NICOTINAMIDE MONONUCLEOTIDE DERIVATIVES FOR THE TREATMENT OF BACTERIAL INFECTIONS

Abstract

A compound of formula (I) or a pharmaceutically acceptable salt and/or solvate thereof;

##STR00001##

in which X, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, Y, and are as described in the claims, for the use thereof in the treatment of bacterial infections including those caused by caused by at least one bacterium of the genus selected from aerobic Gram-positive bacteria; Gram-negative enterobacteria; Gram-negative bacilli; Gram-negative anaerobic bacteria; Gram-positive anaerobic bacteria; mycobacteria and pathogens involved in sexually transmitted infections.

Claims

1-11. (canceled)

12. A method for treating a bacterial infection in a subject in need thereof, said method comprising administering to said subject a therapeutically effective quantity of at least one compound of formula (I) ##STR00069## or a pharmaceutically acceptable salt and/or solvate thereof, wherein: X is selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1 is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected, independently of one another, from H, halogen, azido, cyano, hydroxyl, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 thio-alkyl, C.sub.1-C.sub.12 heteroalkyl, C.sub.1-C.sub.12 haloalkyl and OR; wherein R is selected from H, C.sub.1-C.sub.12 alkyl, C(O)(C.sub.1-C.sub.12)-alkyl, C(O)NH(C.sub.1-C.sub.12)-alkyl, C(O)O(C.sub.1-C.sub.12)-alkyl, C(O)-aryl, C(O)(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)NH(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)O(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl and C(O)CHR.sub.AANH.sub.2; wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R.sub.6 is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.7 is selected from P(O)R.sub.9R.sub.10, P(S)R.sub.9R.sub.10 and ##STR00070## wherein R.sub.9, and R.sub.10 are selected, independently of one another, from OH, OR.sub.11, NHR.sub.13, NR.sub.13R.sub.14, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.12 aryl, (C.sub.5-C.sub.12)-aryl-(C.sub.1-C.sub.8)-alkyl, (C.sub.1-C.sub.8)-alkyl-(C.sub.5-C.sub.12)-aryl, (C.sub.1-C.sub.8)-heteroalkyl, (C.sub.3-C.sub.8)-heterocycloalkyl, (C.sub.5-C.sub.12)-heteroaryl and NHCR.sub.αR.sub.α′C(O)R.sub.12; wherein: R.sub.11 is selected from C.sub.1-C.sub.10 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.5-C.sub.12 aryl, (C.sub.1-C.sub.10)-alkyl-(C.sub.5-C.sub.12)-aryl, C.sub.5-C.sub.12 substituted aryl, C.sub.1-C.sub.10 heteroalkyl, C.sub.1-C.sub.10 haloalkyl, —(CH.sub.2).sub.mC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mOC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mOC(O)O(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mSC(O)(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mC(O)O(C.sub.1-C.sub.15)-alkyl, —(CH.sub.2).sub.mC(O)O(C.sub.1-C.sub.15)-alkyl-aryl; wherein m is an integer selected from 1 to 8; P(O)(OH)OP(O)(OH).sub.2; and an internal or external counter-ion; R.sub.12 is selected from C.sub.1-C.sub.10 alkyl, hydroxy, C.sub.1-C.sub.10 alkoxy, C.sub.2-C.sub.8 alkenyloxy, C.sub.2-C.sub.8 alkynyloxy, halo(C.sub.2-C.sub.10)-alkoxy, C.sub.3-C.sub.10 cycloalkoxy, C.sub.3-C.sub.10 heterocycloalkyloxy, C.sub.5-C.sub.12 aryloxy, (C.sub.1-C.sub.4)-alkyl-(C.sub.5-C.sub.12)-aryloxy, (C.sub.5-C.sub.12)-aryl-(C.sub.1-C.sub.4)-alkyloxy and C.sub.5-C.sub.12 heteroaryloxy; wherein said aryl or heteroaryl groups are optionally substituted by one or two groups selected from halogen, trifluoromethyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and cyano; R.sub.13 and R.sub.14 are selected independently from H, C.sub.1-C.sub.8 alkyl and (C.sub.1-C.sub.8)-alkyl-(C.sub.5-C.sub.12)-aryl; R.sub.