APPLICATIONS THEREOF

FIELD OF THE INVENTION

The present disclosure relates to novel imidazole fused polycyclic systems, methods for their use, and processes for their preparation. The compounds of the present disclosure are particularly useful against a variety of bacteria, including multidrug resistant strains of Staphylococcus aureus, S. saprophyticus, S. haemolyticus, and S. epidermidis.

BACKGROUND

Antibacterial resistance is a global clinical and public health problem that has emerged with alarming rapidity in recent years and, undoubtedly, will increase in the near future. According to the statistics of the Center for Disease Control and Prevention (CDC), infections with antibiotic- resistant bacteria cause more than 2 million illnesses and about 23,000 deaths each year in the United States. Recent research estimates that by 2050, 10 million people may die annually due to infections caused by antibiotic-resistant bacteria. Moreover, mortality due to infection is soon expected to be more than that caused by cancer. Antibiotic resistance will also have a cumulative cost of at least 100 trillion USD, more than one and a half times the annual world Gross Domestic Product (GDP) today. In the United States, there are about 47 million unnecessary antibiotic prescriptions issued each year. These unnecessary prescriptions are made to conditions that do not require antibiotics, such as viral infections which do not respond to antibiotics. Excessive use of antibiotics over the past few decades has led to the development of bacteria that is resistant to currently available antibiotics. Most newly developed antimicrobials are chemical variants of older agents; therefore, resistance can develop quickly. Indeed, bacterial strains resistant to many existing antibiotics are continually being isolated and there is an urgent need to identify new antimicrobial agents with improved activity against both Gram-positive and Gram negative organisms. Staphylococcus is one of most clinically important species of Gram-positive bacteria and methicillin-resistant Staphylococcus aureus (MRSA) causes many severe infections. The World Health Organization has included this bacterium in its list of the top 12 pathogens causing high morbidity and mortality worldwide. Due to the limited therapeutic options currently available to combat this bacterium, patients infected with MRSA are estimated to have 64% more mortality risk than patients infected with other non-resistant forms of the bacteria.

SUMMARY

The present invention relates to novel imidazole fused polycyclic systems, methods for their use, and processes for their preparation. The presently disclosed compounds are particularly useful against a variety of bacteria, including multidrug resistant strains of Staphylococcus aureus, S. saprophyticus, S. haemolyticus , and S. epidermidis. The presently disclosed compounds are more likely to be useful as antibacterial agents as they have potent activity on a variety of multidrug resistant bacteria, including both clinical and environmental isolates. Additionally, as the disclosed compounds are embedded within biologically active ring systems, their applications to serve as pharmaceutical agents to treat different diseases are extendable.

Phenotype -based screening of diverse compound collections generated by privileged substructure -based diversity-oriented synthesis (pDOS) is considered one of the prominent approaches in the discovery of novel drug leads. Indeed, pDOS is a very attractive technique to synthesize small molecule collections with chemical scaffolds that frequently exist in natural products as well as drugs that can target previously unexploited features of disease-relevant proteins. While oxazapine, diazapine, imidazole, and thiazole skeletons and analogs constitute the core scaffold of many bioactive natural products and synthetic drugs, a key challenge is the development of efficient and modular synthetic routes toward small-molecule libraries of compounds with skeletal and stereochemical complexity as well as drug-like properties.

As part of the global effort to fight antibiotic -resistant pathogens, a new series of novel imidazole fused polycyclic compounds is disclosed herein along with demonstrated potent antibacterial activity against several Gram-positive resistant bacteria. In particular, the presently disclosed novel imidazole fused polycyclic compounds have a general formula A, as shown below.

General formula A

In general formula A: denotes a 5, 6, or 7 membered aryl, heteroaryl, alicyclic, or heteroalicyclic ring which may be unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of: a halogen atom, CN, R ³ , OR ³ , SR ³ , N(R ³ ) ₂ , C(0)R ³ , C(0)0R ³ , NR ³ C(0)R ³ , C(0

X denotes

n = 1, 2, or 3, which provides ring B with 7, 8, or 9 members, respectively;

(P denotes a 5, 6, or 7 membered aryl or heteroaryl ring which may be unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of: a halogen atom, CN, R ³ , OR ³ , SR ³ , N(R ³ ) ₂ , C(0)R ³ , C(0)0R ³ , NR ³ C(0)R ³ , C(0)NR ³ , S0 ₂ R ³ , NR ³ S0 ₂ R ³ , and S0 ₂ N(R ³ ) ₂ ;

R ¹ is hydrogen or an unsubstituted or substituted alkyl, aryl, or heteroaryl group;

R ² is selected from the group consisting of: C(0)0R ³ , CN, N(R ³ ) ₂ , C(0)R ³ , C(0)NR ³ , S0 ₂ R ³ , S0 ₂ N(R ³ ) ₂ , and P0 ₂ R ³ ; and

R ³ is independently hydrogen or a substituted group selected from the group consisting of: an alkyl group; a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In addition to compounds having the general formula A, enantiomers, diastereoisomers, racemates, pharmaceutically acceptable salts thereof are also within the scope of the present disclosure. In another embodiment, the present disclosure also provides novel compounds of a general formula B, having the following general chemical formula:

General formula B

In general formula B :

(X) ^ denotes a 5, 6, or 7 membered heteroaryl or heteroalicyclic ring which may be unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of: a halogen atom, CN, R ³ , OR ³ , SR ³ , N(R ³ ) ₂ , C(0)R ³ , C(0)0R ³ , NR ³ C(0)R ³ , C(0)NR ³ , SO2R ³ , NR ³ S0 ₂ R ³ , and S0 ₂ N(R ³ ) ₂ ;

n = 1, 2, or 3 which provides ring B with 6, 7, or 8 members, respectively; denotes a 5, 6, or 7 membered heterocyclic ring which may be unsubstituted or substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of: a halogen atom, CN, R ³ , OR ³ , SR ³ , N(R ³ ) ₂ , C(0)R ³ , C(0)0R ³ , NR ³ C(0)R ³ , C(0)NR ³ , S0 ₂ R ³ , NR ³ S0 ₂ R ³ , and S0 ₂ N(R ³ ) ₂ ;

R ¹ is hydrogen or an unsubstituted or substituted alkyl, aryl, or heteroaryl group;

R ² is selected from the group consisting of: C(0)0R ³ , CN, N(R ³ ) ₂ , C(0)R ³ , C(0)NR ³ , S0 ₂ R ³ , S0 ₂ N(R ³ ) ₂ , and P0 ₂ R ³ ; and

R ³ is independently hydrogen or a substituted group selected from the group consisting of: a C1-6 aliphatic group; a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and a bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In addition to compounds having the general formula B, the present disclosure is also intended to include enantiomers, diastereoisomers, racemates, and pharmaceutically acceptable salts thereof.

Compounds of general formula A can be prepared by any suitable method. For example, in some embodiments, a one-pot reaction between compounds of general formulas I, II and III (illustrated below) provides compounds of the general formula A via Groebke and aza-Michael reactions.

General formula I General formula II General formula III

In general formulas I, II, and III, R ¹ , and R ² are as previously described herein.

Compounds of the general formula B may be prepared by any suitable technique. In some embodiments, a one -pot reaction between compounds of general formulas IV, II, and III (illustrated below) can be used to provide the novel compounds of general formula B via Groebke and aza-Michael reactions.

O

'n

General formula IV General formula II General formula III

In general formula IV, n, R ¹ , and R ² are as previously described herein. In another aspect, the present disclosure also provides pharmaceutical compositions comprising compounds of the general formulas A and/or B.

In yet another aspect, the subject disclosure provides methods of use of the compounds of general formulas A and/or B to treat bacterial infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a photograph of a cell viability assay conducted to screen the disclosed compounds for potential antibacterial activities, in accordance with some embodiments of the subject disclosure.

FIG. 2 shows a graph of experimental data from a time-kill experiment of MRS A- 1 at different time points of exposure to compound lo, in accordance with some embodiments of the subject disclosure.

FIG. 3 shows a graph of experimental data from a time-kill experiment of MRS A- 1 at different time points of exposure to meropenem.

FIG. 4 shows a graph of experimental data from a time-kill experiment of MRS A- 1 at different time points of exposure to ciprofloxacin.

FIG. 5A1 shows an image obtained with atomic force microscopy (AFM) of an untreated MRSA-l strain obtained at a scan size of 10 pm ² .

FIG. 5B1 shows an AFM image of an untreated MRSA-l strain obtained at a scan size of 5 pm ² .

FIG. 5A2 shows an AFM image of a MRSA-l strain treated with compound lo obtained at a scan size of 10 pm ² .

FIG. 5B2 shows an AFM image of a MRSA-l strain treated with compound lo obtained at a scan size of 5 pm ² .

FIG. 6 shows a graph illustrating the effect of various disclosed compounds on the survival of normal human fibroblast cells (F180) at concentrations of 5 and 2 pM, in accordance with some embodiments of the subject disclosure. DETAILED DESCRIPTION

The subject disclosure provides novel imidazole fused polycyclic ring systems of the general formulas A and B, methods of preparing these systems, and uses of such novel compounds for different disease states, including as treatment agents for pathogenic infections. When discussing the disclosed compounds and compositions, the following terms have the following meaning unless otherwise indicated.

The term“aryl” means an aromatic or partially aromatic hydrocarbon group containing 6 to 10 carbon atoms and consisting of one or two rings which may be fused to each other or attached to each other via a single bond. Examples of aryl groups are phenyl, napthyl, biphenyl, and indenyl.

The term“heteroaryl,” as used herein, means an aromatic or partially aromatic group consisting of one or two rings, which may be fused to each other or attached to each other via a single bond. A ring of a heteroaryl group may contain 5 to 10 ring atoms wherein up to four (e.g., one, two, three, or four) ring atoms are heteroatom(s) and the remaining ring atoms are carbon. Examples of heteroaryl groups include, but are not limited to: pyrrolyl, furyl, thienyl, oxazolyl, imidazolyl, purazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyranyl, furazanyl, indolizinyl, indolyl, isoindolinyl, indazolyl, benzofuryl, benzothienyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl, isothiazolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, and the like.

The term“alicyclic” means a saturated or unsaturated aliphatic cyclic ring system consisting of one or more rings, which may be fused to each other or attached to each other via a single bond. A ring of an alicyclic group may contain 5 to 10 atoms (e.g., carbon). Examples of alicyclic groups include cyclopentane, cyclohexane, cycloheptane, cyclooctane, and terpene.

