Bisphosphonate-quinolone conjugates and their uses

Abstract

This application describes bisphosphonate-quinolone conjugates, which may include a bisphosphonate and a quinolone, and in which the quinolone can be separably linked to the bisphosphonate, and pharmaceutical formulations thereof. This application also provides methods of making and using the bisphosphonate-quinolone conjugates and pharmaceutical formulations thereof. [Selected Figure] Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 62 / 345,370, entitled "BONE TARGETED THERAPEUTICS AND DIAGNOSTICS," filed June 3, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] This application also claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 62 / 357,727, entitled "BISPHOSPHONATE QUINOLONE BIOCONJUGATES AND USES THEREOF," filed July 1, 2016, the entire contents of which are incorporated herein by reference.

[0003] This application also claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 62 / 448,060, entitled "BISPHOSPHONATE QUINOLONE BIOCONJUGATES AND USES THEREOF," filed January 19, 2017, the entire contents of which are incorporated herein by reference.

[0004] STATEMENT REGARDING U.S. GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Number 1R41DE025789-01 awarded by the NIH / NIDCR. The U.S. Government has certain rights in the invention. [Background technology]

[0005] Infectious bone disease, also called osteomyelitis, jaw bone infection, and other bone infections, is a significant problem in human and animal health, and the consequences can be severe, ranging from limb loss to death. Due to the unique challenges of bone, treatment of osteomyelitis and other bone infections is typically long and difficult, often requiring surgical intervention. Thus, there is a long felt and unmet need for improved treatment of osteomyelitis and other bone infections of all forms or clinical subtypes. Summary of the Invention

[0006] In some aspects, the present disclosure provides bisphosphonate (BP) quinolone conjugates that may contain a BP that is releasably conjugated to a quinolone, such as ciprofloxacin. In some embodiments, the BP quinolone conjugates can selectively deliver the quinolone to bone, bone grafts, and / or bone graft substitutes in a subject (i.e., can target bone, bone grafts, or bone graft substitutes). In some embodiments, the BP quinolone conjugates can sequester the quinolone. The present disclosure also provides methods of synthesis of BP quinolone conjugates and methods of treating or preventing osteomyelitis or other bone infections using one or more of the BP quinolone conjugates provided herein.

[0007] In some embodiments, the conjugate can be a compound of formula (6).

[0008] [ka]

[0009] The present disclosure also provides a pharmaceutical composition comprising a compound of formula (6) and a pharma- ceutically acceptable carrier.

[0010] The present specification also provides a method of treating a bone infection in a subject in need thereof, which may comprise administering to a subject in need of treatment for a bone infection an amount of the compound of formula (6) or a pharmaceutical formulation containing a compound of formula (6).

[0011] The present specification also provides a compound comprising a bisphosphonate (BP) and a quinolone compound, wherein the quinolone compound is detachably attached to the bisphosphonate via a linker.The BP may be substituted or unsubstituted, and is selected from the group consisting of hydroxyl phenyl alkyl bisphosphonates, hydroxyl phenyl aryl bisphosphonates, hydroxyl phenyl (or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates, hydroxyl phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates, hydroxyl phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, The aryl bisphosphonates may be selected from the group consisting of amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxypyridyl alkyl bisphosphonates, pyridyl alkyl bisphosphonates, hydroxyimidazoyl alkyl bisphosphonates, imidazoyl alkyl bisphosphonates, etidronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate, minodronate, and mixtures thereof.The quinolone compound may be a fluoroquinolone. The quinolone compound may be any of the following: alatrofloxacin, amifloxacin, balofloxacin, besifloxacin, cadazolidine, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ivafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, The medicament may be selected from the group consisting of safloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zavofloxacin, nemonoxacin, and mixtures thereof.

[0012] The quinolone compound can have the structure set forth in Formula A.

[0013] [ka]

[0014] In the formula, R 1is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 and the substituents may include cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 2 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 and the substituents may include cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 3is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 and may be substituents including cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 4 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 The substituents can be cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups.

[0015] In one or more embodiments, the linker can be a carbamate linker. The linker can be an aryl carbamate linker. The linker can be an O-thioaryl carbamate linker. The linker can be an S-thioaryl carbamate linker. The linker can be a phenyl carbamate linker. The linker can be a thiocarbamate linker. The linker can be an O-thiocarbamate linker. The linker can be an S-thiocarbamate linker. The linker can be an R 1 It may be bonded to a group.

[0016] In one or more embodiments, the alpha position of the ethylidene bisphosphonate may be substituted with hydroxy, fluoro, chloro, bromo, or iodo. In some embodiments, the bisphosphonate may include a p-hydroxyphenylethylidene group or a derivative thereof. In some embodiments, the ethylidene bisphosphonate does not include an alpha-hydroxy at the alpha position.

[0017] In some embodiments, the compound has the formula of Formula (12):

[0018] [ka]

[0019] In some embodiments, the compound has the formula of Formula (13):

[0020] [ka]

[0021] In some embodiments, the compound has the formula of Formula (15).

[0022] [ka]

[0023] Also provided herein is a pharmaceutical formulation that may contain a bisphosphonate, a quinolone compound, and a pharma- ceutically acceptable carrier, wherein the quinolone compound is detachably linked to the bisphosphonate via a linker. The bisphosphonate may be substituted or unsubstituted, and may be selected from the group consisting of hydroxyphenyl alkyl bisphosphonates, hydroxyphenyl aryl bisphosphonates, hydroxylphenyl (or aryl) alkyl hydroxyl bisphosphonates, aminophenyl (or aryl) alkyl bisphosphonates, aminophenyl (or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxyl alkyl phenyl (or aryl) alkyl bisphosphonates, hydroxyl phenyl (or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl ... The quinolone compound may be selected from the group consisting of quinolone, aminophenyl (or aryl) alkyl bisphosphonate, aminophenyl (or aryl) alkyl hydroxyl bisphosphonate, hydroxyl alkyl bisphosphonate, hydroxyl alkyl hydroxyl bisphosphonate, hydroxypyridyl alkyl bisphosphonate, pyridyl alkyl bisphosphonate, hydroxyimidazoyl alkyl bisphosphonate, imidazoyl alkyl bisphosphonate, etidronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate, minodronate, and mixtures thereof. The quinolone compound may be a fluoroquinolone.The quinolone compounds include alatrofloxacin, amifloxacin, balofloxacin, besifloxacin, cadazolidine, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ivafloxacin, JNJ-Q2, levofloxacin, lomefloxacin, The medicament may be selected from the group consisting of safloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zavofloxacin, nemonoxacin, and mixtures thereof.

[0024] The quinolone compound can have the structure set forth in Formula A.

[0025] [ka]

[0026] In the formula, R 1 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20Ring group, substituted C3~C 20 and the substituents may include cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 2 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 and the substituents may include cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 3 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20and may be substituents including cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups; R 4 is an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 The substituents can be cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups.

[0027] In one or more embodiments, the linker can be a carbamate linker. The linker can be an aryl carbamate linker. The linker can be an O-thioaryl carbamate linker. The linker can be an S-thioaryl carbamate linker. The linker can be a phenyl carbamate linker. The linker can be a thiocarbamate linker. The linker can be an O-thiocarbamate linker. The linker can be an S-thiocarbamate linker. The linker can be an R 1 It may be bonded to a group.

[0028] In some embodiments, the alpha position of the ethylidene bisphosphonate may be substituted with hydroxy, fluoro, chloro, bromo, or iodo. In some embodiments, the bisphosphonate may include a p-hydroxyphenylethylidene group or a derivative thereof. In some embodiments, the ethylidene bisphosphonate does not include an alpha-hydroxy at the alpha position.

[0029] In some embodiments, the compound has the formula of Formula (12):

[0030] [ka]

[0031] In some embodiments, the compound has the formula of Formula (13):

[0032] [ka]

[0033] In some embodiments, the compound has the formula of Formula (15).

[0034] [ka]

[0035] The amount of the compound in the pharmaceutical formulation may be an amount effective for killing or inhibiting bacteria.The amount of the compound in the pharmaceutical formulation may be an amount effective for treating or preventing osteomyelitis, osteonecrosis, peri-implantitis, and periodontitis.

[0036] The present specification also provides a method of treating osteomyelitis in a subject in need thereof, which can include administering to the subject in need of treatment for osteomyelitis an amount of a compound provided herein or a pharmaceutical formulation thereof.

[0037] The present specification also provides a method of treating peri-implantitis or periodontitis in a subject in need thereof, comprising administering to the subject in need of treatment for peri-implantitis or periodontitis an amount of a compound provided herein or a pharmaceutical formulation thereof.

[0038] The present specification also provides a method of treating diabetic foot in a subject in need thereof, comprising administering to the subject in need of treatment of diabetic foot an amount of a compound provided herein or a pharmaceutical formulation thereof.

[0039] The present disclosure also provides a bone graft composition that may include a bone graft material and a compound or pharmaceutical formulation thereof described herein, wherein the compound or pharmaceutical formulation thereof is attached to, integrated with, chemisorbed to, or mixed with the bone graft material. The bone graft material may be an autograft bone material, an allograft bone material, a xenograft bone material, a synthetic bone graft material, or any combination thereof.

[0040] The present specification also provides methods that can include implanting a bone graft composition described herein in a subject in need thereof.

[0041] The present specification also provides a method for preventing biofilm infection at a bone surgical site or an implant surgical site, or at a surgical site where a bone graft has been performed, which may include administering a compound described herein to a subject in need of prevention of a biofilm infection.

[0042] The present specification also provides a method for preventing biofilm infection at a bone surgical site or an implant surgical site, or at a surgical site where a bone graft has been performed, which may include implanting a bone graft composition described herein into a subject in need of prevention of biofilm infection.

[0043] Other compounds, compositions, formulations, methods, features, and advantages of the present disclosure for manufacturing systems for the synthesis of nanowire templates will become apparent to one of ordinary skill in the art upon examination of the following figures and detailed description, and it is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of this disclosure, and be protected by the accompanying claims.

[0044] Other aspects of the present disclosure will be readily appreciated upon consideration of the detailed description of various embodiments of the present disclosure in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0045] [Figure 1] Scanning electron micrograph (SEM; 100x magnification) of a surgical specimen from a patient with chronic osteomyelitis showing the characteristic stratified matrix-encapsulated biofilm colonizing the inner and outer bone surfaces. The inset in the upper right corner is a higher magnification view (5000x magnification) of the causative Staphylococcus biofilm pathogen. (The specimen was processed for SEM, sputter coated with platinum, and imaged with an XL 30S SEM (FEG; FEI, Hillsboro, OR) operating at 5 kV in secondary electron imaging mode.)

[0046] [Figure 2A] FIG. 1 shows a general synthesis scheme for phenylcarbamate BP-ciprofloxacin conjugates. [Figure 2B] FIG. 1 shows a general synthesis scheme for phenylcarbamate BP-ciprofloxacin conjugates. [Diagram 3] 1 is a table showing AST and MIC results for ciprofloxacin and BP-ciprofloxacin against a panel of clinical S. aureus osteomyelitis pathogens. [Figure 4]1 is a graph showing the results of spectroscopic analysis of BP-ciprofloxacin conjugate in trypticase soy broth microbial medium at 0 and 24 hours for various concentrations of the conjugate used in in vitro antimicrobial susceptibility testing. No degradation was observed after 24 hours, a typical experimental period for in vitro antimicrobial testing, indicating excellent stability of the antimicrobial. (*Results from 0.24 to 3.9 mcg / mL (red bars) are inconclusive due to high "blank" measurements.) [Diagram 5] Figure 1 shows the results of spectrophotometric analysis of one BP-ciprofloxacin complex (BP-carbamate-ciprofloxacin, BCC, compound 6) in trypticase soy broth microbiological medium supplemented with HA spheroids. Since only the supernatant is measured, without the HA spheroids with adsorbed complex, the large decrease from 0 to 24 hours confirms the adsorption of the complex to HA. (All results between 1.95 and 250 mcg / mL are statistically significant (p<0.05, ANOVA; three replicate experiments). *Results between 0.12 and 0.48 mcg / mL (red bars) are inconclusive due to high "blank" measurements.) [Figure 6] FIG. 1 is a graph showing antimicrobial susceptibility testing results of BP-ciprofloxacin against planktonic cultures of Staphylococcus aureus ATCC-6538, showing an improved bactericidal profile at acidic pH (right graph) compared to basic pH (left graph). [Figure 7] FIG. 1 shows time-kill results for BP-ciprofloxacin (conjugate) at 1× (red line) and ½× (black line) the established MIC against Staphylococcus aureus ATCC-6538 (right graph) and MRSA MR4-CIPS (left graph) at 1 hour and up to 24 hours, demonstrating strong bactericidal activity. [Figure 8] 1 is a graph showing the results of antimicrobial susceptibility testing of BP-ciprofloxacin against biofilms of Staphylococcus aureus ATCC-6538 (top graph) and Pseudomonas aeruginosa ATCC-15442 (bottom graph) formed on polystyrene as a substrate. [Figure 9]Figure 1 shows the results of antimicrobial susceptibility testing of BP-ciprofloxacin against S. aureus ATCC-6538 (left graph) and P. aeruginosa ATCC-15442 (right graph) biofilms formed on HA disks as substrates. Statistically significant bactericidal activity was produced against S. aureus by all tested concentrations of the complex (orange bars), including ciprofloxacin alone (red bars) (*p<0.05, Kruskal-Wallis test; three replicate experiments). [Figure 10] This is a graph showing the results of a prophylactic experiment in which HA spheroids were pre-coated with BP-ciprofloxacin and then inoculated with S. aureus. The control (C: red bars) represents bacteria cultured without HA and not treated with the complex, and the expected significant increase in planktonic growth is observed after 24 hours when the supernatant is measured. The control + HA (C + HA bars) represents bacteria cultured with HA but still not treated, and some bacterial growth is observed after 24 hours, but not to the same extent as the HA negative control (red bars), because the bacteria attach to the HA and form a biofilm that is not measured in the HA-free supernatant. Comparing these controls with the treatments, it can be seen that there is a complete inhibition of the bacteria after 24 hours when the complex is between 7.8 and 250 mcg / mL. At the relatively low concentrations of 0.12 to 3.9 mcg / mL, the bacteria grew slightly, but was still greatly inhibited. [Figure 11] 1 is a table showing the viability of biofilm bacteria after 24 hours of incubation in the presence of BP-ciprofloxacin coated HA disks. [Figure 12] 1 is a graph showing the antibacterial results of in vivo animal studies showing the efficacy of test compounds in reducing bacterial load. The complex showed the highest efficacy at a total dose of 0.9 mg / kg administered in multiple doses, with no recoverable bacteria. The complex then showed a single dose of 10 mg / kg, which showed a 10^2-fold reduction (99% bactericidal activity) compared to the negative control, and almost a 10^1-fold higher bactericidal activity compared to multiple dose formulations of ciprofloxacin alone, which showed a 10^1-fold reduction. [Figure 13]FIG. 1 shows an outline of the BP quinolone conjugate targeting strategy. [Figure 14] FIG. 1 shows an outline of the targeting and decoupling strategy for BP-quinolone conjugates. [Figure 15] FIG. 1 shows an embodiment of a BP-FQ complex. [Figure 16] FIG. 1 shows a synthetic scheme for the BP-FQ conjugate. [Figure 17] Figure 1 shows antimicrobial susceptibility testing results of ciprofloxacin, BCC (compound 6) and BP-amide-ciprofloxacin (BAC, compound 11) tested against a panel of clinically relevant S. aureus osteomyelitis pathogens. (MSSA = methicillin-susceptible S. aureus; MRSA = methicillin-resistant S. aureus) [Figure 18] Figure 1 shows the results of spectroscopic analysis of BCC (compound 6) in microbiological medium supplemented with HA spheres. Since only the supernatant is measured, without the HA spheres with adsorbed complex, the adsorption of the complex to HA is confirmed as evident by the large decrease from 0 to 24 hours (all results between 1.95 and 250 mcg / mL are statistically significant (p<0.05, ANOVA; three replicate experiments). *Results between 0.12 and 0.48 mcg / mL (red bars) are inconclusive due to high "blank" measurements). [Figure 19] Graph showing the efficacy of BCC (compound 6) in reducing bacterial load or mean CFU per gram of tissue. The highest efficacy was again observed at a single high dose (10 mg / kg) of the complex compared to the control (*p=0.0005; unpaired t-test; error bars represent standard error). [Figure 20] FIG. 1 shows additional BP-Ab complex designs. [Figure 21] FIG. 1 shows one embodiment of a synthetic scheme for the synthesis of a BP-Ab conjugate containing an O-thiocarbamate linker. [Figure 22] FIG. 1 shows one embodiment of a scheme for the synthesis of an α-OH protected BP ester. [Diagram 23]FIG. 1 shows one embodiment of a scheme for the synthesis of BP3-Linker3-Ciprofloxacin. [Figure 24] Graphs and images showing the results of MIC evaluation of O-thiocarbamate BP complexes against planktonic Staphylococcus aureus ATCC-6538 strain. Negative control = medium + microorganisms not treated with complex; Positive control = sterile medium without microorganisms. [Diagram 25] 1 is a graph showing the results of an evaluation of the antimicrobial activity or bacterial load reduction of the thiocarbamate conjugates against biofilms of Staphylococcus aureus ATCC-6538 formed on polystyrene as a substrate. Negative control = bacterial dilution not treated with the conjugate; Positive control = sterile dilution without microorganisms. [Figure 26] Graph showing the results of the evaluation of the antibacterial activity of O-thiocarbamate BP complexes tested against preformed biofilms of Staphylococcus aureus ATCC-6538 on hydroxyapatite as substrate. Negative control = bacterial dilution not treated with complex. (*p<0.05, Kruskal-Wallis test; three replicate experiments; control drug = control). [Figure 27] Graph showing results of a study using O-thiocarbamate BP complex treated hydroxyapatite disks to evaluate their ability to prevent biofilm formation of Staphylococcus aureus ATCC 6538. Negative control = bacterial dilution not treated with complex. (*p<0.05, Kruskal-Wallis test; three replicate experiments; control drug = control). [Figure 28] Graph showing results of a study using O-thiocarbamate BP complex treated hydroxyapatite powder to evaluate its ability to prevent biofilm formation of Staphylococcus aureus ATCC 6538. Negative control = non-complex treated bacterial dilution. (*p<0.05, Kruskal-Wallis test; three replicate experiments; control drug = control). [Figure 29] FIG. 1 shows α-hydroxy modified risedronate and zoledronate. [Diagram 30]FIG. 1 shows (1) a BP modified by replacing or removing the α-hydroxy group (p-PyrEBP), and (2) a BP modified by making a substitution at the para position of the pyridine ring (p-RIS), where a circled H is attached to the α-carbon of the bisphosphonate-substituted carbon chain.

[0047] [Diagram 31] FIG. 1 shows a synthetic scheme for BP-ciprofloxacin conjugate (BAC, compound 11) that has an amide bond as opposed to a carbamate bond. [Diagram 32] 1 is a graph showing the results of minimum inhibitory concentration (MIC) assays of 6 and 11 against eight strains of Staphylococcus aureus using a microdilution method. [Diagram 33] Antimicrobial susceptibility testing of 6 against biofilms of Staphylococcus aureus ATCC-6538 (top graph) and Pseudomonas aeruginosa ATCC-15442 (bottom graph) formed on HA disks as substrate. Statistically significant bactericidal activity was produced against S. aureus by all tested concentrations of 6 (dotted bars in top graph) and the parent antibiotic ciprofloxacin; c = negative control drug. Against P. aeruginosa, 6 was most effective at a concentration of 8 μg / mL at physiological pH and was also effective at this concentration at acidic pH, whereas ciprofloxacin was inactive in either acidic or physiological conditions compared to the control. (*p<0.05, Kruskal-Wallis test; three replicate experiments). [Diagram 34]Figure 1 shows the results of antimicrobial susceptibility testing of increasing concentrations of 11 against biofilms of S. aureus ATCC-6538 formed on HA as a substrate (top graph). No significant activity was observed at any concentration compared to the concentration control C+ (p>0.05, Kruskal-Wallis test; three replicates). The bottom graph shows a prophylactic experiment in which HA was pretreated with 11 or the parent antibiotic ciprofloxacin and then inoculated with S. aureus, again showing no antimicrobial activity of 11. Only a significant reduction was observed at the relatively high dose of 400 μg / mL of the parent drug (*p<0.05, Kruskal-Wallis test; three replicates). [Diagram 35] FIG. 13 is a graph showing the antibacterial susceptibility of 6 against Aggregatibacter actinomycetemcomitans strain D7S-5 biofilms grown on HA, demonstrating an effective antibacterial profile for complex 6 at concentrations above 15 μg / mL. [Diagram 36] 1 is a graph showing antibacterial performance of in vivo animal studies. The data shows the efficacy of the test compounds in reducing bacterial load. The highest efficacy was observed with a single high dose (10 mg / kg) of 6, which showed a 10-fold reduction (99% bactericidal activity) compared to the negative control. [Figure 37] 1 is a graph showing the antibacterial performance of the second animal study. The data shows the efficacy of 6 in terms of bacterial load reduction or mean CFU reduction per gram of tissue (Y-axis). The highest efficacy was observed when the complex was administered at a single high dose (10 mg / kg) compared to the control and multiple low dose groups (0.3 mg / kg x 3 times) (*p=0.0005; unpaired t-test; error bars represent standard error). [Figure 38] FIG. 1 shows BP-carbamate-moxifloxacin BP conjugates and synthesis scheme. [Figure 39] FIG. 1 shows BP-carbamate-Gatifloxacin BP conjugate and synthesis scheme. [Diagram 40] FIG. 1 shows BP-p-hydroxyphenylacetic acid-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 41] FIG. 1 shows BP-OH-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 42] FIG. 1 shows BP-O-thiocarbamate-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 43] FIG. 1 shows BP-S-thiocarbamate-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 44] FIG. 1 shows BP-resorcinol-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 45] FIG. 1 shows BP-hydroquinone-ciprofloxacin BP conjugate and synthesis scheme. [Diagram 46] FIG. 1 shows one embodiment of the structure of BP-fluoroquinolones. [Figure 47-1] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-2] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-3] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-4] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-5] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-6] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 47-7] FIG. 1 shows various BP-fluoroquinolone conjugates. [Figure 48] FIG. 1 shows one embodiment of the structure of aryl group-containing phosphonates. [Figure 49-1] FIG. 1 shows various BPs where X can be F, Cl, Br, or I. [Figure 49-2] FIG. 1 shows various BPs where X can be F, Cl, Br, or I. [Figure 49-3] FIG. 1 shows various BPs where X can be F, Cl, Br, or I. [Figure 50]FIG. 1 shows various BPs with terminal primary amines. [Figure 51] FIG. 1 shows various BPs coupled to linkers containing terminal hydroxyl and amine functional groups, where R can be risedronate, zoledronate, minodronate, pamidronate, or alendronate. [Figure 52-1] FIG. 1 shows various BP-pamidronate-ciprofloxacin conjugates. [Figure 52-2] FIG. 1 shows various BP-pamidronate-ciprofloxacin conjugates. [Figure 52-3] FIG. 1 shows various BP-pamidronate-ciprofloxacin conjugates.

[0048] [Diagram 53] FIG. 1 shows various BP-alendronate-ciprofloxacin conjugates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Before describing the present disclosure in further detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0050] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. The upper and lower limits of these subranges may independently be included in the subranges and are also included in the disclosure, but are governed by any specifically excluded limit in that stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0051] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0052] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials to which the publications to which they pertain. The mention of any publication is for its disclosure prior to the filing date of the present application and should not be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0053] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct elements and features which may be readily separated from or combined with any of the features of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited methods can be carried out in the order recited or in any other order which is logically possible.

[0054] Embodiments of the present disclosure employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, pharmacology, organic chemistry, biochemistry, botany, and the like, which are within the skill of the art, and such techniques are fully explained in the literature.

[0055] definition Unless otherwise stated herein, the following definitions are provided.

[0056] As used herein, when the terms "about," "approximately," and the like are used in connection with a numerical variable, the term generally refers to the value of that variable, and to all values ​​of that variable that fall within experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or within ±10%, whichever is greater.

[0057] As used interchangeably herein, a "subject," "individual," or "patient" refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sport animals, and pets. The term "pets" includes dogs, cats, guinea pigs, mice, rats, rabbits, ferrets, and the like. The term "livestock" includes horses, sheep, goats, chickens, pigs, cows, donkeys, llamas, alpacas, turkeys, and the like.

[0058] As used herein, a "control" can refer to a surrogate subject or sample used in an experiment for comparative purposes and included to minimize or distinguish the effects of variables other than the independent variable.

[0059] As used herein, an "analog," such as an analog of a bisphosphonate described herein, can refer to a structurally closely related member of the parent molecule or to a parent molecule described elsewhere, such as a bisphosphonate.

[0060] As used herein, "conjugation" can refer to the direct binding of two or more compounds to one another via one or more covalent or non-covalent bonds. As used herein, the term "conjugation" can also refer to the indirect binding of two or more compounds to one another via an intervening compound, such as a linker.

[0061] As used herein, a "pharmaceutical formulation" refers to a combination of active agents, compounds or components with a pharma- ceutical acceptable carrier or excipient that renders the composition suitable for in vitro, in vivo, or ex vivo diagnostic, therapeutic, or prophylactic use.

[0062] As used herein, a "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise harmful and is useful in preparing a pharmaceutical formulation, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, a "pharmaceutically acceptable carrier or excipient" includes both one such carrier or excipient and more than one such carrier or excipient.

[0063] As used herein, "pharmaceutically acceptable salt" refers to any acid or base addition salt having counterions that are non-toxic to subjects to whom such salts are administered in pharmaceutical doses.

[0064] As used herein, "active agent" or "active ingredient" refers to the component or components of a composition that is believed to be responsible, in whole or in part, for an effect.

[0065] As used herein, "dose," "unit dose," or "dosage amount" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a BP conjugate, such as a BP quinolone conjugate, composition, or formulation described herein, calculated to produce a desired response or desired responses in association with its administration.

[0066] As used herein, a "derivative" refers to any compound that has the same or similar core structure as a compound, but has at least one structural difference, including substitution, deletion, and / or addition of one or more atoms or functional groups. The term "derivative" does not imply that the derivative is synthesized from the parent compound, either as a starting material or intermediate, as such. The term "derivative" may include prodrugs or metabolites of the parent compound. Derivatives include compounds in which free amino groups in the parent compound are derivatized to form amine hydrochlorides, p-toluenesulfonamides, benzoxycarboxamides, t-butyloxycarboxamides, thiourethane-type derivatives, trifluoroacetylamides, chloroacetylamides, or formamides. Derivatives include compounds in which carboxyl groups in the parent compound are derivatized to form methyl and ethyl esters, or other types of esters, amides, hydroxamic acids, or hydrazides. Derivatives include compounds in which hydroxyl groups in the parent compound are derivatized to form O-acyl, O-carbamoyl, or O-alkyl derivatives. Derivatives include compounds in which a hydrogen bond donor group in the donor parent compound is replaced by another hydrogen bond donor group, such as OH, NH, or SH. Derivatives include replacing a hydrogen bond acceptor group in the parent compound with another hydrogen bond acceptor group, such as esters, ethers, ketones, carbonates, tertiary amines, imines, thiones, sulfones, tertiary amides, and sulfides. "Derivatives" also include, by way of example, extensions of the cyclopentane ring by replacement with saturated or unsaturated cyclohexane or other more complex rings, such as nitrogen-containing rings, and extensions of these rings by various groups.

[0067] As used herein, "administering" refers to oral, topical, intravenous, subcutaneous, transdermal, transcutaneous, intramuscular, intraarticular, parenteral, intraarteriolar, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, or via an implanted reservoir. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0068] As used herein, the term "substituted" refers to all permissible substituents of the compounds described herein. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms, e.g., 1 to 14 carbon atoms, and may optionally contain one or more heteroatoms, such as oxygen, sulfur, or nitrogen, in a linear, branched, or cyclic structural format. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl group, heteroaryl group, substituted heteroaryl group, halo group, hydroxyl group, alkoxy group, substituted alkoxy group, phenoxy group, substituted phenoxy group, aroxy group, substituted aroxy group, alkylthio group, substituted alkylthio group, phenylthio group, substituted phenylthio group, arylthio group, substituted arylthio group, cyano group, isocyano group, substituted isocyano group, carbonyl group, substituted carbonyl group, carboxyl group, substituted carboxyl group, amino group, substituted amino group, amido group, substituted amido group, sulfonyl group, substituted sulfonyl group, sulfonic acid group, phosphoryl group, substituted phosphoryl group, phosphonyl group, substituted phosphonyl group, polyaryl group, substituted polyaryl group, C3-C 20 Ring group, substituted C3~C 20 These include cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups.

[0069] As used herein, "suitable substituents" refers to chemically and pharma- ceutically acceptable groups, i.e., moieties that do not significantly interfere with the preparation of the compounds of the present invention or that do not significantly impair the efficacy of the compounds of the present invention. Such suitable substituents can be routinely selected by one of ordinary skill in the art. Suitable substituents include the following: halo, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl)C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxy, hydroxy, carboxy, carboxyl ... Suitable substituents include, but are not limited to, si, oxo, sulfanyl, C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, and arylsulfonyl. The groups listed above as suitable substituents are as defined below, with the exception that suitable substituents may be optionally unsubstituted.