α and R.sub.α′ are selected independently from hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.1-C.sub.10 thio-alkyl, C.sub.1-C.sub.10 hydroxylalkyl, (C.sub.1-C.sub.10)-alkyl-(C.sub.5-C.sub.12)-aryl, C.sub.5-C.sub.12 aryl, —(CH.sub.2).sub.3NHC(═NH)NH.sub.2, (1H-indol-3-yl)-methyl, (1H-imidazol-4-yl)-methyl and a side chain selected from a proteinogenic or non-proteinogenic amino acid; wherein said aryl groups are optionally substituted by a group selected from hydroxyl, C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, nitro and cyano; or R.sub.9 and R.sub.10 with the phosphorus atoms to which they are bonded, form a 6-member-ring, wherein —R.sub.9-R.sub.10— represents —O—CH.sub.2—CH.sub.2—CHR—O—; wherein R is selected from hydrogen, C.sub.5-C.sub.6 aryl and C.sub.5-C.sub.6 heteroaryl; wherein said aryl or heteroaryl groups are optionally substituted by one or two groups selected from halogen, trifluoromethyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and cyano; X′ is selected from O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1′ is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.2′, R.sub.3′, R.sub.4′ and R.sub.5′ are selected, independently of one another, from H, halogen, azido, cyano, hydroxyl, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 thio-alkyl, C.sub.1-C.sub.12 heteroalkyl, C.sub.1-C.sub.12 haloalkyl and OR; wherein R is selected from H, C.sub.1-C.sub.12 alkyl, C(O)(C.sub.1-C.sub.12)-alkyl, C(O)NH(C.sub.1-C.sub.12)-alkyl, C(O)O(C.sub.1-C.sub.12)-alkyl, C(O)-aryl, C(O)(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)NH(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl, C(O)O(C.sub.1-C.sub.12)-alkyl-(C.sub.5-C.sub.12)-aryl and C(O)CHR.sub.AANH.sub.2; wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R.sub.6′ is selected from H, azido, cyano, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 thio-alkyl, C.sub.1-C.sub.8 heteroalkyl and OR; wherein R is selected from H and C.sub.1-C.sub.8 alkyl; R.sub.8′ is selected from H, OR, NHR.sub.15′, NR.sub.15′R.sub.16′, NH—NHR.sub.15′, SH, CN, N.sub.3 and halogen; wherein R.sub.15′ and R.sub.16′ are selected, independently of one another, from H, C.sub.1-C.sub.8 alkyl and C.sub.1-C.sub.8 alkyl-aryl; Y′ selected from CH, CH.sub.2, C(CH.sub.3).sub.2 and CCH.sub.3; n is an integer selected from 1 to 3; represents a single or a double bond according to Y′; and represents the alpha or beta anomer according to the position of R.sub.1′; R.sub.8 is selected from H, OR, NHR.sub.15, NR.sub.15R.sub.16, NH—NHR.sub.15, SH, CN, N.sub.3 and halogen; wherein R is selected from H and C.sub.1-C.sub.8 alkyl, and R.sub.15 and R.sub.16 are selected, independently of one another, from H, C.sub.1-C.sub.8 alkyl and C.sub.1-C.sub.8 alkyl-aryl and —CHR.sub.AACO.sub.2H wherein R.sub.AA is a side chain selected from a proteinogenic or non-proteinogenic amino acid; Y is selected from CH, CH.sub.2, C(CH.sub.3).sub.2 and CCH.sub.3; represents a single or a double bond according to Y; and represents the alpha or beta anomer according to the position of R.sub.1.

13. The method according to claim 12, wherein X represents oxygen.

14. The method according to claim 12, wherein R.sub.1 and R.sub.6 each represent hydrogen.

15. The method according to claim 12, wherein R.sub.2, R.sub.3, R.sub.4 and R.sub.5 each represent, independently of one another, hydrogen or OH.