The term“heteroalicyclic” means any five to seven membered monocyclic, saturated, or partially unsaturated ring structure containing at least one heteroatom selected from the group consisting of O, N, and S, and optionally containing one, two, or three additional heteroatoms independently selected from the group consisting of O, N, and S. In some embodiments, the term heteroalicyclic refers to a nine to ten membered saturated, partially unsaturated, partially aromatic bicydic, or spiro-fused ring system containing at least one heteroatom selected from the group consisting of O, N, and S, and optionally containing one to four additional heteroatoms independently selected from the group consisting of O, N, and S. Examples of suitable heteroalicyclic groups include, but are not limited to: pyrrolinyl, pyrrolidinyl, dioxalanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, indolinyl, chromenyl, 3,4- methylenedioxyphenyl, 2,3- dihydrobenzofuryl, 2-aza-spiro[4.5]decyl, and the like. A heterocycloalkyl group may be atached at any heteroatom or carbon atom of a heteroalicyclic ring, in some embodiments.

The term“alkyl,” as used herein, denotes a saturated, linear, or branched chain hydrocarbon group containing 1 to 8 (e.g., 1 to 6 or 1 to 4) carbon atoms. For example, methyl, ethyl, propyl, isopropyl, 1-butyl, 2-butyl, and tert-butyl groups are alkyl groups. In select embodiments,“Cl- C8 alkyl” groups have 1, 2, or 3 carbon atoms.

The term“halogen” means fluorine, chlorine, bromine, or iodine.

The term“substituted” means a group which may be substituted one to three times by a halogen atom, CN, R ³ , OR ³ , SR ³ , N(R ³ ) ₂ , C(0)R ³ , C(0)0R ³ , NR ³ C(0)R ³ , C(0)NR ³ , S0 ₂ R ³ , NR ³ S0 ₂ R ³ , and/or S0 ₂ N(R ³ ) ₂ . With reference to substituents, the term "independently" means that when more than one of such substituents is possible, such substituents may be the same or different from one another.

The term“pharmaceutically acceptable salt” refers to conventional acid-addition salts or base- addition salts that retain the biological effectiveness and properties of the compounds of the underlying chemical formula and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include, for example, those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfonic acid, phosphoric acid, and nitric acid, and those derived from organic acids, such as p- toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide.

The term“polar-protic solvent” means a polar solvent which contains acidic hydrogen and acts as a hydrogen bond donor. Examples of polar-protic solvents include methanol, ethanol, and isopropanol.

The term“polar-aprotic solvent” means a polar solvent which does not contain acidic hydrogen and does not act as a hydrogen bond donor. Examples of polar-aprotic solvents are dimethylsulfoxide, dimethylformamide, hexamethylphosphorotriamide, n-methyl pyrrolidone, tetrahydrofuran, and acetonitrile (ACN).

The disclosed compounds may possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non- racemic mixtures thereof. In some embodiments, the disclosed compounds may be utilized as a single isomer or as a mixture of stereochemical isomers. In embodiments in which diastereoisomers, i.e., nonsuperimposable stereochemical isomers, are used the diastereoisomers can be separated by any known technique, such as chromatography, distillation, crystallization, and/or sublimation. Optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate acids that may be used include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and/or camphorsulfonic acid. A mixture of diastereomers can be separated by crystallization followed by liberation of optically active bases from these salts. An alternative process for separation of optical isomers includes the use of a chiral chromatography column, which may be chosen to maximize separation of the enantiomers.

In some embodiments, the disclosed compounds may be introduced to a patient in pro-drug form (i.e., as a biologically inactive compound that is metabolized in the body to produce a biologically active compound). In some such embodiments, the disclosed pro-drugs include a compound of formulas A and/or B and at least one pharmacologically acceptable protecting group which is removed under physiological conditions. Any suitable pharmacologically acceptable protecting group may be used, such as, for example, an alkoxy-, aralkyloxy-, acyl-, and/or acyloxy group (e.g., an ethoxy, benzyloxy, acetyl, or acetyloxy group).

The present disclosure also relates to the use of one or more of the disclosed compounds (at times referred to herein as“active ingredients”) in the preparation of medicaments. In some embodiments, compounds of the formulas A and/or B are administered either individually or in combination with any other desired therapeutic agent, using suitable delivery methods. The disclosed therapeutically useful agents may be administered, for example, by one of the following routes: orally, for example in the form of dragees, coated tablets, pills, semi-solid substances, soft or hard capsules, solutions, emulsions or suspensions; parenterally, for example, in the form of an injectable solution; rectally in the form of suppositories; by inhalation, for example, in the form of a powder formulation or a spray; and/or trans-dermally or intranasally. For the preparation of such tablets, pills, semi-solid substances, coated tablets, dragees and hard gelatin capsules, the therapeutically usable product may be mixed with pharmacologically inert, inorganic or organic pharmaceutical carrier substances, for example, with lactose, sucrose, glucose, gelatin, malt, silica gel, starch or derivatives thereof, talcum, stearic acid or salts thereof, skimmed milk powder, and the like. For the preparation of soft capsules, pharmaceutical carrier substances such as, for example, vegetable oils, petroleum, animal or synthetic oils, wax, fat, and polyols may be used. For the preparation of liquid solutions and syrups, pharmaceutical carrier substances such as, for example, water, alcohols, aqueous saline solution, aqueous dextrose, polyols, glycerol, vegetable oils, petroleum, and animal or synthetic oils maybe used. For suppositories, pharmaceutical carrier substances such as, for example, vegetable oils, petroleum, animal or synthetic oils, wax, fat, and polyols may be used. For aerosol formulations, compressed gases that are suitable for this purpose, such as, for example, oxygen, nitrogen and carbon dioxide may be used. The pharmaceutically acceptable agents may also comprise additives for preserving and stabilizing, emulsifiers, sweeteners, flavor rings, salts for altering the osmotic pressure, buffers, encapsulation additives and/or antioxidants. Numerous configurations and variations will be apparent to those skilled in the art upon consideration of the subject disclosure. As used herein, the following abbreviations and terms have the definitions provided below, unless otherwise stated.

The term“MIC” means a minimum inhibitory concentration, which is the lowest concentration of a chemical which is required to inhibit visible microbial growth following overnight incubation, usually reported in units of pg/mL.

The term “MBC” refers to a minimum bactericidal concentration, which is the lowest concentration of a chemical causing microbial death as indicated by absence of growth after subculture on antibiotic-free media.

The term“CFU” refers to a colony-forming unit, which is a unit used to estimate the number of viable microbial cells in a sample.

The term“resistant” or“resistance” to a bacterium or of a bacterial infection to an antibiotic includes a complete resistance to the antibiotic or a partial resistance which is defined herein as a circumstance in which the MIC of an antibiotic toward the organism in question has increased.

ACN = Acetonitrile

TFA = Trifluroacetic acid

EtOAc = Ethylacetate

DCM = Dicholoromethane

MeOH = Methanol

NMR = Nuclear magnetic resonance

HRMS = High resolution mass spectroscopy

LCMS = Liquid chromatography-mass spectroscopy

ESI-TOF = Electrosprayionization-time of flight

The presently disclosed compounds exhibited broad antibacterial selectivity against multiple Gram-positive bacteria including Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis. Additionally, the disclosed compounds also exhibited potent activity against multiple species of multidrug resistant Staphylococcus, including S. aureus, S. saprophyticus, S. haemolyticus , and S. epidermidis. Due to the powerful antibacterial properties exhibited, the presently disclosed compounds may also be used for any other medical applications, including as supplement feed for animals.

The presently disclosed compounds may also be used as medicaments or as substances for preserving inorganic and/or organic materials, such as polymers, lubricants, paints, fibers, leather, paper, timber, food stuffs, and/or water. In select embodiments in which the disclosed compounds are used in connection with a polymeric material, the compound(s) present may be covalently bonded to the polymeric material.

The presently disclosed compounds may also be used for the sterilization or disinfection of surfaces possibly contaminated with bacteria, for example, in hospitals, clinics, or in domestic settings. In select embodiments, the disclosed compounds may be used to sterilize medical equipment.

The disclosed compounds may also be used to prevent, alleviate, and/or cure diseases caused by pathogens whose growth is inhibited by these compounds. As explained in detail herein, the instant compounds are particularly active against bacteria and bacteria-like microorganisms. The presently disclosed compounds are thus suitable for use in human and veterinary medicine, for example, for prophylaxis and/or chemotherapy of local and systemic infections caused by these pathogens.

An illustrative (but not exhaustive) list of pathogenic microorganisms that may be possible targets of the presently disclosed compounds is as follows: Gram-positive bacteria Micrococcaceae, such as Staphylococci, for example, Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus aerogenes; Lactobacteriaceae , such as Streptococci, for example, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans; Bacillaceae, for example, Bacillus subtilis, Bacillus anthracis and Bacillus cereus; Corynebacteria, for example, C. diptheriae, C. pseudotuberculosis, and C. ulcerans; Clostridia bacteria, for example, Clostridium difficile, Clostridium botulinum, and Clostridium perfringens; and Listeria bacteria such as Listeria monocytogenes. Fungi, viruses, and parasites (e.g., malaria) are also possible targets for the disclosed compounds.

The disclosed compounds may, in some cases, be used to prevent, alleviate, and/or cure various diseases, such as: diseases of the respiratory passages and of the pharyngeal cavity; otitis; pharyngitis; pneumonia; bronchitis; peritonitis; pyelonephritis; cystitis; endocarditis; gastroenteritis; skin infections; and/or blood stream infections.

The presently disclosed compounds may, in some embodiments, be formulated as pharmaceutical compositions and administered to a patient, such as a human patient, in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intra muscular, topical, and/or subcutaneous routes. In some embodiments, the disclosed antibacterial compounds may be administrated in combination with known antibiotics, including those with a different mechanism of action.

The present disclosure provides novel imidazole fused polycyclic ring systems of the general formulas A and B along with methods of preparing these systems and uses for many disease states, including as antibacterial agents. The following experimental examples are included to provide further detail and are in no way intended to limit the scope of the present disclosure.

EXAMPLE 1

In a first experimental example, novel compounds of the general formula A were prepared and characterized. The methods used to prepare these compounds and the resulting characterization data are provided below.