[0070] The term "alkyl" refers to the radical of a saturated aliphatic group (i.e., an alkane with one hydrogen atom removed), and includes straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.

[0071] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C for straight chain). 30 and for branched chains, C3 to C 30 In other embodiments, straight chain or branched chain alkyls can have up to 20, 15 or 10 carbon atoms in their backbone. Likewise, in some embodiments, cycloalkyls can have from 3-10 carbon atoms in their ring structure. In some of these embodiments, cycloalkyls can have 5, 6 or 7 carbons in the ring structure.

[0072] As used herein, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties.

[0073] Unless the number of carbons is otherwise specified, as used herein, "lower alkyl" refers to an alkyl group as defined above except having from 1 to 10 carbons in its backbone structure. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

[0074] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, where appropriate. For example, substituents of substituted alkyls can include halogens, hydroxy groups, nitro groups, thiol groups, amino groups, azido groups, imino groups, amide groups, phosphoryl groups (including phosphonates and phosphinates), sulfonyl groups (including sulfates, sulfonamides, sulfamoyl and sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls (ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Cycloalkyls can be similarly substituted.

[0075] The term "heteroalkyl" as used herein refers to a linear or branched or cyclic carbon-containing radical, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, where the phosphorus and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heteroalkyl may be substituted as defined above for alkyl groups.

[0076] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term "alkylthio" also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. "Arylthio" refers to an aryl or heteroaryl group. Alkylthio groups can be substituted as defined above for alkyl groups.

[0077] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0078] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, a substituent on an alkyl that makes the alkyl an ether or alkoxyl-like can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. The terms "aroxy" and "aryloxy", as used interchangeably herein, can be represented by -O-aryl or -O-heteroaryl, where aryl and heteroaryl are defined below. Alkoxy and aroxy groups can be substituted as described above for alkyl.

[0079] The terms "amine" and "amino" (and protonated forms thereof) are art recognized and refer to both unsubstituted and substituted amines, such as moieties that can be represented by the general formula:

[0080] [ka]

[0081] In the formula, R, R', and R" are each independently hydrogen, alkyl, alkenyl, or -(CH2) m -R C or R and R' together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure, R Crepresents aryl, cycloalkyl, cycloalkenyl, heterocyclic, or polycyclic; and m is 0 or an integer ranging from 1 to 8. In some embodiments, only one of R or R' can be carbonyl, e.g., R, R', and the nitrogen do not together form an imide. In other embodiments, the term "amine" does not include amides, e.g., where one of R and R' represents carbonyl. In other embodiments, R and R' (and optionally R") each independently represent hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl, or alkynyl. Thus, as used herein, the term "alkylamine" refers to an amine group, as defined above, having attached thereto a substituted or unsubstituted alkyl (as described above for alkyl), i.e., where at least one of R and R' is an alkyl group.

[0082] The term "amide" is art recognized as an amino substituted carbonyl and includes a moiety that can be represented by the general formula:

[0083] [ka]

[0084] where R and R' are as defined above.

[0085] As used herein, "aryl" refers to a C5-C 10It refers to a 1-membered aromatic ring system, heterocyclic ring system, fused aromatic ring system, fused heterocyclic ring system, biaromatic ring system, or biheterocyclic ring system. Broadly defined, "aryl" as used herein includes 5-, 6-, 7-, 8-, 9-, and 10-membered monocyclic aromatic groups that may contain 0-4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Aryl groups having heteroatoms in the ring structure are sometimes referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring may be substituted at one or more ring positions with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, and combinations thereof. The term "aryl" includes phenyl.

[0086] The term "aryl" also includes multiple ring systems having two or more cyclic rings in which two or more carbons are common between two adjacent rings (i.e., "fused rings"), where at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocycle.Examples of heterocyclic rings include benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, isopropyl, ... Midazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl aryl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyridinyl, These include, but are not limited to, rolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.One or more of the rings may be substituted as defined above for "aryl".

[0087] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0088] The term "aralkyloxy" can be represented by -O-aralkyl, where aralkyl is as defined above.

[0089] The term "carbocycle," as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

[0090] The terms "heterocycle" or "heterocyclic" as used herein refer to monocyclic or bicyclic structures containing up to 10 ring atoms, in some embodiments 5-6 ring atoms, which are carbon and 1-4 heteroatoms, each of which is selected from the group consisting of non-peroxide oxygen, sulfur, and Y, H, O, (C1-C2, 10) alkyl, phenyl or benzyl, and may optionally contain 1 to 3 double bonds and may optionally be substituted with one or more substituents. Examples of heterocycles include benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furan, ranyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2 ,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazo allyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,Examples of heterocyclic groups include, but are not limited to, 5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. Optionally, the heterocyclic group may be substituted at one or more positions as defined above for alkyl and aryl with one or more substituents, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The terms "heterocycle" or "heterocyclic" may be used to describe a compound that may contain a heterocycle or heterocyclic ring.

[0091] The term "carbonyl" is art recognized and includes those moieties that can be represented by the general formula:

[0092] [ka]

[0093] where X is a bond or represents oxygen or sulfur, and R and R' are as defined above. When X is oxygen and R or R' is not hydrogen, the formula represents an "ester". When X is oxygen and R is as defined above, the moiety is referred to herein as a carboxyl group, and in particular when R is hydrogen, the formula represents a "carboxylic acid". When X is oxygen and R' is hydrogen, the formula represents a "formate". When the oxygen atom of the above formula is replaced by sulfur, the formula generally represents a "thiocarbonyl" group. When X is sulfur and R or R' is not hydrogen, the formula represents a "thioester". When X is sulfur and R is hydrogen, the formula represents a "thiocarboxylic acid". When X is sulfur and R' is hydrogen, the formula represents a "thioformate". On the other hand, when X is a bond and R is not hydrogen, the formula represents a "ketone" group. Where X is a bond, and R is hydrogen, the above formula represents an "aldehyde" group.

[0094] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Exemplary heteroatoms include, but are not limited to, boron, nitrogen, oxygen, phosphorus, sulfur, silicon, arsenic, and selenium. Heteroatoms such as nitrogen may have hydrogen substituents consistent with the valence of the heteroatom and / or any permissible substituent of organic compounds described herein. It is understood that the term "substituted" or "substituted" includes the implicit proviso that such substitution is consistent with the permissible valence of the substituted atom and substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, and the like.

[0095] As used herein, the term "nitro" refers to -NO2, the term "halogen" refers to -F, -Cl, -Br, or -I, the term "sulfhydryl" refers to -SH, the term "hydroxyl" refers to -OH, and the term "sulfonyl" refers to -SO2-.

[0096] As used herein, the term "carbamate" can be used to refer to compounds derived from carbamic acid (NH2COOH), and can include carbamate esters. A "carbamate" can have the following general structure:

[0097] [ka]

[0098] wherein R1, R2, and R3 can be any permissible substituent.

[0099] As used herein, an "effective amount" can refer to an amount of a composition described herein or a pharmaceutical formulation described herein that will elicit the desired biological or medical response in a tissue, system, animal, plant, protist, bacteria, yeast, or human that is desired by a researcher, veterinarian, physician, or other clinician. The desired biological response can be modulation of bone formation and / or remodeling, including, but not limited to, modulation of bone resorption and / or modulation of uptake of a BP complex, such as a BP quinolone complex described herein. An effective amount will vary depending on the precise chemical structure of the composition or pharmaceutical formulation, the causative agent of the infection and / or the severity of the infection, the disease, disorder, syndrome, or symptoms thereof being treated or prevented, the route of administration, the duration of administration, the rate of excretion, the drug combination, the judgment of the attending physician, the dosage form, and the age, weight, general health, sex, and / or diet of the subject to be treated. An "effective amount" can refer to an amount of a composition described herein that is effective to inhibit the growth or proliferation of microorganisms, including, but not limited to, bacteria or bacterial populations. An "effective amount" can refer to an amount of a composition described herein that kills microorganisms, including but not limited to bacteria or bacterial populations. An "effective amount" can refer to an amount of a composition described herein that is effective to treat and / or prevent osteomyelitis in a subject in need thereof.

[0100] As used herein, the term "therapeutic" may generally relate to the treatment, cure, and / or amelioration of a disease, disorder, condition, or side effect, or the reduction in the rate of progression of a disease, disorder, condition, or side effect. The term also includes within its scope the enhancement of normal physiological function, palliative treatment, and partial treatment of a disease, disorder, condition, side effect, or symptoms thereof.

[0101] As used herein, the terms "treating" and "treatment" may generally relate to obtaining a desired pharmacological and / or physiological outcome. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition thereof.

[0102] As used herein, the terms "synergistic effect," "synergy," or "synergy" refer to an effect that occurs between two or more molecules, compounds, substances, factors, or compositions that is greater than or different from the sum of their individual effects.

[0103] As used herein, "additive effect" refers to an effect that occurs between two or more molecules, compounds, substances, factors, or compositions that is equal to or identical to the sum of their individual effects.

[0104] The term "biocompatible" as used herein refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects in the recipient. Generally speaking, a biocompatible material is one that does not induce a significant inflammatory or immune response when administered to a patient.

[0105] As used herein, the term osteomyelitis can refer to acute or chronic osteomyelitis, and / or diabetic foot osteomyelitis, diabetic chronic osteomyelitis, prosthetic joint infections, periodontitis, peri-implantitis, osteonecrosis, and / or hematogenous osteomyelitis and / or other bone infections.

[0106] Consideration Infectious bone disease, or osteomyelitis, is a major worldwide problem in human and veterinary medicine and can be a serious problem due to the potential for sequelae and mortality associated with limb loss. Therapeutic approaches to osteomyelitis are primarily antimicrobial and are often long-term approaches, with surgical intervention in many cases to control the infection. The causative pathogen in the majority of cases of long bone osteomyelitis is a biofilm of Staphylococcus aureus, which is bone-bound in contrast to their planktonic (free-floating) counterparts. Other bone infections are known to arise from a wide range of both gram-positive and gram-negative bacteria.

[0107] The biofilm-mediated nature of osteomyelitis is important in clinical and experimental settings because many biofilm pathogens are non-culturable and display altered phenotypes in terms of growth rate and antimicrobial resistance (compared to their planktonic counterparts). The reason why relatively successful antimicrobial treatment of infections in the orthopedic field has not yet been achieved in general is partly explained by the occurrence of resistant biofilm pathogens, the low penetration of antimicrobials in bone, and the difficulty of eradicating biofilms with conventional antibiotics, together with adverse events associated with systemic toxicity.

[0108] To overcome the many challenges associated with osteomyelitis treatment, there has been growing interest in drug delivery approaches using bone-targeting complexes to achieve relatively high or relatively sustained local therapeutic concentrations of antibiotics in bone while minimizing systemic exposure. Bisphosphonates (BPs), e.g., fluoroquinolone and non-fluoroquinolone antibiotics complexed to bone-adsorbing BPs, are promising approaches due to the long track record of safety of each component and their favorable biochemical properties. Among the early studies of the fluoroquinolone family in this setting, ciprofloxacin showed the best binding and microbiological properties when bound to BPs. Ciprofloxacin has several advantages for repurposing in this setting: it can be administered orally or intravenously, and the administration is relatively biologically equivalent, ciprofloxacin has a broad spectrum of antibacterial activity including the most commonly encountered osteomyelitis pathogens, ciprofloxacin exhibits bactericidal activity at clinically achievable doses, and ciprofloxacin is the least expensive drug in the fluoroquinolone family.

[0109] The BP family has unique bone-targeting properties that make it an ideal carrier for the delivery of antibiotics to bone in osteomyelitis drug therapy. BPs form strong bidentate and tridentate bonds with calcium, resulting in their enrichment in hydroxyapatite (HA), especially at sites of high metabolic activity or at sites of infection and inflammation. BPs also show exceptional stability against both chemical and biological degradation. The idea of ​​targeting ciprofloxacin to bone via complexation with BPs has been explored in numerous reports over the years.

[0110] Despite these positive attributes of BPs and fluoroquinolones, such as ciprofloxacin, current attempts to create prodrugs containing BPs and fluoroquinolones, such as ciprofloxacin, have not been successful, with most attempts resulting in either systemically unstable prodrugs or non-cleavable conjugates that have been shown to inactivate either component of the conjugate, usually by violating pharmacophore requirements.

[0111] With the shortcomings of current BP fluoroquinolone conjugates in mind, BP quinolone conjugates are described herein that may contain a BP that is releasably conjugable to a quinolone, such as ciprofloxacin. In embodiments, the BP quinolone conjugates can selectively deliver a quinolone to bone, a bone graft, and / or a bone graft substitute in a subject (i.e., can target a bone, a bone graft, or a bone graft substitute). In some embodiments, the BP quinolone conjugates can sequester the quinolone. Also provided herein are methods of synthesis of BP quinolone conjugates and methods of treating or preventing osteomyelitis or other bone infections using one or more of the BP quinolone conjugates provided herein.

[0112] Other compositions, compounds, methods, features, and advantages of the present disclosure will become apparent to one of ordinary skill in the art upon review of the following figures, detailed description, and examples, and all such additional compositions, compounds, methods, features, and advantages are intended to be included within this description and are within the scope of the present disclosure.

[0113] Bisphosphonate (BP) quinolone conjugates and their formulations BP quinolone complex Provided herein are BP quinolone conjugates and their formulations. BP can be conjugated to a quinolone via a linker. In some embodiments, the linker is a detachable linker. The quinolone can be detachably attached to the BP via a linker. Thus, in some embodiments, the BP quinolone conjugates can selectively deliver and detach the quinolone to or near bone, bone graft, or bone graft substitute (FIG. 13). In other words, the BP fluoroquinolone conjugates can provide targeted delivery of fluoroquinolones to bone and / or adjacent areas of bone. The BP of the BP quinolone conjugates provided herein can be any BP, including hydroxyl phenyl alkyl or aryl bisphosphonates, hydroxyl phenyl (or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates, hydroxyl phenyl(or aryl) ... alkyl phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates, hydroxyl alkyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonatesBisphosphonates include, but are not limited to, aminophenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates (all of which are unsubstituted or further substituted), etidronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate, hydroxymethylene bisphosphonates, and combinations thereof. Bisphosphonates may also be substituted for phosphonophosphonic or phosphonocarboxylic acids. In embodiments, the BP may be pamidronate, alendronate, risedronate, zoledronate, minodronate, neridronate, etidronate, which may be unmodified or modified as described herein.

[0114] The BPs can be modified to contain an α-hydroxy group (e.g., α-hydroxy modified risedronate and zoledronate; FIG. 29). Other BPs can be modified similarly. In some embodiments, the BPs can be modified by replacing or removing the α-hydroxy group (FIG. 30; e.g., p-PyrEBP). Removal or replacement of the α-hydroxyl group can reduce or eliminate the bone-resorptive effect of the BP compared to the equivalent unmodified BP. Thus, in some embodiments, the BP conjugates provided herein can contain a BP that does not have the α-hydroxy group or has a substituted α-hydroxy group. Suitable replacements for the α-hydroxy group can include, but are not limited to, H, alkyl, aryl, alkylaryl. Other additional molecules conjugated to the BP can also affect the bone-resorptive effect. For example, when the quinolone and / or linker are attached to the BP with a para-substituted side change, the bone-resorptive effect can be greatly reduced or eliminated. In some embodiments, the BPs can be modified to contain both an α-hydroxyl deletion or substitution and a para-substituted side chain.

[0115] In BPs containing an aryl or phenyl, the aryl or phenyl may be substituted at any position on the ring with a suitable substituent. In some embodiments, the aryl or phenyl ring of the BP is substituted with one or more electron donating species (e.g., F, N, and Cl).

[0116] Pharmacologically inactive BP mutants may be used to deliver fluoroquinolones without BP activity.

[0117] The quinolone can be any quinolone, including, but not limited to, alatrofloxacin, amifloxacin, balofloxacin, besifloxacin, cadazolid, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, ivafloxacin, JNJ-Q2, levofloxacin, , lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zavofloxacin, nemonoxacin, and any combination thereof. The quinolone may be a fluoroquinolone.

[0118] The quinolone can have the general structure of Formula 1, where R 1 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl group, heteroaryl group, substituted heteroaryl group, halo group, hydroxyl group, alkoxy group, substituted alkoxy group, phenoxy group, substituted phenoxy group, aroxy group, substituted aroxy group, alkylthio group, substituted alkylthio group, phenylthio group, substituted phenylthio group, arylthio group, substituted arylthio group, cyano group, isocyano group, substituted isocyano group, carbonyl group, substituted carbonyl group, carboxyl group, substituted carboxyl group, amino group, substituted amino group, amido group, substituted amido group, sulfonyl group, substituted sulfonyl group, sulfonic acid group, phosphoryl group, substituted phosphoryl group, phosphonyl group, substituted phosphonyl group, polyaryl group, substituted polyaryl group, C3-C 20 Ring group, substituted C3~C 20 R can be a substituent including a cyclic group, a heterocyclic group, a substituted heterocyclic group, an amino acid group, a peptide group, and a polypeptide group. 2is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl group, heteroaryl group, substituted heteroaryl group, halo group, hydroxyl group, alkoxy group, substituted alkoxy group, phenoxy group, substituted phenoxy group, aroxy group, substituted aroxy group, alkylthio group, substituted alkylthio group, phenylthio group, substituted phenylthio group, arylthio group, substituted arylthio group, cyano group, isocyano group, substituted isocyano group, carbonyl group, substituted carbonyl group, carboxyl group, substituted carboxyl group, amino group, substituted amino group, amido group, substituted amido group, sulfonyl group, substituted sulfonyl group, sulfonic acid group, phosphoryl group, substituted phosphoryl group, phosphonyl group, substituted phosphonyl group, polyaryl group, substituted polyaryl group, C3-C 20 Ring group, substituted C3~C 20 R can be a substituent including a cyclic group, a heterocyclic group, a substituted heterocyclic group, an amino acid group, a peptide group, and a polypeptide group. 3 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl group, heteroaryl group, substituted heteroaryl group, halo group, hydroxyl group, alkoxy group, substituted alkoxy group, phenoxy group, substituted phenoxy group, aroxy group, substituted aroxy group, alkylthio group, substituted alkylthio group, phenylthio group, substituted phenylthio group, arylthio group, substituted arylthio group, cyano group, isocyano group, substituted isocyano group, carbonyl group, substituted carbonyl group, carboxyl group, substituted carboxyl group, amino group, substituted amino group, amido group, substituted amido group, sulfonyl group, substituted sulfonyl group, sulfonic acid group, phosphoryl group, substituted phosphoryl group, phosphonyl group, substituted phosphonyl group, polyaryl group, substituted polyaryl group, C3-C 20 Ring group, substituted C3~C 20R4 may be a substituent including an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, a phenyl group, a substituted phenyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a halo group, a hydroxyl group, an alkoxy group, a substituted alkoxy group, a phenoxy group, a substituted phenoxy group, an aroxy group, a substituted aroxy group, an alkylthio group, a substituted alkylthio group, a phenylthio group, a substituted phenylthio group, an arylthio group, a substituted arylthio group, a cyano group, an isocyano group, a substituted isocyano group, a carbonyl group, a substituted carbonyl group, a carboxyl group, a substituted carboxyl group, an amino group, a substituted amino group, an amido group, a substituted amido group, a sulfonyl group, a substituted sulfonyl group, a sulfonic acid group, a phosphoryl group, a substituted phosphoryl group, a phosphonyl group, a substituted phosphonyl group, a polyaryl group, a substituted polyaryl group, a C3-C 20 Ring group, substituted C3~C 20 The substituents can be cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups.

[0119] [ka]

[0120] The BP may be conjugated to the fluoroquinolone via a separable linker. In some embodiments, the separable linker may be a phenyl carbamate linker. The separable linker may be an aryl carbamate linker. In some embodiments, the linker may be an aryl thiocarbamate linker. In some embodiments, the linker may be a phenyl thiocarbamate linker. In some embodiments, the thiocarbamate linker may be an O-thiocarbamate linker. In some embodiments, the thiocarbamate linker may be an S-thiocarbamate linker. In some embodiments, the linker may be a carbonate linker. In some embodiments, the linker may be a urea linker. In some embodiments, the linker may be an aryl dithiocarbamate linker.

[0121] BP Quinolone Complex Pharmaceutical Preparation This specification also describes formulations, including pharmaceutical formulations, that may contain an amount of BP quinolone complex described anywhere herein. The amount may be an effective amount. The amount may be effective to inhibit bacterial growth and / or reproduction. The amount may be effective to kill bacteria. Formulations, including pharmaceutical formulations, may be formulated for delivery via various routes and may contain a pharma- ceutically acceptable carrier. Techniques and formulations may generally be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA (20th ed., 2000), the disclosure of which is incorporated herein by reference in its entirety. Injections, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections, are useful for systemic administration. For injection, the therapeutic compositions of the invention may be formulated in a solution, for example, in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. Additionally, the BP quinolone complex and / or components thereof may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. The formulations, including pharmaceutical formulations of the BP-quinolone conjugates, may be characterized as being at least sterile and pyrogen-free. These formulations include those for use in human and veterinary care.

[0122] Suitable pharma- ceutically acceptable carriers include, but are not limited to, water, saline, alcohol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose, or starch, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone, which do not adversely react with the BP quinolone conjugates.

[0123] The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary substances that do not adversely react with the BP quinolone conjugate, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents, and / or aromatic substances.

[0124] Another formulation involves the addition of BP quinolone complexes to bone grafting materials or bone void fillers for the prevention or treatment of osteomyelitis, peri-implantitis or peri-prosthetic infections, and for maintenance of tooth extraction sockets.

[0125] Pharmaceutical preparations can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting the tonicity such as sodium chloride or glucose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0126] Formulations, including pharmaceutical preparations suitable for injectable use, may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Injectable pharmaceutical preparations may be sterile and may be fluid so long as good syringability properties exist. Injectable pharmaceutical preparations may be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a pharma- ceutical acceptable polyol, such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some embodiments, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.

[0127] Sterile injectable solutions can be prepared by incorporating any of the BP quinolone complexes described herein in a specific amount in a suitable solvent containing one of the ingredients listed herein or a combination of ingredients as needed, followed by filter sterilization. In general, dispersions can be prepared by incorporating the BP quinolone complexes into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the ingredients listed herein. In the case of sterile powders for preparing sterile injectable solutions, vacuum drying and freeze-drying are examples of useful preparation methods that yield powders of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0128] Systemic administration may also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fluid acid derivatives for transmucosal administration. Transmucosal administration may be achieved through the use of nasal sprays or suppositories. For transdermal administration, the BP quinolone complex may be formulated into ointments, salves, gels, or creams generally known in the art. In some embodiments, the BP quinolone complex may be applied via a transdermal delivery system, which may slowly release the BP quinolone complex for percutaneous absorption. Permeation enhancers may be used to facilitate transdermal penetration of the active agent in the conditioned medium. Transdermal delivery patches are described, for example, in U.S. Patent Nos. 5,407,713, 5,352,456, 5,332,213, 5,336,168, 5,290,561, 5,254,346, 5,164,189, 5,163,899, 5,088,977, 5,087,240, 5,008,110, and 4,921,475.

[0129] For oral administration, the formulations described herein may be provided as capsules, tablets, powders, granules, or suspensions or solutions.The formulations may contain conventional additives such as lactose, mannitol, corn starch, or potato starch; binders, crystalline cellulose, cellulose derivatives, gum acacia, corn starch, gelatin, disintegrants, potato starch, sodium carboxymethylcellulose, anhydrous dibasic calcium phosphate, or sodium starch glycolate, lubricants, and / or magnesium stearate.

[0130] For parenteral administration (i.e., administration via a route other than the digestive tract), the formulations described herein can be mixed with a sterile aqueous solution that is isotonic with the blood of the subject. Such formulations can be prepared by dissolving the active ingredient (e.g., the BP quinolone conjugate) in water that contains physiologically compatible substances such as sodium chloride, glycine, etc. to produce an aqueous solution, and has a buffered pH that is compatible with physiological conditions, and rendering the solution sterile. The formulations can be provided in unit-dose or multi-dose containers, such as sealed ampoules or vials. The formulations can be delivered by injection, infusion, or other means known in the art.

[0131] For transdermal administration, the formulations described herein can be combined with skin penetration enhancers such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, etc., which increase the permeability of the skin to the nucleic acid vectors of the invention and allow them to penetrate through the skin and into the bloodstream. The formulations and / or compositions described herein can be further mixed with polymeric substances such as ethyl cellulose, hydroxypropyl cellulose, ethylene / vinyl acetate, polyvinylpyrrolidone, etc. to provide a gel-like composition, which can be dissolved in a solvent such as methylene chloride, drained to a desired viscosity, and added to a base material to provide a patch.

[0132] The formulations described herein can be combined with xenograft (bovine), autograft (autologous) or allograft (human cadaveric) materials or synthetic bone substitutes for inclusion in bone graft replacement or bone void filler to prevent local post-operative infection or post-operative graft failure and provide sustained local release of antibiotics at the implantation site. For example, the attending surgeon or clinician can premix the powder formulation with any existing commercially available bone graft replacement or autograft. This formulation may further be combined with any formulation previously described and may be modified with any of the following compositions: hydroxyapatite, tricalcium phosphate, collagen, aliphatic polyesters (polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL), polyhydroxybutyrate (PHB), methacrylates, polymethylmethacrylates, resins, monomers, polymers, cancellous bone allografts, human fibrin, platelet rich plasma, platelet rich fibrin, plaster of Paris, apatite, synthetic hydroxyapatite, coralline hydroxyapatite, wollastonite (calcium silicate), calcium sulfate, bioactive glass, ceramics, titanium, devitalized bone matrix, non-collagenous proteins, collagen, and osteomyelin. The bone graft material may be combined with products containing autolyzed reduced antigenicity human bone. In this embodiment, the bone graft material combined with the BP quinolone conjugate may be in the form of a paste, powder, putty, gel, hydrogel, matrix, granules, particles, lyophilized powder, lyophilized bone, demineralized lyophilized bone, fresh or fresh frozen bone, corticocancellous bone mix, pellets, strips, plugs, membranes, lyophilized powder reconstituted with water to form a moist cake, spheres, sponges, blocks, morsels, sticks, wedges, cements, or formulations consisting of amorphous particles. Many of these may be present in the form of an injectable formulation, or as a combination of two or more of the above formulations (e.g., a sponge and an injectable paste).

[0133] In another embodiment, the BP-quinolone conjugates can be combined with factor-based bone grafts containing natural or recombinant growth factors such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and / or bone morphogenetic proteins (BMPs). In another embodiment, the BP-quinolone conjugates can be combined with cell-based bone grafts used in regenerative medicine and dentistry, including embryonic and / or adult stem cells, tissue-specific stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, gingival stem cells, periodontal stem cells, adipose stem cells, bone marrow stem cells, and blood stem cells. Thus, bone grafts that are osteoconductive, osteoinductive, osteopromoting, osteogenic, or any combination thereof can be combined with the BP-quinolone conjugates for clinical or therapeutic use.

[0134] Dosage form The BP quinolone conjugates and formulations thereof described herein may be provided in unit dosage forms such as tablets, capsules, single-dose injection vials, or single-dose infusion vials, or as a predetermined dose for mixing with bone graft material as in the case of the above formulations. Where appropriate, the dosage forms described herein may be microencapsulated. The dosage forms may also be prepared to prolong or sustain the release of any of the components. In some embodiments, the component that is slow-released may be the conjugated active agent. In other embodiments, the release of an auxiliary component is slowed. Suitable methods for slowing the release of a component include, but are not limited to, coating the component with or embedding it in a material such as a polymer, wax, gel, etc. Delayed release formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et.al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and methods of preparation of tablets and capsules, as well as delayed release dosage forms of tablets, pellets, capsules, and granules. The delayed release can range anywhere from about 1 hour to about 3 months or longer.

[0135] Coatings may be formed with or without water insoluble / water soluble non-polymeric excipients using different ratios of water soluble polymers, water insoluble polymers, and / or pH dependent polymers to produce the desired release profile. Either coating may be performed on dosage forms (matrix or simplified) including, but not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, granular compositions, "ingredients in their natural state" formulated as, but not limited to, suspension or spreadable dosage forms.

[0136] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic acid resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0137] Effective dose The formulation may contain an effective (bacterial inhibiting and / or killing) amount of a BP-quinolone complex described herein. In some embodiments, the effective amount ranges from about 0.001 pg to about 1,000 g or more of the BP-quinolone complex described herein. In some embodiments, the effective amount of the BP-quinolone complex described herein may range from about 0.001 mg / kg to about 1,000 mg / kg of body weight. In yet other embodiments, the effective amount of the BP-quinolone complex may range from about 1% to about 99% or more as a % (wt / wt), % (wt / vol), or % (vol / vol) of the total formulation. In some embodiments, an effective amount of the BP quinolone conjugate is effective to kill bacteria, including but not limited to any strain or species of Staphylococcus, Pseudomonas, Aggregatibacter, Actinomyces, Streptococcus, Haemophilus, Salmonella, Serratia, Enterobacter, Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella, Campylobacter, Peptostreptococcus, Eikenella, Treponema, Dialister, Micromonas, Yersinia, Tannerella, and Escherichia, that are causative agents of osteomyelitis and all its subtypes (e.g., diabetic foot osteomyelitis), osteonecrosis of the jaw, and periodontitis.