16. The method according to claim 12, wherein Y represents CH or CH.sub.2.

17. The method according to claim 12, wherein R.sub.7 represents P(O)(OH).sub.2.

18. The method according to claim 12, wherein R.sub.7 represents ##STR00071## wherein; R.sub.9 is OH or OR.sub.11, wherein R.sub.11 is as defined in formula (I) and X′ is oxygen; R.sub.1′ and R.sub.6′ each represent hydrogen; R.sub.2′, R.sub.3′, R.sub.4′ and R.sub.5′ are independently selected from hydrogen and OH; R.sub.8′ is NH.sub.2; Y′ is selected from CH and CH.sub.2; n is equal to 2; represents a single or a double bond according to Y′; and represents the alpha or beta anomer according to the position de R.sub.1′.

19. The method according to claim 12, wherein the compound of formula (I) is selected from: ##STR00072## ##STR00073## or a pharmaceutically acceptable salt and/or solvate thereof.

20. The method according to claim 12, wherein the bacterial infection is caused by at least one bacterium of the genus selected from aerobic Gram-positive bacteria; Gram-negative enterobacteria; Gram-negative bacilli; Gram-negative anaerobic bacteria; Gram-positive anaerobic bacteria; mycobacteria and pathogens involved in sexually transmitted infections.

21. The method according to claim 20, wherein the aerobic Gram-positive bacteria are selected from the group consisting of Streptococcus, Staphylococcus, Enterococcus and Bacillus; the Gram-negative enterobacteria are selected from the group consisting of Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes, Enterobacter cloacae, Proteus vulgaris, Shigella flexneri, Serratia marcescens, Citrobacter freundii, Yersinia enterocolitica and Salmonella enteritidis; the Gram-negative bacilli are selected from the group consisting of Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia and Stenotrophomonas maltophilia; the Gram-negative anaerobic bacteria are selected from the group consisting of Bacteroides, Fusobacterium and Eubacterium; the Gram-positive anaerobic bacteria are selected from the group consisting of Propionibacterium, Peptococcus, Clostridium, Peptostreptococcus and Veillonella; the mycobacteria are selected from the group consisting of Mycobacterium leprae and Mycobacterium tuberculosis; and wherein the pathogens involved in sexually transmitted infections are selected from the group consisting of Neisseria, Haemophilus, Chlamydia and Mycoplasma.

21. The method according to claim 12, wherein the bacterial infection is selected from bacterial skin and soft tissue infections, sexually transmitted bacterial infections, tetanus, typhoid, tuberculosis, cholera, diphtheria, syphilis, salmonella, pulmonary bacterial infections or sepsis.

22. The method according to claim 12, wherein the bacterial infection is sepsis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0312] FIG. 1 is a histogram showing the percentage of positive bacterial culture in the spleen assessed at 24 h, 48 h and 72 h.

[0313] FIG. 2 is a histogram showing the survival rate of mice after LPC (Caecal ligation and puncture) at 24 h, 48 h, 72 h and 96 h.

[0314] FIG. 3 is a histogram showing the decrease of temperature after LPC, monitored over 48 hours.

[0315] FIG. 4 is a histogram showing the weight (FIG. 4A) and weight loss (FIG. 4B) of mice (FIG. 4B) of mice after LPC at 24 h, 48 h, 72 h and 96 h.

[0316] FIG. 5 is a histogram showing the clinical score of mice after LPC at 24 h, 48 h, 72 h and 96 h.

[0317] FIG. 6 is a histogram showing the bacterial load in blood 24 hours after LPC, assessed by RT-PCR quantification of bacterial DNA (16S).

[0318] FIG. 7 is a histogram showing bacterial load in blood (FIG. 7A) and lungs (FIG. 7B), 5 days after infection with Pseudomonas aeruginosa.

EXAMPLES

[0319] The present invention will be better understood on reading the following examples which illustrate the invention in a non-limiting manner.

I. Synthesis of the Compounds of the Invention

[0320] 1. Material and Methods

[0321] All the chemicals were obtained from commercial suppliers and users without further purification.