Aldehyde (general formula I, 0.5 mmol), 2-aminoazine (general formula II, 0.5 mmol), scandium triflate (20 mol%) and sodium sulfate (1.0 mmol) were mixed in ACN (2 mL) at room temperature. After 45 mins, isocyanide (general formula III, 0.55 mmol) was introduced and stirring was continued at room temperature for 12-15 hours. After completion of step-l (Groebuk reaction), ytterbium triflate was added (30 mol%) and continued the reaction at 70°C for 3 to 12 hours. After completion of step-2 (Michael reaction), ACN was removed and the obtained crude material was purified on flash column chromatography using a gradient of EtOAC/hexane or MeOH/DCM as mobile phase eluents to obtain pure products of general formula A. Compound la had the following chemical formula and characterization data:

Compound la: 14-(tert-butylamino)-7-(2-methoxy-2-oxoethyl)-6,7- dihydrobenzo[f]pyrazino[2',l ':2,3]imidazo[ l,5-d][ 1,4 ] oxazepin-8-ium

trifluoromethanesulfonate

Pale yellow color solid, 137 mg, 52%. NMR (500 MHz, MeOD) d 9.74 (s, 1H), 8.87 (dd, / = 4.5, 1.1 Hz, 1H), 8.56 (d, 7 = 4.5 Hz, 1H), 8.40 (dd, 7 = 8.0, 1.1 Hz, 1H), 7.57 (dd, / = 11.4, 4.1 Hz, 1H), 7.27 (dd, / = 13.5, 8.0 Hz, 2H), 6.04 - 5.95 (m, 1H), 5.00 (s, 0.7H), 4.82 (dd, / = 13.1, 3.7 Hz, 1H), 4.74 - 4.66 (m, 1H), 3.60 (s, 3H), 3.14 - 3.04 (m, 1H), 2.84 - 2.73 (m, 1H), 1.12 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.5, 155.0, 137.9, 133.9, 133.1, 1317, 131.2, 130.0, 127.6, 122.4, 120.8, 117.5, 113.5, 72.5, 56.8, 55.4, 51.3, 33.3, 28.9.HRMS (ESI-TOF): m/z calcd for C22H25F3N4O6S 530.1446, found 381.1962 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lb had the following chemical formula and characterization data:

Compound lb: 16-(tert-butylamino)-7-(2-methoxy-2-oxoethyl)-6,7- dihydrobenzo[6', 7 '][ 1,4 ]oxazepino[ 4 ',5':3,4]imidazo[ 1 ,2-ajquinoH n -8-iun i trifluoromethanesulfonate

Off white color solid, 133 mg, 46%. NMR (500 MHz, MeOD) d 10.14 (d, / = 8.8 Hz, 1H), 8.44 (d, = 9.5 Hz, 1H), 8.21 (d, = 9.3 Hz, 2H), 8.07 (d, / = 7.6 Hz, 1H), 7.99 (t, / = 7.7 Hz, 1H), 7.84 (t, / = 7.4 Hz, 1H), 7.61 (t, / = 7.2 Hz, 1H), 7.42 - 7.27 (m, 2H), 5.96 - 5.84 (m, 1H), 5.16 (s, 1H), 4.71 (dd, / = 12.7, 2.2 Hz, 1H), 3.55 (s, 3H), 2.98 (dd, / = 17.7, 3.8 Hz, 1H), 2.71 (dd, / = 17.7, 10.3 Hz, 1H), 1.01 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.3, 155.0, 136.9, 135.4, 133.6, 132.7, 131.6, 131.4, 130.4, 129.8, 129.4, 127.6, 125.1, 123.7, 121.1, 119.2, 116.9, 108.8, 73.9, 56.8, 54.2, 51.2, 34.9, 28.4. HRMS (ESI-TOF): m/z calcd for C27H28F3N3O6S 579.1650, found 430.2156 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lc had the following chemical formula and characterization data:

Compound lc :15-(tert-butylamino)-7-(2-methoxy-2-oxoethyl)-6,7- dihydrobenzoff] benzo[4' ,5’]thiazolo[2' ^' ,3' :2,3]imidazo[ 1,5-d] [ 1,4] oxazepin-8-ium

trifluoromethanesulfonate

Off white color solid, 152 mg, 52%. NMR (500 MHz, MeOD) d 8.75 (d, / = 8.3 Hz, 1H), 8.19 (d, / = 8.1 Hz, 1H), 8.07 (dd, / = 7.8, 1.4 Hz, 1H), 7.82 - 7.77 (m, 1H), 7.75 - 7.69 (m, 1H), 7.59 - 7.52 (m, 1H), 7.35 - 7.24 (m, 2H), 5.30 (dt, / = 8.0, 3.8 Hz, 1H), 5.07 (s, 0.5H), 4.79 (dd, / = 12.7, 3.2 Hz, 1H), 4.67 (dd, / = 12.7, 4.8 Hz, 1H), 3.66 (s, 3H), 3.08 (dd, / = 18.0, 3.7 Hz, 1H), 2.90 (dd, / = 18.0, 10.2 Hz, 1H), 1.15 - 1.05 (m, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.3, 154.3, 132.4, 132.3, 131.0, 130.9, 130.7, 129.5, 127.5, 127.4, 124.7, 123.4, 121.7, 121.1, 119.1, 117.0, 73.1, 58.3, 56.4, 51.5, 33.50, 28.78. HRMS (ESI-TOF): m/z calcd for C25H26F3N3O6S2 585.1215, found 436.1732 [M-CF ₃ S0 ₃ ] ⁺ . Compound Id had the following chemical formula and characterization data:

Compound Id: 13-(tert-butylamino)-7-(2-methoxy-2-oxoethyl)-10-methyl- 11 -phenyl-6,7- dihydrobenzo[f]thiazolo[2',3':2,3]imidazo[ l,5-d][ 1,4 ] oxazepin-8-ium trifluoromethanesulfonate

Off white color solid, 171 mg, 55%. NMR (500 MHz, MeOD) d 8.06 (dd, / = 7.8, 1.3 Hz, 1H), 7.69 - 7.62 (m, 5H), 7.52 - 7.44 (m, 1H), 7.25 - 7.14 (m, 2H), 5.29 - 5.22 (m, 1H), 4.73 - 4.63 (m, 2H), 3.70 (s, 3H), 3.58 (s, 0.8H), 3.10 (dd, J = 18.1, 3.5 Hz, 1H), 2.89 (dd, J = 18.1, 10.4 Hz, 1H), 2.49 (s, 3H), 0.53 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 172.0, 155.7, 144.9, 133.3, 132.8, 132.5, 132.4, 131.5, 131.1, 131.0, 130.0, 128.8, 128.5, 124.2, 122.0, 117.3, 74.1, 59.9, 56.7, 52.9, 34.76, 29.6, 13.00. HRMS (ESI-TOF): m/z calcd for C28H30F3N3O6S2 625.1528, found 476.2058 [M-CF ₃ S0 ₃ ] ⁺ .

Compound le had the following chemical formula and characterization data:

Compound le: 13-(tert-butylamino)-7-(2-methoxy-2-oxoethyl)-6,7- dihydrobenzo[f]thiazolo[2',3':2,3]imidazo[ l,5-d][ 1,4 ] oxazepin-8-ium trifluoromethanesulfonate

Off white color solid, 120 mg, 45%. NMR (500 MHz, MeOD) d 8.25 (d, / = 8.0 Hz, 1H), 8.15 (d, / = 4.3 Hz, 1H), 7.70 (t, / = 5.8 Hz, 1H), 7.51 - 7.46 (m, 1H), 7.28 - 7.17 (m, 2H), 5.29 - 5.23 (m, 1H), 4.79 (s, 0.5H), 4.68 - 4.64 (m, 2H), 3.67 (s, 3H), 3.09 (dd, J = 18.0, 3.6 Hz, 1H), 2.89 (dd, / = 18.0, 10.3 Hz, 1H), 1.10 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.6, 154.2, 145.1, 131.8, 130.6, 129.4, 128.1, 122.7, 120.8, 120.1, 117.7, 115.9, 72.4, 58.4, 56.0, 51.5, 33.3, 28.7. HRMS (ESI-TOF): m/z calcd for C21H24F3N3O6S2 535.1058, found 386.1058 [M- CF ₃ S0 ₃ ] ⁺ .

Compound If had the following chemical formula and characterization data:

Compound If: 13-( tert-butylamino )-2,4-difluoro-7-( 2-methoxy-2-oxoethyl )-l 0-methyl- 11 - phenyl-6, 7-dihydrobenzo[f]thiazolo[2’,3’:2, 3] imidazo[l,5-d] [1,4] oxazepin-8-ium

trifluoromethanesulfonate

Off white color solid, 148 mg, 45%. ^l U NMR (500 MHz, MeOD) d 7.75 (d, / = 9.3 Hz, 1H), 7.31 - 7.23 (m, 1H), 5.34 - 5.27 (m, 1H), 4.80 - 4.69 (m, 2H), 3.71 (s, 3H), 3.63 (s, 1H), 3.14 (dd, / = 18.0, 3.7 Hz, 1H), 2.97 (dd, / = 18.0, 10.1 Hz, 1H), 2.49 (s, 3H), 0.57 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.4, 157.2 (d, ^C-F = 244.0 Hz), 154.3 (d, ^C-F = 252.0 Hz), 144.1, 139.2, 131.1, 130.9, 130.3, 130.2, 128.7, 127.9, 127.6, 126.9, 112.0, 106.7, 106.5, 73.4, 57.9, 55.7, 51.2, 33.5, 28.3. HRMS (ESI-TOF): m/z calcd for C28H28F5N3O6S2 661.1339, found 512.1819 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lg had the following chemical formula and characterization data:

Compound lg: 11 -(4-bromophenyl )-13-( tert-butylamino )-3-( diethylamino )-7-( 2-methoxy-2- oxoethyl)-10-methyl-6,7-dihydrobenzo[f]thiazolo[2',3':2,3]im idazo[l,5-d][l,4]oxazepin-8-ium trifluoromethanesulfonate

Off white color solid, 185 mg, 48%. NMR (500 MHz, MeOD) d 7.79 (d, / = 8.4 Hz, 2H), 7.74 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 6.55 (dd, J = 9.0, 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 5.21 - 5.14 (m, 1H), 4.65 - 4.55 (m, 2H), 3.70 (s, 3H), 3.44 (q, J = 7.0 Hz, 2H), 3.09 (dd, J = 18.0, 3.3 Hz, 1H), 2.95 (dd, J= 18.0, 10.3 Hz, 1H), 2.48 (s, 3H), 1.21 (t, J = 7.0 Hz, 6H), 0.59 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.8, 155.8, 150.7, 142.2, 133.0, 132.1, 132.0, 131.4, 130.1, 127.6, 127.3, 126.3, 123.9, 106.6, 101.1, 100.9, 72.1, 58.6, 54.8, 51.4, 43.9, 33.0, 28.4, 11.6, 11.4.LCMS (ESI): m/z 626 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lh had the following chemical formula and characterization data:

