Spiro-lactam compounds, process and uses thereof

Abstract

The present disclosure relates to a new class of antimicrobial agents. In particular, this disclosure provides the identification and characterization of novel spiro-lactam compounds as anti-HIV/AIDS and anti-malarial agents. Furthermore, this disclosure also provides the preparation of spiro-β-penicillanic acids, which proved to be potent inhibitors of HIV.

Claims

1. A compound selected from the group consisting of: ##STR00024##

2. The compound of claim 1, wherein the compound exhibits inhibitory activity against human immunodeficiency virus (HIV) and Plasmodium species.

3. The compound of claim 2, wherein the HIV is selected from the group consisting of human immunodeficiency virus-2 group A, human immunodeficiency virus-2 group B, human immunodeficiency virus-1 group M, human immunodeficiency virus-1 group O, human immunodeficiency virus-1 group N, and human immunodeficiency virus-1 group P.

4. The compound of claim 2, wherein the Plasmodium species is selected from the group consisting of: Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi.

5. A pharmaceutical composition comprising the compound of claim 1 in a therapeutically effective amount and as a first active ingredient, a pharmaceutically acceptable carrier or excipient, and a second active ingredient.

6. The pharmaceutical composition of claim 5, further comprising an antiviral agent, an anti-malarial agent, an immunomodulator agent, an analgesic agent, an anti-inflammatory agent, an antibiotic agent or a diuretic agent.

7. The pharmaceutical composition of claim 6, wherein the antiviral agent is an anti-HIV agent.

8. The pharmaceutical composition of claim 5, further comprising a filler, a binder, a disintegrant or a lubricant, or a thereof.

9. The pharmaceutical composition of claim 5, wherein the second active ingredient is a HIV protease inhibitor (PI), a HIV nucleoside reverse transcriptase inhibitor (NRTI), a HIV non-nucleoside reverse transcriptase inhibitor (NNRTI), a HIV integrase inhibitor, or a HIV entry inhibitor, or a mixture thereof.

10. The pharmaceutical composition of claim 6, wherein the anti- malarial agent is selected from the group consisting of: 8 aminoquinoline, amodiaquine, arteether, artemether, artemisinin, artesunate, artesunic acid, artelinic acid, atovoquone, azithromycin, biguanide, chloroquine, chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutyl halofantrine, desipramine, doxycycline, dihydrofolate, reductase inhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors, primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine, quinacrine, sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine, tetracycline, tetrandine, and triazine, or a salt thereof.

11. The pharmaceutical composition of claim 7, wherein the anti-HIV agent is selected from the group consisting of: tenofovir disoproxil fumarate, alafenamide, emtricitabine, atazanavir sulfate, lopinavir-ritonavir, and efavirenz, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of the present disclosure.

(2) FIG. 1. Structure of compounds whose biological evaluation as anti-HIV agents was carried out.

(3) FIG. 2: Compounds identified as potent anti-HIV agents.

(4) FIG. 3: Antiviral activity of compounds BSS-593, BSS-722A and BSS-730A which are selected members of the new class of compounds described in this document. Dose-response curves for different HIV-1 and HIV-2 isolates with the BSS-593 (A), BSS-722A (B) and BSS-730A (C) compounds. Dose-response curves were estimated from the percentage of inhibition of infection (y-axis) against log.sub.10 of concentration of each compound (x-axis) using the sigmoidal dose-response equation (variable slope). IC.sub.50 and IC.sub.90 values were determined from this curve. As stated above, IC.sub.50 and IC.sub.90 values correspond to the concentration of compound that inhibits viral replication by 50% and 90%, respectively.

(5) FIG. 4: Acute toxicity assay of compound BSS-593. Wistar rats (n=5) were inoculated IP with different doses of BSS-593 and effects on liver, renal and lipid homeostasis were assessed by dosing different biochemical parameters 48 h after inoculation. ALT, AST and LDH were determined as markers of liver function and serum level of creatinine were determined as marker of renal function. Serum levels of total cholesterol, low-density cholesterol (LDL), high-density cholesterol (HDL) and triglycerides were measured to evaluate the effect of the drugs on lipid homeostasis. Results were expressed as the means with their standard errors and were compared using a one-factorial ANOVA test, followed by a Bonferroni's multi-comparison post hoc test. There was no statistical difference in the results obtained for the control rats (inoculated with sterile saline) and the rats inoculated with BSS-593, indicating that this compound does not induce liver and renal injury and does not alter lipid metabolism.

(6) FIG. 5: Structure of compounds whose biological evaluation as anti-Plasmodium agents was carried out.

(7) FIG. 6: Compound activity against P. berghei liver stages. Anti-Plasmodial activity (infection axis, bars) and toxicity to hepatoma cells (cell confluency axis, circles) are shown. Infection loads of Huh7 cells, a human hepatoma cell line, were determined by bioluminescence measurements of cell lysates 48 h after infection with luciferase-expressing P. berghei parasites. Luciferase assay in 96 well-plate; 10 000 Huh7 cells/well+10 000 PbA-LuciGFPcon spz/well.

(8) FIG. 7: Representative dose-response curve of compound BSS-730A, in vitro liver stage activity, with a IC.sub.50=0.55 t 0.14 μM. Luciferase assay in 96 well-plate 10 000 Huh7 cells/well+10 000 PbA-LuciGFPcon spz/well.