[0322] Thin layer chromatography was carried out on plastic sheets of TLC silica gel 60 F254 (layer thickness 0.2 mm) from Merck. Purification by column chromatography was carried out on the silica gel 60 (70-230 mesh ASTM, Merck). The melting points were determined either on a digital apparatus (Electrothermal IA 8103) and are not corrected, or on a WME Kofler bench (Wagner & Munz). The IR, .sup.1H, .sup.19F and .sup.13C NMR spectra confirmed the structure of all the compounds. The IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer and the NMR spectra were recorded, using CDCl.sub.3, CD.sub.3CN, D.sub.2O or DMSO-d.sub.6 as solvent, on a BRUKER AC 300 or 400 spectrometer at 300 or 400 MHz for the .sup.1H spectrum, 75 or 100 MHz for the .sup.13C spectrum and 282 or 377 MHz for the .sup.19F spectrum. The chemical shifts (δ) were expressed in parts per million with respect to the signal, indirectly (i) to CHCl.sub.3 (δ 7.27) for .sup.1H and (ii) to CDCl.sub.3 (δ 77.2) for .sup.13C and directly (iii) to CFCl.sub.3 (internal standard) (δ 0) for .sup.19F. The chemical shifts are given in ppm and the multiplicities of peaks are designated as follows: s, singlet; br s, wide singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quadruplet; quint, quintuplet; m, multiplet. High resolution mass spectra (HRMS) were obtained from the “Service central d'analyse de Solaize” (Centre national de la recherche scientifique) and have been recorded on a Waters spectrometer, using electrospray ionisation-TOF (ESI-TOF).

[0323] General Procedure

Step 1: Synthesis of the Compound of Formula A-1

[0324] The compound of formula D (1.0 equiv.) is dissolved in dichloromethane. The nicotinamide of formula E (1.50 equiv.) and TMSOTf (1.55 equiv.) are added at ambient temperature. The mixture is heated with reflux and stirred until the reaction is achieved. The mixture is cooled to ambient temperature and filtered. The filtrate is concentrated to dryness to give tetraacetate A-1.

Step 2: Synthesis of the Compound of Formula A-2

[0325] The tetraacetate A-1 is dissolved in methanol and cooled to −10° C. 4.6 M ammonia in methanol (3.0 equivalents) at −10° C. is added and the mixture is stirred at this temperature until the reaction is complete. Dowex HCR (H+) resin is added to a pH of 6-7. The reaction mixture is heated to 0° C. and filtered. The resin is washed with a mixture of methanol and acetonitrile. The filtrate is concentrated to dryness. The residue is dissolved in acetonitrile and concentrated to dryness. The residue is dissolved in acetonitrile to give a solution of the compound of formula A-2.

Step 3: Synthesis of the Compound of Formula A-3

[0326] The crude solution of the compound of formula A-2 in acetonitrile is diluted with trimethyl phosphate (10.0 equivalents). The acetonitrile is distilled under vacuum and the mixture is cooled to −10° C. Phosphorus oxychloride (4,0 equivalents) is added at −10° C. and the mixture is stirred at −10° C. until the reaction is ended.

Step 4 and 5: Synthesis of the Compound of Formula I-A

[0327] The mixture obtained in step 3 here above is hydrolysed by the addition of a 50/50 mixture of acetonitrile and water, followed by the addition of methyl tert-butyl ether. The mixture is filtered and the solid is dissolved in water. The aqueous solution is neutralised by the addition of sodium bicarbonate and extracted with dichloromethane. The aqueous layer is concentrated to dryness in order to give the crude compound of formula I-A, which is purified on a DOWEX 50wx8 column with elution in water followed by a silica gel chromatograph column.

II. Biological Studies

Example 1: In Vivo Efficacy of the Compound of Formula I-A in a Non-Lethal Model of Escherichia coli (E. coli) Induced Pneumonia

[0328] The purpose of this study is to evaluate the effect of pre-treatment, with an NAD precursor, on the spread of bacterial infection to the spleen in a mouse model of non-lethal Escherichia coli (E. coli) induced pneumonia.

[0329] 1. Material and Methods

[0330] The administration of the compound at 185 mg/kg and the vehicle (physiological buffer) is performed intraperitoneally and/or intratracheally.

[0331] The compound of formula I-A (white powder) is dissolved in the vehicle. The solution is used at room temperature for a maximum of 1 day and freshly prepared for each new experiment.