Compound lh: 13-(tert-butylamino)-3-(diethylamino)-7-(2-methoxy-2-oxoethy l)-10,H-bis(4- methoxyphenyl )-6, 7-dihydrobenzo[f]thiazolo[2',3':2,3]imidazo[ l,5-d][ 1,4 ]oxazepin-8-ium trifluoromethanesulfonate Pale brown color solid, 184 mg, 44%. ¹ H NMR (500 MHz, MeOD) d 7.84 (d, / = 9.0 Hz, 1H), 7.49 - 7.40 (m, 2H), 7.27 - 7.22 (m, 2H), 7.07 (d, / = 8.1 Hz, 2H), 6.96 - 6.92 (m, 2H), 6.58 - 6.52 (m, 1H), 6.38 (d, J = 2.5 Hz, 1H), 5.25 - 5.18 (m, 1H), 4.66 - 4.59 (m, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 3.69 (s, 3H), 3.45 (q, / = 7.0 Hz, 2H), 3.11 (dd,/ = 18.1, 3.4 Hz, 1H), 2.98 (dd, / = 18.1, 10.3 Hz, 1H), 1.21 (t, / = 7.0 Hz, 7H), 0.61 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 172.3, 162.7, 162.3, 157.1, 152.0, 143.7, 134.3, 133.6, 132.4, 132.2, 131.4, 130.8, 129.4, 122.4, 120.6, 115.6, 115.4, 108.0, 102.4, 102.1, 73.3, 60.1, 56.5, 56.0, 55.9, 52.9, 45.3, 34.4, 29.9, 12.8.LCMS (ESI): m/z 626 [M-CF ₃ S0 ₃ ] ⁺ .

Compound li had the following chemical formula and characterization data:

Compound li: 15-( tert-butylamino )-9-(2-methoxy-2-oxoethyl )-l 2-methyl- 13 -phenyl-8, 9- d i hyd ronaph ih o / 1 ,2-flthiazoloj 2 ',3':2,3]imidazo[ l,5-d][ 1,4 ]oxazepin-l 0-ium

trifluoromethanesulfonate

Off white color solid, 162 mg, 48%. Mixture of diastereoisomers (64:36). ^X H NMR (500 MHz, CDCl ) d 8.06 (d, J = 8.4 Hz, 0.36H), 8.01 (d, J = 8.8 Hz, 0.36H), 7.97 (d, J = 8.8 Hz, 0.66H), 7.93 - 7.86 (m, 1.8H), 7.71 - 7.50 (m, 6H), 7.36 -7.29 (m, 1H), 5.46 - 5.36 (m, 0.66H), 5.14 - 5.05 (dd, J = 12.4, 6.2 Hz, 0.66H), 5.05 - 4.97 (m, 0.36H), 4.66 - 4.56 (m, 0.66H), 4.46 (d, J = 12.4 Hz, 0.66H), 3.84 (dd, / = 16.7, 5.8 Hz, 0.36H), 3.71 (s, 3H), 3.57 (s, 2H), 3.17 (dd, / = 16.7, 8.5 Hz, 0.36H), 2.70 - 2.48 (m, 2.2H), 2.42 (two s, 3H), 0.18 (two s, 9H). LCMS (ESI): m/z 526 [M-CF ₃ S0 ] ⁺ .

Compound lj had the following chemical formula and characterization data:

Compound lj: 15-( tert-butylamino)-9-(2-methoxy-2-oxoethyl)-l 3 -phenyl-8, 9- d i hyd ronaph th o / 1 ,2-flthiazoloj 2 ',3':2,3]imidazo[ l,5-d][ 1,4 ]oxazepin-l 0-ium

trifluoromethanesulfonate

Off white color solid, 99 mg, 30%. Mixture of diastereoisomers (64:36). ^X H NMR (500 MHz, CDCls) d 8.15 - 7.99 (m, 2H), 7.94 (dd, 7 = 8.1, 2.6 Hz, 1H), 7.84 - 7.72 (m, 3H), 7.64 - 7.54 (m, 4H), 7.43 - 7.30 (m, 2H), 5.52 - 5.39 (m, 0.64H), 5.19 - 5.05 (m, 1H), 4.68 - 4.60 (m, 0.72H), 4.54 - 4.47 (m, 0.64H), 3.90 - 3.80 (m, 0.6H), 3.73 (s, 1.2H), 3.60 (s, 1.8H), 3.24 - 3.17 (m, 0.4H), 2.85- 2.58 (m, 2H), 2.66 (t, J = 18.0 Hz, 1H), 0.21 (two s, 9H). LCMS (ESI): m/z 512 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lk had the following chemical formula and characterization data:

Compound lk: 13-( tert-butylamino )-7-(2-methoxy-2-oxoethyl )-l 0-methyl- 11 -phenyl-6, 7- dihydronaphtho[ 2, 1 -f]thiazolo[ 2 ',3':2,3]imidazo[ 1, 5-d][ 1,4 ] oxazepin-8-ium trifluoromethanesulfonate

Pale yellow color solid, 172 mg, 51%. NMR (500 MHz, CDCl ) d 8.30 (dd, J = 8.2, 0.8 Hz, 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.79 - 7.76 (m, 1H), 7.68 - 7.51 (m, 7H), 7.47 (d, J = 8.9 Hz, 1H), 5.47 - 5.40 (m, 1H), 5.03 - 4.94 (m, 2H), 3.69 (s, 3H), 3.06 - 2.91 (m, 2H), 2.43 (s, 3H), 0.56 (s, 9H). ¹³ C NMR (125 MHz, CDCb) d 171.2, 150.0, 144.7, 135.0, 130.8, 130.0, 129.80, 128.3, 128.2, 128.0, 127.5, 127.3, 127.3, 126.7, 126.3, 123.2, 121.9, 108.4, 72.9, 59.8, 57.3, 52.6, 33.5, 29.6, 13.2. LCMS (ESI): m/z 526 [M-CF ₃ S0 ₃ ] ⁺ .

Compound 11 had the following chemical formula and characterization data:

Compound 11: 14-(tert-butylamino)-5-(2-methoxy-2-oxoethyl)-2-methyl-l -phenyl-5,6- d i hyd rona ph tho / 2,3-f]thiazolo[ 2 ',3':2,3]imidazo[ l,5-d][ 1,4 ] oxazepin-4-ium trifluoromethanesulfonate

Off white color solid, 185 mg, 55%. NMR (500 MHz, CDCb) d 8.60 (s, 1H), 7.97 (d, / = 8.1 Hz, 1H), 7.79 (d, /= 8.1 Hz, 1H), 7.70 - 7.45 (m, 7H), 7.28 (s, 1H), 5.30 - 5.21 (m, 1H), 4.95 - 4.86 (m, 1H), 4.58 - 4.50 (m, 1H), 3.67 (s, 3H), 3.14 (s, 0.9H), 3.02 (dd, / = 17.7, 8.3 Hz, 1H), 2.81 (dd, / = 17.7, 3.9 Hz, 1H), 2.45 (s, 3H), 0.47 (s, 9H). ¹³ C NMR (126 MHz, CDCb) d 170.4, 151.4, 142.9, 135.1, 132.2, 131.5, 131.4, 130.7, 130.4, 130.2, 129.5, 129.2, 128.8, 128.3, 127.1, 126.9, 126.8, 126.2, 119.2, 117.9, 74.8, 56.8, 56.1, 52.7, 35.9, 29.6, 13.3. LCMS (ESI): m/z 526 [M-CF ₃ S0 ] ⁺ .

Compound lm had the following chemical formula and characterization data:

Compound lm: 12-( tert-butylamino )-6-(2-methoxy-2-oxoethyl )-9-methyl-l 0-phenyl- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2", 3": 2 ',3 '] imidazo[ l ',5':4,5][l,4] oxazepino[ 7,6-f]quinolin-7-ium

trifluoromethanesulfonate

Pale yellow color solid, 194 mg, 54%. NMR (500 MHz, MeOD) d 7.65 - 7.58 (m, 5H), 7.30 (s, 1H), 5.17 - 5.10 (m, 1H), 4.63 (dd, J = 12.3, 4.3 Hz, 1H), 4.49 (dd, J = 12.3, 4.7 Hz, 1H), 3.68 (s, 3H), 3.28 - 3.21 (m, 4H), 3.03 - 2.89 (m, 2H), 2.80 - 2.72 (m, 4H), 2.46 (s, 1H), 2.02 - 1.92 (m, 4H), 0.53 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.5, 150.3, 145.6, 141.5, 132.0, 131.21, 131.19, 129.9, 128.3, 127.7, 127.4, 127.2, 126.3, 117.1, 111.9, 104.1, 72.6, 57.0, 54.8, 51.4, 49.42, 48.9, 34.0, 28.4, 26.9, 21.5, 20.8, 20.7, 11.5. HRMS (ESI-TOF): m/z calcd for C34H39F3N4O6S2 720.2263, found 571.2742 [M-CF ₃ S0 ₃ ] ⁺ .