[0138] How to use BP quinolone conjugate A specific amount, including an effective amount, of the BP quinolone conjugates and formulations thereof described herein can be administered to a subject in need thereof. In some embodiments, the subject in need thereof may have a bone infection, bone disease, bone disorder, or a symptom thereof. In some embodiments, the subject in need thereof may be suspected of having or otherwise prone to having a bone infection, bone disease, bone disorder, or a symptom thereof. In some embodiments, the subject in need thereof may be at risk of developing osteomyelitis, osteonecrosis, peri-prosthetic infection, and / or peri-implantitis. In some embodiments, the disease or disorder may be osteomyelitis and all its variants, osteonecrosis, peri-implantitis, or periodontitis. In some embodiments, the subject in need thereof has bone infected with a microorganism, such as bacteria. In some embodiments, the bacteria can be any strain or species of Staphylococcus, Pseudomonas, Aggregatibacter, Actinomyces, Streptococcus, Haemophilus, Salmonella, Serratia, Enterobacter, Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella, Campylobacter, Peptostreptococcus, Eikenella, Treponema, Dialister, Micromonas, Yersinia, Tannerella, or Escherichia. In some embodiments, the bacteria can form a biofilm. In some embodiments, osteomyelitis can be treated in a subject in need thereof by administering to the subject an amount, e.g., an effective amount, of a BP quinolone conjugate or formulation thereof described herein. In some embodiments, the compositions and compounds provided herein can be used for the treatment and / or prevention of osteonecrosis, distraction bone procedures, cleft lip and palate repair, repair of severe supraglomerular defects, jaw bone reconstruction, and any other reconstruction or repair of bone and / or joint.

[0139] The administration of the BP quinolone complex is not limited to a single route, but may include administration by multiple routes. For example, exemplary administration by multiple routes includes, among others, a combination of intradermal and intramuscular administration, or a combination of intradermal and subcutaneous administration. The multiple administrations may be sequential or simultaneous. Other application modes by multiple routes will be apparent to those skilled in the art.

[0140] The pharmaceutical agent can be administered to the subject by any suitable method that allows the pharmaceutical agent to exert its effect on the subject in vivo. For example, the formulations and other compositions described herein can be administered to the subject by known methods, including, but not limited to, oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, inhalation, nasal delivery, intravaginal administration, intrarectal administration, and intramuscular administration. The formulations or other compositions described herein can be administered parenterally by epifascial, intracapsular, intradermal, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymal, and / or sublingual delivery. Delivery can be by injection, infusion, catheter delivery, or some other means, such as a tablet or spray. Delivery can also be by a carrier, such as hydroxyapatite, or bone in the case of anti-infective bone graft material at the surgical site. Delivery can be by binding or other contact with bone graft material. EXAMPLES

[0141] Having described the embodiments of the present disclosure above, the following examples generally describe some additional embodiments of the present disclosure. The embodiments of the present disclosure are described in the following examples and in conjunction with the corresponding text and figures, but are not intended to limit the embodiments of the present disclosure to the description. On the contrary, they are intended to embrace all alternatives, modifications, and equivalents that fall within the spirit and scope of the embodiments of the present disclosure.

[0142] Example 1 Introduction Infectious bone disease, or osteomyelitis, is a major problem worldwide in human and veterinary medicine and can be a serious problem due to the sequelae and potential mortality associated with limb loss (Lew, et al., Osteomyelitis. Lancet 2004;364:369-79; Desrochers, et al, Limb amputation and prosthesis. Vet Clin North Am Food Anim Pract 2014;30:143-55; Stoodley, et al., Orthopaedic biofilm infections. Curr Orthop Pract 2011;22:558-63; Huang, et al., Chronic osteomyelitis increases long-term mortality risk in the elderly: a nationwide population-based cohort study. BMC Geriatr 2016;16:72). Therapeutic approaches to osteomyelitis are primarily antimicrobial and often long-term approaches, with surgical intervention to manage the infection in many cases. The causative pathogen in most cases of long bone osteomyelitis is Staphylococcus aureus biofilm; by definition, these organisms are bound to bone (Figure 1) in contrast to their planktonic (free-floating) counterparts (Wolcott, et al., Biofilms and chronic infections. J Am Med Assoc 2008;299:2682-2684).

[0143] The biofilm-mediated nature of osteomyelitis is important in clinical and experimental settings because many biofilm pathogens are non-culturable and have altered growth rates and antimicrobial resistance phenotypes (compared to their planktonic counterparts) (Junka, et al., Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro. J Oral Maxillofac Surg 2015;73:451-64; Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials. J Med Chem 2002;45:2338-41). The lack of generally successful antibiotic treatment of infections in the orthopedic field is partly explained by the development of resistant biofilm pathogens, the low penetration of antibiotics in bone, and the difficulty of eradicating biofilms with conventional antibiotics, along with adverse events associated with systemic toxicity (Buxton, et al., Bisphosphonate-ciprofloxacin bound to Skelite is a prototype for enhancing experimental local antibiotic delivery to injured bone. Br J Surg 2004;91:1192-6).

[0144] To overcome the many challenges associated with osteomyelitis treatment, there has been growing interest in drug delivery approaches using bone-targeting complexes to achieve relatively high or relatively sustained local therapeutic concentrations of antibiotics in the bone while minimizing systemic exposure (Panagopoulos, et al., Local Antibiotic Delivery Systems in Diabetic Foot Osteomyelitis: Time for One Step Beyond? Int J Low Extrem Wounds 2015;14:87-91; Puga, et al., Hot melt poly-epsilon-caprolactone / poloxamine implantable matrices for sustained delivery of ciprofloxacin. Acta biomaterialia 2012;8:1507-18). Fluoroquinolone antibiotics conjugated to bone-adsorbing bisphosphonates (BPs) are a promising approach because of the long track record of safety of each component and their favorable biochemical properties (Buxton, et al., Bisphosphonate-ciprofloxacin bound to Skelite is a prototype for enhancing experimental local antibiotic delivery to injured bone. Br J Surg 2004;91:1192-6). Among early studies of the fluoroquinolone family in this setting, ciprofloxacin showed the best binding and microbiological properties when bound to BPs (Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials. J Med Chem 2002;45:2338-41). Ciprofloxacin has several advantages for repurposing in this setting.Ciprofloxacin can be administered orally or intravenously and is relatively biologically equivalent when administered intravenously; it has broad spectrum antibacterial activity that includes the most commonly encountered osteomyelitis pathogens; it exhibits bactericidal activity at clinically achievable doses; and it is the least expensive member of the fluoroquinolone family (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J Med Chem 2008;51:6955-69).

[0145] The specific bone targeting properties of the BP family make them ideal carriers for the delivery of antibiotics to bone in osteomyelitis drug therapy (Zhang S,et al.,'Magic bullets' for bone diseases: progress in rational design of bone-seeking medicinal agents.Chem Soc Rev 2007;36:507-31). BPs form strong bidentate and tridentate bonds with calcium, resulting in their enrichment in hydroxyapatite (HA), especially at sites of high metabolic activity or at sites of infection and inflammation (Cheong,et al.,Bisphosphonate uptake in areas of tooth extraction or periapical disease.J Oral Maxillofac Surg 2014;72:2461-8). BP also exhibits exceptional stability against both chemical and biological degradation (Russell, et al., Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 2008;19:733-59).The concept of targeting ciprofloxacin to bone via complexation with BP has been discussed in a number of reports over the years (David,et al.,Methylene-bis[(aminomethyl)phosphinic acids]: synthesis, acid-base and coordination properties.Dalton Trans 2013;42:2414-22; Fardeau,et al.,Synthesis and antibacterial activity of catecholate-ciprofloxacin conjugates.Bioorg Med Chem 2014;22:4049-60; EUCAST: European Committee on Antimicrobial Susceptibility Testing breakpoint tables for interpretation of MICs and zone diameters.2015.http: / / www.eucast.org / fileadmin / src / media / PDFs / EUC; CLSI.M100-S25 performance standards for antimicrobial susceptibility testing,Twenty-fifth informational supplement,2015; Tanaka,et al., Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis.Bioorg Med Chem 2008;16:9217-29; McPherson, et al., Synthesis of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials.Eur J Med Chem 2012;47:615-8).However, early attempts resulted in either systemically unstable prodrugs or non-cleavable conjugates that were found to inactivate either component of the conjugate, usually by violating pharmacophore requirements. In the field of fluoroquinolones, a striking example was described by Herczegh et al., where the important anti-Gram-positive bacterial properties of the ciprofloxacin moiety were lost when complexed with a stable BP-linked analogue (Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials. J. Med. Chem 2002; 45:2338-41). Subsequent work in this field has demonstrated that these conjugates alone are incapable of exerting significant antibacterial activity without cleavage of the parent antibiotic (Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibiotics. J. Med. Chem 2002; 45:2338-41; Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69).Houghton et al., for example, synthesized and tested various BP-fluoroquinolone conjugates and found that phenylpropanone gatifloxacin prodrugs and acyloxyalkylcarbamate gatifloxacin prodrugs, once bound to bone, were likely able to regenerate the parent drug and therefore exhibited greater antibacterial activity than simpler conjugates such as bisphosphonoethyl, bisphosphonopropionyl, and amide derivatives, which are unable to detach the antibiotic (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69).

[0146] In summary, current research findings in the field indicate that the antibacterial activity of BP-fluoroquinolones is complex and related to the specific strain of pathogen tested, the choice of antibiotic and covalently attached BP moiety, the tether length between the two components, the bone-binding affinity of the BP, the adsorption-desorption equilibrium of the BP, and the stability / lability and kinetics of the conjugation scheme used for the complexation (Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials. J. Med. Chem 2002; 45:2338-41; Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69; Tanaka, et al., Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2009; 52:281-282; (McPherson,et al.,Synthesis of osteotropic hydroxybiphosphonate derivatives of fluoroquinolone antibiotics.Eur J.Med Chem 2012:47:615-8). Accumulating evidence suggests that more opportunities for optimization and success may be offered by a “target-release” linker strategy in this setting. We therefore hypothesized that conjugation of ciprofloxacin to a phenyl BP moiety via a metabolically hydrolyzable carbamate linker should alleviate the problems seen with antibiotic delivery to bone in osteomyelitis drug therapy.The cleavable carbamate bond and structural motif are key features in many drugs designed for targeting and sequestration in specific tissues, providing pharmacokinetic advantages such as stability in serum and labile on infected bone surfaces in the presence of acidic and enzymatic environments (Ossipov,et al.,Bisphosphonate-modified biomaterials for drug delivery and bone tissue engineering. Expert Opin Drug Deliv 2015;12:1443-58; Guo,et al.,pH-triggered intracellular release from actively targeting polymer micelles. Biomaterials 2013;34:4544-54; Ghosh,et al.,Organic carbamates in drug design and medicinal chemistry. J Med Chem 2015;58:2895-940).

[0147] One recent clear success story using a bone-targeted release strategy has been recognized, in which Morioka et al. designed an estradiol analog to target and sequester in bone by using a cleavable variant (carbamate) of a relatively stable amide peptide bond (Morioka, et al., Design, synthesis, and biological evaluation of novel estradiol-bisphosphonate conjugates as bone-specific estrogens. Bioorg Med Chem 2010;18:1143-8). Several versions of this conjugate were attempted before a pharmacologically active variant (phenylcarbamate) was found. Importantly, they demonstrated that a single dose of a similarly linked BP-estradiol conjugate produced effects on bone similar to those of estradiol administered alone, but at a dose 1000-fold lower (Morioka, et al., Design, synthesis, and biological evaluation of novel estradiol-bisphosphonate conjugates as bone-specific estrogens. Bioorg Med Chem 2010; 18:1143-8). The conjugates also offered a higher therapeutic index or improved safety, as there were minimal effects in uterine tissue. Consistent with this dramatic increase in efficacy associated with phenylcarbamate-linked BP-prostaglandins is the pharmacokinetic study completed by Arns et al. (Arns, et al., Design and synthesis of novel bone-targeting dual-action pro-drugs for the treatment and reversal of osteoporosis. Bioorg Med Chem 2012; 20:2131-40). A synthetic example of this approach in the antibacterial field has been reported for macrolides.However, only alkyl carbamates have been examined, and there have been no other successful examples, suggesting that targeted release strategies are likely to be biochemical target-dependent as well as chemical class-dependent (considering compatibility of the functional groups of each component), and that the strategy must be designed to suit any particular chemical class (Tanaka,et al.,Synthesis and in vitro evaluation of bisphosphonated glycopeptide prodrugs for the treatment of osteomyelitis.Bioorg Med Chem Lett 2010;20:1355-9).

[0148] This example presents a systematic evaluation of phenylcarbamate BP-ciprofloxacin conjugates and their antimicrobial activity against common osteomyelitis pathogens in vitro, whereby in vivo safety and efficacy were evaluated in an animal model of peri-implant osteomyelitis. Importantly, the in vitro and in vivo studies presented herein are based on biofilm models and biofilm methodologies that have not been performed in the field to date in addition to planktonic cultures and that should provide relatively high clinical relevance. The studies are specifically designed to address unmet medical needs in the treatment of infectious bone disease and thus meet technology transfer priorities.

[0149] Results and Discussion Chemistry: The overall synthetic route for one BP-ciprofloxacin conjugate (BCC, compound 6) is shown in the scheme in Figures 2A-2B. As a starting point, our project team identified an inactive 4-hydroxyphenylethylidene BP for this conjugation. The rationale for this BP design was to retain its bone-seeking ability while suppressing the antiresorptive activity of the unwanted BP moiety, minimizing confounding factors, and focusing on the evaluation of the antibacterial activity uniquely attributable to the parent ciprofloxacin compound. BP ligands can be designed to have antiresorptive functionality (of various potencies) if required to provide a dual effect of bone tissue protection in addition to antibacterial activity at the anatomical site of infection. We also selected this phenyl BP for its bone-binding affinity and tether length because previous studies have demonstrated that weak binding affinity reduces targeting efficiency and that increasing the distance between the fluoroquinolone and the BP functional group can slow down hydrolysis and regeneration of the parent compound (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69; Tanaka, et al., Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis, Bioorg Med Chem 2008,16:9217-29; McPherson, et al., Synthesis of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials, Eur J Med Chem 2008,16:9217-29). 2012,47:615-8).Most importantly, we believe that the use of aryl carbamates as linkers may provide optimized stability in plasma and adequate separation in bone for this biochemical target compared to previous BP-F quinolone conjugates. Thus, the tetraethyl ester of 4-hydroxyphenylethylidene BP (4) was prepared as previously described (David, et al., Methylene-bis[(aminomethyl)phosphinic acids]: synthesis, acid-base and coordination properties. Dalton Trans 2013;42:2414-22). The phenolic group of BP (4) was then activated with p-nitrophenyl chloroformate to form compound (5) for conjugation with protected ciprofloxacin (7) (Fardeau, et al., Synthesis and antibiotic activity of catecholate-ciprofloxacin conjugates. Bioorg Med Chem 2014;22:4049-60). Ciprofloxacin (6) was protected with a benzyl (Bn) group via di-t-butyl dicarbonate (BocO) reaction. Hydrolysis and final deprotection of the conjugate (8) with bromotrimethylsilane (TMSBr) yielded our first fluoroquinolone phenylcarbamate BP-ciprofloxacin prodrug (9), which was ready for biochemical and antibacterial evaluation.

[0150] Microbiology: The first study we undertook aimed to evaluate the antibacterial activity of the conjugate against a panel of 14 S. aureus clinical strains (methicillin-susceptible: MSSA and methicillin-resistant: MRSA) associated with bone infections in a standard laboratory planktonic culture system. According to EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines, the results of the disk zone assay showed diameters ranging from 25 to 40 mm (mean: 31.5, SD: ±5), and all strains showed antimicrobial susceptibility according to EUCAST breakpoints (EUCAST: European Committee on Antimicrobial Susceptibility Testing breakpoint tables for interpretation of MICs and zone diameters. 2015. http: / / www.eucast.org / fileadmin / src / media / PDFs / EUC). The results of the MICs of BP-ciprofloxacin tested against all 14 strains using microdilution are shown in Figure 3. The MICs of the parent compound ciprofloxacin alone were determined simultaneously (represented in Table 1) to serve as a reference, and their MICs were compared with those of established clinical breakpoints. 26 This class of prodrugs has been shown to lose significant antibacterial activity of the prodrug itself, and any BP-associated antibacterial activity has been shown to be negligible, and therefore isolation of the parent drug is a prerequisite for observing any appreciable antibacterial activity, such as that reported herein (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem. 2008, 51:6955-69).

[0151] AST and MIC data indicate that both the conjugate and ciprofloxacin have bactericidal activity against planktonic S. aureus pathogens, and that complexation affects the antibacterial activity of ciprofloxacin in vitro, with slightly higher concentrations of the conjugate required to reach the MIC than ciprofloxacin alone. This result is expected, as it is well established that complexation is based on chemical modifications of both the BP and the antibiotic to be delivered to bone, and as a consequence the properties of the parent drug, including its therapeutic effect, may be altered by such modifications. Our results are consistent with previous literature in the field showing that successfully conjugated functional conjugates retain their antibacterial activity, albeit at slightly lower levels than that of the parent compound (Herczegh, et al., Osteoadsorptive bisphosphonate derivatives of fluoroquinolone antibacterials. J. Med. Chem 2002,45:2338-41; Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008,51: 6955-69; Zhang, et al., 'Magic Bullets' for bone diseases: progress in rational design of bone-seeking medicinal agents). Importantly, in the context of osteomyelitis treatment, where the pathogen is not planktonic (as in these standard assays) but rather a biofilm and is bound to bone as a substrate, the enhanced bone-targeting properties of the BP-ciprofloxacin conjugate should result in higher antibiotic concentrations in bone than are appropriate for antibacterial action (as supported by biofilm-related in vitro and in vivo data described below), and therefore greater efficacy.

[0152] Since the microbial media used for in vitro antibacterial testing contains proteins, carbohydrates, enzymes, and salts / metals, the possibility exists for degradation, denaturation, or chelation of BP-ciprofloxacin during antibacterial testing. This degradation, denaturation, or chelation could have a detrimental effect on antibiotic activity but could be unrelated to the chemical complexation itself. Based on our AST and MIC results and the apparent antibacterial effect of the complex, this is not at all likely. Nevertheless, we attempted to objectively evaluate the stability of the complex by introducing BP-ciprofloxacin into trypticase soy broth microbial media and performing quantitative spectroscopic analysis as shown in Figure 4. The results showed good stability of the antibacterial agent, with no evidence of degradation or denaturation in the microbial media after 24 hours. Thus, the microbial media likely has no or little detrimental effect on the activity and efficacy of the complex.

[0153] After determining the antibacterial effect and chemical stability of the complex, we next sought to evaluate its HA binding ability. When HA spheres were added to our microbial medium and various concentrations of BP-ciprofloxacin were introduced, similar to those used in our antibacterial studies, significant adsorption and retention of the complex by HA was confirmed by quantitative spectroscopic analysis of the supernatant (containing no HA spheres) (Figure 5). These results are consistent with previously reported analogs of this class containing the BP moiety that have similar bone affinity (Tanaka, et al., Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis. Bioorg Med Chem 2008;16:9217-29; McPherson, et al., Synthesis of osteotropic hydroxybisphosphonate derivatives of fluoroquinolone antibacterials. Eur J Med Chem 2012;47:615-8). Bone adsorption also appeared to be a concentration-dependent phenomenon.

[0154] As S. aureus ATCC-6538 strain showed the least susceptibility and the lowest MIC profile (Figure 3) to both ciprofloxacin and the conjugate compared to the other test strains, we next selected this strain for further testing. This strain is also a well-known robust biofilm-forming pathogen compared to the other test strains. Therefore, we had the potential to test and optimize our conjugate against the most virulent pathogens to limit bias and overestimation of results, while also facilitating testing of antibacterial activity in a biofilm-based, clinically relevant model. Therefore, we performed AST against planktonic S. aureus ATCC-6538 strain with BP-ciprofloxacin under both acidic and alkaline conditions to evaluate the effect of pH on the conjugate activity. We found an overall improvement in antibacterial activity under acidic conditions and an improvement in MIC under alkaline conditions. 50 The MIC at half the conjugate concentration required to reach 50 Quantitative results from standard microdilution techniques showed that the MICs reached 100% (Figure 6). This could be useful for clinical applications in osteomyelitis, where biofilm pathogens create an acidic local environment along with host inflammation and osteoclast formation. However, other researchers have suggested that although the local acidic environment created by infectious organisms and inflammation may be associated with some drug elution in bone, the efficiency of such processes in providing sufficient concentrations of the antimicrobial agent is questionable, and prodrug design and conjugation schemes may play a larger role (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69). Finally, the AST data also showed that the MICs of ciprofloxacin and the conjugates were equal to their mean bactericidal concentrations (MBC), respectively.

[0155] The conjugate was then used in a time-kill assay performed according to CLSI (Clinical Laboratory Standards Institute) methodology, and the results showed that the conjugate was bactericidal within 1 hour and up to 24 hours with 100% growth inhibition of methicillin-susceptible (ATCC-6538) and methicillin-resistant (MR4-CIPS) isolates of planktonic S. aureus at previously established MICs. These kinetic studies showed bactericidal activity within 1 hour and growth inhibition up to 24 hours (50%) at half the MIC compared to the control (Figure 7) (CLSI.M100-S25 performance standards for antimicrobial susceptibility testing; Twenty-fifth informational supplement; 2015). The kinetic results showed a time-dependent potency of the conjugate against the test bacteria and sustained bactericidal activity over 24 hours, supporting the cleavage activity in the presence of the test bacteria.

[0156] We then tested the complex against preformed bacterial biofilms on two different substrates (polystyrene and HA disks) to assess for the first time the antibacterial effect against biofilms in this context and also to determine whether substrate specificity plays a role. Biofilms of Staphylococcus aureus (ATCC-6538) and additionally Pseudomonas aeruginosa (ATCC-15442) were exposed to BP-ciprofloxacin and the antibacterial activity was assessed. We also tested P. aeruginosa, as it is the second most common clinical pathogen of osteomyelitis, although it is much less frequent than S. aureus cases in terms of prevalence. Figure 8 shows the results for polystyrene as a substrate for biofilm growth and the minimum biofilm inhibitory concentration (MBIC) of BP-ciprofloxacin. 50 ) for Staphylococcus aureus ATCC-6538 ranged from 15.6 to 31.2 mcg / mL, which corresponded to the MIC for this strain in planktonic cultures. 50 was not recognized.

[0157] However, when HA disks were used as biofilm substrates, a significant improvement in bactericidal activity was observed as shown in Figure 9, where all tested concentrations of the complex produced statistically significant bactericidal activity and reduced colony forming units (CFU). 50 is 8mcg / mL, and MBIC 90 The MBIC of the parent drug, ciprofloxacin, against this pathogen was 50 mcg / mL. 90 The concentration of ciprofloxacin was 8 mcg / mL. However, ciprofloxacin had no inhibitory or bactericidal activity against P. aeruginosa ATCC-15442 strain, whereas the complex was bactericidal at a concentration of 50 mcg / mL under acidic and basic conditions, and showed improved antibacterial activity under basic conditions in contrast to S. aureus, which showed improved antibacterial activity under acidic conditions. Taken together, these results suggest that the complex is more effective against biofilm pathogens in the presence of HA as a substrate than polystyrene as a substrate, and that substrate specificity plays a role in antibacterial activity in addition to factors such as the strain of pathogen tested and bacterial growth mode (planktonic vs. biofilm). This has not been demonstrated before, and adds insight into the antibacterial potential of these compounds for clinical application against biofilm pathogens.

[0158] Finally, we performed antibacterial testing using the complex in a prophylactic experimental setup using planktonic and biofilm cultures that may have clinical relevance in antibiotic prophylaxis scenarios for osteomyelitis, where HA spheroids were introduced into various concentrations of BP-ciprofloxacin and incubated with S. aureus for 24 hours, and quantitative evaluation showed no bacterial growth at concentrations of the complex as low as 7.8mcg / mL up to 250mcg / mL, and strongly inhibited minimal bacterial growth at complex concentrations ranging from 0.12 to 3.9mcg / mL, as shown in Figure 10.

[0159] We next used the HA disks again as substrates for growing S. aureus biofilms, but this time the disks were washed with medium after incubation with either BP-ciprofloxacin or ciprofloxacin, but before inoculating and growing biofilms. Figure 11 shows the results of quantitative biofilm cultures and CFU after 24 h of growth, with ciprofloxacin at a concentration of 100 mcg / mL inhibiting all biofilm growth, while BP-ciprofloxacin at a concentration of 10 mcg / mL inhibiting all growth. As the molecular mass of ciprofloxacin is approximately half that of the complex, the complex was 20-fold more active in achieving complete bactericidal activity compared to ciprofloxacin alone. These findings support an efficient mechanism for enzymatic cleavage and time-dependent separation of the parent drug ciprofloxacin from the prodrug. Efficient binding to HA and cleavage or regeneration of the parent antibiotic are prerequisites for this class of complexes to exhibit substantial antibacterial efficacy comparable to or greater than that of the parent antibiotic alone (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69 (Tanaka, et al., Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis. Bioorg Med Chem 2008; 16:9217-29; McPherson, et al., Synthesis of osteotropic hydroxybiphosphonate derivatives of fluoroquinolone antibiotics).

[0160] In vivo safety and efficacy: Since this BP-ciprofloxacin conjugate is new and has not been previously tested in vivo, we performed the first safety and efficacy study in an animal model of peri-implant osteomyelitis. This model is a unique home-grown jaw peri-implant osteomyelitis model that was specifically developed to be of technology transfer value for studying biofilm-mediated disease and host responses in vivo (Freire, et al, Development of animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study. J Periodontol 2011;82:778-89). Briefly, biofilms of the jaw osteomyelitis pathogen Aggregatibacter actinomycetemcomitans (Aa; wild-type R-strain D7S-1; a serotype), which is not a resident bacterium, were inoculated into the normal flora of rats for 10 min. 9 CFU were precultured on small titanium implants. Prior to our animal studies, to confirm the susceptibility of Aa to the parent drug, ciprofloxacin, we performed AST and MIC assays as performed for previously described pathogens of long bone osteomyelitis. Disk zone of inhibition assay revealed diameters of >40 mm and MICs of >100 μg / mL were not observed. 90Aa was 2 mcg / mL, indicating strong susceptibility of this organism to the parent drug, ciprofloxacin. Aa was also previously tested for susceptibility to a pH-sensitive biotinylated ciprofloxacin prodrug and was found to be susceptible to the parent antibiotic (Manrique, et al., Perturbation of the indigenous rat oral microbiome by ciprofloxacin dosing. Mol Oral Microbiol 2013; 28:404-14). After biofilms were established on the implants in vitro, they were surgically implanted into the jawbone of each rat. The animals were anesthetized, the cheeks were retracted with a hook, and a transmucosal osteotomy was performed so that the implants could be inserted and secured manually into the osteotomy. Two biofilm-inoculated implants were placed on either side of the palatine bone of each rat (n=12 rats and 24 implants). This model results in standard and reproducible amounts of viable bacteria forming a well-established biofilm on each implant, and we have previously demonstrated that the biofilm persists in vivo for several weeks after implant placement and causes local infection, inflammation, and bone destruction (Freire, et al, Development of animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study. J Periodontol 2011;82:778-89).

[0161] One week after surgery, once peri-implant infection was established, the animals were administered BP-ciprofloxacin, ciprofloxacin alone as a positive control, and sterile endotoxin-free saline as a negative control, according to the dosing schedule specified in the experimental section. To determine the appropriate dosing concentration, we used a similar target-release strategy and calculated the approximate initial dose of the conjugate based on previous studies in rodents and pharmacokinetic data (Houghton,et al.,Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis.J Med Chem 2008;51:6955-69; Morioka,et al.,Design,synthesis,and biological evaluation of novel estradiol-bisphosphonate conjugates as bone-specific estrogens.Bioorg Med Chem 2010;18:1143-8). Based on our sample size estimates and previous experience with this animal model, we predicted that the molar equivalents of BP-ciprofloxacin at gradually increasing doses of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg would allow us to determine antibacterial activity in two experimental animals per group (Freire, et al, Development of animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study. J Periodontol 2011;82:778-89). Animals were dosed by intraperitoneal injection under general anesthesia, and all compounds were made up in sterile injectable saline at the appropriate pH.All animals were sacrificed one week after drug treatment, peri-implant tissue excision was performed, and tissues were immediately homogenized and processed for quantitative assessment of microbial burden. Animals were monitored throughout the study for any local or systemic adverse effects of drug treatment.

[0162] All animals tolerated the drug therapy well, with no cutaneous injection site reactions or inflammation, and no systemic adverse events reported by the attending veterinarian over the course of the study. Treatment efficacy was measured quantitatively in terms of the log amount of viable bacteria (mean log CFU per gram of tissue), as shown in Figure 12.