[0332] The mice are weighed daily in order to adapt the volume of compound to be administered.

[0333] 1.1. Escherichia coli Induced Pneumonia

[0334] Immediately before use, the E. coli culture was washed twice with 0.9% NaCl. After the second wash, the pellet was re-suspended in sterile saline and the dose calibrated by nephelometry.

[0335] Female BALB/c mice (20-24 g) were then inoculated using intratracheal insertion of a gavage needle (24 G) for injection of 75 μL of the bacterial suspension.

[0336] 1.2. Administration of the Compounds

[0337] The compound of formula I-A and the vehicle are administered to the animals intraperitoneally and/or intratracheally. The injection of the compound of formula I-A is performed 24 hours prior to the infection with Escherichia coli. Simulated animals are given a physiological buffer by intraperitoneal administration.

[0338] 1.3. Bacterial Load

[0339] At 24 hours, 48 hours, and 72 hours postoperatively, spleens from sacrificed animals were weighed and homogenized in 1 mL of saline. These solutions were then used for quantitative agar gel cultures during 24 hours of incubation at 37° C. Viable bacterial colony counts are expressed as Log 10 CFU per gram of organ.

[0340] 2. Results and Discussion

[0341] FIG. 1 shows the percentage of positive bacterial culture in the spleen assessed at 24 h, 48 h and 72 h. As shown, pre-treatment with the compound of formula I-A reduced the percentage of positive cultures in the spleen compared to untreated mice (control) after intraperitoneal administration. Notably, no bacteria were found in the spleen 48 h after intraperitoneal administration and 72 h after intratracheal administration.

[0342] 3. Conclusion

[0343] After induction of non-lethal pneumonia by E. coli in mice, the spleens were collected and analyzed.

[0344] The percentage of animals with bacteria in the spleen after 24 hours was greatly reduced in the group treated with compound of formula I-A compared to the control group. Furthermore, no bacteria were found in the spleen of mice pre-treated with compound of formula I-A 48 hours after intraperitoneal administration while animals in the control group were positive.

[0345] Pre-treatment with compound of formula I-A prevented the spread of bacteria from the lungs to the spleen after intraperitoneal and/or intratracheal administration demonstrating a potential effect of compound of formula I-A in the prevention of sepsis during a bacterial infection.

Example 2: In Vivo Evaluation of Compounds of Formula I-A and I-B on the Course of Sepsis in a Lethal Model of Caecal Ligation and Puncture

[0346] The purpose of this study is to evaluate the effect of administration of compounds according to the invention (I-A and I-B) on the survival rate of mice in a caecal ligation and puncture (LPC) induced sepsis model.

[0347] All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (revised 1996 and 2011, 2010/63/EU) and French laws.

[0348] 1. Material and Methods

[0349] 1.1. Protocol of the Study

[0350] The study consists of creating a sepsis model in mice by caecal ligation and puncture (LPC) and evaluating the impact of compounds I-A and I-B on the development of sepsis for 4 days.

[0351] The test compounds are administered at 185 mg/kg, intraperitoneally, immediately after the LPC procedure, and then once daily at 24, 48, and 72 hours. The remaining mice are euthanized at 96 hours post-LPC. Vehicle (physiological buffer) is used as a control and administered under the same conditions.

[0352] Three groups are formed and a total of 36 mice are included in the study:

[0353] Group 1: LPC+vehicle i.p. (n=12);

[0354] Group 2: LPC+compound I-A i.p. (n=12);

[0355] Group 3: LPC+compound 1-B i.p. (n=12).

[0356] In each group, survival, temperature, body weight and clinical score were assessed over the 4 days of the study. Bacterial load assessed at 24 hours after LPC.

[0357] 1.1. Ligation and Puncture of the Caecum

[0358] Mice are anesthetized with 3% Vetoflurane. A laparotomy is performed to exteriorize the caecum. For induction of medium grade sepsis, the caecum is ligated between the distal pole and the base of the caecum. A through puncture with a 21-gauge needle is performed. A small amount of excreta is extruded from the mesenteric and antimesenteric penetration holes to ensure patency. The caecum is repositioned over the abdominal cavity. The peritoneum, fascia, abdominal musculature and then the skin are closed by applying simple sutures. The animals are resuscitated by injecting pre-warmed normal saline (37° C.; 5 ml per 100 g body weight) subcutaneously.