(9) FIG. 8: Inhibitory activity of BSS730A against multidrug resistant HIV isolates. BSS-730A dose-response curves in TZM-bI cells are shown for several HIV-2 primary isolates that show resistance to multiple antiretroviral drugs (see Table 3). 03PTHCC19 is a drug sensitive clinical isolate of HIV-2. Bars indicate the standard error from the mean.

DETAILED DESCRIPTION

(10) In an embodiment, the synthesis of spiro-3H-pyrazole-β-lactams was carried out as follows.

(11) In an embodiment, studies on the deprotection of carboxylic ester of spiro-β-lactams 10 and 12/13 to afford the corresponding penicillanic acid derivatives were carried out. The benzhydryl ester derivatives were selected for the synthesis of the free acids, since it is known that the removal of this protective group is usually easier than from benzyl esters. Deprotection of benzhydryl esters of penicillanates can be achieved by treatment with anisole or with phenol in the presence of trifluoroacetic acid (TFA). In most cases, a large excess of acid is required to complete the reaction. However, the synthesis of penicillanic acids from the corresponding benzhydryl esters under free acid conditions by gentle heating in the presence of m-cresol has been reported, said reaction conditions being particularly useful for the cleavage of esters of compounds unstable under acidic conditions.

(12) In this context, in an embodiment, a solution of spiro-β-lactam 10a in m-cresol was heated at 50° C. for 3 h (Method A). However, under these reactions conditions spiro-β-lactam 10a afforded spiro-3H-pyrazole-β-lactam 11a in only 26% yield. It was observed, that by carrying out the reaction with m-cresol in the presence of TFA (10 equiv) at 0° C. for 16 h (Method B), β-lactam 11a could be obtained in good yield (64%). These reactions conditions when applied to benzhydryl ester 10b furnished spiro-β-lactam 11b in 34% yield. It was concluded that deprotection of spiro-β-lactams 10 requires TFA catalysis. Finally, the deprotection of the benzhydryl esters 10a and 10b with anisole and TFA (25 equiv) at −5° C. for 4 h (Method C) proved to be the more efficient methodology, affording the free acids 10a (96%) and 10b (97%) in high yield (Scheme 3).

(13) ##STR00016##

(14) Surprisingly, attempts to convert the benzhydryl esters 12 and 13 into the free acids using Method C were unsuccessful, resulting only in decomposing products. On the other hand, it was observed that the reaction of these spiro-β-lactams with m-cresol in the presence of TFA at 0° C. for 16 h (Method B), resulted in the target free acids 14a and 14b, respectively, in good yield (67-76%) (Scheme 4). These results indicate that spiro-β-lactams 14 are more acid-labile than spiro-β-lactams 11. Thus, the optimized reaction conditions for deprotection of benzhydryl esters of penicillantes are strongly dependent on the type of β-lactam derivative.

(15) It is worth emphasizing that the deprotection of spiro-β-lactams 12/13 was carried starting from a mixture of isomers, but after work-up spiro-β-lactams 14 were isolated as single products.

(16) ##STR00017##

(17) In an embodiment, the spiro-β-lactams antimicrobial activity was determined as follows.

(18) In an embodiment, having access to a range of new spiro-β-lactams, the biological evaluation of 17 compounds as anti-HIV and anti-malarial agents was carried out (FIG. 1).

(19) In an embodiment, the spiro-β-lactams anti-HIV activity evaluation was determined as follows.

(20) In an embodiment, the biological evaluation as anti-HIV agents of the spiro-β-lactams presented in Table 1, resulted in the identification of three compounds with potent anti-HIV activity, namely BSS-593, BSS-722A and BSS-730A (FIGS. 2 and 3, Table 1). It is observed that compound BSS-593 was only active against HIV-1, however compounds BSS-722A and BSS-730A exhibited potent activity against HIV-1 and HIV-2. In fact, results show that BSS-730A leads to inhibition for both viral strains of about 100% (concentration of 15.21 μM).

(21) TABLE-US-00001 TABLE 1 IC.sub.50, IC.sub.90, CC.sub.50 and TI of compounds BSS-593, BSS-722A and BSS-730A. Compound Virus IC50 (μM) IC90 (μM) CC50 (μM) TI MPI (%) BSS-593 01PTHDECJN 0.035 na 158.00 4553.31 58 SG3.1 0.012 na 13144.76 84 03PTHCC19 wa wa Na wa BSS-722A 93AOHDC249 0.332 0.7008 53.70 161.80 99 SG3.1 0.650 1.0909 82.64 97 03PTHCC19 0.510 1.1819 105.29 90 BSS-730A 93AOHDC249 0.026 0.1180 76.84 2946.32 99 93AOHDC250 0.004 0.0197 20247.69 94 SG3.1 0.014 0.0252 5584.30 99 03PTHCC19 0.005 0.1454 14029.58 99 na—not applicable; wa—without antiviral activity; IC.sub.50—inhibitory concentration 50%; IC.sub.90—inhibitory concentration 90%; CC.sub.50—cytotoxic concentration 50%; TI—in vitro therapeutic index (TI = CC.sub.50/IC.sub.50); MPI—maximum percentage of inhibition; 01PTHDECJN, 93AOHDC249 and 93AOHDC249—CCR5 tropic HIV-1 primary isolates (uses the CCR5 co-receptor to enter cells); SG3.1—CXCR4 tropic HIV-1adapted isolate (uses the CXCR4 co-receptor to enter cells); 03PTHCC19—CCR5 tropic HIV-2 isolate.