Compound In had the following chemical formula and characterization data:

Compound In: 12-( tert-butylamino)-10-(4-fluorophenyl)-6-(2-methoxy-2-oxoethyl )-9-methyl-

2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ G,5':4,5] [1,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 202 mg, 55%. ^X H NMR (500 MHz, Acetone) d 7.83 - 7.76 (m, 1H), 7.43 - 7.37 (m, 2H), 5.30 - 5.24 (m, 1H), 4.79 (dd, J = 12.5, 3.5 Hz, 1H), 4.69 (dd, J = 12.5, 5.0 Hz, 1H), 3.69 (s, 0.8H), 3.67 (s, 3H), 3.27 - 3.21 (m, 4H), 3.15 (dd, 7 = 18.0, 3.9 Hz, 1H), 3.04 (dd, J = 18.0, 10.0 Hz, 1H), 2.78 - 2.69 (m, 4H), 2.53 (s, 3H), 2.00 - 1.89 (m, 4H), 0.60 (s, 9H). ¹³ C NMR (126 MHz, Acetone) d 170.4, 163.5, 150.4, 145.5, 141.9, 133.9, 132.5, 130.3, 128.1, 127.7, 127.6, 126.8, 123.7, 116.6, 115.5, 111.3, 103.1, 72.6, 57.8, 55.0, 54.9, 51.7, 49.5, 48.9, 33.9, 26.9, 21.6, 21.0, 20.9, 12.2. HRMS (ESI-TOF): m/z calcd for C34H38F4N4O6S2 738.2168, found 589.2635 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lo had the following chemical formula and characterization data:

Compound lo: 10-(4-bromophenyl)-12-(tert-butylamino)-6-(2-methoxy-2-oxoet hyl)-9-methyl-

2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ G,5':4,5] [1,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 231 mg, 58%. NMR (500 MHz, MeOD) d 7.77 (d, / = 8.5 Hz, 2H), 7.55 (d, / = 8.4 Hz, 2H), 7.26 (s, 1H), 5.17 - 5.09 (m, 1H), 4.63 (dd, / = 12.3, 4.5 Hz, 1H), 4.47 (dd, J = 12.3, 4.7 Hz, 1H), 3.75 (s, 0.5H), 3.67 (s, 3H), 3.33 (dt, J = 3.2, 1.6 Hz, 1H), 3.29 - 3.21 (m, 2H), 3.03 - 2.88 (m, 2H), 2.82 - 2.72 (m, 4H), 2.47 (s, 3H), 2.07 - 1.90 (m, 4H), 0.56 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.5, 150.32, 145.7, 141.5, 133.2, 132.6, 131.3, 130.1, 127.6, 127.4, 126.9, 126.2, 123.9, 117.2, 112.0, 104.1, 72.7, 56.9, 54.7, 51.4, 49.4, 48.9, 34.1, 28.5, 26.9, 21.5, 20.8, 20.7, 11.6. HRMS (ESI-TOF): m/z calcd for C ₃₄ H ₃₇ BrF ₃ N ₄ 0 ₆ S ₂ 797.1290, found 649.1700 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lp had the following chemical formula and characterization data:

Compound lp: 12-( tert-butylamino )-l 0-(2, 3-dihydrobenzo[b ][ 1,4 ]dioxin-5-yl )-6-( 2-methoxy-2- oxoethyl )-2, 3, 5, 6,15,16-hexahydro- 1H, 14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2’,3’] imidazo[ l ',5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate Light green color solid, 183 mg, 48%. NMR (500 MHz, MeOD) d 7.40 (s, 1H), 7.34 (s, 1H), 7.25 (d, / = 1.9 Hz, 1H), 7.17 (dd, / = 8.3, 2.0 Hz, 1H), 7.02 (d, / = 8.3 Hz, 1H), 5.20 - 5.11 (m, 1H), 4.63 (dd, / = 12.3, 4.4 Hz, 1H), 4.48 (dd, / = 12.3, 4.6 Hz, 1H), 4.38 - 4.30 (m, 4H), 3.87 (s, 0.3H), 3.66 (s, 3H), 3.30 - 3.21 (m, 4H), 3.04 - 2.94 (m, 2H), 2.82 - 2.73 (m, 4H), 2.04 - 1.91 (m, 4H), 0.63 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.5, 150.4, 145.6, 144.3, 143.5, 136.2, 132.7, 127.9, 127.4, 123.3, 120.7, 119.4, 117.19, 117.16, 116.8, 112.9, 111.9, 104.1, 72.6, 64.6, 64.3, 57.0, 55.0, 51.4, 49.4, 48.9, 34.1, 28.4, 26.9, 21.5, 20.8, 20.7. HRMS (ESI-TOF): m/z calcd for C35H39F3N4O8S2 764.2161, found 615.2616 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lq had the following chemical formula and characterization data:

Compound lq: 10-(4-bromophenyl)-12-( tert-butylamino)-6-(2-methoxy-2-oxoethyl)- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ V,5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Off white color solid, 199 mg, 51%. NMR (500 MHz, MeOD) d 7.74 (d, / = 8.3 Hz, 1H), 7.66 (d, / = 8.3 Hz, 1H), 7.56 (s, 1H), 7.29 (s, 1H), 5.22 - 5.14 (m, 1H), 4.65 (dd, / = 12.3, 4.4 Hz, 1H), 4.49 (dd, / = 12.3, 4.6 Hz, 1H), 3.66 (s, 3H), 3.31 - 3.22 (m, 4H), 3.04 - 2.91 (m, 2H), 2.84 - 2.72 (m, 4H), 2.02 - 1.94 (m, 4H), 0.58 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.5, 150.4, 145.8, 144.4, 135.4, 133.5, 132.1, 131.1, 127.7, 127.3, 127.2, 124.0, 117.3, 114.5, 112.1, 104.1, 72.7, 57.0, 54.9, 51.4, 49.4, 48.9, 34.1, 28.4, 26.9, 21.4, 20.8, 20.7. FCMS (ESI): m/z 635 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lr had the following chemical formula and characterization data:

Compound lr: I2-(tert-butylamino)-9-( ethoxy carbonyl )-6-(2-methoxy-2-oxoethyl )- 10-phenyl-

2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ V,5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 210 mg, 54%. NMR (500 MHz, MeOD) d 7.78 - 7.54 (m, 5H), 7.31 (s, 1H), 5.28 - 5.18 (m, 1H), 4.66 (dd, / = 12.5, 4.9 Hz, 1H), 4.52 (dd, / = 12.5, 4.2 Hz, 1H), 4.33 - 4.21 (m, 2H), 3.69 (s, 3H), 3.44 (s, 0.6H), 3.29 - 3.21 (m, 1H), 3.02 (dd, 7 = 18.1, 3.8 Hz, 1H), 2.91 (dd, 7 = 18.1, 10.1 Hz, 1H), 2.80 - 2.70 (m, 4H), 2.03 - 1.92 (m, 4H), 1.20 (t, 7 = 7.1 Hz, 3H), 0.55 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.7, 159.4, 150.4, 145.8, 143.4, 139.8, 133.0, 132.4, 130.6, 128.3, 127.8, 127.7, 126.4, 121.1, 117.0, 111.6, 103.0, 72.3, 62.5, 57.6, 54.9, 51.5, 49.4, 48.9, 34.0, 28.5, 28.3, 26.8, 21.4, 20.8, 12.7. LCMS (ESI): m/z 629 [M-CF ₃ S0 ₃ ] ⁺ .

Compound Is had the following chemical formula and characterization data:

Compound Is: 12-(tert-butylamino)-10-(3,4-difluorophenyl)-6-(2-methoxy-2- oxoethyl)- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2", 3": 2 ',3 '] imidazo[ l ',5':4,5][l,4] oxazepino[ 7,6-f]quinolin-7-ium

trifluoromethanesulfonate

Off white color solid, 210 mg, 54%. NMR (500 MHz, MeOD) d 7.81 - 7.74 (m, 1H), 7.63 - 7.43 (m, 3H), 7.28 (s, 1H), 5.22 - 5.14 (m, 1H), 4.66 (dd, / = 12.3, 4.5 Hz, 1H), 4.48 (dd, / = 12.3, 4.6 Hz, 1H), 4.19 (s, 0.8H), 3.66 (s, 3H), 3.31 - 3.22 (m, 4H), 3.08 - 2.88 (m, 2H), 2.85 - 2.71 (m, 4H), 2.06 - 1.92 (m, 4H), 0.61 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 171.8, 152.9, 151.8 (d, ^C-F = 243 Hz), 150.9 (d, ^C-F = 249.0 Hz), 147.2, 145.7, 135.7, 135.0, 129.2, 128.8, 128.7, 126.6, 121.5, 118.7, 118.3, 116.5, 113.6, 105.5, 74.1, 58.4, 56.3, 52.8, 50.8, 50.3, 35.5, 29.8, 28.3, 22.8, 22.2, 22.1. LCMS (ESI): m/z 593 [M-CF ₃ S0 ₃ ] ⁺ .

Compound It had the following chemical formula and characterization data:

Compound It: 12-( tert-butylamino)-10-(2,5-dimethoxyphenyl)-6-(2-methoxy-2-oxo ethyl)- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ G,5':4,5] [1,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Off white color solid, 183 mg, 48%. NMR (500 MHz, MeOD) d 7.47 (s, 1H), 7.35 (s, 1H), 7.18 (ddd, / = 16.5, 8.2, 2.7 Hz, 3H), 5.20 - 5.12 (m, 1H), 4.62 (dd, / = 12.3, 4.0 Hz, 1H), 4.55 - 4.46 (m, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.68 (s, 3H), 3.40 (s, 0.3H), 3.29 - 3.19 (m, 4H), 3.09 - 2.90 (m, 2H), 2.83 - 2.72 (m, 4H), 2.05 - 1.93 (m, 4H), 0.60 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.5, 154.0, 151.8, 150.4, 145.6, 143.7, 132.6, 131.7, 127.7, 127.5, 117.9, 117.8, 117.0, 116.8, 114.8, 112.2, 111.7, 103.8, 72.4, 55.5, 55.1, 54.9, 51.4, 49.4, 48.9, 28.5, 26.9, 21.5, 20.85, 20.77. LCMS (ESI): m/z 617 [M-CF ₃ S0 ] ⁺ .

Compound lu had the following chemical formula and characterization data:

Compound lu: 10-(benzo[d] [l,3]dioxol-4-yl)-12-(tert-butylamino)-6-(2-methoxy-2-oxoeth yl)-9- methyl-2, 3, 5, 6,15,16-hexahydro-lH, 14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ G,5':4,5] [1,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 194 mg, 5l%. ^{: 1} H NMR (500 MHz, MeOD) d 7.30 (s, 1H), 7.13 (d, / = 1.5 Hz, 1H), 7.11 - 7.03 (m, 2H), 6.14 - 6.07 (m, 2H), 5.15 - 5.08 (m, 1H), 4.62 (dd, / = 12.3, 4.0 Hz, 1H), 4.48 (dd, / = 12.3, 4.7 Hz, 1H), 3.67 (s, 3H), 3.28 - 3.20 (m, 4H), 3.03 - 2.86 (m, 2H), 2.80 - 2.73 (m, 4H), 2.46 (s, 3H), 2.03 - 1.92 (m, 4H), 0.62 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.5, 150.3, 149.4, 148.0, 145.6, 141.4, 131.8, 131.0, 127.7, 127.5, 126.0, 125.5, 120.3, 117.1, 111.9, 111.4, 107.9, 104.1, 101.9, 72.6, 57.0, 54.8, 51.4, 49.4, 48.9, 28.6, 26.9, 21.5, 20.8, 20.7, 11.6.