[0359] 1.2. Administration of the Compounds

[0360] Administration of Compound I-A at 185 mg/kg, Compound I-B at 185 mg/kg and vehicle (physiological buffer) is performed intraperitoneally immediately after LPC surgery and then once daily at 24, 48 and 72 hours post LPC.

[0361] Preparation of the formulations: the powder of compounds I-A and I-B are dissolved in the vehicle. Storage conditions: The solution is used at room temperature for a maximum of 1 day and freshly prepared for each new experiment.

[0362] Mice are weighed daily to adapt the volume of compound to be administered.

[0363] 1.3. Survival

[0364] Survival is assessed every 12 hours for the first two days, then once a day until euthanasia.

[0365] 1.4. Temperature

[0366] Rectal temperature is checked every 2 to 4 hours for the first day, then twice for the second day.

[0367] 1.5. Body Weight and Clinical Score

[0368] These two parameters are monitored once a day.

[0369] The clinical score is defined according to the criteria defined in Table 3:

TABLE-US-00003 TABLE 3 Murine sepsis score Score 0 1 2 3 Appearance Smooth Slightly rough Most of the back is Pilo erection coat coat ruffled may or may not be present, the mouse appears “puffy”. Level of Active Active, avoids The mouse activity Activity is consciousness standing is noticeably reduced. The slowed. The mouse mouse only is still ambulatory moves when provoked, movements have a tremor Activity Normal Suppression of Suppressed activity. No activity. The eating, drinking The mouse is mouse is or running stationary and stationary occasionally moves to investigate Response to Normal Slow response No response to No response to stimuli to auditory or auditory stimulus; auditory tactile stimuli moderate response stimulus; slight to touch (moves a response to few steps) touch (no locomotion) Eyes Open Not fully open, Eyes at least half Eyes half closed potential closed, possibly or more, secretion with secretions possibly with secretions Breathing Normal Brief periods of Labored, without Brief periods of quality labored panting labored breathing breathing Labored, without panting Labored with intermittent panting

[0370] 1.6. Bacterial Load

[0371] 24 hours after surgery, mice were anesthetized with Vetoflurane and blood was collected from the retroorbital sinus to quantify the bacterial load. Bacterial DNA (16S) is extracted and quantified by RT-PCR.

[0372] 2. Results and Discussion

[0373] 1.1. Survival Rate

[0374] FIG. 2 shows the survival rate of mice after LPC at 24, 48, 72 and 96 hours. Table 4 specifies the number of surviving mice after LPC at 24, 48, 72 and 96 hours.

TABLE-US-00004 TABLE 4 Number of surviving mice Time (h) Vehicle I-A I-B 24 11/11  12/12 12/12  48 5/11 11/12 9/12 72 3/11 10/12 8/12 96 0/11  9/12 6/12

[0375] The results in FIG. 2 and Table 4 show that the survival rate in mice treated with compound I-A or I-B is improved compared with the control group.

[0376] 1.2. Temperature

[0377] As shown in FIG. 3, a significant decrease in temperature is observed in the first 6 hours after LPC. Mice treated with compound I-A or 1-B regain their basal temperature 24 hours after surgery while the temperature of the control group continues to decrease.

[0378] 1.3. Weight Loss

[0379] FIG. 4A shows the weight of mice after LPC at 24, 48, 72, and 96 hours, and FIG. 4B illustrates their weight loss over this period. Induced sepsis leads to a decrease in the weight of mice in all three groups. However, the weight loss is less in the group treated with compound I-B.

[0380] 1.4. Clinical Score

[0381] FIG. 5 shows the clinical score of mice after LPC at 24, 48, 72, and 96 hours. The clinical score is very significantly improved in the groups treated with compound I-A or I-B, compared to the control group.