(22) In an embodiment, the cellular cytotoxicity of 17 spiro-β-lactams was investigated in TZM-bl cells using the AlamarBlue assay as recommended by the manufacturer (Invitrogen, USA). TZM-bl, previously designated JC53-bl (clone 13) is a HeLa cell line. The parental cell line (JC.53) stably expresses large amounts of CD4, CCR5 and CXCR4. The TZM-bl cell line was generated from JC.53 cells by introducing separate integrated copies of the luciferase and 8-galactosidase genes under control of the HIV-1 promoter. The TZM-bl cell line is highly sensitive to infection with diverse isolates of HIV.

(23) In an embodiment, the results presented in Table 2 show that these compounds did not possess in vitro cytotoxicity, however exhibited high in vitro therapeutic index.

(24) In an embodiment the cytotoxic concentration at 50% (CC.sub.50) was determined for the following spiro-β-lactams (Table 2).

(25) TABLE-US-00002 TABLE 2 Cytotoxicity of spiro-β-lactams Compound CC.sub.50 (μM) Compound CC.sub.50 (μM) BSS-591 163.76 BSS-796 98.91 BSS-597 151.79 BSS-973C 79.80 BSS-587 135.83 BSS-974C 80.79 BSS-1026 82.01 BSS-730A 76.84 BSS-593 158.14 BSS-730B 74.45 BSS-452 104.73 BSS-793B 47.69 BSS-708 83.80 BSS-794B 49.02 BSS-971S 81.98 BSS-722A 53.74 BSS-974S 91.93

(26) In an embodiment, the spiro-β-lactams antibacterial activity of the spiro-β-lactams BSS-591, BSS-597, BSS-587, BSS-593 and BSS-730A was determined. These spiro-β-lactams tests showed no inhibitory activity against different species of Gram.sup.+ and Gram.sup.− bacteria, namely Escherichia coli ATCC 10536, Escherichia coli (Clinical strain), Staphylococcus aureus ATCC 6538, Bacillus subtillis ATCC 6633, Pseudomonas aeruginosa (Clinical strain), Enterococcus faecalis ATCC 29212, Lactobacillus rhamnosus and Lactobacillus plantarum at the concentration of 1 mg/ml.

(27) In an embodiment, the dose-responsive curve of compound BSS-730A was determined (FIG. 7).

(28) In an embodiment, the acute toxicity assay of spiro-β-lactam BSS-593 was carried out. A study was conducted in Wistar rats (220-250 g) to evaluate the effects of single administration of compound BSS-593 on organ function and survival over a period of 48 h. Animals received the test drug intraperitoneally (lower dose 0.1 mg/kg; n=5 animals; intermediate dose 1 mg/kg; n=5; higher dose 10 mg/kg; n=5) and were monitored regularly for visible stress or toxicity signs. Control animals were administered with sterile saline (1 mL/kg; n=4). No animal died during the test period of 48 h (animal survival rate 100%). No abnormal behavior or signs of abnormal toxicity were noticed. There were no significant differences between the biochemical markers of injury used to characterize liver function, kidney function and general cell injury analyzed parameters (as determined by the levels of ALT, AST, creatinine and LDH in the blood) in animals administered with compound BSS-593 and control animals (FIG. 4). Similarly, there were no changes in the blood lipid profiles as determined by the levels of cholesterol, LDL, HDL and TG in the blood. After blood collection animals were necropsied to identify macroscopic signs of organ injury. No significant differences were identified between animals.

(29) In an embodiment, the anti-Plasmodium activity of the three spiro-β-lactams compounds with higher anti-HIV (BSS-593, BSS-730A and BSS-722A) and of the two non-active derivatives (BSS-452 and BSS-1026) was also evaluated (FIGS. 2 and 5). Results show that the two compounds with no anti-HIV activity also did not display anti-Plasmodium activity, whereas the ones active against HIV also presented activity against Plasmodium bergheiliver stages. Spiro-β-lactam BSS-730A was identified as the compound with the highest anti-Plasmodium activity (IC.sub.50=0.55 0.14 μM). These results demonstrate a very interesting mimetic activity for HIV and Plasmodium, possibly suggesting a similar mechanism of action. It is conceivable that the compounds may act as inhibitors of pathogen invasion, interfering with the entry of the virus/parasite into the cell, by acting on specific cell receptors.

(30) In an embodiment, the general procedure for the synthesis of spiro-3H-pyrazole-β-lactams 10 and 12 was carried out in the following way:

(31) In an embodiment, Method A, a solution of the corresponding spiro-3H-pyrazole-β-lactam 10 (0.52 mmol) in m-cresol (2.7 mL, 26 mmol) was stirred at 50° C. under nitrogen for 3 h. The mixture was cooled in an ice bath and then ethyl acetate (10 mL) was added. The organic layer was extracted with saturated aqueous NaHCO.sub.3 (3×10 mL) and then with deionized water (10 mL). The combined aqueous layers were extracted with ethyl acetate. The aqueous layer was then cooled in an ice bath to 0-5° C. and acidified to pH 1 with 10% aqueous HCl. The mixture was stirred for 30 minutes. The mixture was then extracted with ethyl acetate (3×10 mL), and the combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure.