[0163] In vivo, the animals administered the complex at a dose of 0.3 mg / kg in multiple doses (3 times) over a one week period showed no recovery of Aa or 100% killing. A single dose of BP-ciprofloxacin at a dose of 10 mg / kg also showed high potency with a 10^2-fold reduction or 99% killing, and an order of magnitude higher activity than ciprofloxacin alone at the same total concentration, albeit in multiple doses. Ciprofloxacin alone in a multiple dose formulation produced a 10^1-fold reduction or 90% killing, which was expected and is why we chose ciprofloxacin as a positive control given the known potency of this compound, its antibacterial activity, and the fact that this compound is the parent drug of the complex. Complex concentrations of 0.1 mg / kg and 1 mg / kg had little effect, suggesting that further optimization is possible in this situation. Nonetheless, given the targeting and segregation capabilities of the prodrug, it is reasonable to expect that an effective dose would be achievable in a clinical setting that takes into account the safety profile and oral or intravenous administration capabilities of the constituent compounds. It is interesting to note that the multiple dose group of the conjugate showed evidence of yeast-like morphology in our cultures and no recoverable Aa. Contamination could be one explanation for this phenomenon, but this explanation is entirely unrealistic, since we performed similar and simultaneous methods, did not culture yeast in our laboratory, and only had animal samples in which Aa was not recovered in this same multiple dose group and two separate animals. Therefore, a more plausible explanation is that the killing and dissipation of Aa occurred in vivo, and that other organisms, such as yeast, that are less sensitive to the parent drug, ciprofloxacin, grew in our cultures.In fact, rats are used as a well-established model of oral candidiasis, and the rat equivalent of the yeast of the normal human oral flora is Candida pintolopessi, which can be an unexpected cause of disease in antibiotic-treated or immunocompromised rodents (Junqueira. Models hosts for the study of oral candidiasis. Adv Exp Med Biol. 2012;710:95-105). This overgrowth of yeast or candidiasis due to suppression of the flora that normally competes with yeast in vivo is also a well-known phenomenon in human patients treated with antibiotics.

[0164] The resolution of infection over time in vivo by the conjugate compared to a negative control and also compared to a positive control parent drug further supports that the conjugate effectively binds to bone and sequester the parent antimicrobial agent. Lack of efficacy in this model would suggest either that the prodrug is not binding or that the prodrug is not sequestering the parent drug. This at least provides an indirect method to understand the pharmacokinetics of the prodrug in vivo (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69). A similar study examined the activity of BP-fluoroquinolones in a rat tibial osteomyelitis model and found evidence of similar efficacy and greatly enhanced antibacterial activity of the test conjugates, but in a prophylactic context in which a single intravenous injection of the prodrug was administered 1-2 days prior to bone infection (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69). Infection in this model was produced by bolus injection of planktonic bacteria into the surgically exposed tibia, and animals were sacrificed 24 hours after infection.Although this study was not a biofilm-mediated osteomyelitis treatment study, it is consistent with the in vitro data presented herein showing that biofilm growth can be prevented by pretreatment with BP-ciprofloxacin (Houghton, et al., Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69). Our experiments confirm the ability of a safe and adequate single dose of BP-ciprofloxacin prodrug to yield sufficient concentrations of its parent drug to maintain bactericidal activity against established biofilms when the activity of the parent antibiotic alone has already disappeared. The conjugate will be further evaluated for its ability to treat long bone osteomyelitis in animal models, and comprehensive pharmacokinetic and pharmacodynamic studies will also be performed in vivo. The results of these studies will be presented subsequently.

[0165] Consideration This example presents the successful design and synthesis of a phenylcarbamate BP-ciprofloxacin conjugate utilizing a targeted release strategy, whereby the functionality of each component of this compound (as well as the entire conjugate) was systematically evaluated in vitro and in vivo. In vitro antibacterial investigations of BP-ciprofloxacin tested against common osteomyelitis pathogens revealed a strong bactericidal profile, and safety and efficacy were demonstrated in vivo in an animal model of periprosthetic osteomyelitis. In vivo, the animals receiving the conjugate at a dose of 0.3 mg / kg (total 0.9 mg / kg) in multiple doses over a one-week period showed the highest efficacy and no recoverable bacteria. A single dose of the conjugate at a dose of 10 mg per kg (5 mg ciprofloxacin considering that the molecular mass of the conjugate is twice that of the parent drug) also showed strong antibacterial activity, resulting in the killing of 99% of bacteria. Multiple doses of the conjugate and the highest single dose of the conjugate were superior to multiple doses of the parent antibiotic, ciprofloxacin, at a dose of 30 mg / kg. Lower single dose concentrations of the conjugate (0.1 mg / kg and 1 mg / kg) were not effective.

[0166] These findings indicate that a minimal dose is necessary for the in vivo efficacy of the conjugate when provided as a single dose, but much lower concentrations of the conjugate can provide the highest efficacy when administered routinely, at less than one-tenth the concentration of the parent antibiotic. For translation to the clinic, this targeting strategy may prove useful by lowering the administered concentration to patients and improving the therapeutic index, as well as by limiting systemic exposure. Importantly, these results, along with other studies in the field, show that direct comparisons between these prodrugs and their parent compounds are somewhat arbitrary, since the conjugates have unique pharmacokinetic parameters. Any future pharmacokinetic modeling of this class of conjugates will need to mathematically include backbone compartments for distribution, which is not commonly done in antibiotic pharmacokinetic studies. This will provide new pharmacological data that also have in vivo implications.

[0167] BP-ciprofloxacin was also tested here for the first time against clinically relevant biofilms and showed potent antibacterial activity both in vitro and in vivo when the biofilm was attached to bone as a substrate. The antibacterial activity of the complexes appears to be related to a number of parameters, including the species and strain of the pathogen tested, its growth mode (biofilm vs. planktonic), the substrate for biofilm colonization, pH, concentration, bone-binding affinity, and detachment kinetics. Optimization of this class of complexes using BP as a biochemical vector for delivery of antibacterial agents to bone (where biofilm pathogens reside) by a targeted release strategy should be an advantageous approach for the treatment of osteomyelitis and provide improved pharmacokinetics while minimizing systemic toxicity. Materials and Methods Chemistry:

[0168] [ka]

[0169] 1(Benzyloxy)-4(bromomethyl)benzene(1)

[0170] 4-Benzyloxybenzyl alcohol (1.00 g, 4.67 mmol) was dissolved in anhydrous diethyl ether (25 ml) in an oven-dried flask under nitrogen. The flask was cooled in an ice bath. Bromotrimethylsilane (BTMS, 1.26 ml, 9.52 mmol) was added via syringe. The flask was allowed to warm slowly to room temperature. After stirring for 17 hours, the reaction mixture was poured into water (50 ml) and the organic phase separated. The aqueous phase was washed with diethyl ether (2×20 ml) and the combined organic phase was washed with brine (2×20 ml) and dried over sodium sulfate. Evaporation of the ether gave the product as a white crystalline solid (1.23 g, 95% yield). 1 H NMR(400MHz,Chloroform-d) δ7.47 - 7.28(m,7H),6.98 - 6.90(m,2H),5.07(s,2H),4.50(s,2H).

[0171] [ka]

[0172] Tetraisopropyl(2(4(benzyloxy)phenyl)ethane-1,1-diyl)bis(phosphonate)(2)

[0173] Under nitrogen protection, anhydrous THF (2 ml) was added to sodium hydride (57-63% dispersion) in mineral oil. Tetraisopropyl methylenediphosphonate (0.57 ml, 1.8 mmol) was added dropwise with stirring at room temperature. Gas was evolved and the grey suspended solid was consumed leaving a mostly clear solution. The mixture was stirred for a further 10 min. Solid 1 was added in one portion under nitrogen counterflow. The solution was clear for 1 min and then became cloudy. Stirring was continued for 2 h, then the reaction was checked by TLC (100% EtOAc; visualized by UV or cerium ammonium molybdate (CAM) staining) and two new spots appeared at RF = 0.37 and 0.58. Some 1 (RF > 0.9) remained and the reaction was heated to 50 °C for 30 min, but the TLC revealed little progress. The reaction mixture was poured into 5% aqueous citric acid, extracted with ether (2×30 ml), washed with brine, and evaporated. The residue was purified by flash chromatography on 230-400 mesh silica using 10% to 100% EtOAc in hexane as eluent. The desired compound was obtained as a colorless oil (0.508 g, 52% yield).

[0174] [ka]

[0175] Tetraisopropyl(2(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate)(3)

[0176] Compound 2 (0.508 g, 0.925 mmol) was dissolved in 13 ml of methanol and 70 mg of 10% palladium on carbon was added. The flask was flushed with nitrogen, then with hydrogen, and a hydrogen balloon was attached and stirred overnight. TLC (10% MeOH in EtOAc, visualized by UV or CAM stain) showed the disappearance of the starting material (RF=0.63) and the appearance of a new spot with RF=0.49. The reaction mixture was filtered through Celite with the aid of 100 ml of methanol. Evaporation of the filtrate gave the desired compound as a light yellow oil that was used without further purification (0.368 g, 88% yield). 1 H NMR(400MHz,Chloroform-d) d 7.07(d,J=8.2Hz,2H),6.69(d,J=8.2Hz,2H),4.71(m,4H),3.11(td,J=16.9,6.0 Hz,2H),2.47(tt,J=24.4,6.0 Hz,1H),1.32 - 1.21(m,24H).31P NMR(162MHz,Chloroform-d) δ21.06 .

[0177] [ka]

[0178] 4(2,2-bis(diisopropoxyphosphoryl)ethyl)phenyl(4-nitrophenyl)carbonate(4)

[0179] Compound 3 (0.171 g, 0.380 mmol) was dissolved in 8 ml of dichloromethane, followed by the addition of triethylamine (159 μl, 1.14 mmol), followed by p-nitrophenyl chloroformate (0.086 g, 0.418 mmol) in one portion. The solution quickly turned from colorless to yellow. After stirring for 2.5 h, TLC (5% MeOH in EtOAc; visualized by UV) showed only traces of starting material (RF=0.31) and the appearance of a high intensity spot at RF=0.59. The compound was purified by flash chromatography using a 1:1 ethyl acetate:hexane mixture as the eluent to remove one impurity (RF=0.88) before eluting the product with pure ethyl acetate. 1 H NMR(400MHz,Chloroform-d) δ8.29(d,J=9.1Hz,2H),7.46 (d,J=9.1Hz,2H),7.33(d,J=8.5Hz,2H),7.15(d,J=8.6Hz,2H),4.84-4.58(m,4H) ,3.22(td,J=16.5,6.2Hz,2H),2.47(tt,J=24.1,6.2Hz,1H),1.33-1.14(m,24H).

[0180] [ka]

[0181] 7(4((4(2,2-bis(diisopropoxyphosphoryl)ethyl)phenoxy)carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5)

[0182] Ciprofloxacin (46.5 mg, 0.140 mmol) was suspended in 1.4 ml of water in a plastic vial. 151 μl of 1 M HCl was added and the vial was vortexed to dissolve the ciprofloxacin to give a clear, colorless solution. The pH was adjusted to 8.5 by addition of Na2CO3, resulting in a thick white precipitate. The vial was placed in an ice bath and compound 4 (71.9 mg, 0.117 mmol) dissolved in 1.4 ml of THF was added dropwise over approximately 5 min. The vial was then removed from the ice bath, protected from light, and stirred overnight at room temperature. The reaction mixture turned bright yellow as the solids were stirred. TLC (5% MeOH in EtOAc) showed the disappearance of starting material 4 and the appearance of a blue spot under fluorescent light (RF=0.51) and a yellow spot under visible light (RF=0.816) due to the p-nitrophenol by-product. The reaction mixture was diluted with 10 mL of water and filtered through a fine glass frit. The remaining solids were washed with water until no yellow color remained. The solids were then dissolved in DCM and rinsed through the frit, the solution was loaded onto a flash silica column and eluted with increasing concentrations of MeOH up to 5% in DCM to elute a band with bright blue fluorescence. The combined fractions were evaporated to give the title compound as a white solid. 1 H NMR (400MHz, Methanol-d4) 1 H NMR(400MHz,Methanol-d4) δ8.79(s,1H),7.93(d,J=13.3Hz,1H),7.54(s,1H),7.30(d,J=8.4Hz,2H ),7.05(d,J=8.5Hz,2H),4.70(dpd,J=7.4,6.2,1.3Hz,4H),3.90(s,5H), 3.75(s,3H),3.39(s,4H),3.18(td,J=16.6,6.4Hz,2H),2.65(tt,J=24. 3,6.3Hz,1H),1.43-1.34(m,1H),1.34-1.19(m,24H),1.18-1.10(m,2H).

[0183] [ka]

[0184] 1-Cyclopropyl-7(4((4(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6), also referred to herein as Formula 2

[0185] Compound 5 (10.0 mg, 0.0124 mmol) was dissolved in DCM (0.2 ml) in a 1.5 ml vial, bromotrimethylsilane (BTMS) (0.2 ml) was added, and the vial was immediately capped and immersed in a 35° C. oil bath. After stirring for 24 h, the solvent and BTMS were removed by evaporation, 1 ml MeOH was added, and the vial was stirred overnight. Evaporation of the solvent left 6.82 mg (0.107 mmol) of a pale yellow solid with green fluorescence. Approximately 0.2 mg of sample was removed for HPLC analysis. Suspended in water, the pH was measured to be 2.5, and then it was adjusted to 6.7 to give a pale yellow solution with blue fluorescence. HPLC analysis (Luna C 18 ; buffer system: 0.1 M NH4OAc buffer pH 7.1; A: 20% acetonitrile, B: 70% acetonitrile. A gradient of 0-7 min: 100% A, 7-25 min: 0-100% B) gave a major peak at RT = 14.8 min, and minor peaks at 5.76 min (assigned to ciprofloxacin) and 18.8 min. Ciprofloxacin standard (2-fold dilution of saturated solution in buffer A; 5 μl injected) gave a RT of 5.68 min.

[0186] Microbiology: Experimental strains: Twelve S. aureus clinical osteomyelitis strains with methicillin susceptibility profiles and one clinical methicillin-resistant strain (MR-CIPS) were tested. These pathogens are part of the strain collection of the Department of Pharmaceutical Microbiology and Parasitology, Medical University in Wroclaw, Poland. In addition, the following ATCC collection strains were selected for experimental purposes: S. aureus 6538 and P. aeruginosa 15442.

[0187] HA disks: Commercially available HA powder was used for the fabrication of the customized disks. Powder pellets with a diameter of 9.6 mm were pressed without the use of binders. Sintering was performed at 900 °C. The tablets were compressed using a Universal Testing System (Instron model 3384; Instron, Norwood, MA) for static tensile, compression, and bending tests. The quality of the fabricated HA disks was examined by confocal microscopy and microtomography (microCT) using a LEXT OLS4000 microscope (Olympus, Center Valley, PA) and a Metrotom 1500 microtomograph (Carl Zeiss, Oberkochen, Germany), respectively.

[0188] Disk diffusion test to assess susceptibility of test strains to ciprofloxacin: The method was carried out according to EUCAST guidelines. Briefly, 0.5 McFarland (MF) bacterial dilutions were spread on Mueller-Hinton (MH) agar plates. A disk containing 5 mg of ciprofloxacin was inserted and the plate was incubated at 37 °C for 24 h. The zone of inhibition was then recorded using a ruler. The obtained values ​​(mm) were compared with the appropriate zone of inhibition values ​​derived from EUCAST tables.

[0189] Evaluation of MICs of test compounds against analyzed planktonic clinical Staphylococcus strains: To evaluate the effect of BP-ciprofloxacin and ciprofloxacin on microbial growth, 1 × 10 5100 μl of microbial solution with a density of cfu / ml was placed in the wells of a 96-well test plate together with the appropriate concentration of test compound. The absorbance of the solution was measured immediately using a spectrophotometer (Thermo Scientific Multiscan GO) at a wavelength of 580 nm. The plate was then incubated at 37°C in a shaker for 24 hours to obtain optimal conditions for bacterial growth and to prevent bacterial biofilm formation. After incubation, the absorbance was measured again. The following controls were prepared: negative control sample 1: sterile medium without microorganisms, negative control sample 2: sterile medium without microorganisms with DMSO (dimethyl sulfoxide; Sigma-Aldrich) added to a final concentration of 1% (vol / vol), positive control sample 1: medium + microorganisms without test compound, positive control sample 2: medium + microorganisms without test compound but with DMSO added to a final concentration of 1% (vol / vol). The rationale for using 1% DMSO was that ciprofloxacin dissolves efficiently in this solvent, but DMSO concentrations above 1% can be harmful to microbial cells. The following calculation was performed to evaluate the relative number of cells: the absorbance value of the control sample (medium + microorganisms in the case of BP-ciprofloxacin, medium + microorganisms + DMSO for ciprofloxacin) was evaluated as 100%. The relative number of cells incubated with the test compound was then counted as follows: control sample absorbance value / test sample value x 100%.

[0190] Spectrophotometric Analysis of BP-Ciprofloxacin Complex in Trypticase Soy Broth (TSB) Microbiological Medium to Examine Stability: BP-ciprofloxacin at final concentrations of 0.24-250 mg / L in TSB microbial medium was placed into wells of a 96-well plate. The absorbance of the solution was measured immediately using a spectrophotometer (Thermo Scientific Multiscan GO) at a wavelength of 275 nm. The solution was then left at 37°C for 24 h with stirring. The absorbance was measured again after incubation. The absorbance values ​​measured at 0 and 24 h were compared to assess the degradation of the complex.

[0191] Spectroscopic analysis of BP-ciprofloxacin complex in trypticase soy broth microbiological medium supplemented with HA spherules: Various concentrations of BP-ciprofloxacin were introduced into HA powder (spheroids) suspended in TSB microbial medium. Solutions containing BP-ciprofloxacin and HA spheroids were placed into the wells of a 24-well plate. The final concentration of the powder was 10 mg / 1 mL, while the final concentration of the complex ranged from 0.24 to 250 mg / L. The absorbance of the solution was measured immediately using a spectrophotometer (Thermo Scientific Multiscan GO) at a wavelength of 275 nm. The plate was automatically stirred in the spectrophotometer before the measurement. The plate was then left at 37°C for 24 hours with stirring. The absorbance was measured again after 24 hours. The absorbance values ​​measured at the beginning and end of the experiment were compared to evaluate the relative concentrations of the complex at 0 and 24 hours.

[0192] Antimicrobial susceptibility testing of BP-ciprofloxacin against planktonic cultures of Staphylococcus aureus ATCC-6538 at acidic and basic pH: The experimental set-up was performed as previously described for the disk diffusion test, except that the microbial medium was adjusted to pH 7.4 and pH 5 using KOH or HCl solutions and the microbial medium was measured using a universal pH indicator (Merck, Poland).

[0193] Time-kill assay of BP-ciprofloxacin conjugate against Staphylococcus aureus ATCC-6538 (MSSA) and clinical MRSA MR4-CIPS strain: The experiment was performed as previously described under the subheading "Evaluation of the MICs of test compounds against analyzed planktonic clinical Staphylococcus strains" except that absorbance measurements (wavelength of 580 nm) were taken at 0, 1, 2, 4, 8, 16, and 24 hours.

[0194] Antimicrobial susceptibility testing of BP-ciprofloxacin performed on biofilms of Staphylococcus aureus ATCC-6538 and Pseudomonas aeruginosa ATCC-15442: Strains cultured on appropriate agar plates (Columbia agar plates for S. aureus; MacConkey agar plates for P. aeruginosa) were transferred to liquid microbiological medium and incubated at 37°C for 24 hours under anaerobic conditions. After incubation, strains were diluted to a density of 1MF. The microbial dilutions were placed in wells of a 24-well plate containing HA disks as substrate or directly in polystyrene wells whose bottom surface served as substrate for biofilm formation. Strains were incubated for 4 hours at 37°C. The microorganism-containing solution was then removed from the wells. The surfaces, HA disks, and polystyrene plates were gently washed to remove plaquetonic or loosely bound microorganisms while leaving attached cells. The surfaces thus prepared were immersed in fresh TSB medium containing 0.24-125 mg / L of BP-ciprofloxacin complex. After 24 h of incubation at 37 °C, the surfaces were washed using saline solution and transferred to 1 mL of 0.5% saponin (Sigma-Aldrich, St. Louis, MO). The surfaces were vigorously mixed on a vortex mixer for 1 min to detach the cells. All microbial suspensions were then diluted with 10–10 9 2-fold dilutions were made. Each dilution (100 mL) was plated on the appropriate stable medium (MacConkey and Columbia for P. aeruginosa and S. aureus, respectively) and incubated at 37°C for 24 hours. After this, the microbial colonies were counted and the number of cells forming a biofilm was assessed. Results were expressed as the mean number of CFU per square millimeter surface ± standard error of the mean. X-ray tomography analysis was applied to estimate the exact surface area of ​​the HA disks. The formula for the area of ​​a circle, πr 2 was applied.

[0195] Inhibitory ability of BP-ciprofloxacin complex to inhibit adhesion of Staphylococcus aureus 6538 strain to HA spherules: Various concentrations of BP-ciprofloxacin were introduced into HA powder (spheroids) suspended in TSB microbial medium. The solutions containing the complex and HA spheroids were placed in the wells of a 24-well plate. The final concentration of the powder was 10 mg / 1 mL, while the final concentration of the complex ranged from 0.12 to 250 mg / L. The suspensions were left at 37°C for 24 hours with stirring. After 24 hours, the suspensions were removed from the wells and centrifuged momentarily to precipitate the HA powder. The supernatant was then very gently discarded and a fresh 1 mL of Staphylococcus aureus with a density of 105 cfu / mL was added to the HA spheroids. The solution was then stirred and the absorbance was measured using a wavelength of 580 nm, and left at 37°C for 24 hours with stirring. The absorbance was measured again after incubation and the values ​​at 0 and 24 hours were compared to evaluate the reduction in bacterial growth relative to control sample 1 (bacterial suspension without spheroids) and control sample 2 (bacterial suspension + spheroids without the addition of complex). The solution was then centrifuged briefly and the supernatant gently discarded, while the bacteria-containing HA spheroids were plated and cultured as before and quantitatively assessed.

[0196] Survival of S. aureus after 24 hours of incubation in the presence of complex-coated HA disks: HA disks were immersed in 2 mL of solutions containing various concentrations of BP-ciprofloxacin or ciprofloxacin alone, and the disks were left at 37°C for 24 hours. HA disks incubated in DMSO or phosphate buffer served as control samples. The disks were then washed three times with sterile water. After washing, 2 mL of 0.5 MF of S. aureus ATCC6538 strain was placed into the wells containing the HA disks as substrates for biofilm formation, and biofilms were formed in the same manner as before.

[0197] Animal testing: All animal experimental protocols and methods were approved and performed in accordance with the recommendations of the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC) and the American Veterinary Medical Association Euthanasia Study Group. USC is registered with the United States Department of Agriculture (USDA), has a fully licensed charter (No. A3518-01) registered with the National Institutes of Health (NIH), and is accredited by the American Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). USC's animal welfare assurance number is A3518-01. The title of our IACUC-approved protocol is "Bone-targeted antimicrobial agents for the treatment of biofilm-mediated osteolytic infections" and its protocol number is 20474. Twelve 5-month-old, virgin female Sprague-Dawley rats, each weighing approximately 200 g, were used in this study. Animals were housed two per cage in a small animal holding room at 22°C under a 12-h light / dark cycle and fed a soft diet (Purina Laboratory Rodent Chow) ad libitum. All animals were handled in accordance with the guidelines and regulations for the use and care of animals at USC. Animals were under the supervision of a dedicated veterinarian on call 24 hours a day who directly evaluated the animals daily. All animal studies were described using the ARRIVE guidelines for reporting animal studies to ensure the quality, reliability, validity, and reproducibility of results (Kilkenny, et al., Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. Vet Clin Pathol 2012;41:27-31).

[0198] This animal model is a homemade jaw bone peri-implant osteomyelitis model specifically designed to study biofilm-mediated disease and host response in vivo (Freire, et al, Development of animal model for Aggregatibacter actinomycetemcomitans biofilm-mediated oral osteolytic infection: a preliminary study. J Periodontol 2011;82:778-89). Biofilms of the jaw bone osteomyelitis pathogen Aa were cultured for 10 min. 9 CFU were preformed on small titanium implants. To confirm the susceptibility of Aa to the parent drug ciprofloxacin prior to our animal studies, we performed AST and MIC assays as previously performed for the long bone osteomyelitis pathogen described previously. After the biofilms were established on the implants in vitro, they were surgically implanted in the jawbone of each rat. For surgery, animals were first anesthetized using 4% isoflurane inhalant followed by intraperitoneal injection of ketamine (80–90 mg / kg) and xylazine (5–10 mg / kg). Local anesthesia was then added by infiltration injection of 0.25% bupivacaine into the surgical site. Preemptive analgesia was then administered subcutaneously with sustained release buprenorphine (1.0–1.2 mg / kg) before the first incision. Once anesthetized, the buccal mucosa of each rat was retracted with a hook and a transmucosal osteotomy was performed using a pilot drill on the alveolar ridge in the natural midline diastema of the anterior palate. The implant was then manually inserted into the osteotomy until the platform was at mucosal level and fixed to the bone. Two biofilm-inoculated implants were placed on either side of the palatine bone of each rat.

[0199] One week after surgery, rats were lightly anesthetized with 4% isoflurane again to ensure implant stability and to note clinical findings at the implant and infection sites. The animals were then administered BP-ciprofloxacin (0.1 mg / kg, 1 mg / kg, or 10 mg / kg as single doses and 0.3 mg / kg three times per week for multiple dose groups) by intraperitoneal injection or ciprofloxacin alone (10 mg / kg three times per week, also for multiple dose groups) as a positive control and sterile endotoxin-free saline as a negative control. Allocation of animals to treatment and control groups was done via a randomization process. Animals in the multiple dose groups were anesthetized as previously described before each additional injection over the week. All compounds were pharmacological grade and made up in sterile injectable saline of the appropriate pH. One week after drug treatment, all animals were placed in a CO2 chamber (60-70% concentration) for 5 min and then euthanized by cervical dislocation. Peri-implant tissue (1cm 2 ) were extracted en bloc and the implants were removed. Peri-implant tissues were immediately homogenized and processed for quantitative assessment of microbial burden. Rats assigned to treatment and control groups were anonymized and concealed from subsequent investigators analyzing their microbial data. For microbial analysis, peri-implant soft tissues and bone were processed in 1 mL of 0.5% saponin, vortexed for 1 min, directly transferred to agar plates, and cultured. The medium for culturing Aa consisted of modified TSB, and frozen stocks were maintained at -80°C in 20% glycerol, 80% modified TSB. All cultures were performed at 37°C in 5% CO2. CFU counts (CFU per gram) of homogenates were determined by plating serially diluted aliquots of homogenates on TSA plates. The mean base 10 log reduction in CFU per gram as a function of treatment was recorded.

[0200] Statistical analysis: Statistical calculations were performed using the SigmaStat package version 2.0 (SPSS Inc., Chicago, IL). Power analysis was performed using G Power 3 software to estimate sample sizes for in vitro and in vivo studies prior to the experiment (Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Meth 2009;41:1149-60). Quantitative data derived from experimental results were analyzed using Kruskal-Wallis test or one-way ANOVA, and statistical significance was accepted at p<0.05 when comparing treated to control groups.

[0201] Example 2

[0202] [ka]

[0203] Methyl 4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoate (7)

[0204] THF (3 mL) was added to a 60% NaH dispersion (0.122 g, 3.05 mmol) in mineral oil in a 50 mL 2-neck round bottom flask under nitrogen. The suspension was cooled to 0° C. with stirring and tetraisopropyl methylenediphosphonate (0.69 mL, 2.18 mmol) was added slowly. The reaction was allowed to reach ambient temperature and when hydrogen gas stopped bubbling from the reaction mixture, the solution was cooled again to 0° C. Methyl 4(bromomethyl)benzoate (0.5 g, 2.18 mmol) was dissolved in THF (2 mL) and added dropwise to the reaction. The resulting solution was stirred overnight while slowly coming to ambient temperature. The reaction mixture was then cooled to 0° C. and quenched with H2O (1 mL). A 5% aqueous solution of citric acid (30 mL) was added, extracted with EtO (3 × 30 mL), and the combined organics were washed with brine (50 mL), dried over NaSO, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography using an EtOAc:Hex gradient (10-100%) to give 7 as a faintly yellow oil (0.323 g, 30% yield). 1 H NMR(400MHz,CDCl3) δ7.93(d,J=8.0 Hz,2H),7.33(d,J=8.4,6.0 Hz,2H),4.79-4.683(m,4H),3.88(s,3H),3.24(td,J=16.0,6.4Hz,2H),2.50(tt,J=24.0,6.2Hz,1H),1.34-1.24(m,24H). 31 P NMR(162MHz,Chloroform-d) δ20.57.

[0205] [ka]

[0206] 4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoic acid (8)

[0207] To a solution of 7 (0.278 g, 0.583 mmol) in MeOH (3 mL) in an 8-dram glass vial, LiOH·HO (0.122 g, 2.914 mmol) was added and the resulting solution was stirred overnight at room temperature. The reaction mixture was evaporated to dryness, the residue was dissolved in water (30 mL), and HCl(aq) (1 M) was added slowly to reach pH 3. The resulting mixture was diluted with CHCl 3( The mixture was extracted with 3×30 mL of ethyl acetate (HCl). The combined organics were dried over MgSO4 and concentrated under reduced pressure to give a thick, clear oil. Yield: quantitative. 1 H NMR(400MHz,CDCl3): δ=7.96 (d,J 6.4,2H),7.36 (d,J 6.4,2H),4.78(sex,J 5.0,4H),3.27(td,J 14.0,4.8,2H),2.60(tt,J 20.0,4.8,1H),1.43-1.26 (m,24H). 31 P NMR(162MHz,Chloroform-d) δ20.57.