Compound lv had the following chemical formula and characterization data:

Compound lv: 14-bromo-18-(tert-butylamino)-10-(2-methoxy-2-oxoethyl)-2,3, 6,7,9,10- hexahydro-lH,5H-benzo[4",5”]thiazolo[ 2”,3”:2’,3’]imidazo[ 1 ',5':4,5][ 1,4 ]oxazepino[7,6- f]pyrido[ 3,2, 1 -ij]quinolin-l 1 -ium trifluoromethanesulfonate Pale yellow color solid, 185 mg, NMR (500 MHz, MeOD) d 8.65 (d, = 8.8 Hz, 1H), 8.40 (d, = 1.8 Hz, 1H), 7.92 (dd, / = 8.8, 1.9 Hz, 1H), 7.30 (s, 1H), 5.24 - 5.14 (m, 1H), 4.71 (dd, / = 12.1, 4.7 Hz, 1H), 4.47 (dd, / = 12.1, 4.5 Hz, 1H), 3.63 (s, 3H), 3.31 - 3.21 (m, 4H), 3.03 - 2.87 (m, 2H), 2.87 - 2.73 (m, 4H), 2.07 - 1.93 (m, 4H), 1.11 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.3, 150.3, 145.9, 142.8, 133.3, 131.6, 131.0, 130.6, 128.7, 127.3, 127.0, 119.9, 118.0, 117.5, 112.4, 104.3, 72.8, 57.1, 56.0, 51.4, 49.4, 48.9, 34.2, 28.9, 26.9, 21.4, 20.7, 20.6. LCMS (ESI): m/z 609 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lw had the following chemical formula and characterization data:

Compound lw: 10-( 4-acetamidophenyl )-12-(tert-butylamino)-6-( 2-methoxy-2-oxoethyl )- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ V,5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 171 mg, 45%. NMR (500 MHz, MeOD) d 7.79 (d, / = 8.6 Hz, 1H), 7.67 (d, / = 8.6 Hz, 1H), 7.45 (s, 1H), 7.32 (s, 1H), 5.22 - 5.13 (m, 1H), 4.64 (dd, / = 12.3, 4.4 Hz, 1H), 4.49 (dd, / = 12.3, 4.7 Hz, 1H), 3.94 (s, 0.3H), 3.66 (s, 3H), 3.29 - 3.21 (m, 4H), 3.05 - 2.90 (m, 2H), 2.84 - 2.72 (m, 4H), 2.19 (s, 3H), 2.05 - 1.94 (m, 4H), 0.58 (s, 9H). ¹³ C NMR (126 MHz, MeOD) d 170.5, 170.4, 150.4, 145.7, 144.3, 140.5, 136.3, 133.0, 130.8, 127.8, 127.4, 123.2, 118.8, 117.2, 113.3, 112.0, 104.1, 72.6, 57.0, 55.0, 51.4, 49.4, 48.9, 34.1, 28.4, 26.9, 22.6, 21.5, 20.8, 20.7. LCMS (ESI): m/z 609 [M-CF ₃ S0 ] ⁺ .

Compound lx had the following chemical formula and characterization data:

Compound lx: 12-(tert-butylamino)-10-(2,4-dichlorophenyl)-6-(2-methoxy-2- oxoethyl)-9- methyl-2, 3, 5, 6,15,16-hexahydro-lH, 14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ V,5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate

Pale yellow color solid, 228 mg, 58%. NMR (500 MHz, MeOD) d 7.77 (d, / = 1.9 Hz, 1H), 7.73 - 7.66 (m, 1H), 7.59 (dd, / = 8.3, 1.9 Hz, 1H), 7.21 (s, 1H), 5.13 - 5.07 (m, 1H), 4.60 (dd, / = 11.9, 4.6 Hz, 2H), 4.46 (dd, / = 11.9, 6.0 Hz, 1H), 3.69 (s, 3H), 3.62 (s, 0.4 H), 3.29 - 3.21 (m, 4H), 3.07 - 3.00 (m, 2H), 2.82 - 2.73 (m, 4H), 2.41 (s, 3H), 2.03 - .93 (m, 4H), 0.59 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 171.6, 151.5, 147.2, 142.0, 138.4, 137.6, 136.2, 134.5, 130.7, 130.3, 128.8, 128.8, 128.5, 128.2, 126.6, 118.8, 113.9, 106.1, 74.5, 57.7, 55.8, 52.9, 50.8, 50.3, 34.9, 30.1, 28.3, 22.8, 22.1, 22.0, 13.0. LCMS (ESI): m/z 639 [M-CF ₃ S0 ₃ ] ⁺ .

Compound ly had the following chemical formula and characterization data:

Compound ly: 18-(tert-butylamino)-10-(2-methoxy-2-oxoethyl)-14-(methylsul fonyl)-

2, 3, 6, 7, 9, 10-hexahydro-lH, 5H- benz.oj 4",5" Irhiaz.oloj 2",3":2',3'l imidaz.oj / ',5':4,5 U 1 ,4]oxaz.epinol 7,6-fjpyridol 3,2, 1- ij]quinolin-l 1 -ium trifluoromethanesulfonate

Pale yellow color solid, 94 mg, 25%. NMR (500 MHz, MeOD) d 8.96 (d, = 8.7 Hz, 1H), 8.83 (d, / = 1.5 Hz, 1H), 8.31 (dd, / = 8.7, 1.7 Hz, 1H), 8.21 (d, / = 1.7 Hz, 1H), 7.81 (dd, / = 8.5, 1.9 Hz, 1H), 7.52 (d, / = 8.5 Hz, 1H), 7.32 (s, 1H), 5.27 - 5.20 (m, 1H), 4.73 (dd, / = 12.2, 4.7 Hz, 1H), 4.50 (dd, / = 12.2, 4.4 Hz, 1H), 3.64 (s, 3H), 3.31 - 3.25 (m, 4H), 3.13 (s, 3H), 3.04 - 2.91 (m, 2H), 2.86 - 2.76 (m, 4H), 2.06 - 1.95 (m, 4H), 1.12 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 171.7, 151.7, 147.4, 145.8, 141.1, 137.0, 135.2, 131.9, 130.3, 128.4, 127.9, 126.4, 126.2, 118.9, 113.8, 105.5, 74.1, 58.7, 57.5, 52.8, 50.8, 50.3, 44.4, 35.6, 30.3, 28.3, 22.8, 22.1, 22.1. LCMS (ESI): m/z 609 [M-CF ₃ S0 ₃ ] ⁺ .

Compound lz had the following chemical formula and characterization data:

Compound lz: 12-( tert-butylamino )-6-(2-methoxy-2-oxoethyl )-9, 10-bis( 4-methoxyphenyl )- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2',3 '] imidazo[ G,5':4,5] [1,4] oxazepino[ 7,6-f ] quinolin-7-ium

trifluoromethanesulfonate

Pale yellow color solid, 227 mg, 54%. NMR (500 MHz, MeOD) d 7.50 - 7.41 (m, 2H), 7.34 (s, 1H), 7.24 (d, / = 8.2 Hz, 1H), 7.06 (d, / = 7.6 Hz, 1H), 6.93 (d, / = 8.2 Hz, 1H), 5.23 - 5.13 (m, 1H), 4.71 - 4.63 (m, 1H), 4.52 (dd, / = 12.1, 3.8 Hz, 1H), 3.87 (s, 3H), 3.81 9s, 3H), 3.66 (s, 3H), 3.20 - 3.2l(m, 4H), 3.07 - 2.97 (m, 2H), 2.84 - 2.72 (m, 4H), 2.05 - 1.93 (m, 4H), 0.58 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 172.0, 162.7, 162.3, 151.8, 147.0, 143.0, 134.5, 133.2, 132.3, 131.5, 130.4, 129.4, 128.9, 122.4, 120.6, 118.5, 115.6, 115.2, 113.2, 105.4, 74.0, 58.5, 56.4, 56.0, 55.9, 52.8, 50.8, 50.3, 35.5, 30.0, 28.3, 22.9, 22.2, 22.1.LCMS (ESI): m/z 693 [M- CF ₃ S0 ] ⁺ . Compound 2a had the following chemical formula and characterization data:

Compound 2a: 12-( tert-butylamino )-6-( 2-methoxy-2-oxoethyl )-9, 10-diphenyl-2, 3,5,6,15,16- hexahydro- 1H,14H-pyrido[ 3,2,1-ij] thiazolo[ 2",3":2',3 '] imidazo[ l ',5':4,5] [1,4] oxazepino[ 7,6- flquinolin- 7-ium trifluoromethanesulfonate

Pale yellow color solid, 218 mg, 56%. NMR (500 MHz, MeOD) d 7.63 - 7.47 (m, 5H), 7.46 - 7.28 (m, 6H), 5.26 - 5.17 (m, 1H), 4.68 (dd, / = 12.3, 4.1 Hz, 1H), 4.53 (dd, / = 12.3, 4.4 Hz, 1H), 3.67 (s, 1H), 3.60 (s, 0.3H), 3.30 - 3.20 (m, 4H), 3.07 - 2.91 (m, 2H), 2.83 - 2.71 (m, 4H), 2.05 - 1.93 (m, 4H), 0.55 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.6, 150.4, 145.7, 142.0, 132.2, 131.8, 130.8, 130.0, 129.9 (2C), 129.6, 129.0, 128.8, 128.4, 128.0, 127.6, 127.3, 117.1, 111.8, 103.8, 72.5, 57.3, 54.8, 51.4, 49.4, 48.9, 34.0, 28.5, 26.9, 21.5, 20.8, 20.7.LCMS (ESI): m/z 633 [M-CF ₃ S0 ₃ ] ⁺ .

Compound 2b had the following chemical formula and characterization data:

Compound 2b: 12-amino-10-(4-fluorophenyl)-6-(2-methoxy-2-oxoethyl)-9-meth yl- 2, 3, 5, 6,15,16-hexahydro- 1H,14H-pyrido[ 3,2,1- ij ] thiazolo[ 2",3":2’,3’] imidazo[ l ',5':4,5][l,4] oxazepino[ 7 ,6-f] quinolin-7 -ium

trifluoromethanesulfonate Pale brown color solid, 184 mg, 44%. NMR (500 MHz, MeOD) d 7.67 (dd, / = 8.8, 5.2 Hz, 1H), 7.36 (t, / = 8.7 Hz, 1H), 7.04 (s, 1H), 5.13 - 5.05 (m, 1H), 4.60 (dd, / = 12.4, 3.8 Hz, 1H), 4.53 - 4.45 (m, 1H), 4.48 (dd, / = 12.4, 4.7 Hz, 1H), 3.68 (s, 3H), 3.27 - 3.16 (m, 4H), 3.03 - 2.98 (m, 1H), 2.95 - 2.88 (m, 1H), 2.78 - 2.70 (m, 4H), 2.39 (s, 3H), 2.03 - 1.91 (m, 4H). ¹³ C NMR (125 MHz, MeOD) d 172.0, 166.4, 164.4, 151.4, 146.2, 140.6, 134.7, 130.4, 130.2, 127.7, 127.0, 123.9, 123.9, 121.9, 118.5, 117.1, 116.9, 113.5, 104.6, 73.4, 58.6, 52.8, 50.9, 50.3, 35.2, 28.3, 22.9, 22.3, 22.3, 12.8. LCMS (ESI): m/z 533 [M-CF ₃ S0 ₃ ] ⁺ .