[0382] 1.5. Bacterial Load

[0383] FIG. 6 shows the bacterial load in the blood, 24 hours after LPC. The bacterial load is significantly decreased in the groups treated with compound I-A or I-B, compared to the control group.

[0384] 3. Conclusion

[0385] Treatment with compound I-A or I-B increased survival in treated mice in a lethal sepsis model. The treatment also minimized temperature drop, delayed infection progression and reduced bacterial load.

Example 3: In Vivo Evaluation of Compound I-A in a Mouse Model of Respiratory Tract Infection Induced by Pseudomonas aeruginosa PAO.SUB.1

[0386] The purpose of this study is to evaluate the antibacterial effect of the administration of compound I-A in a lethal mouse model of Pseudomonas aeruginosa airway infection and its effect on sepsis.

[0387] 1. Material and Methods

[0388] 1.1. Protocol of the Study

[0389] The study consists in creating a model of respiratory infection in mice, by intranasal administration of 1.10.sup.4 CFU of Pseudomonas aeruginosa PAO.sub.1, and to evaluate the effect of compound I-A on the bacterial load.

[0390] Compound I-A was administered at 185 mg/kg, intraperitoneally, 1 h after infection, and then once a day until euthanasia. The vehicle (physiological buffer) is used as a control and is administered under the same conditions. Ciprofloxacin is used as a reference compound and is administered under the same conditions.

[0391] Four groups (10 mice/group) are included in the study: [0392] Vehicle group: PAO.sub.1 infection+physiological buffer; [0393] Ciprofloxacin group: PAO.sub.1 infection+ciprofloxacin (2 mg/kg); [0394] Group I-A: PAO.sub.1 infection+compound I-A (185 mg/kg); [0395] Group I-A+ciprofloxacin: PAO.sub.1 infection+compound I-A (185 mg/kg)+ciprofloxacin (2 mg/kg).

[0396] In each group, the bacterial load in the blood and lungs is assessed on day 5 post infection.

[0397] 1.1. Leukopenia and Infection

[0398] Leukopenia is induced by intraperitoneal injections of cyclophosphamide 4 days and 1 day before infection, with doses of 150 mg/kg and 100 mg/kg, respectively. P. aeruginosa PAO.sub.1 was obtained from ATCC. One vial is thawed and diluted in sterile PBS on the day of infection. Mice are infected by intranasal administration of 1 10.sup.4 CFU of Pseudomonas aeruginosa PAO.sub.1.

[0399] 1.2. Administration of the Compounds

[0400] Compound I-A at 185 mg/kg and vehicle (physiological buffer) are administered intraperitoneally (i.p.) 1 hour after infection and then once a day until euthanasia.

[0401] Preparation of the formulations: The powder of compound I-A is dissolved in the vehicle. Storage conditions: The powder is stored at +4° C. until use. The solution should be used at room temperature for maximum 1 day and freshly prepared for each new experiment.

[0402] Mice are weighed regularly to adjust the volume of compound to be administered.

[0403] For reference, ciprofloxacin is also administered at sub-active dose (2 mg/kg) to mice intraperitoneally under the same conditions.

[0404] A co-administration of ciprofloxacin and compound I-A is also performed, under the same conditions.

[0405] 1.3. Bacterial Load

[0406] Bacterial load is assessed in blood and lungs on day 5. Quantitative cultures on agar gel are performed during 24 hours of incubation at 37° C. Viable bacterial colony counts are expressed as Log 10 CFU per mL of blood or per gram of organ.

[0407] 2. Results and Discussion

[0408] FIG. 7 shows the bacterial load in blood (FIG. 7A) and lungs (FIG. 7B), 5 days after infection with Pseudomonas aeruginosa.

[0409] Evaluation of the pulmonary bacterial load showed an antibacterial effect of treatment with compound I-A, ciprofloxacin, or a combination of ciprofloxacin and compound I-A. All 3 treatment groups showed significant efficacy compared with the vehicle group. Blood bacterial load results showed that P. aeruginosa triggered sepsis in all 4 groups. The three treated groups showed a reduced bacterial load compared to the vehicle group by approximately 2 log.

[0410] Thus, treatment with compound I-A limited the bacterial load during infection and limited sepsis.