[0208] [ka]

[0209] Tetraisopropyl(2(4(chlorocarbonyl)phenyl)ethane-1,1-diyl)bis(phosphonate) (9)

[0210] Compound 8 (0.162 g, 0.339 mmol) was dissolved in chloroform (1 ml) under nitrogen atmosphere and a catalytic amount of DMF (1.3 μL, 0.017 mmol) was added. Thionyl chloride (49.2 μL, 0.678 mmol) was added slowly and the reaction was stirred at room temperature for 2 h. The solvent was removed under vacuum to give a clear oil. The product was used immediately in the next step without further manipulation. Yield: quantitative.

[0211] [ka]

[0212] 7(4(4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (10)

[0213] Ciprofloxacin (0.112 g, 0.339 mmol) was suspended in chloroform (1 mL) and N,N-diisopropylethylamine (DIPEA) (354.3 μL, 2.034 mmol) was added. The freshly made compound 9 was dissolved in chloroform (1 mL) and slowly added to the ciprofloxacin:DIPEA suspension. The reaction mixture was covered with foil and stirred at room temperature overnight. The next day, the solvent was removed under vacuum and the resulting crude was dissolved in DCM (5 mL), filtered through a medium fritted funnel, and washed with more DCM (3×5 mL). The filtrate was concentrated under vacuum and further purified by silica gel column chromatography using a MeOH:DCM gradient (0-10%) to give 10 (0.226 g, 84% yield, 1.8 equiv. DIPEA salt) as a viscous oil that gradually solidified. 1 H NMR(400MHz,CDCl3) δ=8.79(s,1H),8.06 (d,J 12.8,1H),7.38(m,5H),4.80-4.73(m,4H),4.00(s,br,4H),3.56-3.53(m,1H),3.33-3.20(m,6H) 2.50(m,1H),1.45-1.38(m,2H),1.32-1.25(m,24H),1.23-1.19(m,2H). 31 P NMR(162MHz,Chloroform-d) δ20.77.

[0214] [ka]

[0215] 1-Cyclopropyl-7(4(4(2,2-diphosphonoethyl)benzoyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (11)

[0216] Compound 10 (0.108 g, 0.136 mmol) was dissolved in DCM (700 μL) in an 8-dram glass vial and BTMS (686.0 μL, 5.200 mmol) was added. The vial was capped, covered with foil, and heated at 35° C. overnight with stirring. The next day, the solvent was removed under vacuum and the crude was quenched with MeOH (2 mL). The resulting solution was stirred at room temperature for 30 minutes. The solvent was removed under vacuum to give an orange oil. A few drops of water were added to precipitate a yellow solid. More MeOH (2 mL) was added and the resulting suspension was filtered using a medium fritted funnel. The resulting solid was further washed with MeOH to give a yellow powder (0.070 g, 82% yield). 1 H NMR(400MHz,D2O,pH 7.5): δ=8.54(s,br,1H),7.89(m,1H),7.64(m,1H),7.54(d,J 8.0,2H),7.44(d,J 8.0,2H),4.79(m,overlap with D2O,4H),4.00(s,br,2H),3.79(s,br,2H),3.47(s,br,2H),3.34(s,br,2H),3.21(td,J 14.0,6.4,2H),2.30(tt,J 22.0,6.6,1H). 31 P NMR(162MHz,Chloroform-d) δ19.12.ESI-MS m / z (-): 622.24 [MH].

[0217] Example 3 Non-limiting examples of quinolones that can be included in the BP complex Fluorinated quinolones

[0218] [ka]

[0219] [ka]

[0220] [ka] JPEG0007602217000029.jpg34117 JPEG0007602217000030.jpg38126 JPEG0007602217000031.jpg40117

[0221] [ka] JPEG0007602217000033.jpg36125 JPEG0007602217000034.jpg38124 JPEG0007602217000035.jpg36118 JPEG0007602217000036.jpg32123

[0222] [ka] JPEG0007602217000038.jpg36124 JPEG0007602217000039.jpg39136 JPEG0007602217000040.jpg46125

[0223] [ka] JPEG0007602217000042.jpg52138 JPEG0007602217000043.jpg47143

[0224] The following are examples of non-fluorinated quinolones:

[0225] [ka]

[0226] Example 4 The following are non-limiting examples of BP-quinolone conjugates described herein.

[0227] [ka] JPEG0007602217000046.jpg62141

[0228] [ka] JPEG0007602217000048.jpg4474 JPEG0007602217000049.jpg46114,

[0229] [ka] JPEG0007602217000051.jpg103132

[0230] [ka] JPEG0007602217000053.jpg42141 JPEG0007602217000054.jpg40146

[0231] [ka] JPEG0007602217000056.jpg35143 JPEG0007602217000057.jpg40100 JPEG0007602217000058.jpg45139

[0232] [ka] JPEG0007602217000060.jpg43147 JPEG0007602217000061.jpg4374 JPEG0007602217000062.jpg4359

[0233] [ka] JPEG0007602217000064.jpg3975 JPEG0007602217000065.jpg50143 JPEG0007602217000066.jpg50147

[0234] [ka] JPEG0007602217000068.jpg8889

[0235] Example 5 Infectious bone disease, or osteomyelitis, is a common disease in human medicine. 1 and veterinary medicine 2 It is a major global problem in the United States, and the sequelae associated with limb loss 3 and death 4 This can be a serious problem due to the possibility of osteomyelitis. Current approaches to treat osteomyelitis are primarily antimicrobial, often intravenous long-term approaches, with surgical intervention in many cases to control the infection. The causative pathogen in the majority of cases of long bone osteomyelitis is Staphylococcus aureus biofilm, and these microorganisms are bound to bone (Figure 1) in contrast to their planktonic (free-floating) counterparts.5 .

[0236] The biofilm-mediated nature of osteomyelitis is important in clinical and experimental settings because many biofilm pathogens are nonculturable and display altered phenotypes with respect to growth rate and antimicrobial resistance. 5、6 The reasons why highly successful antibiotic treatment of infections in orthopedic surgery has not yet been achieved in general are the occurrence of resistant biofilm pathogens, the low penetration of antibiotics into bone, and adverse events associated with systemic toxicity. 3 This is partly explained by the difficulty in eradicating biofilms with conventional antibiotics.

[0237] Many difficulties in treating osteomyelitis 7 To overcome this, there has been growing interest in drug delivery approaches that use bone-targeting complexes to achieve relatively high or relatively sustained local therapeutic concentrations of antibiotics in bone while minimizing systemic exposure. 8 Conjugation of fluoroquinolone antibiotics to bone-adsorbing bisphosphonates (BPs) (Figure 13) is a promising approach due to the long track record of safety of each component and their favorable biochemical properties. 9、10 Ciprofloxacin (Figure 13) has several advantages for repurposing in this context: 1) it can be administered orally or intravenously and the administration is relatively biologically equivalent, 2) ciprofloxacin is already FDA approved and indicated for bone and joint infections caused by P. aeruginosa and several other pathogens, and 3) ciprofloxacin is effective against the most commonly encountered osteomyelitis pathogens such as Staphylococcus aureus (methicillin-susceptible) and P. aeruginosa for long bone osteomyelitis. 11 , and Aggregatibacter actinomycetemcomitans for osteomyelitis of the jaw. 12 4) ciprofloxacin has bactericidal activity at clinically achievable doses. 13 , 5) Ciprofloxacin is the cheapest drug in the fluoroquinolone family.

[0238] However, like most antibiotics, fluoroquinolones suffer from reduced activity against biofilms compared to the same bacteria in planktonic form. This reduced activity has been specifically shown for ciprofloxacin against Staphylococcus aureus, as well as many other bacterial strains and antibiotic classes. 14~17 Such studies have previously shown that biofilms can be one to several orders of magnitude more resistant to the same antimicrobial agents compared to their planktonic counterparts. This highlights the importance of a bone-targeted approach to osteomyelitis treatment to achieve relatively high local concentrations of antibiotics to the causative biofilm and overcome potential resistance.

[0239] The unique bone-targeting properties of the BP family make these agents ideal carriers for targeting antibiotics to bone in osteomyelitis therapy. 18~20 BP forms strong bidentate and tridentate bonds with calcium phosphate minerals, resulting in their enrichment in hydroxyapatite (HA), especially in skeletal sites of high metabolic activity, including sites of infection and inflammation. 21 BP also exhibits exceptional stability against both chemical and biological degradation. 22 The antibacterial activity of BP-fluoroquinolones is complex and related to the particular strain of pathogen tested, the choice of antibiotic and covalently attached BP moiety, the tether length between the two components, the bone-binding affinity of the BP, the adsorption-desorption equilibrium of the BP, and the stability / lability and kinetics of the conjugated moiety used for conjugation. 18~20Accumulating evidence therefore suggests that a "target-release" linker strategy (Figure 13) in this setting may offer more opportunities for optimization and success when the conjugate is stable in the circulation but unstable at the bone surface. We therefore hypothesized that conjugation of ciprofloxacin to a phenyl BP moiety via a metabolically hydrolyzable carbamate linker should alleviate the problems associated with antibiotic administration in osteomyelitis drug therapy. The cleavable carbamate bond is a key feature in many drugs designed for targeting and release in specific tissues. 23、24 , providing pharmacokinetic advantages such as stability in serum in the presence of acidic and enzymatic environments (e.g., inflammation or infection) and labile on infected bone surfaces. 25 .

[0240] One recent clear success story using a bone-targeted release strategy is Morioka et al. 26 The theory has been presented by et al., who designed an estradiol analogue conjugate using a cleavable variant (carbamate) of a relatively stable amide peptide bond. Several versions of this conjugation were attempted before a pharmacologically active variant (aryl carbamate) was found. Importantly, they demonstrated that effects on bone similar to those of estradiol administered alone (nearly 5,600-fold lower than the total dose of estradiol alone) were produced by a single dose of a similarly linked BP-estradiol conjugate. 26 The conjugate also offered a higher therapeutic index, as it had minimal effects in the whole body and uterine tissues compared to estradiol alone. A pharmacokinetic study completed by Arns et al. 27 This is consistent with the dramatic potency enhancement in studies based on BP-prostaglandins that contain relatively labile linkers. Other synthetic examples of this approach in the antibacterial field have been reported for macrolides. 28However, only alkyl carbamates have been examined, and the lack of other successful examples suggests that targeted release strategies are likely to be biochemical target-dependent as well as chemical class-dependent (considering compatibility of the functional groups of each component), and that their use requires that they be designed to suit any particular chemical class.

[0241] This example describes an aryl carbamate BP-carbamate-ciprofloxacin conjugate 6 (BV600022) and evaluates the conjugate for antimicrobial activity against common osteomyelitis pathogens, as well as in vivo safety and efficacy in an animal model of periprosthetic osteomyelitis. To provide greater clinical or technology transfer relevance, the study in this example utilizes a biofilm model and biofilm methodology in addition to planktonic cultures.

[0242] This compound may sometimes be referred to herein as "Compound 6," "Complex 6," or simply as "6." Similarly, other compounds or complexes may be referred to, for example, as "Compound 11," e.g., "Complex 11," or simply by the compound's field code (e.g., 11).

[0243] result chemistry The overall synthetic scheme for 6 starting from the relatively pharmacologically inert 4-hydroxyphenylethylidene BP (3) is shown in Figure 16. The reagents in the scheme presented in Figure 16 were as follows: Reagents and conditions: (a) BTMS (2 equiv), Et2O, 0 °C to room temperature, 17 hours, 95% yield. (b) i) tetraisopropyl methylene bisphosphonate (1 equiv), NaH (1 equiv), THF, room temperature, 10 min; ii) 1 (1 equiv), room temperature, 2 hours, 52% yield. (c) Pd / C (catalyst) (0.07 equiv), H2, MeOH, room temperature, overnight, 88% yield. (d) 4-nitrophenyl chloroformate (1.1 equiv), Et3N (3 equiv), DCM, room temperature, 2.5 hours, 44% yield. (e) Ciprofloxacin (1.2 equiv), water (pH 8.5), THF, 0° C. to room temperature, overnight, 52% yield. (f) i) BTMS (excess), DCM, 35° C., 24 hours, ii) MeOH, room temperature, overnight, 86% yield.

[0244] The rationale for this BP design was to retain its bone-seeking ability while suppressing the antiresorptive activity of the unwanted BP moiety, minimizing confounding factors and focusing on evaluating the antibacterial activity attributed to the parent ciprofloxacin compound. BP ligands can also be designed to have antiresorptive functionality (of various potencies) if required to provide a dual antibacterial effect plus bone tissue protection at the anatomical site of infection. Previous studies have shown that weak binding affinity reduces targeting efficiency. 13、14 This phenyl BP was selected considering bone-binding affinity and tether length because of its proven ability to bind to bone. The use of an aryl carbamate as a linker was believed to provide optimal plasma stability and adequate separation on bone for this biochemical target compared to previously obtained BP-fluoroquinolone conjugates.

[0245] Additionally, a similar BP-ciprofloxacin conjugate with an amide bond as opposed to a carbamate bond was synthesized as outlined by the scheme shown in Figure 31 as control conjugate 11 (BV600026). The reagents in the scheme presented in Figure 31 were as follows: Reagents and conditions: (a) i) NaH (1.4 equiv), THF, 0°C to room temperature, 1 hour; ii) methyl 4(bromomethyl)benzoate (0.7 equiv), THF, 0°C to room temperature, overnight, 37% yield; (b) LiOH·HO (5 equiv), MeOH, room temperature, overnight, 91% yield; (c) SOCl 2( (d) Ciprofloxacin (1 eq), DIPEA (6 eq), CHCl3, rt, overnight, 65% yield. (e) i) BTMS (excess), DCM, 35°C, overnight, ii) MeOH, rt, 30 min, 82% yield. Previous studies have shown that the amide conjugate is unable to disassociate the parent antibiotic and is therefore less effective in vitro and in vivo, which in this example sought to be confirmed for 11.

[0246] Antibacterial properties of BP-ciprofloxacin complex Minimum inhibitory concentration (MIC) assay: The antibacterial activity of both the complex (6 and 11) and the parent antibiotic ciprofloxacin against a panel of clinical strains of S. aureus, including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), associated with bone infections, was evaluated in a standard laboratory planktonic culture system according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines. 29 According to the results of the disk zone assay, the diameters ranged from 25 to 40 mm (mean: 31.5, SD: ±5) and all strains showed antimicrobial susceptibility according to the EUCAST clinical breakpoints for the parent antibiotic, ciprofloxacin. The minimum inhibitory concentration (MIC) results of 6 and 11 against eight strains of S. aureus using the microdilution method are shown in Figure 32. For reference, the MICs of the parent compound, ciprofloxacin, were determined simultaneously (see Figure 32) and their MICs were consistent with the established clinical breakpoints.29 was found to be inconsistent with

[0247] Hydroxyapatite (HA) binding assay: Having established the antibacterial effect of 6, we next sought to assess its HA binding ability. HA spheroids were added to the microbial medium and various concentrations of 6 were introduced, similar to those used in the antibacterial tests. Significant adsorption and retention of the complex by HA was confirmed by quantitative spectroscopic analysis of the supernatant (containing no HA spheroids) (Figure 18 and Figure 5).

[0248] Effect of pH on planktonic Staphylococcus aureus ATCC-6538 in antimicrobial susceptibility testing (AST): S. aureus ATCC-6538 strain was selected for further investigation as it showed the lowest MIC profile against both ciprofloxacin and 6 compared to the other test strains (see FIG. 32). This ATCC strain is also a well-known and robust biofilm-forming pathogen. Therefore, the conjugate was tested against a challenging pathogen to limit bias and overestimation of results while also facilitating the evaluation of antibacterial activity in a biofilm-based and clinically relevant model. Antimicrobial susceptibility testing (AST) against planktonic S. aureus ATCC-6538 strain was performed with 6 at both acidic and physiological pH to evaluate the effect of pH on the conjugate activity. Quantitative results from standard microdilution methods showed an overall improved antibacterial activity of 6 under acidic conditions (pH 5) as the MIC50 was reached at half the conjugate concentration required to reach the MIC50 under physiological conditions (FIG. 6 and FIG. 4). In these results and those presented elsewhere herein, the terms minimum inhibitory concentration, MIC50 or MIC90, refer to a 50% or 90% reduction in bacterial load, respectively, and the biofilm-related terms for minimum biofilm inhibitory concentration (MBIC50 or MBIC90) refer to a similar reduction (50% or 90%), except that it is a reduction in biofilm bacterial load.

[0249] Time-kill assay of compound 6: Next, the kinetic assay was performed according to CLSI (Clinical Laboratory Standards Institute) method 6. 30 The experiments were carried out according to the method described in the literature. The results showed that the conjugate killed methicillin-susceptible (ATCC-6538) and methicillin-resistant (MR4-CIPS) isolates of planktonic Staphylococcus aureus within 1 hour and up to 24 hours at previously established MICs, with 100% inhibition of bacterial growth. These kinetic studies also showed that the inhibition of bacterial growth was evident after 2 hours at half the MIC values, and that the inhibition of growth was 50% of the control after 24 hours (e.g., Figure 7).

[0250] Evaluation of the antibacterial effect of 6 on biofilms: Compound 6 was then tested against preformed bacterial biofilms on two different substrates (polystyrene and HA disks) to evaluate the antibacterial effect against biofilms and also to determine whether substrate binding properties play any role in the observed antibacterial effect. Biofilms of Staphylococcus aureus (ATCC-6538) and additionally Pseudomonas aeruginosa (ATCC-15442) were grown on polystyrene or HA as substrates and exposed to various concentrations of 6 for evaluation of antibacterial activity. P. aeruginosa was chosen here because it is a gram-negative pathogen and is the second most common clinical pathogen of osteomyelitis, although less frequent than gram-positive S. aureus in terms of prevalence. For example, Figure 8 shows the results for polystyrene as substrate for biofilm growth, where the minimum biofilm inhibitory concentration (MBIC50) of 6 was 15.6-31.2 μg / mL for S. aureus ATCC-6538, which corresponded to the MIC for this strain in planktonic culture state. No MBIC50 was observed for P. aeruginosa strain ATCC-15442 in the concentration range tested, and no MBIC90 was observed for either pathogen.

[0251] However, significant bactericidal activity was observed for 6 when HA disks were used as biofilm substrates. As shown in Figure 33, all tested concentrations of this complex produced statistically significant (p<0.05, Kruskal-Wallis test) bactericidal activity and reduced colony forming units (CFU). The MBIC50 of 6 was 16 μg / mL and the MBIC90 was 100 μg / mL against S. aureus ATCC-6538. The MBIC90 of the parent drug ciprofloxacin against this pathogen was 8 μg / mL. However, against P. aeruginosa ATCC-15442, ciprofloxacin had no inhibitory or bactericidal activity in this setting, whereas the complex was bactericidal at a concentration of 50 μg / mL under acidic and physiological conditions, and showed improved bactericidal activity in physiological conditions in contrast to S. aureus, where improved antibacterial activity was observed under acidic conditions.

[0252] Prophylactic antibacterial assay: Next, antibacterial testing was performed with 6 in a preventive experimental setup using planktonic and biofilm cultures that may have clinical relevance in antibiotic prophylaxis scenarios for osteomyelitis chemotherapy, where HA spheroids were introduced into various concentrations of 6 and incubated with S. aureus for 24 hours, and quantitative evaluation showed no bacterial growth at concentrations of 6 as low as 15.6 μg / mL up to 250 μg / mL, and strongly inhibited minimal bacterial growth at complex concentrations ranging from 0.24 to 7.8 μg / mL, as shown, for example, in Figure 10.

[0253] The amide conjugate (11) was then tested under experimental conditions similar to those used to test the carbamate conjugate 6 for its ability to treat S. aureus ATCC-6538 biofilms. The activity of 11 was assessed against established S. aureus biofilms grown on HA and against established S. aureus biofilms grown on HA that had been pretreated with 11 prior to biofilm growth in a prophylactic experimental setting; even at doses higher than those used to test 6, the antibacterial activity of 11 was marginal in both cases, as shown in FIG.

[0254] When 6 was tested for its ability to prevent S. aureus ATCC-6538 biofilm formation on pretreated HA, the complex showed superior antibacterial activity compared to the parent antibiotic and in contrast to 11, which showed no significant antibacterial activity. Figure 11 shows the results of quantitative biofilm culture and CFU counts after 24 hours of growth, where the parent drug ciprofloxacin inhibited all biofilm growth at a concentration of 100 μg / mL, while 6 inhibited all growth at a concentration of 10 μg / mL. As the molecular mass of ciprofloxacin is approximately half that of 6, 6 was 20 times more active in achieving complete bactericidal activity compared to ciprofloxacin alone.

[0255] In vivo safety and efficacy: Since 6 showed promising activity in vitro, we sought to evaluate the drug safety and efficacy in vivo in an animal model of periprosthetic osteomyelitis. This model is a unique home-grown jaw bone peri-implant osteomyelitis model specifically developed to serve technology transfer value for studying biofilm-mediated disease and host responses in vivo. 31Since osteolysis resulting from infection is important for the attraction (targeting) of high concentrations of the BP complex, as in any highly metabolic site in bone, and subsequently for isolating the active ciprofloxacin component of said complex at the diseased bone surface, this assay is also useful for modeling any infected bone surface where a systemic treatment regimen is utilized. Briefly, biofilms of the jaw osteomyelitis pathogen Aggregatibacter actinomycetemcomitans (Aa; wild-type R-strain D7S-1; a serotype), a bacterium specific for jaw bone infections but not a resident bacterium in the normal flora of rats, were cultured for 10 min at 4°C. 9 CFU were pre-inoculated onto small titanium implants. To confirm the susceptibility of Aa to the parent drug, ciprofloxacin, prior to our animal studies, AST and MIC assays were performed as performed for previously described pathogens of long bone osteomyelitis. Disk zone of inhibition assay revealed a diameter of more than 40 mm and an MIC90 of 2 μg / mL, indicating the strong susceptibility of this organism to the parent drug, ciprofloxacin. Aa was also previously tested for susceptibility to a pH-sensitive biotinylated ciprofloxacin prodrug and was found to be susceptible to the parent antibiotic. 32 Like the previous pathogens in this study, we tested Aa biofilm pathogens grown on HA for susceptibility to 6 and found that our complex exhibited effective antibacterial activity as shown in Figure 35.

[0256] After Aa biofilms have been established on the implants in vitro, they are surgically implanted into the jawbone of each rat. The animals are anesthetized, the cheeks are retracted with a hook, and a transmucosal osteotomy is performed to allow the implants to be inserted and secured into the osteotomy by hand. Two biofilm-inoculated implants are placed on either side of the palate bone of each rat (n=12 rats, 24 implants total). This model results in standard and reproducible amounts of live bacteria forming a well-established biofilm on each implant, which we have previously demonstrated can persist in vivo for weeks after implant placement and cause localized infection, inflammation, and bone destruction. 31 .

[0257] One week after surgery, once peri-implant infection had been established, the animals were administered ciprofloxacin alone as a positive control and sterile endotoxin-free saline as a negative control, according to the dosing regimen specified in the experimental section. To determine the appropriate dosing concentration, an approximate initial dose of the conjugate was calculated based on previous studies using a similar target-release strategy in rodents and on pharmacokinetic data. 13、26 Based on sample size estimates and previous experience with this animal model, six molar equivalents corresponding to gradually increasing doses of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg may allow the determination of antibacterial activity in two experimental animals per group. 32 Animals were dosed by intraperitoneal injection under general anesthesia, and all compounds were made up in sterile injectable saline at the appropriate pH. Intraperitoneal injection was utilized because it is simple to administer in small rodents compared with other parenteral methods such as tail vein injection, and because the pharmacokinetics of ciprofloxacin after gastrointestinal administration indicate good bioavailability. Serum drug levels achieved after such administration are slightly lower but comparable to those achieved by intravenous administration, with virtually no loss after first-pass metabolism. 33One week after drug treatment, all animals were sacrificed, and en bloc excision of peri-implant hard and soft tissues was performed and homogenized for quantitative assessment of microbial burden.

[0258] All animals tolerated the drug therapy well, without any skin injection site reactions or irritation. There were no indications of major tolerability issues during treatment. Treatment efficacy was quantitatively measured as the log reduction in viable bacteria (mean log base 10 of CFU per gram of tissue) as shown in Figure 36.

[0259] In vivo, a single dose of 6 at 10 mg / kg showed the highest efficacy with a 10^2 reduction in bacterial counts (99% kill) and activity nearly an order of magnitude higher than ciprofloxacin alone (30 mg / kg total dose) administered multiple times at the same concentration per dose (mg / kg). Thus, taking into account the larger molecular weight of 6 (approximately twice that of ciprofloxacin), a single dose of 6 administered at 10 mg / kg can deliver roughly 5 mg / kg of effective ciprofloxacin assuming complete dissociation, which is one-sixth the molar dose of ciprofloxacin in the control ciprofloxacin group (30 mg / kg total). Ciprofloxacin alone in a multiple dose formulation produced a 10^1 reduction in bacterial counts (90% kill). Concentrations of 0.1 mg / kg and 1 mg / kg of 6 had little effect, suggesting that a minimum dose is required for clinical efficacy and that further chemical optimization may be possible in this setting.

[0260] To confirm the findings in the animal study and to provide stronger statistical power and a larger sample size for statistical analysis, we conducted a second animal study that was nearly identical to the first, except for the dosing regimen assignment. Based on the dosing data and antibacterial performance from our first animal study above, we focused this second animal study on three treatment groups: negative control (n=5 rats), 6 (n=5 rats) with a single high dose of 10 mg / kg, and 6 (n=2 rats) with multiple low doses of 0.3 mg / kg three times per week. The 0.1 mg / kg and 1 mg / kg dose groups were excluded because they had not shown efficacy to date, and the parent antibiotic alone was also excluded because of the strong track record data on the efficacy of ciprofloxacin that was also confirmed in our first animal study. The multiple dose regimen was again utilized to determine whether the absence of recoverable bacteria could be attributed to a therapeutic effect or to experimental and sampling error. All other experimental parameters were identical to the first animal study, and as before, two implants were placed in each animal, thereby allowing for two results per animal and providing sufficient statistical power for statistical analysis as determined by sample size estimates.

[0261] All animals again tolerated the treatment and medication well, with no indication of major tolerability issues during treatment. Clinically, it was observed at anesthesia and surgical removal that the majority of animals in the control group showed evidence of localized periprosthetic inflammation compared to the majority of animals in the treatment group who had non-inflamed peri-implant tissue. Implant retention was high, with 23 of 24 implants (96%), providing strong statistical power for subsequent analyses. Quantitative antibacterial performance from this second animal study is shown in Figure 37. A one-way ANOVA test (α=0.05) comparing CFU between treatment groups yielded a p-value of 0.006 for significance between groups, and post-hoc tests utilizing independent t-tests (p=0.0005; df=20) and Dunnett's multiple comparison test (p<0.05) showed significance of the 6 single high-dose treatments compared to the control, but not the multiple low-dose treatments compared to the control or single high-dose treatment groups (p>0.05).

[0262] Consideration Targeting antibiotics to bone by conjugation to the BP moiety (via a detachable carbamate linker) is a promising approach for the treatment of osteomyelitis biofilms. The AST test results and MIC data presented herein indicate that ciprofloxacin and 6 have effective bactericidal activity against planktonic S. aureus, and that the conjugation linkage affects the antibacterial activity of the parent drug, as evidenced by the relatively weak activity of 11 (Figure 32). A relatively high concentration of 6 was required to reach the MIC. This is expected, since conjugation is a chemical modification that may alter the biochemical interactions of the antibiotic before it is separated from the conjugate. As a result, the properties of the parent drug, including the pharmacodynamic effects, may be altered by such modifications. The MIC results for 6 indicate that this class of conjugates may retain its antibacterial activity, albeit at a slightly lower level than the antibacterial activity of the parent compound, as previously reported in previous literature. 9、10 It matched.

[0263] It was interesting to note that, compared to ciprofloxacin alone, which showed little difference in antibacterial efficacy against the multiple S. aureus strains tested, the MIC values ​​of both conjugates against the same strains were widely distributed (Figure 17). There are several possible explanations for these results. It is known that different bacterial strains within the same species show significant differences in terms of virulence and drug susceptibility / resistance to antibiotics. It is well documented that strain-specific variations exist with regard to antibiotic transport efflux mechanisms, bacterial cell wall density, enzyme activity levels, resistance mechanisms, and ability to modify the pH of the environment. 34 The bactericidal activity of ciprofloxacin results from intracellular inhibition of topoisomerase II and IV, enzymes required for DNA replication. 35 .

[0264] The complete complex of this class has largely lost any significant intrinsic antibacterial activity. 18、19 , and any BP-associated antibacterial activity has proven to be negligible, therefore at least partial dissociation of the parent drug is a prerequisite for significant antibacterial activity as observed for 6. This is consistent with the low antibacterial activity of 11, which differs in its relatively stable amide bond, which achieved the same antibacterial activity in the assay at concentrations 2-64 times higher than the relatively unstable carbamate-linked conjugate 6.