EXAMPLE 2

In a second experimental example, novel compounds of general formula B were prepared and characterized. In this experimental example, an aldehyde (general formula IV, 0.5 mmol), 2- aminoazine (general formula II, 0.5 mmol), scandium triflate (20 mol%), and sodium sulfate (1.0 mmol) were mixed in ACN (2 mL) at room temperature. After 45 mins, isocyanide (general formula III, 0.55 mmol) was introduced and stirring was continued at room temperature for 12- 15 hours. After completion of step-l (Groebuk reaction), ytterbium triflate was added (30 mol%) and continued the reaction at 70°C for 3 to 12 hours. After completion of step-2 (Michael reaction), ACN was removed and the obtained crude material was purified on flash column chromatography using a gradient of EtO AC/hexane or MeOH/DCM as mobile phase eluents to obtain pure products of general formula B.

Compound 2c had the following chemical formula and characterization data:

Compound 2c: 12-(tert-butylamino)-6-(2-methoxy-2-oxoethyl)-9-methyl-10-ph enyl-5,6- dihydropyrrolo[J2-a]thiazolo[2',3':2,3]imidazo[5,l-c]pyrazin -7-ium trifluoromethanesulfonate Pale yellow color solid, 167 mg, 55%. NMR (500 MHz, MeOD) d 7.64 - 7.57 (m, 5H), 7.02 - 6.98 (m, 1H), 6.86 (d, J = 2.9 Hz, 1H), 6.36 - 6.27 (m, 1H), 5.41 - 5.35 (m, 1H), 4.60 - 4.48 (m, 2H), 3.92 (s, 0.7H), 3.71 (s, 3H), 2.92 (dd, / = 17.4, 4.5 Hz, 1H), 2.70 (dd, / = 17.4, 8.5 Hz, 1H), 2.48 (s, 3H), 0.70 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.6, 141.8, 131.4, 131.3, 129.8, 128.1, 127.0, 126.7, 125.3, 124.3, 122.9, 117.0, 109.4, 109.2, 56.0, 52.8, 51.5, 48.1, 35.1, 28.8, 11.8. HRMS (ESI-TOF): m/z calcd for C26H29F3N4O5S2 598.1531, found 449.2060 [M- CF ₃ S0 ₃ ] ⁺ .

Compound 2d had the following chemical formula and characterization data:

Compound 2d: 12-( tert-butylamino)-6-(2-methoxy-2-oxoethyl)-9-methyl-10-phenyl -5,6- dihydroimidazol 1 ,2-ajthiaz.oloj2 3':2,3 /imidazolS, ] -cjpyrazin-7-ium trifluoromethanesulfonate

Off white color solid, 152 mg, 51%. NMR (500 MHz, MeOD) d 7.66 - 7.57 (m, 5H), 7.43 (d, / = 3.1 Hz, 1H), 7.30 - 7.25 (m, 1H), 5.57 - 5.48 (m, 1H), 4.78 - 4.65 (m, 2H), 3.72 (s, 3H), 2.97 (dd, / = 17.5, 3.6 Hz, 1H), 2.77 (dd, / = 17.5, 8.3 Hz, 1H), 2.50 (s, 3H), 0.70 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.4, 143.6, 134.0, 131.6, 131.5, 129.8, 129.2, 129.1, 127.8, 127.7, 126.5, 121.0, 119.9, 56.3, 52.5, 51.6, 35.20, 28.10, 12.02. HRMS (ESI-TOF): m/z calcd for C2 ₅ H28F ₃ N ₅ 0 ₅ S2 599.1483, found 450.1976 [M-CF ₃ S0 ] ⁺ .

Compound 2e had the following chemical formula and characterization data:

Compound 2e: 13-(tert-butylamino)-5-(2-methoxy-2-oxoethyl)-2-methyl-l -phenyl-5, 6- dihydrothiazolof 2 ",3":2’,3 ']imidazo[5',E:3,4]pyrazino[ 1 ,2-aj indol-4-ium

trifluoromethanesulfonate

Off white color solid, 145 mg, 45%. NMR (500 MHz, MeOD) d 7.70 - 7.60 (m, 6H), 7.53 (d, / = 8.2 Hz, 1H), 7.32 (t, / = 7.2 Hz, 1H), 7.27 (s, 1H), 7.18 (t, / = 7.2 Hz, 1H), 5.58 - 5.52 (m, 1H), 4.95 (dd, J = 13.5, 1.4 Hz, 1H), 4.53 (dd, J = 13.5, 3.8 Hz, 1H), 3.65 (s, 3H), 2.97 (dd, J = 17.3, 4.4 Hz, 1H), 2.77 (dd, / = 17.3, 8.4 Hz, 1H), 2.51 (s, 3H), 0.75 (s, 9H). ¹³ C NMR (125 MHz, MeOD) d 170.6, 142.7, 136.6, 131.4, 131.3, 129.9 (2C), 128.2, 128.0, 127.3, 126.9, 123.5, 123.4, 122.6, 121.1, 120.7, 109.0, 102.1, 56.6, 53.1, 51.4, 35.4, 28.8, 11.9. HRMS (ESI-TOF): m/z calcd for C30H31F3N4O5S2 648.1687, found 499.2147 [M-CF ₃ S0 ₃ ] ⁺ .

EXAMPLE 3

In a third experimental example, the compounds prepared in Examples 1 and 2 were tested for potential antibacterial activities against the Gram-negative bacteria Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) and the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), and Bacillus subtilis (environmental isolate). The results of the assay used to test the disclosed compounds for antibacterial effects on Staphylococcus aureus (ATCC 25923) are shown in FIG. 1. Specifically, the compounds shown in FIG. 1 were each tested in duplicate and pink and blue colors were used to differentiate viable from dead bacterial suspensions, respectively. Two compounds (lo and 2a) in wells A6 and B9 (red arrows) exhibited positive antibacterial activities. The background color of the test compound is responsible for the dark pink to red color in A7 wells (no antibacterial activity). Two standard antibiotics (ciprofloxacin and amikacin) were used in serial dilution as positive controls. Blank (media only), growth (untreated), and vehicle (DMSO) controls were also included in each experiment. Positive controls included two standard antibiotics (ciprofloxacin and amikacin) used in 2-fold serial dilutions (10 concentrations started from 8 and 32 pg/mF for ciprofloxacin and amikacin, respectively). The black circles represent MICs for ciprofloxacin and amikacin (0.5 and 4 pg/mF, respectively). Cost-effective and fast methods for high throughput simultaneous screening of multiple compounds were used to screen the disclosed compounds. While broth microdilution is the standard method for testing minimum inhibitory concentrations, this method was modified to be used as a method for screening of multiple compounds simultaneously in a single 96-well microplate. As some of the tested compounds were colored, causing turbidity in the culture media, it was not possible to conclude the absence of bacterial growth by visual inspection of the media or by measuring the optical density. Therefore, a colorimetric assay“CellTiter-Blue® Cell Viability Assay (Promega, USA)” was used to test bacterial cell viability.

The Cell Viability Assay solution (Promega, USA) used contained resazurin dye, which was used to measure the metabolic capacity of cells as an indicator of cell viability. In microbiology, resazurin has been used as a growth indicator for Mycobacterium tuberculosis to facilitate MIC determination in this slowly growing microbe.

Resazurin has been used in some cell cytotoxicity studies. According to manufacturer instructions, the CellTiter-Blue ^® Cell Viability Assay (Promega, USA) is intended for cell cytotoxicity testing in both absorbance and fluorescence modes. However, it was used in the disclosed experiments to screen for potential antibacterial activities and MIC determination using colorimetric (visual) methods.

Resazurin is dark blue in color, but when it is reduced to resorufin, the solution becomes pink in color and highly fluorescent. Viable bacterial cells have the ability to reduce resazurin into resorufin, which is highly fluorescent; thus, viable cells can be differentiated from dead cells which do not reduce the indicator dye. A change of color from blue to pink indicates reduction of resazurin and therefore bacterial growth and viability. Absence of bacterial growth can be detected by absence of any color change (i.e., a solution that remains dark blue in color). In summary, a pink color (resazurin reduced to resorufin) indicates bacterial viability and blue color (non-reduced resazurin) indicates bacterial death.

This screening method can be applied for testing the antimicrobial activities of any colored chemical (including natural products) or chemicals causing turbidity in the culture media, mimicking microbial growth. The use of the growth indicator resazurin facilitates differentiation between live and dead microbes in response to a test chemical. Table 1 (below) shows all the tested compounds that were active against all the three tested Gram-positive bacteria ( Staphylococcus aureus, Enterococcus faecalis , and Bacillus subtilis).

Bacterial species

Compound Staphylococcus Enterococcus Bacillus Escherichia Pseudomonas aureus faecalis subtilis coli aeruginosa lh Positive Positive Positive Negative Negative

In Positive Positive Positive Negative Negative lo Positive Positive Positive Positive Negative lp Positive Positive Positive Negative Negative

Iq Positive Positive Positive Negative Negative lr Positive Positive Positive Negative Negative

Is Positive Positive Positive Negative Negative

It Positive Positive Positive Negative Negative lu Positive Positive Positive Negative Negative lv Positive Positive Positive Negative Negative lw Positive Positive Positive Negative Negative lx Positive Positive Positive Positive Negative lz Positive Positive Positive Negative Negative

2a Positive Positive Positive Negative Negative

2b Positive Positive Positive Negative Negative

Table 1- List of active compounds tested against the identified Gram-positive and Gramnegative bacteria

Each compound was tested at a single high concentration of 1 mg/mL. A positive result indicates antibacterial activity expressed as growth inhibition and a negative result indicates no antibacterial activity expressed as bacterial growth at the tested concentration. As shown in Table 1, two of compounds tested (lo and lx) were also active against E. coli at a test concentration of 1 mg/mL.

The compounds exhibiting potential antibacterial activities in the screening phase were further tested for the minimum inhibitory concentrations (MIC), which is the lowest concentration of a chemical required to inhibit visible microbial growth following overnight incubation. The results of this testing are shown in Table 2 (below).