(32) In an embodiment, Method B, to a mixture of the corresponding spiro-3H-pyrazole-β-lactam 10 or spiro-2-pyrazoline-f-lactams 12/13 (0.38 mmol) and m-cresol (0.8 mL, 7.4 mmol) at 0° C. was added TFA (0.3 mL, 3.7 mmol). The reaction mixture was stirred at 0° C. under nitrogen for 16 h. The mixture was diluted with ethyl acetate (10 mL) and was extracted with saturated aqueous NaHC.sub.3 (3×10 mL). The organic layer was extracted with deionized water (10 mL). The combined aqueous layers were extracted with ethyl acetate (3×10 mL). The aqueous layer was then cooled in an ice bath to 0-5° C. and acidified to pH 1 with 10% aqueous HCl. The mixture was stirred for 30 minutes. The mixture was then extracted with ethyl acetate (3×20 mL), and the combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The solid obtained was washed for 3 times with diethyl ether/petroleum ether and the acid was recovered by decantation.

(33) In an embodiment, Method C, the corresponding spiro-3H-pyrazole-β-lactam 10 or spiro-2-pyrazoline-β-lactams 12/13 (0.26 mmol) was dissolved and stirred in anhydrous CH.sub.2Cl.sub.2 (2 mL) at −5° C. Anisole (0.2 mL, 1.82 mmol) and TFA (0.5 mL, 6.5 mmol) were added, and the reaction mixture stirred for 4 h. The mixture was diluted with cold diethyl ether (10 mL), and the solvent was evaporated. The residue was dissolved in THF (5 mL) and treated at 0° C. for 15 min with half saturated aqueous NaHCO.sub.3 solution (15 mL). After addition of deionized water (5 mL) and ethyl acetate (20 mL), the two layers were separated and the aqueous layer extracted with ethyl acetate (2×20 mL). The aqueous layer was acidified to pH 3 in an ice bath with HCl (1 N) and extracted with ethyl acetate (3×20 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give the desired acid.

(34) In an embodiment, Method A, (2,3′R,5R)-5′-(methoxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-azaspiro[bicyclo[3.2.0]heptane-6,3′-pyrazole]-2-carboxylic acid (11a-BSS-593) was obtained from benzhydryl penicillanate 10a (253 mg, 0.53 mmol) as a yellow solid (45 mg, 0.14 mmol, 26%).

(35) In an embodiment, Method B, (2,3′R,5R)-5′-(methoxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-azaspiro[bicyclo[3.2.0]heptane-6,3′-pyrazole]-2-carboxylic acid (11a-BSS-593) was obtained from benzhydryl penicillanate 10a (310 mg, 0.65 mmol) as a yellow solid (129 mg, 0.41 mmol, 64%).

(36) In an embodiment, Method C: (2,3′R,5R)-5′-(methoxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-azaspiro[bicyclo[3.2.0]heptane-6,3′-pyrazole]-2-carboxylic acid (11a-BSS-593) was obtained from benzhydryl penicillanate 10a (122 mg, 0.26 mmol) as a yellow solid (79 mg, 0.25 mmol, 96%).

(37) In an embodiment, the data for compound 11a (BSS-593) is as follows: mp 90-91° C. ν.sub.max/cm.sup.−1 (film) 3469, 1783 (β-lactam), 1732 (ester), 1567; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 1.64 (s, 3H), 1.67 (s, 3H), 3.97 (s, 3H), 4.69 (s, 1H), 4.85 (brs, 1H), 6.33 (s, 1H), 6.87 (s, 1H); .sup.13CNMR (100 MHz, CDCl.sub.3) δ.sub.C 26.2, 31.2, 52.7, 60.3, 61.8, 69.0, 104.4, 145.4, 149.6, 151.5, 161.8, 170.7; HRMS (ESI) m/z 312.06378 (C.sub.12H.sub.14N.sub.3O.sub.5S [MH.sup.+], 312.06487). [α].sub.20.sup.D=+235 (c=1, CH.sub.3OH).

(38) In an embodiment, Method B, (2,3′R,5R)-4′,5′-bis(methoxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-azaspiro[bicyclo[3.2.0]heptane-6,3′-pyrazole]-2-carboxylic acid (11b-BSS-587) was obtained from benzhydryl penicillanate 10b (205 mg, 0.38 mmol) as a yellow solid (49 mg, 0.13 mmol, 34%).

(39) In an embodiment, Method C, (2,3′R,5R)-4′,5′-bis(methoxycarbonyl)-3,3-dimethyl-7-oxo-4-thia-1-azaspiro[bicyclo[3.2.0]heptane-6,3′-pyrazole]-2-carboxylic acid (11b-BSS-587) was obtained from benzhydryl penicillanate 10b (167 mg, 0.31 mmol) as a yellow solid (111 mg, 0.30 mmol, 97%).

(40) In an embodiment, the data for compound 11b (BSS-587) is as follows: mp 89-91 T. ν.sub.max/cm.sup.−1 (film) 3477, 1793 (0-lactam), 1744 (ester), 1589; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 1.61 (s, 3H), 1.68 (s, 3H), 3.89 (s, 3H), 4.00 (s, 3H), 4.75 (s, 1H), 6.02 (brs, 1H), 6.48 (s, 1H); .sup.13CNMR (100 MHz, CDCl.sub.3) δ.sub.C 26.2, 32.1, 52.5, 53.2, 60.9, 61.4, 69.0, 110.5, 148.9, 150.0, 150.5, 160.1, 170.0, 171.0; HRMS (ESI) m/z 370.07154 (C.sub.14H.sub.16N.sub.3O.sub.7S [MH.sup.+], 370.07035). [α].sub.20.sup.D=+241 (c=1.7, CH.sub.3OH).

(41) In an embodiment, Method B, Spiro[penicillanic-6,5′-(3-ethoxycarbonyl-2-pyrazoline)] acid (14a-BSS-97) was obtained from benzhydryl penicillanates 12a/13a (282 mg, 0.57 mmol) as a yellow solid (142 mg, 0.43 mmol, 76%).