[0265] After evaluating the antibacterial effect of 6, we sought to evaluate the bone-binding functionality of the BP moiety and found effective adsorption and retention to HA spheroids by the complex in a concentration-dependent manner. These results are consistent with previously reported analogs of this class containing BP moieties with similar bone affinity. 13、19Next, the activity of 6 was investigated under different pH conditions, and a slightly improved profile was found in acidic conditions. This can be explained at least in part by the fact that the linker is less stable at pH 5 than at pH 7.4, and therefore more ciprofloxacin is detached at the relatively low pH. This could be useful for clinical applications in osteomyelitis, where biofilm pathogens create an acidic local environment along with host inflammation and osteoclast formation. However, others have suggested that although the acidic pH generated by infectious organisms and inflammation may cause some drug elution in bone, the effectiveness of such a process in providing significant concentrations of the antimicrobial agent is questionable, and that the prodrug design, conjugation scheme, and susceptibility to local enzymatic hydrolysis may play a larger role in linker cleavability and efficacy. 13 The data in this example also support such a conclusion.

[0266] A 6-hour kill kinetics study demonstrated efficient bactericidal activity rates against the test bacteria with sustained bactericidal activity over 24 hours, supporting the cleavage activity of the parent antibiotic with a stable and sustained dissociation profile over time. The antibiotic dissociation kinetics observed herein may differ from those observed for biodegradable and non-biodegradable delivery systems currently used for osteomyelitis treatment, which generally show an initial high release of antibiotic at the administration site, with the remaining antibiotic disappearing at a smaller percentage over an extended period of time. 36、37 .

[0267] This example provides evidence of the antibacterial efficacy of complexes such as 6 in in vitro and in vivo biofilm-associated models for osteomyelitis treatment. When osteomyelitis biofilms (S. aureus and P. aeruginosa) were grown in vitro on different substrates such as polystyrene or HA and subsequently treated with 6, the complex was more effective against the biofilms in the presence of HA than polystyrene. This indicates that in addition to factors such as the strain of pathogen tested and the bacterial growth mode (planktonic vs. biofilm), substrate binding properties play a role in antibacterial activity. The fact that 6 was effective against osteomyelitis pathogens on HA but not against the same strains on polystyrene as substrate indicates that binding to the substrate (e.g., HA) and releasing the antibiotic directly under or within the biofilm is necessary to effectively treat osteomyelitis biofilms rather than simply shedding the antibiotic along the biofilm surface (as in the case of the parent antibiotic alone or 6 on polystyrene where no substrate binding occurred and no activity against established surface biofilms was observed). The improved activity of 6 found in the experimental setup using HA disks compared to the setup using polystyrene as substrate is likely due to the fact that the BP moiety of the complex has a high affinity for HA structures and therefore localization of 6 to the disks could expose HA-adherent bacteria to relatively high concentrations of the parent antibiotic. Also, cleavage of 6 in bone underlying biofilm bacterial cells was previously shown to be associated with increased activity of 6. 22 This may be similar to carbamate cleavage in osteoclasts, suggesting that the local environment plays a role in this situation, and further indicating that the environment in the bacteria causing osteolysis has similarities to that in the osteoclasts present in bone, as both of these environments appear to be capable of cleaving the aryl carbamate bond to release the active ciprofloxacin, possibly by a combination of pH and enzymatic hydrolysis. Previous studies by Arns et al. using BP (radiolabeled) prostaglandin complexes 27The half-life of the complex in the bloodstream is the half-life of most BP. 38 suggests that the half-life for dissociation of the active drug (in this case a prostaglandin) from the BP on bone surfaces with bonds similar to our carbamate is between 5 and 28 days. The bonds shown here must dissociate relatively close to the 5-day half-life to achieve the exciting in vivo results reported here. Arns et al. 27 speculate that in the presence of bone cells the cleavage mechanism is most likely enzymatic. In the presence of bacteria on mineral surfaces the cleavage mechanism may also be enzyme-based. As already noted in the manuscript during in vitro antibacterial testing in the absence of osteoclasts, our carbamate-based conjugates are active, whereas our non-cleavable amide-based conjugates have very low activity.

[0268] The conjugates were also tested in osteomyelitis prevention experiments against S. aureus and found that 6 was 20-fold more active than ciprofloxacin alone in achieving complete bactericidal activity (Figure 11), whereas 11 did not show any antibacterial activity (Figure 34). These findings support an efficient mechanism of cleavage of the parent antibiotic from 6 over time compared to 11. Efficient binding to HA and release of the parent antibiotic are prerequisites for this class of conjugates to show substantial antibacterial efficacy.

[0269] Finally, we sought to test the in vivo safety and efficacy of 6 in a rat model of peri-implant osteomyelitis of the jaw bone using the model jaw bone pathogen Aa. To confirm the susceptibility of Aa to the parent drug ciprofloxacin prior to our animal studies, we performed in vitro AST and MIC assays as performed for the long bone osteomyelitis pathogens in this study. Aa showed strong susceptibility to the parent drug ciprofloxacin. We also tested Aa biofilms grown on HA (as well as S. aureus and P. aeruginosa) for susceptibility to 6 and found that our complex exhibited effective antibacterial activity (Figure 35). Therefore, two consecutive animal studies were performed utilizing the peri-implant jaw bone osteomyelitis model. In the first in vivo study, a single dose of 10 mg / kg of 6 produced a 10-fold reduction in CFU or 99% kill, with maximal efficacy with activity nearly an order of magnitude higher than ciprofloxacin alone given multiple doses at the same concentration (mg / kg) per dose (Figure 36), which was not observed with the relatively stable 11 even at high doses, but was more potent than the parent antibiotic alone as observed with the relatively unstable 6. 18~20 The results showed equal or greater efficacy than either of the two, confirming that cleavage contributes to, and in some instances may be necessary for, the antibacterial effect. Lower concentrations of 6 were ineffective in this experiment. To confirm these results, we performed a second, larger and more statistically powerful in vivo study focusing on an effective dosing regimen of 6 (10 mg / kg) compared to controls and a multiple-dose formulation of 6. The greatest CFU reduction and efficacy was again observed with the single high dose (10 mg / kg) of the conjugate.

[0270] In vivo experiments confirmed the ability of 6 to target infected peri-implant bone and produce sufficient concentrations of parent antibiotic for bactericidal activity against established Aa biofilms when the activity of the parent antibiotic alone had already disappeared, in a safe and adequate single dose. Since microbial quantification involved homogenates of en bloc excised tissue (as the methodology did not include surface scraping for plating and evaluation), the analysis included not only surface pathogens but also biofilm bacteria within the tubules of the 3D bone construct. This suggests effective BP absorption / adsorption and sequestration of the antibiotic to the peri-prosthetic bone as evidenced by a significant reduction in CFU of biofilm bacteria.

[0271] These results, along with other studies in the field, also indicate that direct comparisons between these conjugates and their parent compounds are somewhat arbitrary, since the conjugates have unique efficacy parameters and are primarily localized in bone due to the BP moiety. This is due to the fact that in humans, uptake is much higher in muscle and tendon than in bone. 39 This is in contrast to parent antibiotics (generally of the fluoroquinolone class) which are therefore correlated with adverse events such as Achilles tendon rupture in susceptible populations. Any future pharmacokinetic modeling and pharmacokinetic studies of this class of complex will need to mathematically include the skeletal compartment of distribution, which is not commonly done in pharmacokinetic studies of ciprofloxacin and most other antibiotics. The importance of such an approach to accurately determine the bone pharmacokinetics of BP drugs in human populations has been demonstrated. 40 Such an approach would provide more precise and necessary pharmacological data in this setting and also inform clinical dosing approaches.

[0272] Materials and Methods All operations were carried out under a nitrogen atmosphere unless otherwise stated. Anhydrous ethyl ether, anhydrous tetrahydrofuran, anhydrous citric acid, chloroform, and magnesium sulfate were purchased from EMD. 4-Benzyloxybenzyl alcohol, bromotrimethylsilane, 4-nitrophenyl chloroformate, hydrochloric acid (37%), absolute ethanol, anhydrous N,N-dimethylformamide, and thionyl chloride were purchased from Sigma-Aldrich. Sodium sulfate was purchased from Amresco. Sodium hydride (57-63% oil dispersion), tetraisopropyl methylenediphosphonate, 10% palladium on activated carbon, 4(bromomethyl)benzoate, lithium hydroxide monohydrate, and N-ethyldiisopropylamine were purchased from Alfa Aesar. Ethyl acetate, hexane, and dichloromethane were purchased from VWR. Anhydrous methyl alcohol, trimethylamine, and sodium carbonate were purchased from Macron. Hydrogen gas was purchased from Airgas. Ciprofloxacin was purchased from Enzo Life Sciences. Acetonitrile (HPLC grade) was purchased from Spectrum. All reagents were used as received unless otherwise stated. 3 Å molecular sieves (20% (w / v)) 41 All solvents were dried using HPLC. Silica gel (SilicaFlash P60, 40-63 Å, 40-63 μm, 230-400 mesh) was purchased from Silicycle.

[0273] Nuclear magnetic resonance spectra were recorded on a Varian 400-MR dual channel NMR spectrometer equipped with a 96-spinner sample changer and analyzed using TopSpin and MestReNova. 1H chemical shifts (δ, ppm) were referenced to the residual solvent peak. Mass spectra were acquired on a Thermo-Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source under positive and / or negative mode using Tune Plus version 2.0 software for data acquisition and Xcalibur® 2.0.7 for data processing, and reported in m / z format. Organic elemental analysis was performed by Thermo Fisher Scientific on a Flash 2000 Elemental Analyzer.

[0274] The purity of the final compounds 6 and 11 and the commercially available ciprofloxacin was 95% or higher. 1 H and 31 The P content was determined using NMR spectroscopy, HPLC, and elemental analysis. Analytical HPLC of the final compound was performed on a SHIMADZU HPLC system equipped with a diode array detector. Lab Solutions software was used for both data collection and analysis. HPLC Method A: A Phenomenex Luna 5μ C18(2) 100 Å analytical column (250 × 4.6 mm) operated at a flow rate of 1.0 mL / min was used. The following solvent gradient was used: (Buffer A = 20% ACN in 0.1 M NH4OAc (pH 7.53), Buffer B = 70% ACN in 0.1 M NH4OAc (pH 7.16)) 0-7 min: 0% B, 7-25 min: 100% B, 25-100 min: 100% B.

[0275] Synthesis of 1(benzyloxy)-4(bromomethyl)benzene(1) 4-Benzyloxybenzyl alcohol (1.00 g, 4.67 mmol) was dissolved in anhydrous diethyl ether (25 mL) in an oven-dried flask under nitrogen. The flask was cooled in an ice bath. Bromotrimethylsilane (BTMS) (1.26 mL, 9.52 mmol, 2 equiv.) was added via syringe. The flask was allowed to warm slowly to room temperature. After stirring for 17 h, the reaction mixture was poured into water (50 mL) and the organic phase was separated. The aqueous phase was washed with diethyl ether (2×20 mL) and the combined organic phase was washed with brine (2×20 mL) and dried over sodium sulfate. Evaporation of the solvent afforded compound 1 as a white crystalline solid (1.23 g, 95% yield). 1H NMR (400MHz, Chloroform-d) δ7.47 - 7.28(m,7H),6.98 - 6.90(m,2H),5.07(s,2H),4.50(s,2H).

[0276] Tetraisopropyl(2(4(benzyloxy)phenyl)ethane-1,1-diyl)bis(phosphonate)(2) Anhydrous THF (2 mL) was added to sodium hydride (57-63% dispersion in mineral oil) (75 mg, 1.80 mmol, 1 equiv.) under nitrogen protection. Tetraisopropyl methylenediphosphonate (570 μL, 1.80 mmol, 1 equiv.) was added dropwise with stirring at room temperature. Gas was evolved and the grey suspended solid was consumed leaving a clear solution. The mixture was stirred for another 10 min. Compound 1 (500 mg, 1.80 mmol, 1 equiv.) was added in one portion under nitrogen counterflow. The solution was clear for 1 min and then became cloudy. Stirring was continued for 2 h and the progress of the reaction was monitored by TLC (100% EtOAc; visualized by UV and cerium ammonium molybdate (CAM) stain). The reaction mixture was poured into 5% aqueous citric acid (30 mL), extracted with ether (2×30 mL), washed with brine (30 mL), and evaporated. The residue was purified by flash chromatography using an EtOAc:hexane gradient (10-100%) to give 2 as a colorless oil (0.508 g, 52% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.44-7.27 (m, 5H), 7.18 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.04 (s, 2H), 4.86-4.63 (m, 4H), 3.15 (td, J = 16.6, 6.1 Hz, 2H), 2.44 (tt, J = 24.2, 6.1 Hz, 1H), 1.48-1.01 (m, 24H). 31P NMR (162 MHz, Chloroform-d) δ 21.11.

[0277] Tetraisopropyl(2(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate)(3) Compound 2 (0.508 g, 0.925 mmol) was dissolved in 13 mL of methanol and 10% palladium on activated carbon (70 mg, 0.066 mmol, 0.07 equiv.) was added. The flask was flushed with nitrogen, then with hydrogen, and a hydrogen balloon was attached and stirred overnight. The reaction mixture was filtered through Celite with the aid of 100 mL of methanol. Evaporation of the filtrate gave the desired compound 3 as a pale yellow oil that was used without further purification (0.368 g, 88% yield). 1H NMR(400MHz,Chloroform-d) δ7.07(d,J=8.2Hz,2H),6.69(d,J=8.2Hz,2H),4.71(m,4H),3.11(td,J=16.9,6.0 Hz,2H),2.47(tt,J=24.4,6.0 Hz,1H),1.32 - 1.21(m,24H).31P NMR(162MHz,Chloroform-d) δ21.06.

[0278] 4(2,2-bis(diisopropoxyphosphoryl)ethyl)phenyl(4-nitrophenyl)carbonate(4) Compound 3 (7.91 g, 15.9 mmol) was dissolved in 150 mL of dichloromethane, followed by the addition of triethylamine (6.70 mL, 47.9 mmol, 3 equiv.), followed by p-nitrophenyl chloroformate (3.54 g, 17.6 mmol, 1.1 equiv.) in one portion. The reaction mixture was stirred for 2.5 h while being monitored by TLC (5% MeOH in EtOAc; visualized by UV). After disappearance of the starting material, the reaction was stopped and the target compound was purified by flash chromatography (1:1 ethyl acetate:hexane mixture) to give compound 4 (4.33 g, 44% yield). 1H NMR(400MHz,Chloroform-d) δ8.29(d,J=9.1Hz,2H),7.46 (d,J=9.1Hz,2H),7.33(d,J=8.5Hz,2H),7.15(d,J=8.6Hz,2H),4.84-4.58(m,4H) ,3.22(td,J=16.5,6.2Hz,2H),2.47(tt,J=24.1,6.2Hz,1H),1.33-1.14(m,24H).

[0279] 7(4((4(2,2-bis(diisopropoxyphosphoryl)ethyl)phenoxy)carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5) Ciprofloxacin (2.76 g, 8.34 mmol, 1.2 equiv.) was suspended in 74.7 mL of water in a flask. Then, 8.30 mL of 1 M HCl was added and the flask was stirred to dissolve the ciprofloxacin, resulting in a clear, colorless solution. The pH was adjusted to 8.5 by adding Na2CO3, resulting in a thick white precipitate. The flask was placed in an ice bath and compound 4 (4.28 g, 6.95 mmol, 1 equiv.) dissolved in 83 mL of THF was added slowly over a period of about 5 min. The flask was then removed from the ice bath, protected from light, and stirred overnight at room temperature. The reaction mixture was concentrated under vacuum to about half of its original volume and filtered through a fine glass fritted funnel. The remaining solid was washed with water until no yellow color remained. The solids were then dissolved in DCM and washed through the frit, and the solution was loaded onto a flash silica column and eluted with a MeOH:DCM gradient (2-5%) to give compound 5 as a white solid (3.47 g, 51.5% yield). 1H NMR(400MHz,Methanol-d4) δ8.79(s,1H),7.93(d,J=13.3Hz,1H),7.54(s,1H),7.30(d,J=8.4Hz,2H), 7.05(d,J=8.5Hz,2H),4.70(dpd,J=7.4,6.2,1.3Hz,4H),3.90(m,4H),3.65 (s,br,1H),3.39(s,br,4H),3.18(td,J=16.6,6.4Hz,2H),2.65(tt,J=24.3 ,6.3Hz,1H),1.43-1.34(m,2H),1.34-1.19(m,24H),1.18-1.10(m,2H).31P NMR(162MHz,Methanol-d4) δ20.71.MS (ESI+) m / z: 808.2(M+H),830.2(M+Na) calc.for C38H53FN3O11P2+: 808.3.

[0280] 1-Cyclopropyl-7(4((4(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6) 42、43 Compound 5 (10.0 mg, 1.24 μmol) was dissolved in DCM (200 μL) in a 1.5 mL glass vial, BTMS (200 μL, 1.52 mmol, 122 equiv.) was added, and the vial was immediately capped and immersed in a 35° C. oil bath. After stirring for 24 h, the solvent and BTMS were removed under vacuum, 1 mL of MeOH was added, and the vial was stirred overnight. The solvent was removed under vacuum to give pure compound 6 (6.82 mg, 86.1% yield) as a light yellow solid with green fluorescence. 1H NMR(400MHz, Deuterium Oxide) δ8.51(s,1H),7.92(d,J=12.2Hz,1H),7.67(s,1H),7.47(d,J=8.3Hz,2H),7. 10(d,J=8.3Hz,2H),3.98(s,2H),3.79(s,2H),3.67(s,1H),3.42(s,4H),3.16 (td,J=15.5,6.8Hz,2H),2.21(tt,J=6.9,21.6Hz,1H),1.37(d,J=6.9Hz,2H),1.15(s,2H).31P NMR(162MHz,Deuterium Oxide) δ19.16 MS (ESI-) m / z: 638.06 (MH) calc.for C26H27FN3O11P2-: 638.1.HPLC (Method A,UV 190,274,330nm):tr=11.62 min.

[0281] Methyl 4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoate (7) 44 THF (5 mL) was added to a 57-63% sodium hydride dispersion in mineral oil (0.163 g, 4.07 mmol, 1.4 equiv) in a 25 mL round bottom flask under nitrogen. The suspension was cooled to 0 °C with stirring and tetraisopropyl methylenediphosphonate (0.926 mL, 2.90 mmol, 1 equiv) was added slowly. The reaction was allowed to reach ambient temperature and when hydrogen gas stopped bubbling from the reaction mixture, the solution was cooled again to 0 °C. Methyl 4-(bromomethyl)benzoate (0.465 g, 2.03 mmol, 0.7 equiv) was dissolved in THF (2 mL) and added dropwise to the reaction. The resulting solution was stirred overnight while slowly approaching ambient temperature. The reaction mixture was then cooled to 0 °C and quenched with EtOH (1 mL). A 5% aqueous solution of citric acid (30 mL) was added, the mixture was extracted with EtO (3 × 30 mL), and the combined organics were washed with brine (50 mL), dried over NaSO, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography using an EtOAc:Hex gradient (10-100%) to give 7 as a faintly yellow oil (0.371 g, 37.0% yield). 1H NMR (400MHz, Chloroform-d) δ7.93(d,J=8.0 Hz,2H),7.33(d,J=8.4,2H),4.79-4.68(m,4H),3.88(s,3H),3.24(td,J =16.0,6.4Hz,2H),2.50(tt,J=24.0,6.2Hz,1H),1.34-1.24(m,24H).31P NMR(162MHz,Chloroform-d) δ20.57.

[0282] 4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoic acid (8) 44 To a solution of 7 (0.131 g, 0.278 mmol) in MeOH (1.5 mL) in an 8-dram glass vial, LiOH·HO (0.058 g, 1.39 mmol, 5 equiv.) was added and the resulting solution was stirred overnight at room temperature. The reaction mixture was evaporated to dryness, the residue was dissolved in water (30 mL), and HCl(aq) (1 M) was added slowly to reach pH 3. The resulting mixture was diluted with CHCl 3( The mixture was extracted with 100 mL of 1,000 sq. m. (3 × 30 mL). The combined organics were dried over MgSO4 and concentrated under reduced pressure to give 8 as a thick clear oil (0.115 g, 90.6% yield). 1H NMR (400 MHz, Chloroform-d): δ = 7.96 (d, J = 8.0, 2H), 7.37 (d, J = 8.0, 2H), 4.82-4.74 (m, 4H), 3.28 (td, J = 16.6, 6.1, 2H), 2.60 (tt, J = 24.2, 6.2, 1H), 1.33-1.26 (m, 24H). 31P NMR (162 MHz, Chloroform-d) δ 20.57.

[0283] Tetraisopropyl(2(4(chlorocarbonyl)phenyl)ethane-1,1-diyl)bis(phosphonate) (9) Compound 8 (0.162 g, 0.339 mmol) was dissolved in chloroform (1 mL) under nitrogen atmosphere and a catalytic amount of DMF (1.30 μL, 0.017 mmol, 0.05 equiv.) was added. Thionyl chloride (49.2 μL, 0.678 mmol, 2 equiv.) was added slowly and the reaction was stirred at room temperature for 2 h. The solvent was removed in vacuo to give 9 as a clear oil. The product was used immediately in the next step without further manipulation (quantitative yield).

[0284] 7(4(4(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (10) Ciprofloxacin (0.112 g, 0.339 mmol, 1 equiv.) was suspended in chloroform (1 mL) and N,N-diisopropylethylamine (DIPEA) (354 μL, 2.03 mmol, 6 equiv.) was added. Freshly made compound 9 (168 mg, 0.338 mmol, 1 equiv.) was dissolved in chloroform (1 mL) and slowly added to the ciprofloxacin:DIPEA suspension. The reaction mixture was covered with foil and stirred overnight at room temperature. The next day, the solvent was removed under vacuum and the resulting crude was dissolved in DCM (5 mL), filtered through a medium fritted funnel, and washed with more DCM (3×5 mL). The filtrate was concentrated under vacuum and further purified by silica gel column chromatography using a MeOH:DCM gradient (0–10%) to give 10 (0.226 g, 65.1% yield, 1.8 equiv. of DIPEA salt) as a viscous oil that gradually solidified. 1H NMR (400MHz, Chloroform-d) δ=8.79(s,1H),8.06 (d,J=12.8,1H),7.38(m,5H),4.80-4.73(m,4H),4.00(s,br,4H),3.56-3.53(m,1H),3.33-3.20(m,6H) 2.50(m,1H),1.45-1.38(m,2H),1.32-1.25(m,24H),1.23-1.19(m,2H).31P NMR(162MHz,Chloroform-d) δ20.77.

[0285] 1-Cyclopropyl-7(4(4(2,2-diphosphonoethyl)benzoyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (11) 42、43 Compound 10 (0.108 g, 0.136 mmol) was dissolved in DCM (700 μL) in an 8-dram glass vial and BTMS (686 μL, 5.20 mmol, 38 equiv.) was added. The vial was capped, covered with foil, and heated at 35° C. overnight with stirring. The next day, the solvent was removed under vacuum and the crude was quenched with MeOH (2 mL). The resulting solution was stirred at room temperature for 30 minutes. The solvent was removed under vacuum to give an orange oil. A few drops of water were added to produce a yellow solid. More MeOH (2 mL) was added and the resulting suspension was filtered using a medium fritted funnel. The resulting solid was further washed with MeOH to give 11 as a yellow powder (0.070 g, 82.0% yield). 1H NMR (400MHz, Deuterium Oxide, pH 7.5): δ=8.54(s,br,1H),7.90-7.87(m,1H),7.65-7.63(m,1H),7.54(d,J=8.0,2H),7.44(d,J=8.0,2H),4.79(m,overlap with D2O,4H),4.00(s,br,2H),3.79(s,br,2H),3.47(s,br,3H),3.34(s,br,2H),3.21(td ,J=14.0,6.4,2H),2.30(tt,J=22.0,6.6,1H),1.38-1.33(m,2H),1.15(s,br,2H).31P NMR(162MHz,Deuterium Oxide,pH 7.5) δ19.12.MS (ESI -) m / z: 622.24(MH) calc.for C26H27FN3O10P2-: 622.12.HPLC (Method A,UV 190,274,330nm):tr=4.43 min.

[0286] Antibacterial properties of bisphosphonate-ciprofloxacin conjugates Experimental strains: Seven S. aureus clinical osteomyelitis strains with a methicillin susceptibility profile and one strain with a methicillin resistance profile were tested. These pathogens are part of the strain collection of the Department of Pharmaceutical Microbiology and Parasitology, Medical University in Wroclaw, Poland. In addition, the following American Type Culture Collection (ATCC) strains were selected for experimental purposes: S. aureus 6538 and P. aeruginosa 15442.

[0287] HA disks: Commercially available HA powder was used for the fabrication of the customized disks. Powder pellets with a diameter of 9.6 mm were pressed without the use of binders. Sintering was performed at 900 °C. The tablets were compressed using a Universal Testing System (Instron model 3384; Instron, Norwood, MA) for static tensile, compression, and bending tests. The quality of the fabricated HA disks was examined by confocal microscopy and microtomography (microCT) using a LEXT OLS4000 microscope (Olympus, Center Valley, PA) and a Metrotom 1500 microtomograph (Carl Zeiss, Oberkochen, Germany), respectively.

[0288] Disk diffusion test to assess susceptibility of test strains to ciprofloxacin: EUCAST 29 The method was carried out according to the guidelines. Briefly, 0.5 McFarland (MF) bacterial dilutions were spread on Mueller-Hinton (MH) agar plates. A disk containing 5 mg of ciprofloxacin was inserted and the plate was incubated at 37°C for 24 hours. The zone of inhibition was then recorded using a ruler. The obtained values ​​(mm) were then calculated according to the EUCAST table 29 The inhibition zone values ​​were compared with the appropriate inhibition zone values ​​derived from the

[0289] Evaluation of MICs of test compounds against analyzed planktonic clinical Staphylococcus strains: To evaluate the effect of parent antibiotics and conjugates on microbial growth, 1 × 10 5 100 μl of microbial solution with a density of CFU / ml was placed into the wells of a 96-well test plate together with the appropriate concentration of test compound. The absorbance of the solution was measured immediately using a spectrophotometer (Thermo Scientific Multiscan GO) at a wavelength of 580 nm. The plate was then incubated at 37°C in a shaker for 24 hours to obtain optimal conditions for bacterial growth and to prevent bacterial biofilm formation. After incubation, the absorbance was measured again. The following controls were prepared: negative control sample 1: sterile medium without microorganisms, negative control sample 2: sterile medium without microorganisms to which DMSO (dimethyl sulfoxide; Sigma-Aldrich) was added to a final concentration of 1% (vol / vol), positive control sample 1: medium + microorganisms without test compound, positive control sample 2: medium + microorganisms without test compound but to which DMSO was added to a final concentration of 1% (vol / vol). The rationale for using 1% DMSO was that ciprofloxacin dissolves efficiently in this solvent, but DMSO concentrations above 1% can be harmful to microbial cells. The following calculations were performed to assess the relative number of cells: the absorbance value of the control sample (medium + microorganism for the complex, medium + microorganism + DMSO for ciprofloxacin) was assessed as 100%. The relative number of cells incubated with the test compound was then counted as follows: control sample absorbance value / test sample value x 100%.

[0290] To confirm the results obtained by spectrophotometric evaluation, the treated bacterial solution was transferred to 10 mL of fresh medium and left at 37°C for 48 hours. The presence or absence of turbidity formation in the medium was evidence of the presence or absence of pathogen growth, respectively. Furthermore, the bacterial solution was cultured on an appropriate stable medium. The presence or absence of bacterial colony growth along with the above results derived from the liquid culture served as confirmation of the results obtained by spectrophotometry.

[0291] Spectroscopic analysis of 6 and 11 in trypticase soy broth (TSB) microbial medium supplemented with HA spheroids: Various conjugate concentrations were introduced into HA powder (spheroids) suspended in TSB microbial medium. Solutions containing BP-ciprofloxacin and HA spheroids were placed into wells of a 24-well plate. The final concentration of the powder was 10 mg / 1 mL, while the final concentration of the conjugates ranged from 0.24 to 250 mg / L. The absorbance of the solutions was measured immediately using a spectrophotometer (Thermo Scientific Multisca GO) at a wavelength of 275 nm. The plates were automatically stirred in the spectrophotometer before measurements. The plates were then left at 37°C for 24 hours with stirring. The absorbance was measured again after 24 hours. The absorbance values ​​measured at the beginning and end of the experiment were compared to evaluate the relative concentrations of the conjugates at 0 and 24 hours. The excitation slit, emission slit, integration time, and increment were optimized based on the concentration of the samples.

[0292] Six antimicrobial susceptibility tests for planktonic cultures of Staphylococcus aureus ATCC-6538 at acidic and physiological pH: The experimental set-up was performed as previously described for the disk diffusion test, except that the microbial medium was adjusted to pH 7.4 and pH 5 using KOH or HCl solutions and the microbial medium was measured using a universal pH indicator (Merck, Poland).

