6 group, each of which contains at least one C=C bond; said heteroaryl independently represents an aromatic monocyclic or bicyclic C _1-11 heterocyclic group containing from 1 to 4 ring heteroatoms selected from N, S and O; And said heteroalkyl independently represents a linear or branched, open C _1-5 heteroalkyl group containing from 1 to 4 heteroatoms selected from N, S and O, or alternatively A is selected from the group consisting of 2-methoxyphenyl, 3-methoxyphenyl, 4-propoxyphenyl, 4-methoxyphenyl, 4-isopropoxyphenyl, 4-benzyloxyphenyl, 3,4-dimethoxyphenyl, 2,3-dihydro-1,4-benzodioxin-6 -yl, 2,2-difluoro-1,3-benzodioxol-5-yl, 7-methoxy-1,3-benzodioxol-5-yl, 2,6-difluoro-4-methoxyphenyl, 3-(1-piperidyl) phenyl, 4-morpholinophenyl, [1,1'-biphenyl]-3-yl, [1,1'-biphenyl]-4-yl, 3-phenoxyphenyl, 4-phenoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4- chlorophenyl, 3,4-dichlorophenyl, 3,4,5-trifluorophenyl, 4-(2-pyridyl)phenyl, 4-(2-carbamimidamidoethyloxy)phenyl, 4-(2-aminoethoxy)phenyl, 4-(2-morpholinoethoxy) phenyl, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl and 3-thienyl. 2. The compound according to claim 1, which is represented by the following Formula Ia: where A is selected from optionally substituted aryl and optionally substituted heteroaryl; And R ¹ , R ^a and R ^b are H. 3. Connection according to any one of paragraphs. 1, 2, where R ^a and R ^b are independently selected from ¹ H and D. 4. Connection according to any one of paragraphs. 1-3, characterized in that A is replaced by at least one substituent represented by the following structure B: where n = 1, 2, 3, 4, 5 or 6; And R ⁷ and R ⁸ are independently selected from alkyl, aryl, heteroalkyl, heteroaryl, carbamidoyl, formyl, alkylacyl and arylacyl, each of which may optionally be further substituted, and wherein R ⁷ and R ⁸ may optionally be combined together to form a carbocyclic or heterocyclic rings. 5. The compound according to claim 4, characterized in that R ⁷ is represented by the following structure E: where M is selected from Fe ²⁺ and Fe ³⁺ or is absent; R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH; And X is selected from covalent bond, alkylene, aralkylene, heteroalkylene, carbocyclene, heterocyclene, heteroarylene, heteroaralkylene, aminoalkylene, alkylamino and carbonylalkylamino groups; where each of them may have up to 12 carbon atoms and up to 11 heteroatoms in the main chain and may be replaced by one or more C _1-6 alkyl group(s), C _1-6 heteroalkyl group(s), C _1-6 carbocyclyl group (s), C _1-5 heterocyclyl group(s), C _1-5 heteroaryl group(s), halogen atom(s), hydroxyl group(s), cyano group(s), primary, secondary or tertiary amino group(s) , carboxyl group(s) and side chain(s) derived from proteinogenic or non-proteinogenic amino acids. 6. The compound according to claim 5, where X is represented by the following structure: where m = 0, 1, 2 or 3, and R ^e is a side chain derived from a proteinogenic amino acid. 7. Connection according to any one of paragraphs. 1-6, characterized in that A is replaced by a substituent represented by the following structure F: where M is selected from Fe ²⁺ and Fe ³⁺ or is absent; And R ^c and R ^d are independently selected from –H, –CH ₃ , –CH=CH ₂ , –SO ₃ H and –CH(OH)CH ₂ OH. 8. A compound according to claim 1, selected from the group consisting of: 9. A pharmaceutical composition having inhibitory activity against bacterial glutaminyl cyclases (bacQC), containing an effective amount of a compound according to one of claims. 1-8 and a pharmaceutically acceptable carrier. 10. Use of the compound according to any one of paragraphs. 1-8 or a pharmaceutical composition according to claim 9 in the treatment and/or prevention of a bacterial infection, where: (i) the bacterial infection is caused by a bacterium that expresses bacterial glutamyl cyclase (bacQC), wherein bacQC is a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or an amino acid sequence having 90% or more sequence identity with any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and/or (ii) the bacterial infection is caused by a bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia; and/or (iii) the compound selectively inhibits the growth of bacteria in the biofilm. 11. Use of the compound according to any one of paragraphs. 1-8 or a pharmaceutical composition according to claim 9 in the treatment or prevention of acute, chronic or recurrent periodontal disease. 12. Application according to claim 11, characterized in that the disease is selected from the group consisting of gum diseases caused by plaque, chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of a systemic disease, necrotic periodontal diseases, periodontal abscesses, periodontitis associated with endodontic lesions, peri-implant mucositis, peri-implantitis and endodontic infections.

Images