(42) In an embodiment, the data for compound 14a (BSS-597) is as follows: mp 114-115° C. ν.sub.max/cm.sup.−1 (film) 3334, 1774, 1731, 1716; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 1.34 (t, J=7.0 Hz, 3H), 1.54 (s, 3H), 1.56 (s, 3H), 3.30 (d, J=18.8 Hz, 1H), 3.64 (d, J=18.8 Hz, 1H), 4.29-4.34 (m, 2H), 4.47 (s, 1H), 5.36 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ.sub.C 14.2, 26.0, 33.1, 37.0, 61.6, 63.6, 68.6, 77.2, 82.3, 140.0, 161.8, 170.5, 173.0; HRMS (ESI) m/z 328.09679 (C.sub.13H.sub.18N.sub.3O.sub.5S [MH.sup.+], 328.09617). [α].sub.20.sup.D=+140 (c=0.5, CH.sub.3OH).

(43) In an embodiment, Method B, Spiro[penicillanic-6,5′-(3-acetyl-2-pyrazoline)] acid (14b-BSS-591) was obtained from benzhydryl penicillanates 12b/13b (266 mg, 0.57 mmol) as a brown solid (112 mg, 0.38 mmol, 67%).

(44) In an embodiment, the data for compound 14b (BSS-591) is as follows: mp 152-153° C. ν.sub.max/cm.sup.−1 (film) 3338. 1766, 1736; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 1.54 (s, 3H), 1.57 (s, 3H), 2.41 (s, 3H), 3.23 (d, J=18.4 Hz, 1H), 3.61 (d, J=18.4 Hz, 1H), 4.48 (s, 1H), 5.33 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ.sub.C 25.6, 25.9, 33.2, 35.7, 63.6, 65.9, 68.5, 82.5, 148.2, 170.5, 173.0, 193.8; HRMS (ESI) m/z 298.08627 (C.sub.12H.sub.16N.sub.3O.sub.4S [MH.sup.+], 298.08560). [α].sub.20.sup.D=+145 (c=1, CH.sub.3OH).

(45) In an embodiment, the synthesis of spiro-3H-pyrazole-γ-lactams and spiro-3H-pyrazole-6-lactams was carried out in a similar way to the synthesis of spiro-β-lactams.

(46) In an embodiment, the spiro-γ-lactams and spiro-δ-lactams structures are:

(47) ##STR00018##

(48) wherein:

(49) R.sup.1 is an ester group or an acetyl group.

(50) In an embodiment, the spiro-γ-lactams and spiro-δ-lactams structures are:

(51) ##STR00019##

(52) wherein:

(53) R.sup.1 and R.sup.2 are independently selected from each other;

(54) R.sup.1 and R.sup.2 are ester substituents, or R.sup.1 is an ester substituent and R.sup.2 is a hydrogen.

(55) In an embodiment, the spiro-γ-lactams and spiro-δ-lactams structures are:

(56) ##STR00020##

(57) wherein:

(58) R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently selected from each other;

(59) R.sup.1 is selected from an acetyl substituent, abenzoyl substituent, R.sup.2 is ahydrogen; R.sup.3 is an aryl substituent, R.sup.4 is ahydrogen; or R.sup.2/R.sup.3 is a succinimide-ring fused system.

(60) In an embodiment, the spiro-γ-lactams and spiro-δ-lactams structures are:

(61) ##STR00021##
wherein:
R.sup.1 is an alkyl substituent or an aryl substituent.

(62) In an embodiment, the spiro-γ-lactams and spiro-δ-lactams structures are:

(63) ##STR00022##
wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected from each other;
R.sup.1 is selected from an ester group or an acetyl group;
R.sup.2 is selected from a hydrogen or an ester substituent;
R.sup.3 is an aryl substituent,
R.sup.4 is a hydrogen; or R.sup.2/R.sup.3 is a succinimide-ring fused system.

(64) In an embodiment, the synthetic strategy described in Schemes 1 and 2 for the preparation of spiro-beta-lactams can be applied to the synthesis of the corresponding spiro-γ-lactams and spiro-δ-lactams by replacing the starting 6-diazopenicillanates 3 or 6-alkylidenepenicillanates 1 by the corresponding diazolactams or 6-alkylidenelactams. The required compounds, derived from nicillamine are prepared as outlined in the Scheme 5.

(65) ##STR00023##

(66) In an embodiment the anti-HIV activity of the compounds now disclosed was evaluated.

(67) In an embodiment, TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health, USA) were cultured in complete growth medium that consists of Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin-streptomycin (Gibco/Invitrogen, USA), 1 mM of sodium pyruvate (Gibco/Invitrogen, USA), 2 mM of L-glutamine (Gibco/Invitrogen, USA) and 1 mM of non-essential amino acids (Gibco/Invitrogen, USA).

(68) In an embodiment, peripheral blood mononuclear cells (PBMCs) from healthy individuals (blood donors) were separated by Ficoll-Paque PLUS (GE Healthcare, Waukesha, Wis., USA) density gradient centrifugation and stimulated for 3 days with 5 g/ml of phytohemaglutinin (PHA; Sigma-Aldrich, St. Louis, Mo., USA). PBMCs cultures were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml of penicillin-strepotmycin, 2 mM of L-glutamine (Gibco/Invitrogen, USA) 0.3 mg/ml of gentamicin (Gibco/Invitrogen, Carlsbad, Calif., USA), 5 μg/ml of polybrene (Sigma-Aldrich, St. Louis, Mo., USA) and 20 U/ml units of recombinant interleukin-2 (Roche, Basel, Switzerland). All cell cultures were maintained at 37° C. in 5% CO.sub.2.