[0293] Six-hour kill assay against Staphylococcus aureus ATCC-6538 (MSSA) and clinical MRSA strains (MR4-CIPS): The experiment was performed as previously described under the subheading "Evaluation of the MICs of test compounds against analyzed planktonic clinical Staphylococcus strains" except that absorbance measurements (wavelength of 580 nm) were taken at 0, 1, 2, 4, 8, 16, and 24 hours.

[0294] Six antimicrobial susceptibility tests performed on biofilms of Staphylococcus aureus ATCC-6538 and Pseudomonas aeruginosa ATCC-15442: Strains cultured on the appropriate agar plates (Columbia agar plates for Staphylococcus aureus; MacConkey agar plates for Pseudomonas aeruginosa) were transferred to liquid microbiological medium and incubated at 37°C for 24 hours under anaerobic conditions. After incubation, strains were diluted to a density of 1MF. The microbial dilutions were placed in wells of a 24-well plate containing HA disks as a substrate or directly in polystyrene wells whose bottom surface served as a substrate for biofilm formation. Strains were incubated for 4 hours at 37°C. The microorganism-containing solution was then removed from the wells. The surfaces, HA disks, and polystyrene plates were gently washed to remove plaquetonic or loosely bound microorganisms while leaving attached cells. The surfaces thus prepared were immersed in fresh TSB medium containing 0.24-125 mg / L 6 and ciprofloxacin as a control. After 24 h of incubation at 37°C, the surfaces were washed using saline solution and transferred to 1 mL of 0.5% saponin (Sigma-Aldrich, St. Louis, MO). The surfaces were vigorously mixed on a vortex mixer for 1 min to detach the cells. All microbial suspensions were then transferred to 10 -1 ~10 -9 100-fold dilutions were made. Each dilution (100 mL) was plated on the appropriate stable medium (MacConkey and Columbia for P. aeruginosa and S. aureus, respectively) and incubated at 37°C for 24 hours. After this, microbial colonies were counted and the number of cells forming a biofilm was assessed. Results were expressed as the mean number of CFU per square millimeter surface ± standard error of the mean. X-ray tomography analysis was applied to calculate the surface area of ​​the HA disks. The formula for the area of ​​a circle, πr 2 was applied.

[0295] Inhibitory ability of 6 and 11 to inhibit adhesion of Staphylococcus aureus strain 6538 to HA: Various concentrations of 6 and 11 were introduced into HA powder (spheroids) suspended in TSB microbial medium. The solutions containing 6 and HA spheroids were placed into wells of a 24-well plate. The final concentration of the powder was 10 mg / 1 mL, while the final concentration of the complex ranged from 0.12 to 250 mg / L. The suspensions were left at 37°C for 24 hours under stirring. After 24 hours, the suspensions were removed from the wells and centrifuged briefly to precipitate the HA powder. The supernatant was then very gently discarded and 10 5 A fresh 1 mL of S. aureus with a density of 1000 CFU / mL was added to the HA spheroids. The solution was then stirred, the absorbance was measured using a wavelength of 580 nm, and left at 37°C for 24 hours with stirring. After incubation, the absorbance was measured again and the values ​​at 0 and 24 hours were compared to evaluate the reduction in bacterial growth relative to control sample 1 (bacterial suspension without spheroids) and control sample 2 (bacterial suspension + spheroids with no complex added). Furthermore, the solution was centrifuged momentarily and the supernatant gently discarded, while the bacteria-containing HA spheroids were plated and cultured as before and quantitatively evaluated. For the test of 11, solutions containing HA spheroids and relatively high concentrations of 11 ranging from 1 to 400 μg / mL and ciprofloxacin ranging from 0.5 to 400 μg / mL were prepared and the solutions were again compared to the control sample (bacterial suspension without HA) for their ability to inhibit biofilm formation. Higher concentrations of 11 were tested due to the weaker activity of the amide conjugate compared to the carbamate conjugate.

[0296] Survival of S. aureus after 24 h incubation on HA pretreated with 6: HA disks were immersed in 2 mL of solutions containing various concentrations of BP-ciprofloxacin or ciprofloxacin alone, and the disks were left at 37°C for 24 hours. HA disks incubated in DMSO or phosphate buffer served as control samples. The disks were then washed three times with sterile water. After washing, 2 mL of 0.5 MF of S. aureus ATCC6538 strain was placed into the wells containing the HA disks as substrates for biofilm formation, and biofilms were formed in the same manner as before.

[0297] Ethics statement: All animal experimental protocols and methods were approved and performed in accordance with the recommendations of the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California (USC) and the American Veterinary Medical Association Euthanasia Study Group. USC is registered with the United States Department of Agriculture (USDA), has a fully licensed charter (No. A3518-01) registered with the National Institutes of Health (NIH), and is accredited by the American Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The title of our IACUC-approved protocol is "Bone-targeted antimicrobial agents for the treatment of biofilm-mediated osteolytic infections" and its protocol number is 20474. All animal experimental protocols and researchers and personnel involved in the animal experiments presented herein strictly adhered to the Guide for the Care and Use of Laboratory Animals in the USDA Animal Welfare Regulations (CFR 1985) and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals (1996).

[0298] In vivo animal studies: Twelve 5-month-old, virgin female Sprague-Dawley rats, each weighing approximately 200 g, were used in this study. Animals were housed 2-3 per cage in a small animal holding room at 22°C under a 12-h light / dark cycle and were allowed to consume soft food (Purina Laboratory Rodent Chow) ad libitum. All animals were handled in accordance with the guidelines and regulations for the use and care of animals at USC. The animals were under the supervision of a dedicated veterinarian on-call 24 hours a day who directly assessed the animals daily. All animal experiments were performed in accordance with the ARRIVE guidelines for reporting animal experiments to ensure the quality, reliability, validity, and reproducibility of the results. 45 It is described using.

[0299] This animal model is a home-made jaw bone peri-implant osteomyelitis model specifically designed to study biofilm-mediated disease and host responses in vivo. 31 Biofilms of the jaw osteomyelitis pathogen Aa were grown for 10 min. 9CFU were preformed on small titanium implants. To confirm the susceptibility of Aa to the parent drug ciprofloxacin prior to our animal studies, AST and MIC assays were performed against planktonic Aa in addition to biofilm HA assays as described for long bone osteomyelitis pathogens. After biofilms were established on the implants in vitro, they were surgically implanted in the jawbone of each rat. For surgery, animals were first anesthetized using 4% isoflurane inhalant followed by intraperitoneal injection of ketamine (80–90 mg / kg) and xylazine (5–10 mg / kg). Local anesthesia was then added by infiltration injection of 0.25% bupivacaine into the surgical site. Preemptive analgesia was then administered subcutaneously with sustained release buprenorphine (1.0–1.2 mg / kg) before the first incision. Once anesthetized, the buccal mucosa of each rat was retracted with a hook and a transmucosal osteotomy was performed on the alveolar ridge in the natural midline diastema of the anterior palate using a pilot drill. The implant was then manually inserted into the osteotomy until the platform was at mucosal level and fixed to the bone. Two biofilm-inoculated implants were placed on either side of the palatine bone of each rat (n = 12 rats).

[0300] One week after surgery, rats were lightly anesthetized again with 4% isoflurane to check the stability of the implants and to note clinical findings such as the presence or absence of inflammation at the implants and infection sites.The animals were then administered BP-ciprofloxacin (0.1 mg / kg, 1 mg / kg, or 10 mg / kg as single doses, and 0.3 mg / kg three times per week for multiple dose groups6) by intraperitoneal injection or ciprofloxacin alone (10 mg / kg three times per week, also for multiple dose groups) as a positive control, and sterile endotoxin-free saline as a negative control.

[0301] Animals were assigned to treatment and control groups via a randomization process. Animals in the multiple dose groups were anesthetized as previously described before each additional injection over the week. All compounds were pharmacological grade and made up in sterile injectable saline of appropriate pH. One week after drug treatment, all animals were placed in a CO2 chamber (60-70% concentration) for 5 min and then euthanized by cervical dislocation. Peri-implant tissue (1 cm 2 ) were extracted en bloc, and the implants were removed. Clinical parameters, including the presence or absence of periprosthetic inflammation, were noted at the time of surgery and extraction. Rats assigned to treatment and control groups were anonymized and kept confidential from subsequent investigators analyzing their microbiological data.

[0302] For microbial analysis, the excised peri-implant soft tissue and bone were homogenized and processed in 1 mL of 0.5% saponin immediately after surgical removal, and serially diluted after vortexing for 1 min. 0 ~10 -9 Serial dilutions were prepared ranging from 0.1 mL of saponin solution to 0.9 mL of 0.9% sterile isotonic saline solution, with a dilution factor of 10, and 0.1 mL of each of these dilutions was plated using the spread plate method. The medium for culturing Aa consisted of modified TSB, and frozen stocks were maintained at -80°C in 20% glycerol, 80% modified TSB. All cultures were performed at 37°C in 5% CO2 for 48 hours. The number of live Aa bacteria cultured (CFUs per gram of tissue) was manually counted, and the mean log base 10 reduction in CFUs per gram as a function of treatment was recorded. To confirm the morphotype of Aa bacteria and exclude contamination, colonies were sampled from the plates once CFU counting was completed to perform Gram staining and histological evaluation.

[0303] statistical analysis Statistical calculations were performed using SPSS 22.0 (IBM, Armonk, NY) and Excel 2016 (Microsoft, Redmond, WA). G Power 3 software was used to estimate sample sizes for in vitro and in vivo studies prior to the experiments. 46 A power analysis was performed using the ANOVA. Quantitative data from the experimental results of each group were first analyzed by descriptive statistics to understand the distribution of the data (parametric or non-parametric) and generate the mean, standard error, standard deviation, kurtosis and skewness, and 95% confidence level. The data were then analyzed using the Kruskal-Wallis test or one-way ANOVA, as appropriate, and statistical significance was accepted at p<0.05 when comparing treated to control groups. Additionally, post-hoc tests utilizing independent t-tests and Dunnett's multiple comparison tests were performed for in vivo experiments.

[0304] Abbreviations used Aa, Aggregatibacter actinomycetemcomitans; AAALAC, American Association for Assessment and Accreditation of Laboratory Animal Care; ANOVA, analysis of variance; ARRIVE, Reporting of in vivo animal studies; AST, antibiotic susceptibility testing; ATCC, American Culture Collection; BP, bisphosphonate; BTMS, bromotrimethylsilane; CFU, colony forming unit; CLSI, Clinical Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; HA, hydroxyapatite; IACUC, Institutional Animal Care and Use Committee committee; MBC, mean bactericidal concentration; MBIC50, minimum biofilm inhibitory concentration required to inhibit the growth of 50% of organisms; MF, McFarland; MH, Mueller-Hinton; MIC50, minimum inhibitory concentration required to inhibit the growth of 50% of organisms; MSSA, methicillin-susceptible Staphylococcus aureus; Pd / C, palladium-activated carbon; SD, standard deviation; BTMS, bromotrimethylsilane; DCM, dichloromethane; SOCl2, thionyl chloride; SEM, scanning electron microscopy.

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Bandyopadhyay,S.; Das,P.; Samanta,I.; Mukherjee,P.; Roy,S.; Kundu,B.Understanding osteomyelitis and its treatment through local drug delivery system.Biotechnol.Adv.2016,34,1305-1317. 38.Lin,J.H.Bisphosphonates: a review of their pharmacokinetic properties.Bone 1996,18,75-85. 39.Fong,I.W.; Ledbetter,W.H.; Vandenbroucke,A.C.; Simbul,M.; Rahm,V.Ciprofloxacin concentrations in bone and muscle after oral dosing.Antimicrob.Agents Chemother.1986,29,405-408. 40.Sedghizadeh,P.P.; Jones,A.C.; LaVallee,C.; Jelliffe,R.W.; Le,A.D.; Lee,P.; Kiss,A.; Neely,M.Population pharmacokinetic and pharmacodynamic modeling for assessing risk of bisphosphonate-related osteonecrosis of the jaw.Oral Surg.Oral Med.Oral.Pathol.Oral Radiol.2013,115,224-232. 41.Williams,D.B.; Lawton,M.Drying of organic solvents: quantitative evaluation of the efficiency of several desiccants.J.Org.Chem.2010,75,8351-8354. 42.McKenna,C.E.; Higa,M.T.; Cheung,N.H.; McKenna,M.-C.The facile dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane.Tetrahedron Lett.1977,18,155-158. 43.McKenna,C.E.; Schmidhuser,J.Functional selectivity in phosphonate ester dealkylation with bromotrimethylsilane.J.Chem.Soc.,Chem.Commun.1979,739-739. 44.David,T.; Kotek,J.; Kubicek,V.; Tosner,Z.; Hermann,P.; Lukes,I.Bis(phosphonate)- building blocks modified with fluorescent dyes.Heteroat.Chem.2013,24,413-425. 45.Kilkenny,C.; Browne,WJ; Cuthi,I.; Emerson,M.; Altman,DGImproving bioscience research reporting: the ARRIVE guidelines for reporting animal research.Vet.Clin.Pathol.2012,41,27-31. 46.Faul,F.; Erdfelder,E.; Buchner,A.; Lang,AGStatistical power analyzes using G*Power 3.1: tests for correlation and regression analyses.Behav.Res.Methods 2009,41,1149-1160.

[0306] Example 6 It is demonstrated herein that carbamate-linked bisphosphonate-ciprofloxacin is a potent antibacterial conjugate specifically for the targeted treatment of infectious bone diseases (Figure 14).

[0307] Bisphosphonates (BPs) form strong bidentate and tridentate interactions with calcium, and thus can target bone or hydroxyapatite (HA) surfaces (where biofilm pathogens also reside). A bisphosphonate-carbamate-ciprofloxacin (BCC, compound 6) conjugate was designed, synthesized, and successfully tested in vitro and in vivo against common bone biofilm pathogens, demonstrating the feasibility of a bone-biofilm targeted antimicrobial approach. Our results showed that BCC (compound 6) has a potent bactericidal profile against common long bone and jaw osteomyelitis organisms in vitro, especially when using a biofilm model with HA as a substrate for microbial growth and antimicrobial testing. Biofilm growth on HA was inhibited by chemisorption of the conjugate in an osteomyelitis prophylaxis experimental setting, where BCC (compound 6) showed predictable rates of sustained detachment and was 20-fold more active in achieving complete bactericidal activity compared to the parent drug ciprofloxacin alone. We have demonstrated efficacy and safety of BCC (compound 6) against Aggregatibacter actinomycetemcomitans biofilms in an animal model of peri-implantitis in vivo. In vivo, a single intraperitoneal dose of 10 mg / kg (15.6 μmol / Kg) of the complex produced 99% peri-implant bactericidal activity, an order of magnitude higher than the parent antibiotic ciprofloxacin alone (90.6 μmol / Kg; a total overall dose of ciprofloxacin six times higher) administered in multiple doses. At this single dose of 10 mg / kg, BCC (compound 6) demonstrated greater efficacy and disease resolution than the parent antibiotic ciprofloxacin administered in multiple higher doses, without the potential for systemic toxicity or side effects due to the pharmacokinetic advantages of bone targeting / bone biodistribution at the site of biofilm infection and the pharmacodynamic advantages of sustained antibiotic sequestration.

[0308] Dental implants are an important part of modern dental practice, with up to 35 million Americans estimated to be missing all of their teeth in one or both jaws. The overall market for these implants for tooth replacement and reconstruction is expected to reach $4.2 billion by 2022. While the majority of implants are successful, some of these prostheses fail due to peri-implantitis, leading to destruction of the supporting bone. The occurrence of peri-implantitis is bimodal, including early (<12 months) failure and late (>5 years) failure. Both of these failure thresholds are primarily the result of bacterial biofilm infection on and around the implant. Peri-implantitis is a common reason for implant failure. Dental implant failure is commonly caused by biomechanical or biological / microbiological reasons. The prevalence of peri-implantitis, the most severe form of microbial-associated implant disease leading to destruction of the supporting bone, is difficult to ascertain from the current literature. However, recent studies have shown that peri-implantitis is becoming a problem with an increasing prevalence. 4 A recent study of 150 patients followed for 5 to 10 years showed rates of peri-implantitis of approximately 17% and 30%, respectively, indicating that peri-implantitis is a significant problem. 5 Early implant failure or lack of osseointegration is an isolated problem that occurs in roughly 9% of implant-treated jaws. 6 This is more common in the upper jaw. 6 , associated with bacterial infection during surgery or from nearby sites (e.g., periodontitis), as well as other well-recognized and reversible risk factors such as smoking, diabetes, excess cement, and poor oral hygiene. 2 .

[0309] Biofilm infections may be involved in the pathogenesis of peri-implantitis. They pose unique treatment challenges, are often difficult to diagnose, are resistant to standard antibiotic therapy, are resistant to the host immune response, and cause persistent, intractable infections. 7Early explanations that bacteria live in matrix-supported communities 8、9 Since then, the biofilm infection hypothesis has steadily developed; it is now proven that over 65% of chronic infections are caused by bacteria living in biofilms. 7 This means that approximately 12 million people in the United States suffer from these infections and nearly 500,000 people die in the United States each year. Peri-implantitis and periodontitis are among the most common biofilm infections. Peri-implantitis has a less diverse community (and predominant pathogens) than periodontitis infections. 10 It has been found to be a relatively simple infection in terms of infection rate, with Gram-negative species typically dominating. 11 Other orthopedic or bone infections, including jaw infections, are also caused by bacterial biofilm communities. 12 , the techniques developed here could be used in these diseases as well.

[0310] Currently, therapeutic approaches for peri-implantitis are limited. Peri-implantitis has several causes, but the primary etiology is bacterial biofilm. There are no universally accepted guidelines or protocols for the treatment of peri-implantitis, and most clinical protocols for the treatment of bacterial peri-implantitis involve local and systemic antibiotic delivery. 13 , as well as surgical resection of the lesion, including reconstructive grafting using bone graft substitutes. 14、15 However, it has been difficult to advance a local antibiotic delivery device to the bottom of the deep peri-implant pocket and into the infected jawbone, or even to adequately penetrate the infected jawbone with systemically administered antibiotics to kill biofilm pathogens. 16 Clinical experience has shown that this is difficult, as the latter is due in part to the inherent poor bone (and peri-implant) biodistribution or pharmacokinetics of antibiotics. 17 Previous long-term studies have shown that even when infected implants were cleaned locally with antiseptics and treated with systemic antibiotics, there was additional loss of supporting bone in more than 40% of advanced peri-implantitis lesions. 15.

[0311] Furthermore, long-term systemic antibiotic therapy can result in systemic toxicity or side effects, and resistance can also occur. Therefore, it has become the practice of clinicians to use local delivery systems to achieve relatively high therapeutic antibacterial concentrations in bone. For example, dentists use bone graft materials for local delivery. 18 The clinical mix of antibiotics with minocycline or doxycycline powders (e.g., Arestin®) or chlorhexidine solutions (e.g., Periochip®) is used. Such approaches are simply slurries and do not imply a strong bond between the antibiotic and the bone substitute as in the BioVinc approach, and therefore suffer from relatively early washout and less efficient pharmacokinetics as previously discussed. Additionally, researchers have developed several biodegradable and non-biodegradable local antibiotic delivery systems. 19 have also been used. However, these approaches have several limitations, for example, non-biodegradable approaches (e.g., polymethylmethacrylate cement) require a second surgery to remove the antibiotic-loaded device, are incompatible with certain antibiotics, and suffer from inefficient dissociation kinetics, with less than 10% of the total antibiotic delivered being dissociated in some cases. 17 There has been increasing interest in biodegradable materials, including fibers, gels, and beads, however, their clinical efficacy for the treatment of peri-implantitis is unclear. 3 Even when effective antimicrobial / antiseptic agents, such as topical chlorhexidine delivery, are used to treat peri-implantitis in the jaw, there is little impact on treatment outcomes as demonstrated in promising animal and human studies. 15、17 Taken together, these data further support the previously mentioned poor pharmacokinetics of antibiotics in bone and highlight the need for osteointegrative / bone-targeted sustained antibiotic sequestration strategies.

[0312] BP-complex Given the limitations of current therapeutic approaches, developing bone / biofilm targeted antimicrobial agents is a significant advance in the field. The BP-antibiotic (BP-Ab) conjugates provided herein can overcome many of the challenges associated with poor antibiotic pharmacokinetics or bioavailability within bone and bone-associated biofilms. These compounds can reduce infection via a "targeted release approach," which can reduce concerns regarding systemic toxicity and / or drug exposure in other (e.g., non-infected) tissues. The BP-Ab conjugates can be incorporated into bone graft substitutes. The BP-Ab can be a BP-fluoroquinolone conjugate. In some examples, the BP-Ab can be a bisphosphonate-carbamate-ciprofloxacin (BCC, compound 6), as shown in FIG. 15. The exemplary structure of FIG. 15 is also referred to herein as BCC (compound 6). When incorporated into a bone graft, the BP-Ab bone graft material can be referred to as a BP-Ab-bone graft. For example, it can be referred to as a BP-FQ-bone graft when the antibiotic is a fluoroquinolone. These compounds can effectively adsorb to hydroxyapatite (HA) / bone and achieve sustained detachment over time and antibacterial effects against biofilm pathogens. The compounds provided herein and implant materials incorporating the compounds can be used as anti-infective bone graft substitutes for adjunctive treatment or prevention of peri-implantitis. The complexes are locally released from the implant material with sustained detachment kinetics and will be cleaved in the presence of bacterial or osteoclastic activity, as we have previously demonstrated in vitro and in vivo in other results provided elsewhere herein. In this manner, the implants can provide high local concentrations of FQs, such as ciprofloxacin, compared to current delivery routes. In summary, the compounds and bone graft materials provided herein can contain antibiotics complexed to safe or pharmacologically inactive (non-resorptive) BP moieties that are bound to calcium / HA in the implant material through strong multidentate electrostatic interactions, and the antibiotics detach over time.It does not simply represent a topical antibiotic that is simply mixed into a slurry with existing bone graft materials as some current clinical approaches in this setting. Thus, this chemisorbed drug bound to calcium phosphate mineral (HA) represents a major advance in the field, overcoming many of the limitations in antibiotic delivery to peri-implant bone for effective bactericidal activity against biofilm pathogens.

[0313] The general idea of ​​targeting bone by linking active drug molecules to BPs is discussed in a review article. 30 However, at this time no FDA-approved drugs had been developed, and early attempts had resulted in either systemically unstable prodrugs or non-cleavable conjugates that were found to inactivate both components of the conjugate, usually by violating pharmacophore requirements. In the quinolone field, a notable example was described by Herczegh where the antibacterial properties of fluoroquinolones were lost when conjugated to stable BP-linked analogues. 31~32 Therefore, a target-release linker strategy is necessary.

[0314] In recent years, medicinal chemistry strategies have begun to emerge that exploit less stable linking techniques. Other investigators have linked fluoroquinolones to several different BP moieties via carboxylic acid groups. They found that glycolamide ester prodrugs of the antibiotics moxifloxacin and gatifloxacin suppressed infection when used prophylactically in a rat osteomyelitis model. 33 The same group used acyloxycarbamate- and phenylpropanone-based linkers to tether the same antibiotics to simple BP systems via amine functions. 34 Using the same prophylactic rat model, they show that these conjugates are also better than the parent antibiotics at preventing the establishment of infection. 33 have used some of these prodrug strategies for the glycopeptide antibiotic oritavancin. 35This dual function drug appears to be somewhat effective in preventing infection. However, to date they have not published studies showing that it is possible to treat established infections, nor have they published the pharmacokinetics of the prodrug. Since their drug candidate selection was based in part on plasma instability, it is believed that these analogs are too unstable in the bloodstream for this therapeutic approach to be completely successful. Thus, it is believed that the compounds developed by these groups are unable to achieve effective local concentrations of the antibiotic.

[0315] BCC compounds (Figure 15) allow the incorporation of the phenyl moiety of a phenyl carbamate linker directly into the BP portion of the molecule. It is possible to alter or tune the dissociation kinetics through modification of the phenyl ring with electron withdrawing or donating groups that can alter the propensity of the linker. Furthermore, the BP core lacks the efficacy as a bone resorption inhibitor and therefore is unable to inhibit jaw drug treatment-related osteonecrosis like the relatively potent nitrogen-containing BP drugs (e.g., zoledronate). 39、40 This targeted release strategy using a phenylcarbamate linker has been demonstrated herein and elsewhere herein to have a high potential for releasing active drug directly to bacterial biofilms in the bone environment. Bone targeting is so effective that the compound outperforms ciprofloxacin against biofilms grown on HA bone matrix substitutes rather than planktonic cultures grown in plastic containers. A similar conjugate made using a non-cleavable amide bond that excludes the phenolic oxygen of the carbamate (bisphosphonate-amide-ciprofloxacin; BAC; compound 11) was found to have little effect on bacterial growth under all conditions, indicating that active cleavage of the conjugate is required for antibacterial activity.

[0316] The synthesis scheme of BCC (compound 6) is shown in Figure 16. The properties of the compound are 1 H, 13 C, and 31The compound was analyzed by P NMR and mass spectrometry. To help determine whether the antibacterial activity was primarily due to the isolated ciprofloxacin, we decided to synthesize an amide-linked conjugate as well that was designed to avoid isolating ciprofloxacin from the conjugate. The synthesis of this compound went smoothly (Figure 31) and afforded the control compound BAC (compound 11) in reasonable yield.

[0317] A series of assays were performed to determine the minimum inhibitory concentrations (MICs) of BCC (compound 6) (also referred to as “BCC(6)”), BAC (compound 11) (also referred to as “BAC(11)”), and the parent drug, ciprofloxacin, against a panel of Staphylococcus aureus (SA) strains. These experiments were performed using dilution assays following the European Committee on Antibiotic Susceptibility Testing guidelines (ref. 43). Testing of the three compounds (FIG. 17) indicates that the BCC (compound 6) conjugate retains significant bactericidal activity against these pathogens, while the BAC (compound 11) has lost most of its activity. These antibiotic susceptibility testing (AST) and MIC data indicate that ciprofloxacin and BCC (compound 6) have potent bactericidal activity against planktonic and clinically relevant SA pathogens, with the conjugation linkage affecting the antibacterial activity of the parent drug, as evidenced by the weak activity of BAC (compound 11). Our testing of ciprofloxacin against these strains was consistent with established clinical breakpoints.

[0318] Since HA is the main inorganic component of bone, the compounds were tested for adsorption to suspended HA beads as a bone substitute. Our BCC (compound 6) is indeed absorbed by HA beads as shown by the measurement of the residual complex in the supernatant after removal of the beads (Figure 18). The complex in the supernatant was measured at 0 and 24 hours by spectrophotometry using absorbance at a wavelength of 275 nm, the λmax determined for BCC (compound 6). This clearly shows that BCC (6) is bound to this bone substitute. We did not measure the binding of BAC (compound 11) because the data for BCC (compound 6) did not match the binding of this type of BP, which would cause the binding of BAC (compound 11) as well (refs. 35, 44).

[0319] The next in vitro test was to combine the bone substitute targeting activity of BCC(6) with the rapid release of ciprofloxacin as demonstrated by its MIC activity against SA strains. For this experiment, HA disks were pretreated with solutions of the complex or ciprofloxacin at the indicated concentrations, and then the disks were washed to remove the compound solutions. Biofilms of SA (strain ATCC-6538) were grown according to our published method. Quantitative counts of colony forming units (CFU) were performed after 24 hours of growth. Disks pretreated with DMSO and PBS were used as controls. The results showed that BCC(6) inhibited all bacterial growth at 10 μg / mL (FIG. 11), while pure ciprofloxacin became completely inhibitory at 100 μg / mL. Since the molecular weight of the complex is approximately half that of pure ciprofloxacin, this indicated that BCC (compound 6) was roughly 20-fold more potent than the parent drug in completely inhibiting bacterial biofilm growth. This supports the dissociation of the drug from the complex over time in the bone matrix environment. The amide complex BAC (11) did not inhibit biofilm growth on bone substitutes even at very high concentrations (data not shown), and dissociation of ciprofloxacin is important for this activity, as previously reported in the literature and in tests with planktonic cultures (Figure 18). 30、34、35、44 was shown to be consistent with

[0320] Given the above results showing that our BCC (6) has bactericidal activity, we sought to test its activity against biofilm infections in animal models. Briefly, we inoculated rats with Aggregatibacter actinomycetemcomitans (Aa; wild-type R-strain D7S-1; a serotype), a bacterium specific for jaw bone infections but not a resident bacterium in the normal flora of rats, at 10 °C. 9 CFU were precultured on small titanium implants. To confirm the susceptibility of Aa to the parent drug ciprofloxacin prior to our animal studies, we performed AST and MIC assays on Aa as performed for previously described pathogens of long bone osteomyelitis. Disk zone of inhibition assay revealed diameters of >40 mm and MICs of >100 μg / mL were not observed. 90The dose of BCC(6) was 2 mg / mL, indicating the strong susceptibility of Aa to the parent drug, ciprofloxacin. Inoculated implants carrying Aa biofilms were placed in 12 rats (2 implants per animal). This model reliably developed a well-characterized biofilm infection on the surrounding jawbone, causing inflammation and associated peri-implantitis disease (Ref. 45). After biofilm development, animals were randomized into three treatment groups: 5 animals receiving a single dose of BCC(6) at a dose of 10 mg / kg, 2 animals receiving BCC(6) at a dose of 0.3 mg / kg three times a week, and 5 animals receiving a control treatment with sterile saline. Pilot experiments (2 animals / group) showed that a single dose of BCC(6) at 10 mg / kg was nearly as effective as ciprofloxacin administered at a total dose of 30 mg / kg in a 10 mg three times a week regimen (data not shown). Therefore, we decided not to include a ciprofloxacin control in the larger study to reduce animal utilization. All animals tolerated the treatment and medication well and there were no adverse events. Clinically, we observed that the majority of animals in the control group showed evidence of localized periprosthetic inflammation at the time of anesthesia and surgical extraction compared with the majority of animals in the treatment group who had non-inflamed peri-implant tissue, and the overall implant retention rate was 23 of 24 implants (96%), providing a robust statistical analysis.