(69) In an embodiment, the primary isolates 93AOHDC249, 93AOHDC250, 01PTHDECJN and 03PTHCC9, used in this study were previously isolated, titrated and characterized for co-receptor usage. HIV reference strain SG3.1 was obtained by transfection of HEK293T cells with pSG3.1 (HIV-1) plasmid using Fugene 6 reagent (Roche, Switzerland) according to manufacturer's instructions.

(70) In an embodiment, the 50% tissue culture infectious dose (TCID.sub.50) of the virus was determined in a single-round viral infectivity assay using a luciferase reporter gene assay in TZM-bl cells and calculated using the statistical method of Reed and Muench.sup.27.

(71) In an embodiment, the cellular viability assay was carried out as follows: the potential in vitro cytotoxicity of all BSS compounds was evaluated in TZM-bl cells. TZM-bl cells (10000 cells/well in 96-well plates) were incubated in absence or presence of serial-two-fold dilutions of the compounds in growth medium (GM), with starting concentration of 100 μg/ml and a final concentration of 0.781 μg/ml. After 48 hours, cell viability was examined with alamarBlue reagent (Invitrogen, USA) according to manufacturer's instructions. Briefly, 10 μl of alamarBlue reagent was added to each well. After 4 hours at 37° C., the fluorescence was measured in Tecan Infinite M200 (excitation wavelength 550 nm and emission wavelength 600 nm).

(72) In an embodiment, at least two independent experiments have been performed for each cytotoxicity analysis. Each dilution of each compound has been performed in duplicate wells.

(73) In an embodiment, for each assay there were medium controls (only growth medium), cell controls (cells without test compound) and cytotoxicity controls (a compound that kill cells—SDS).

(74) In an embodiment, the cytotoxicity of each test compound has been expressed by the 50% cytotoxic concentration (CC.sub.50), which is the concentration of compound causing 50% death of the cells as measured by a 50% decrease of fluorescence in the compound-treated cells.

(75) In an embodiment, the antiviral activity of the compounds was determined in a single-round assay with TZM-bl cells as previously described .sup.26. Cells were infected with 200 TCID50 of virus in the presence of serial fold dilutions of the BSS compounds in GM, supplemented with DEAE-dextran (19.7 μg/ml). After 48 h of infection, luciferase expression was quantified with the One-Glow luciferase assay substrate reagent (Promega, USA) according to manufacturer's instructions. Briefly, a volume of reagent equal to that of the culture medium in each well was added. For 96-well plates, typically 100 μl of reagent is added to the cells grown in 100 μl of medium. After an incubation of 3 minutes at room temperature to allow complete cell lysis luminescence was measured in Tecan Infinite M200. Background luminescence was measured by using control wells that contained only target cells and GM. Maraviroc (CCR5 antagonist), T1249 (fusion inhibitor) and AMD3100 (CXCR4 antagonist) were used as positive controls of the antiviral activity. At least two independent experiments were performed for each analysis and each assay was set up in triplicate wells.

(76) In an embodiment, dose-response curves were estimated from the percentage of inhibition of infection (y-axis) against logo of concentration of each compound (x-axis) using the sigmoidal dose-response equation (variable slope) in GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego Calif. USA, www.graphpad.com). IC.sub.50 and IC.sub.90 values were determined from this curve. IC.sub.50 and IC.sub.90 values correspond to the concentration of compound that inhibits viral replication in 50% and 90%, respectively.

(77) In an embodiment, anti-bacterial activity of the compounds now disclosed was determined according to CLSI guidelines. The following bacteria were used: reference strains E. coli ATCC 10536, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 and Enterococcus faecalis ATCC 29212; clinical strains of E. coli and Pseudomonas aeruginosa isolated in our laboratory; and strains of Lactobacillus rhamnosus and Lactobacillus plantarum also isolated in our laboratory. The Minimum Inhibitory Concentration (MIC) was determined by the agar diffusion method, in plates of Mueller-Hinton agar or Rogosa agar (for Lactobacilli). Briefly, 10.sup.8 cfu/ml bacterial suspensions were prepared in sterile water and spread in the culture media. Sterile disks containing different concentrations of P3 were placed on the inoculated surface. Plates were incubated at 37° C. for 24 h or 48 h. Lactobacilli were incubated at microaerophilic conditions. A negative control made of sterile water was used. Disks of amoxicillin and imipenem were used as positive controls. The maximum concentration tested for each compound was 1 mg/ml.

(78) In an embodiment, an acute toxicity assay was carried out. Female Wistar (220-250 g) rats were randomly divided in 4 groups. Treated animals received 3 different doses of the BSS-593, comprising a lower dose (0.1 mg/kg; n=5), an intermediate dose (1 mg/kg; n=5), and a higher dose (10 mg/kg; n=5). A fourth group of control animals was administered with sterile saline (B. Braun, Portugal) (1 mL/kg; n=4). Animals received the test drug via the intraperitoneal route and were monitored regularly for visible stress or toxicity signs such as death. All rats were anesthetized with sodium pentobarbital (Eutasil, 60 mg/kg i.p; Sanofi VeterinAria, Alges, Portugal) prior to intracardiac puncture for blood collection. There were no significant differences in the time to anaesthesia between treated and control animals, reflecting no apparent effects related to the CNS. Blood was collected into a serum SST gel and clot activator tube (Becton Dickinson, Le Pont de Claix, France) and was centrifuged (1000×g for 10 min at room temperature) to separate serum. Serum was analyzed within 24 h on a laboratory bench Clinical Chemistry Analyser c111 (Roche Diagnostics, Lda). Uver injury was assessed by measuring the rise in the serum levels of alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury), aspartate aminotransferase (AST, a nonspecific marker for hepatic injury) and lactate dehydrogenase (LDH, a marker of nonspecific cellular injury). Serum levels of creatinine was determined as marker of renal injury. Serum levels of total cholesterol, low-density cholesterol (LDL), high-density cholesterol (HDL) and triglycerides were also measured to evaluate the effect of the drugs on lipid homeostasis. Results were expressed as the means with their standard errors and were compared using a one-factorial ANOVA test, followed by a Bonferroni's multi-comparison post hoc test. A P value <0.05 was considered to be statistically significant.

(79) In an embodiment, the in vitro activity against the liver stage of Plasmodium infection was also carried out: inhibition of liver stage Plasmodium infection by test compounds was determined by measuring the luminescence intensity in Huh-7 cells infected with a firefly luciferase-expressing P. berghei line, as previously described. Briefly, Huh-7 cells, a human hepatoma cell line, were cultured in 1640 RPMI medium supplemented with 10% v/v fetal bovine serum, 1% v/v nonessential amino acids, 1% v/v penicillin/streptomycin, 1% v/v glutamine, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7, and maintained at 37° C. with 5% CO.sub.2. For infection assays, Huh-7 cells (1.0×10.sup.4 per well) were seeded in 96-well plates the day before drug treatment and infection. The medium was replaced by medium containing the appropriate concentration of each compound approximately 1 h prior to infection with sporozoites freshly obtained through disruption of salivary glands of infected female Anopheles stephensi mosquitoes. Sporozoite addition was followed by centrifugation at 1700×g for 5 min. Parasite infection load was measured 48 h after infection by a bioluminescence assay (Biotium). The effect of the compounds on the viability of Huh-7 cells was assessed by the AlamarBlue assay (Invitrogen, U.K.) using the manufacturer's protocol.

(80) In an embodiment, the antimicrobial activity, anti-HIV activity, the cellular viability of spiro-3H-pyrazole-γ-lactams and spiro-3H-pyrazole-δ-lactams was carried out in a similar way to the synthesis of spiro-β-lactams.

(81) According to the latest WHO HIV Drug Resistance Report.sup.14, HIV drug resistance is rising globally. Levels of pretreatment resistance to efavirenz or nevirapine, the most widely used drugs in first-line ART, reached≥10% in six out of 11 countries that reported pretreatment drug resistance survey data. Likewise, NNRTI resistance among people retained on ART ranged from 4% to 28%, while among people with unsuppressed viral load on first-line NNRTI regimens, it ranged from 47% to 90%.

(82) In an embodiment, the synergistic interaction between BSS-730A and AMD3100 studies on the activity of BSS-730A was carried out as follows. The interaction between BSS-730A and AMD3100, a CXCR4 antagonist, was examined in a single-round viral infectivity assay using TZM-b reporter cells. Cells were incubated for one hour with compounds and then were infected with 200 TCID50 of HIV-1 strain SG3.1. After 48 h of infection, luciferase expression was quantified. Serial two-fold dilutions of a fixed combination of BSS-730A and AMD3100 were tested. Each concentration of BSS-730A and AMD3100 was also tested alone. Duplicate cultures were maintained for each compound concentration and for infected and uninfected controls. The synergism was determined by using CalcuSyn software (Biosoft, Cambridge, UK). Combination indices (Cls) were calculated based on the median-effect principle.sup.15,16, where Cl<0.9 indicates a synergistic effect (Cl values were interpreted as follows: 0.9>Cl>0.85: slight synergism, 0.85>Cl>0.7: moderate synergism, 0.7>Cl>0.3: synergism, 0.3>Cl>0.1: strong synergism, Cl<0.1: very strong synergism), 0.9<Cl<1.1 indicates an additive effect, and C>1.1 indicates an antagonism effect. Because high effect degrees are more important to the treatment than low effect degrees, the weighted average C value was assigned as Cl.sub.wt═[Cl.sub.50+2Cl.sub.75+3Cl.sub.90]/10, where Cl.sub.50, Cl.sub.75 sand Cl.sub.90 are the Cl values at 50, 75 and 90 inhibition, respectively.sup.15,16.

(83) In an embodiment, the BSS-730A is active against multidrug resistant isolates. The activity of the spiro-β-lactam with the higher anti-HIV activity (BSS-730A) was evaluated against eight drug resistant HIV-2 primary isolates and a control isolate that is sensitive to all antiretroviral drugs (isolate 03PTHCC9) (Table 3). BSS-730A was highly active against all isolates with a median IC.sub.50 fold-change of 2.39 and median IC.sub.90 fold-change of 1.09 relative to the control isolate 03PTHCC19 (Table 3 and FIG. 8). These results suggest that BSS-730A could be used to treat infections caused by multidrug resistant isolates.sup.17.