[0321] CFU of Aa from excised peri-implant tissues (23 of 24 implants) after euthanasia were quantitatively determined, and results show that in this study a single dose of BCC(6) at a dose of 10 mg / kg demonstrated approximately 10^6 units of kill, and even multiple doses at lower doses demonstrated 10^2-3 units (99%-99.9%) of kill, as shown in Figure 19. A single dose of BCC(6) at a dose of 10 mg / kg demonstrated the highest efficacy for this study, which was highly statistically significant when compared to the control group (p=0.0005).

[0322] In both of these animal models, BCC (6) was delivered by intraperitoneal injection to ensure exposure to the compound, since there is relative bioequivalence with intravenous or gastrointestinal routes of administration for fluoroquinolones. We believe these results indicate that a separable BP-antibiotic (BP-Ab) complex could be used as a drug for the treatment of peri-implant disease and associated osteomyelitis. Here, we propose to take advantage of these results and incorporate the BP-Ab complex into a dental bone substitute graft material for localized oral delivery and release.

[0323] Example 7 Design and synthesis of other BP-Ab conjugates (Figure 20) For example, it is possible to design additional BP-Ab conjugates using ciprofloxacin and moxifloxacin conjugated to BPs (e.g., 4-hydroxyphenylethylidene BP (BP1, FIG. 20), its hydroxy-containing analog (BP2, FIG. 20; with relatively high bone affinity), and pamidronate (BP3, FIG. 20) via carbamate-based linkers (e.g., carbamate, S-thiocarbamate, and O-thiocarbamate). FIG. 21 shows an exemplary synthetic scheme for the synthesis of BP-Ab conjugates containing O-thiocarbamate linkers. The S-thiocarbamate bond (slightly more labile) can be obtained by isomerization of the conjugate with the O-thiocarbamate bond via the Newman-Kuwart rearrangement. (References 47, 48). Preliminary chemistry has already been performed to demonstrate the feasibility of rapid synthesis of these targets. Thus, the addition of bone affinity has been well demonstrated using α-OH-containing BPs (49). The added bone affinity increases the concentration of the conjugates at the bone surface, and relatively high local drug concentrations are easily achieved over short and long periods of time. Since the α-OH bisphosphonate esters are easily rearranged to phosphonophosphates, their α-OH can be protected by a tert-butyldimethylsilyl (TBS) group (Scheme 2, Figure 22) (50) for the synthesis of conjugates with α-OH-containing BPs (BP2 and pamidronate, Figure 20). Then, the α-O-TBS BP2 ester is activated by 4-nitrophenyl chloroformate and reacts with ciprofloxacin or moxifloxacin similarly as shown in Figure 21. For α-O-TBS BP3 ester, a linker with a phenol group (e.g., Linker 1 (resorcinol), Linker 2 (hydroquinone), Linker 3 (4-hydroxyphenylacetic acid), Figure 20) is used to tether the BP and the antibacterial agent. Here, the synthetic route using Linker 3 is shown as an example (Scheme 3, Figure 23). To identify all BP-Ab complexes, their properties are analyzed. 1 H, 31 P, and 13 Analyze by C NMR, MS, HPLC, and elemental analysis.

[0324] The mineral binding affinity of the BP-Ab complex is determined. Briefly, inorganic bovine bone of large particle size (uniformly 1-2 mm) can be accurately weighed (1.4-1.6 mg) and suspended in 4 mL clear vials containing an appropriate amount of assay buffer (0.05% (wt / vol) Tween 20, 10 μM EDTA, and 100 mM HEPES pH=7.4) for 3 hours. The bone material can then be incubated with increasing amounts of BP-Ab (0, 25 μM, 50 μM, 100 μM, 200 μM, and 300 μM). Samples can be gently shaken in the assay buffer for 3 hours at 37° C. After the equilibration period, the vials can be centrifuged at 10,000 rpm for 5 minutes to separate the solids and the supernatant. The supernatant (0.3 mL) can be collected, and the concentration of the equilibrated solution is measured using a Shimadzu UV-VIS spectrophotometer (wavelength of 275 nm). Fluorescence emission can also be used to calculate binding parameters. Non-specific binding can be measured using the same method in the absence of HA as a control. The amount of parent drug / BP-Ab complex bound to HA is inferred from the difference between the amount input and the amount recovered in the supernatant after binding. The binding parameters (K d It is possible to calculate the K and Bmax (equilibrium dissociation constant and maximum number of binding sites, respectively) and measure their binding parameters in five independent experiments. d ) is lower than the equilibrium dissociation constant (K d ) may be preferred. A two-sample t-test can be used to evaluate the binding parameters of the BP-Ab. A sample size of n=5 in each group can be used to detect an effect size of 1.72 with 80% statistical power and a one-sided type I error of 0.05.

[0325] The binding stability of the BP-Ab conjugates can be determined. Briefly, the linker stability of each BP-Ab conjugate can be tested in PBS buffer with different pH (pH=1, 4, 7.4, 10) and human or dog serum. BP-Ab can be suspended in 400 μL of the above PBS or 400 μL of 50% (volume / volume in PBS) human or dog serum. The suspension / solution can be incubated at 37° C. for 24 hours and centrifuged at 13000 rpm for 2 minutes, and the supernatant can be collected. Methanol (5 times the volume of the supernatant) can be added to each supernatant, and the mixture can be vortexed for 15 minutes to extract the separated fluoroquinolones. The mixture can then be centrifuged at 10000 rpm for 15 minutes to precipitate insoluble materials. The supernatant containing the extracted fluoroquinolones can be collected and evaporated to dryness. The dried precipitates can be resuspended in PBS and the amount of separated fluoroquinolone can be determined by UV-VIS measurement as previously described. The percentage of separated fluoroquinolone drug can then be calculated based on the input amount and the measured amount of separated drug. The identity of the separated drug can be confirmed by LC-MS analysis and / or NMR if the concentration is sufficient.

[0326] In vitro inhibition of biofilm growth on HA discs can be determined. Briefly, commercially available HA powder can be used for the production of custom discs. Powder pellets with a diameter of 9.6 mm can be pressed without the use of binders. Sintering can be performed at 900°C. The tablets can be compressed using a Universal Testing System (Instron model 3384; Instron, Norwood, MA) for static tensile, compression, and bending tests. The quality of the produced HA discs can be examined by confocal microscopy and microtomography (microCT) using a LEXT OLS4000 microscope (Olympus, Center Valley, PA) and a Metrotom 1500 microtomograph (Carl Zeiss, Oberkochen, Germany), respectively. The HA disks can then be placed in the following concentrations of BP-Ab complex and ciprofloxacin / moxifloxacin: 800 mg / mL, 400 mg / mL, 200 mg / mL, 100 mg / mL, 50 mg / mL, 25 mg / mL, 10 mg / mL, 5 mg / mL, and 1 mg / mL, and left at 37° C. for 24 hours. After incubation, the HA disks can be removed and placed in 1 mL of PBS and left in a light rocking shaker for 5 minutes. Three subsequent washes are performed in this manner. After washing, 1 mL of the Aa suspension can be placed on the disks and left at 37° C. for 24 hours. The disks can then be washed to remove unbound bacteria, and the disks can be vigorously agitated on a vortex mixer. Serial dilutions of the resulting suspension can then be plated on modified TSB agar plates, and colony growth counted after 24 hours.

[0327] The osseointegration effect of the BP-FQ-bone graft on critical size can be evaluated in a superior alveolar peri-implant defect model for bone grafting. Briefly, in this split-mouth design, six beagle dogs (three males, three females) are extracted from the mandibles at PM2-PM4 on both sides and allowed to heal for 12 weeks. An alveolar crest incision is made followed by mucoperiosteal flap reflection. An osteotomy is performed to create a 6 mm superior alveolar defect. Implant site osteotomy preparations are created in each of the premolars by sequential cuts of gradually increasing diameters while copiously irrigating using a drill with a built-in irrigator. Implants (Astra Tech Osseospeed Tx® 3×11 mm) are placed in the PM2-PM4 positions on both sides such that the implants are positioned 4 mm toward the alveolar crest and the same distance from the buccal cortical bone plate with respect to the created defect. The dogs are randomly divided into three different groups (two dogs per group).

[0328] Group 1: inorganic bovine bone (large particle size of 1-2 mm in 1 g) with BP-fluoroquinolone chemisorbed is used on the right side, and a collagen plug (negative control) is used on the left side.

[0329] Group 2: inorganic bovine bone (large particle size of 1-2 mm in 1 g; positive control) is used on the right side and a collagen plug (negative control) is used on the left side.

[0330] Group 3: Bio-Oss® (large particle size of 1-2 mm per gram) with BP-fluoroquinolone chemisorbed is used on the right side, and Bio-Oss® (large particle size of 1-2 mm per gram, positive control) is used on the left side.

[0331] The results of the chemical and antibacterial assays of the above experiments can inform the calculation of the ideal standard amount of the complex for adsorption to the implant material to be used in all in vivo experiments described here. Initial calculations predicted based on the preliminary results indicate that up to 5 mg of the complex adsorbed to 1 g of implant material provides bactericidal activity 2-3 orders of magnitude above the MIC of the test pathogen. Our BP-fluoroquinolone complexes are applicable to a wide range of bone graft materials, including commercial bone graft materials, such as Bio-Oss®, and therefore we select homemade anorganic bovine bone and BioOss as positive controls in the study to demonstrate the broad applicability of the complexes. Fill all defects (according to the above groups) up to the platform of each implant on both sides with a standard amount of biomaterial and cover the grafts and implants using Bio-Gide® membrane to improve stability. Close the flap without tension using periosteal tension reducing incisions, inner mattresses, and finally edge line simple interrupted sutures (PTFE 4,0, Cytoplast, USA). At this point, a micro-CT scan is obtained and the animals are clinically monitored for inflammation and adverse events. Additionally, the animals undergo PK studies to assess any systemic exposure to components within the implant material (e.g., complete complex, BP, antibiotic, or linker) as described in subsequent experiments. After 12 weeks, the animals are sacrificed and the mandibles are removed and examined with micro-CT followed by preparation for histology. A baseline micro-CT scan of the jaw is taken for comparison with post-study scans. Quantitative 3D volumetric micro-CT and histomorphometric analysis is performed to investigate the volume of new bone present at the peri-implant site, as well as the first bone-implant interface, total defect area, regenerated area, regenerated area within the total defect area, regenerated bone, residual substitute bone material, percentage of mineralized tissue, percentage of soft tissue, and percentage of void space. Finally, a necropsy is performed to post-mortem examine the internal organs and systems for gross and microscopic signs of tolerability issues from the local oral therapy.

[0332] The antimicrobial efficacy of the BP-FQ-bone graft can be evaluated in a canine peri-implantitis model. Briefly, PM2-PM4 are extracted bilaterally from the mandibles of eight beagle dogs (4 males, 4 females; 48 teeth total) using a minimally traumatic technique in this split-mouth design. After 3 months of healing, the mucoperiosteal flap is elevated on both sides of the jaw, and osteotomy preparation sites are created in each of the premolar regions by sequential cuts of gradually increasing diameters while copiously irrigating using a irrigation-equipped drill. Implants (Astra Tech Osseospeed Tx® 3×11 mm) are placed in each site using a non-impacting technique. The sequence of implant placement is identical on both sides but randomized between dogs by a computer-generated randomization scheme. Healing abutments are connected to the approximating implants and flaps by absorbable sutures. A plaque control regimen is then initiated, including brushing the teeth with toothpaste, four times per week. Twelve weeks after implant placement, just before starting the experimental peri-implantitis, microbial samples are obtained from all peri-implant sites using sterile paper points (Dentsply, size 35; Maillefer, Vallèges, Switzerland) and immediately placed into Eppendorf tubes (Starlab, Ahrensburg, Germany) for microbial analysis. Microbial analysis is performed via DNA extraction and PCR amplification of 16S rRNA as we have previously described in detail (55). PCR amplicons are sequenced using a Roche 454 GS FLX platform and data are analyzed by the Quantitative Insights into Microbial Ecology (QIIME) software package (56). Colony forming unit counts (CFU / mL) are determined from samples as determined in our previously described phase I study. At this point, experimental peri-implantitis is initiated as follows:As performed in our previous experiments in rat animal models and in our previous animal peri-implantitis studies, biofilms of Aggregatibacter actinomycetemcomitans (Aa), an important periodontal pathogen but not present in the canine microbiota, are established on the healing abutments in vitro. The biofilm-inoculated healing abutments are placed on the implants and cotton ligatures are placed in a submarginal position around the implant neck. After 10 weeks of bacterial infection, bacterial sampling and analysis are performed as before, and micro-CT scans are taken as a baseline of the peri-implantitis defect. Treatment of this experimental peri-implantitis model is initiated by surgically excising all implant sites by lifting the full-thickness buccolingual flap, removing any existing calculus from the implant surface using an air abrasion device, and swabbing the implant surface with gauze soaked in 0.12% chlorhexidine gluconate. The animals are divided into 4 groups (2 dogs per group) as anabolic groups.

[0333] Group 1: inorganic bovine bone (large particle size of 1-2 mm in 1 g) with BP-fluoroquinolone chemisorbed is used on the right side, and a collagen plug (negative control) is used on the left side.

[0334] Group 2: inorganic bovine bone (large particle size of 1-2 mm in 1 g; positive control) is used on the right side and a collagen plug (negative control) is used on the left side.

[0335] Group 3: BP-fluoroquinolone chemisorbed inorganic bovine bone (large particle size of 1-2 mm in 1 g) is used on the right side, and an antimicrobial releasing device (100 mg topical minocycline, positive control) is used on the left side.

[0336] Group 4: Bio-Oss® (large particle size of 1-2 mm per 1 g) chemisorbed with BP-fluoroquinolone (positive control) is used on the right side, and an antimicrobial releasing device (100 mg topical minocycline, positive control) is used on the left side.

[0337] The allocation to the treatment groups will be concealed to the investigator who will perform the data analysis. Standard and equivalent amounts of antimicrobial agents will be used in the treatment groups. After treatment, the valves will be repositioned and sutured (PTFE 4,0, Cytoplast, USA) and oral hygiene measures will be resumed one week after removal of the sutures. Clinical and micro-CT scan examinations will be performed again at 3 months after surgery, and microbiological samples will also be obtained at this time for analysis as described above. Six months after the peri-implantitis surgery, the animals will be euthanized, micro-CT scans will be performed, and the jaws will be removed for evaluation of histological parameters as detailed in the section "Critical Size Supraalveolar Peri-Implant Defect Model". Inflammation scores will be determined from histological sections as detailed previously (ref. 57) for correlation with clinical and radiographic findings.

[0338] Statistical analysis: Statistical calculations were performed using SPSS 22.0 (IBM Corp., Armonk, NY) and Excel 2016 (Microsoft Corp., Redmond, WA). G Power 3 software was used to estimate sample size for all animal studies. 58 A power analysis was performed using the Statistical Power Analysis (SPA) method. After data collection from these animal studies, quantitative results are first analyzed by descriptive statistics to understand the distribution of the data (parametric or non-parametric) and generate the mean, standard error, standard deviation, kurtosis and skewness, and 95% confidence level. The data are analyzed using Kruskal-Wallis tests, ANOVA, or mixed linear models as appropriate, and statistical significance is performed at the α=0.05 level when comparing groups. Post-hoc tests utilizing independent t-tests and Dunnett's multiple comparison tests are also performed to further confirm findings. All animal studies are conducted in accordance with the ARRIVE guidelines for reporting animal studies to ensure the quality, reliability, validity, and reproducibility of the results. 59 It is described using.

[0339] The stability of the drug compound and components and the in vitro ADME of BCC(6) can be evaluated. This data can help determine if significant differences may exist between human metabolism and experimental animals. Incubation of 6 with human, rat, and dog liver microsomes and hepatocytes followed by LC / MS analysis of the metabolic mixture will be performed. Metabolic profile of Ciprofloxacin 62、63 We focus on the BP portion of the molecule and any metabolites of the parent (e.g. piperazine ring cleavage as known for ciprofloxacin) since these are known. Where metabolites have been determined in vitro, we will determine these compounds in vivo at steady state using plasma samples from the other in vivo experiments mentioned above.

[0340] The toxicology of BCC(6) can be evaluated in rats and dogs to determine the NOAEL. We will first conduct a dose-ranging study to determine the NOAEL and maximum tolerated dose (MTD) in rats and dogs. Groups of six rats (three males, three females) will receive a single intravenous dose of 10 mg / kg of 6, or based on our best estimate at the time. The dose will be doubled until acute toxicity is observed (MTD), and then this dose will be successively decreased by 20% until no effect is observed, which becomes the NOAEL for the compound. Toxicity will be rated as mild, moderate, or severe, with moderate toxicity in two or more animals or severe toxicity in one or more animals defined as the MTD. 64 Animals are followed for weight and clinical observations over a 5-day period. After 5 days, animals are euthanized and necropsies are performed to assess visceral weights and histology (15 sections to include liver and kidneys based on clinical BP toxicology). A similar dose-range-finding study is performed in dogs (one per sex; starting with an equivalent dose of 4 mg / kg determined from allometric growth ratios assuming rats weighing 250 g and dogs weighing 10 kg) and will include the same terminal studies as rats, plus hematology and clinical chemistry. This study can use 4-6 cohorts in total.

[0341] Toxicokinetics and extended acute toxicity studies including recovery studies at NOAEL and MTD can be performed on the animal groups. Groups of 48 rats can be used, including 10 rats per sex for toxicity evaluation at each dose, 9 rats per sex for toxicokinetics, and 5 rats per sex for recovery studies. Toxicokinetics is performed at 6 time points (3 rats per time point randomly selected from males or females) followed by administration of each dose. The time points are 5, 30, 60, 120, 12, and 24 hours after administration. Animals are observed for recovery over a period of 14 days, after which visceral weights and histology are evaluated as in the above studies. PK parameters including Cmax, AUC, and half-life are determined from the toxicokinetic studies by non-compartmental analysis (NCA). An identical experiment will be performed in dogs, except that a total of 10 animals (3 per sex for dosing and 2 per sex for recovery study) will be included, and blood will be collected from each animal at the same time points as in rats. The AUC at the dog NOAEL will be used to calculate the maximum tolerable exposure of the bone graft / BP-fluoroquinolone conjugate described in Objective 2, and PK studies in dogs will be used to determine if there is systemic exposure greater than 1 / 100th of this level.

[0342] A unique three-compartment (blood / urine / bone) mathematical model of BP pharmacokinetics clinically validated for population analyses 65 is applied to the current project. From the dog study, we sample bone (jaw and femur), tendon (gastrocnemius) in each animal at the time of euthanasia for determination of BP and fluoroquinolone concentrations. We combine these data and our model to describe the time course in dogs. From this model we can simulate the expected exposure of bone and cartilage to both alternating or repeated doses of BP and fluoroquinolone, which can inform subsequent human dosing. As previously described 66~68The nonparametric adaptive grid (NPAG) algorithm with adaptive gamma, implemented within the Pmetrics package for R (Laboratory of Applied Pharmacokinetics and Bioinformatics, Los Angeles, CA), is used for all PK model fitting methods. The rationale for using an error polynomial as a function of measured concentration is the assay error (SD), and comparative performance assessment is completed using Akaike's information criterion, regression of observed concentration against expected concentration, visual plots of PK parameter covariate regressions, and parsimony rules.

[0343] Example 8 The BP-Ab complexes can be incorporated into implants and implant devices. In embodiments, one or more of the BP-Ab complexes can be incorporated into already approved bone graft products, such as BioOss® (Geistlich Pharma AG, Switzerland) or MinerOss® (BioHorizons, Birmingham, Alabama) bovine bone materials, to name just a few. The BP-Ab complexes can be mixed with a support material used as a dental bone graft substitute. The product will contain the complex adsorbed to an inorganic bovine bone material. This material provides localized delivery of antibiotics to the bone graft implant area, reducing bacterial infection rates and associated dental pathologies such as peri-implantitis and other infections. Dental applications of our products could include treatment of peri-implantitis as well as maintenance of tooth extraction sockets, ridge or sinus floor elevation, prevention or treatment of periodontitis, treatment or prevention of osteomyelitis or osteonecrosis, or other oral periodontal surgical applications in which such bone grafts may be beneficial. The BP-fluoroquinolone conjugate material will essentially be adsorbed onto the bone graft substitute and our preliminary data shows sustained release to the area of ​​bone destruction in the case of infection, thereby allowing our product to more effectively deliver antibiotics to the site of infection with negligible systemic exposure to either component of the conjugate compound.

[0344] The implant material may also be beneficial for non-dental implant procedures, such as sinus floor implants.

[0345] References for Examples 6 to 8 1. http: / / www.aaid.com / about / press_room / dental_implants_faq.html 2. Quirynen M, De Soete M, van Steenberghe D. Infectious risks for oral implants: a review of the literature. Clinical Oral Implants Research. 2002;13(1):1-19. doi: 10.1034 / j.1600-0501.2002.130101.x. PubMed PMID: 12005139. 3. Norowski PA, Jr., Bumgardner JD. Biomaterial and antibiotic strategies for peri-implantitis: a review. J Biomed Mater Res B Appl Biomater. 2009;88(2):530-43. doi: 10.1002 / jbm.b.31152. PubMed PMID: 18698626. 4. Salvi GE, Cosgarea R, Sculean A. Prevalence and Mechanisms of Peri-implant Diseases. J Dent Res. 2016. doi: 10.1177 / 0022034516667484. PubMed PMID: 27680028. 5. Meijer HJA,Raghoebar GM,de Waal YCM,Vissink A.Incidence of peri-implant mucositis and peri-implantitis in edentulous patients with an implant-retained mandibular overdenture during a 10-year follow-up period.Journal of Clinical Periodontology.2014;41(12):1178-83.doi: 10.1111 / jcpe.12311.PubMed PMID: 25229397. 6. Jemt T,Olsson M,Stenport VF.Incidence of First Implant Failure: A Retroprospective Study of 27 Years of Implant Operations at One Specialist Clinic.Clinical Implant Dentistry and Related Research.2015;17:E501-E10.doi: 10.1111 / cid.12277.PubMed PMID: 25536273. 7. Wolcott RD,Ehrlich GD.Biofilms and chronic infections.Jama-Journal of the American Medical Association.2008;299(22):2682-4.doi: 10.1001 / jama.299.22.2682.PubMed PMID: 18544729. 8. Costerton JW,Cheng KJ,Geesey GG,Ladd TI,Nickel JC,Dasgupta M,Marrie TJ.Bacterial biofilms in nature and disesae.Annual Review of Microbiology.1987;41:435-64.PubMed PMID: 3318676. 9. Costerton JW,Geesey GG,Cheng KJ.How bacteria stick.Scientific American.1978;238(1):86-95.PubMed PMID: 635520. 10. Kumar PS,Mason MR,Brooker MR,O’Brien K.Pyrosequencing reveals unique microbial signatures associated with healthy and failing dental implants.J Clin Periodontol 2012;39(5):425-33.doi: 10.1111 / j.1600-051X.2012.01856.x.PubMed PMID: 22417294; PubMed Central PMCID: PMC3323747. 11. Shibli JA,Melo L,Ferrari DS,Figueiredo LC,Faveri M,Feres M.Composition of supra and subgingival biofilm of subjects with healthy and diseased implants.Clin Oral Implants Res.2008;19(10):975-82.doi: 10.1111 / j.1600-0501.2008.01566.x.PubMed PMID: 18828812. 12. Stoodley P,Ehrlich GD,Sedghizadeh PP,Hall-Stoodley L,Baratz ME,Altman DT,Sotereanos NG,Costerton JW,DeMeo P.Orthopaedic Biofilm Infections.Curr Orthop Pract.2011;22(6):558-63.PubMed PMID: 22323927; PubMed Central PMCID: PMC3272669. 13. Javed F,AlGhamdi AST,Ahmed A,Mikami T,Ahmed HB,Tenenbaum HC.Clinical efficacy of antibiotics in the treatment of peri-implantitis.International Dental Journal.2013;63(4):169-76.doi: 10.1111 / idj.12034.PubMed PMID: 23879251. 14. Smeets R,Henningsen A,Jung O,Heiland M,Hammacher C,Stein JM.Definition,etiology,prevention and treatment of peri-implantitis - a review.Head Face Med.2014;10.doi: 10.1186 / 1746-160x-10-34.PubMed PMID: 25185675; PubMed Central PMCID: PMC4164121. 15. Leonhardt A,Dahlen G,Renvert S.Five-year clinical,microbiological,and radiological outcome following treatment of peri-implantitis in man.J Periodontol.2003;74(10):1415-22.doi: 10.1902 / jop.2003.74.10.1415.PubMed PMID: 14653386. 16. Mombelli A.Microbiology and antimicrobial therapy of peri-implantitis.Periodontol 2000.2002;28:177-89.PubMed PMID: 12013341. 17. Levison ME,Levison JH.Pharmacokinetics and Pharmacodynamics of Antibacterial Agents.Infect Dis Clin North Am.2009;23(4):75-89.doi: 10.1016 / j.idc.2009.06.008.PubMed PMID: 24484576; PubMed Central PMCID: PMC4079031 18. Kaur K,Sikri P.Evaluation of the effect of allograft with doxycycline versus the allograft alone in the treatment of infrabony defects: A controlled clinical and radiographical study.Dent Res J (Isfahan).2013;10(2):238-46.PubMed PMID: 23946743; PubMed Central PMCID: PMC3731967. 19. Inzana JA,Schwarz EM,Kates SL,Awad HA.Biomaterials approaches to treating implant-associated osteomyelitis.Biomaterials.2016;81:58-71.doi: 10.1016 / j.biomaterials.2015.12.012.PubMed PMID: 26724454. 20. Schmitt CM,Moest T,Lutz R,Neukam FW,Schlegel KA.Anorganic bovine bone (ABB) vs.autologous bone ...

Claims

1. A compound which is a conjugate of a bisphosphonate and a quinolone moiety, The quinolone moiety is releasably attached to the bisphosphonate via a carbamate, O-thiocarbamate, or S-thiocarbamate, as represented by the formula: 【Chemistry 1】 (In the formula, X is O and W is O, X is O and W is S, or X is S and W is O, Y is H, CH 3 , NO 2 , F, Cl, Br, I, or CO 2 H, Z is H, CH 3 , OH, NH 2 , SH, F, Cl, Br, or I, and n is 1 to 5.) the quinolone moiety is selected from the group consisting of alatrofloxacin, amifloxacin, balofloxacin, besifloxacin, ciprofloxacin, clinafloxacin, delafloxacin, enoxacin, finafloxacin, gatifloxacin, gemifloxacin, grepafloxacin, JNJ-Q2, lomefloxacin, moxifloxacin, norfloxacin, orbifloxacin, pazufloxacin, pradofloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zavofloxacin, and nemonoxacin; compound.

2. The compound according to claim 1, represented by formula (12), formula (13) or formula (15): 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 。

3. The compound according to claim 1, represented by formula (6): 【Chemistry 5】 。

4. The compound according to claim 1, represented by any one of the following formulas: 【Chemistry 6】 In the formula, R 5 is hydrogen or a hydroxy group. 【Chemistry 7】 【Chemistry 8】

5. A compound according to any one of claims 1 to 4, and Pharmaceutically Acceptable Carriers 23. A pharmaceutical composition comprising:

6. The pharmaceutical composition of claim 5, wherein the amount of the compound is an amount effective for treating or preventing osteomyelitis, osteonecrosis, peri-implantitis, or periodontitis, or an amount effective for killing or inhibiting the growth of bacteria.

7. A medicament for treating or preventing bone infections, comprising the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.

8. A medicament for treating or preventing osteomyelitis, comprising the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.

9. A medicament for treating or preventing diabetic foot disease, comprising the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.

10. A medicament for treating or preventing bacterial infection or biofilm infection at a bone surgery site or an implant surgery site, or at a surgery site where a bone graft has been performed, comprising a compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5.

11. Bone graft material; A compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5, A bone graft composition, wherein the compound or pharmaceutical composition is attached to, integrated with, chemically adsorbed to, or mixed with the bone graft material.

12. 12. The bone graft composition of claim 11, wherein the bone graft material is an autograft bone material, an allograft bone material, a xenograft bone material, a synthetic bone graft material, or a combination thereof.

13. 13. A pharmaceutical product comprising the bone graft composition of claim 11 or claim 12 for treating or preventing bacterial or biofilm infection at a bone surgical site or an implant surgical site, or at a surgical site where a bone graft has been performed.

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