(84) TABLE-US-00003 TABLE 3 Activity of BSS-730A against drug-resistant HIV primary isolates Susceptibility to IC.sub.50 IC.sub.90 IC.sub.50 Fold IC.sub.90 Fold Virus Tropism antiretroviral drugs.sup.1 (μM) (μM) change.sup.2 change.sup.3 03PTHCC19 R5 Sensitive 0.008 0.064 00PTHCC20 X4 Resistant to ABC, 0.018 0.073 2.25 1.14 ZDV, d4T, ddl, LPV 03PTHCC20 X4 Resistant to ABC, 0.019 0.095 2.38 1.48 ZDV, d4T, ddl, LPV 00PTHDECT R5/X4 Resistant to DTG 0.023 0.057 2.88 0.89 03PTHSM9 X4 Resistant to SQV, 0.016 0.116 2.00 1.81 LPV, DRV and TAF 10PTHSJIG R5 Resistant to RAL, 0.012 0.032 1.50 0.50 DTG, LPV, SQV, DRV and all NRTIs 15PTHSJIG R5 Resistant to RAL, 0.018 0.056 2.25 0.88 DTG, 3TC and FTC 15PTHCEC X4 Resistant to RAL, 0.017 0.051 2.13 0.79 DTG, LPV, SQV, DRV, ABC, ddl, TDF, TAF, 3TC, d4T and FTC .sup.1Susceptibility profile to antiretroviral drugs. .sup.2Relative to IC.sub.50 of wild type isolate 03PTHCC19; .sup.3Relative to IC.sub.90 of wild type isolate 03PTHCC19; ND—not determined; ABC, abacavir; ZDV, zidovudine; d4T, stavudine; ddl, didanosine; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir disoproxil fumarate; TAF, tenovovir alafenamide; LPV, lopinavir SQV, saquinavir; DRV, darunavir; DTG, dolutegravir; RAL, raltegravir; NRTIs, nucleoside reverse transcriptase inhibitors.

(85) In an embodiment, BSS-730A displays a synergistic interaction with AMD3100. The activity of BSS-730A was preliminarily assessed in a combination experiment with AMD3100, an entry inhibitor that binds to the CXCR4 co-receptor, in a single cycle assay in TZM-bl cells against HIV-1 SG3.1. Different ratios were tested: AMD3100+BSS-730A (1:1.4), AMD3100+BSS-730A (1:4.21) and AMD3100+BSS-730A (2.14:1). Combination indices (CI) were calculated to determine whether synergistic, additive or antagonistic effects occurred after these combinations. C calculations showed synergism or strong synergism at 50, 75, and 90% inhibition of HIV-1 (CI: 0.27-0.39) for the first and second drug combinations where BSS-730A was in a higher concentration relatively to AMD3100 (Table 4). When AMD3100 was in a higher concentration, the Cl calculations showed an additive effect at 50 and 75% inhibition of HIV-1 (Cl:0.93-0.97) and a slight synergism at 90% inhibition of HIV-1 (CI: 0.88). The strongest synergistic interactions were observed with AMD3100+BSS-730A (1:1.4) and AMD3100+BSS-730A (1:4.21) combinations that presented Clwt of 0.21 and 0.18, respectively. The strong synergy between BSS-730A and AMD3100 suggest that BSS-730A could be used in combination with entry inhibitors to treat or prevent HIV infection.

(86) TABLE-US-00004 TABLE 4 Synergistic interaction between BSS-730A and AMD3100. Drug combination CI values at inhibition of.sup.a: (combination ratio) 50% 75% 90% CI.sub.wt-values.sup.c AMD3100 + BSS-730A 0.38572 0.36094 0.33801 0.21 (1:1.4) +++ +++ +++ ++++ AMD3100 + BSS-730A 0.37132 0.31846 0.27351 0.18 (1:4.21) +++ +++ ++++ ++++ AMD3100 + BSS-730A 0.97482 0.92757 0.8829  0.55 (2.14:1) ad ad + +++ .sup.aCI > 1.1 indicates antagonism (—), 1.1 > CI > 0.9 indicates the additive effect (ad) and CI < 0.9 indicates a synergistic effect; .sup.bSynergy levels: 0.9 > CI > 0.85: + (slight synergism); 0.85 > CI > 0.7: ++ (moderate synergism); 0.7 > CI > 0.3: +++ (synergism); 0.3 > CI > 0.1: ++++ (strong synergism); CI < 0.1; +++++ (very strong synergism); .sup.cBecause high degree effects are more important to the treatment than the low degree effects, the weighted average CI value was assigned as CI.sub.wt = [CI.sub.50 + 2CI.sub.75 + 3CI.sub.90]/10, where CI.sub.50, CI.sub.75 and CI.sub.90 are the CI values at 50, 75 and 90, inhibition, respectively.

(87) Therefore, the combinations of BSS-730A with the entry inhibitor AMD3100 showed significant synergistic effect indicating this combination of drugs might be useful to treat and/or prevent infection caused by CXCR4-using HIV isolates which are usually found in late stage disease and are associated with disease progression.sup.18.

(88) Furthermore, the present disclosure also shows that BSS-730A was highly active against HIV isolates that were resistant to several protease inhibitors (LPV, SQV, DRV), integrase inhibitors (RAL, DTG) and to all NRTIs. These results indicate that BSS-730A should be useful to treat or prevent infection by multidrug resistant HIV isolates.

(89) The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

(90) It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

(91) Flow diagrams of particular embodiments of the presently disclosed methods are depicted in figures. The flow diagrams do not depict any particular means, rather the flow diagrams illustrate the functional information one of ordinary skill in the art requires to perform said methods in accordance with the present disclosure.

(92) The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

(93) The above described embodiments are combinable.

(94) The following claims further set out particular embodiments of the disclosure.

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