Table 2 - MIC values (pg/mL) for selected compounds tested on S. aureus 25923), 3 clinical isolates (MRSA) and 7 environmental isolates of the genus Staphylococcus

Bacteria of the genus Staphylococcus were selected for MIC investigation due to its clinical importance. Compounds showing activity against S. aureus (ATCC 25923) were tested against 10 multidrug-resistant strains of Staphylococcus species, including: clinical isolates methicillin- resistant Staphylococcus aureus (MRS A) strains MRSA-l, MRSA-2, and MRSA-3, and environmental isolates S. saprophyticus (strains UDH-l, UDH-2, UDH-3 and UDH-4), S. haemolyticus (strains UDH-6 and UDH-7), and S. epidermidis strain UDH-5.

All these strains were resistant to at least three antibiotics from different classes, as confirmed by a disk diffusion assay. The assay was performed according to the Clinical and Laboratory Standards Institute guidelines using the following antibiotic disks (Himedia, India): ceftriaxone (30 m g), cefoxitin (30 m g), cefepime (30 pg), cefpodoxime (10 pg), cefpodoxime/ clavulanic acid (10 pg/5 pg), meropenem (10 pg), gentamicin (50 pg), ciprofloxacin (30 pg) and trimethoprim/sulphamethoxazole (1.25/23.75 pg). The results of this experiment are shown in Table 2.

MIC values of two quality control antibiotics (amikacin and ciprofloxacin) are also shown in Table 2. As shown in Table 2, the tested compounds exhibited bactericidal activity against all the tested strains (MBC/MIC ratio < 4).

The above results indicate that some of the disclosed compounds are 10-20 times more potent than ciprofloxacin, a commonly used antibiotic. For example, compounds lo and lu showed MICs of 3.125 pg/mL for the clinical strain MRSA-l ( Staphylococcus aureus), which is approximately 10 times lower than the MIC of ciprofloxacin (32 pg/mL). For the environmental strain UDH-l (S. saprophyticus), compounds lh, lo, lq, It, lu, lx, and 2a showed MICs of 6.25 pg/mL, which are approximately 10 times lower than MIC of ciprofloxacin (64 pg/mL). For the environmental strain UDH-3 (S. saprophyticus), compounds lh, lo, lu, and 2a showed MICs of 3.125 pg/mL, which are approximately 20 times lower than MIC of ciprofloxacin (64 pg/mL). For the environmental strains UDH-6 and UDH-7 (S. haemolyticus), compounds lo and lu showed MICs of 6.25 pg/mL, which are approximately 10 times lower than MIC of ciprofloxacin (64 pg/mL).

EXAMPLE 4

A time-kill experiment for compound lo revealed fast bactericidal activity with complete elimination of MRSA growth within 30 minutes of exposure to the compound at concentration > 50 pg/mL. The results of this experiment are shown in FIG. 2. As shown in FIG. 2, compound lo was tested at concentrations between 3.125-100 pg/mL and killing was achieved at concentrations between 50-100 mg/mL after 30 minutes of exposure to the compound and at concentrations of 25 and 12.5 pg/mL after 2 and 6 hours of exposure to the compound, respectively. At lower concentrations (3.125-6.25 pg/mL), killing was achieved after 24 hours of exposure to the compound.

In comparison, meropenem, a carbapenem which acts by inhibiting cell wall synthesis, was able to completely inhibit bacterial growth after 4 hours of exposure to the antibiotic at a concentration between 32-64 pg/mL (see FIG. 3). As shown in FIG. 3, meropenem was tested at concentrations between 2-64 pg/mL and killing was achieved at concentrations between 32-64 pg/mL after 4 hours of exposure to the drug and at concentrations of 16 and 8 pg/mL after 6 and 8 hours of exposure to the drug, respectively. At lower concentrations (2-4 pg/mL), killing was achieved after 24 hours of exposure to the compound.

Ciprofloxacin, a fluoroquinolone which acts intracellularly by inhibiting DNA synthesis, exhibited delayed killing effect with complete growth inhibition after 24 hours of exposure to the antibiotic at concentration between 32-256 pg/mL (see FIG. 4). As shown in FIG. 4, ciprofloxacin was tested at concentrations between 8-256 pg/mL and killing was achieved after 24 hours of exposure to the drug at concentrations between 32 and 256 pg/mL.

EXAMPLE 5

Atomic force microscopy (AFM) provides three-dimensional images of the surfaces of living microbial cells in real time and in high resolution with minimal sample preparation. Compound lo was tested against the bacterial strain MRSA-l and the slides were examined under AFM. Specifically, FIGS. 5A1 and 5B1 show an untreated MRSA-l strain at scan sizes of 10 pm ² and 5 pm ² , respectively, and FIGS. 5A2 and 5B2 show a MRSA-l strain treated with compound lo at scan sizes of 10 pm ² and 5 pm ² , respectively. Bacterial cell shape distortion and leakage of the intracellular contents are evident in FIGS. 5A2 and 5B2, suggesting cell wall damage caused by compound lo. Rapid bacterial killing coupled with cell wall damage as evidenced in the AFM images indicate the action of compound lo on the cell wall, as reported for the known antibiotics like polymyxin, which rapidly breaks the cell membrane of Gram-negative bacteria, causing fast cell death. EXAMPLE 5

The effect of the disclosed compounds on the survival of normal human fibroblast cells (F180) at different concentrations (2 and 5 mM) was tested by a sulforhodamine B (SRB) assay. The anticancer drug doxorubicin was used as a positive control and cells treated with DMSO served as a negative control. The antibiotics ciprofloxacin and meropenem were also included in the assay as controls. A surviving fraction of each compound was calculated in reference to the negative control (DMSO). The compounds were found safe because they had a lower effect on the survival of normal fibroblasts compared to the anticancer drug doxorubicin. FIG. 6 shows the experimental results of the surviving fraction of normal human fibroblast cells tested with some of the safe and presently disclosed compounds at concentration of 5 and 2 mM.

In the disclosed compositions, methods, and experimental examples, the compounds discussed were prepared as follows unless otherwise stated.

Chemistry: Purchased chemical reagents and anhydrous solvents were used without further purification. Solvents for extraction and column chromatography were distilled prior to use. TLC analysis were performed with silica gel plates (0.25 mm, 60 F254) using iodine and a UV lamp for visualization. ¹ H and ¹³ C NMR experiments were performed on a 400 MHz instrument, respectively. Chemical shifts are reported in parts per million (ppm) downstream from the internal tetramethylsilane standard. Spin multiplicities are described as s (singlet), bs (broad singlet), d (doublet), dd (doubledoublet), t (triplet), q (quartet), or m (multiplet). Coupling constants are reported in Hertz (Hz). ESI mass spectrometry was performed on a Q-TOF high resolution mass spectrometer or Q-TOF Ultim LC-MS.

Screening the compounds by the cell viability assay: Each test compound was dissolved in DMSO at a concentration of 100 mg/mL. A 96-well Microtiter™ microplate was filled with sterile Mueller-Hinton broth at a volume of 49 pL/well. Each compound was tested in duplicate by adding 1 pL of the compound solution into two adjacent wells. Then the volume was adjusted to 100 pL by adding 50 pL of the bacterial suspension containing 10 ⁶ CFU/mL in Mueller- Hinton broth. Final compound concentration per well was 1 mg/mL and the final bacterial concentration was 5 X10 ⁵ CFU/mL. Two standard antibiotics (ciprofloxacin and amikacin) were used as positive controls. Blank (media only), growth (untreated) and vehicle (DMSO) controls were also included in each experiment.

A volume of 20 pL of the Cell Viability Assay solution was added to each well of the 96-well microplate containing bacteria pre-incubated with the test compounds ( 1 mg/mL) for 20 hours at 37°C. After the addition of the Cell Viability Assay solution, the 96-well microplate was incubated at 37°C for 4 more hours. Pink and blue colors were used to differentiate viable from dead bacterial suspensions, respectively.

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC):

A broth microdilution method was used to measure MIC according to the Clinical and Laboratory Standards Institute guidelines. Two-fold serial dilutions of the test compound (8 concentrations ranged between 100-0.78 pg/mL) were tested. Two reference antibiotics were used as quality controls (ciprofloxacin and amikacin). In each experiment, solvent (DMSO) was used as a negative control, un-treated control (composing of bacteria in Mueller-Hinton broth) was used as a growth control, blank (culture media alone) was used as sterility control. The 96- well microplate with the test compounds was incubated for 20 hours at 37°C. Turbidity in wells indicated bacterial growth. MIC was recorded as the lowest concentration of a compound that completely inhibited any visible bacterial growth after overnight incubation. For the definite determination of MIC, CellTiter-Blue ^® Cell Viability Assay (Promega, USA) was used (as above). The MIC was defined as the lowest compound concentration that prevented the color change of the Cell Viability solution (absence of bacterial growth; thus, suspension remained blue in color). For the definite determination of MIC, CellTiter-Blue ^® Cell Viability Assay (Promega, USA) was used (as above). To each well of the 96-well microplate, 20 pL of the Cell Viability Assay solution was added to the overnight (20 hours culture) and incubated at 37°C for 4 hours. After the end of the incubation, pink and blue colors were used to differentiate viable from dead bacterial suspensions, respectively. The MIC was defined as the lowest compound concentration that prevented the color change. To confirm bacterial death in response to the test compound and to determine the minimum bactericidal concentration (MBC), 50 pL of bacterial suspension in the well representing MIC and wells with X2 MIC, X4 MIC, and X8 MIC were spread on the surface of Mueller-Hinton agar plates and incubated at 37°C for 24 hours. MBC was defined as the lowest concentration of the test compound that needed to kill >99.9% of the microorganism. Thus, the lowest concentration showing no growth on the agar plate was considered as the MBC. To determine whether a test compound is bactericidal or bacteriostatic, MBC/MIC ratio was calculated. Antimicrobials are usually regarded as bactericidal if the MBC/MIC ratio is < 4 and bacteriostatic if >4.

Time-kill study: Compound lo was tested for the time required to kill the bacterial strain MRSA-l. A time -kill assay was performed by the broth micro-dilution method in accordance with the CLSI guidelines. Inoculum suspensions with approximately 5 X 10 ⁵ CFU/mL of exponentially growing bacterial cells were incubated in each well of a 96-well plate. Two-fold serial dilutions of the test compound were added to each well at concentration ranged between X¼ MIC (0.78 pg/mL) to X32 MIC (100 pg/mL). Aliquots of 20 pL were removed from the inoculum culture after timed intervals of incubation (i.e. 0, 30 minutes, 1, 2, 4, 6, 8, and 24 hours), and serial lO-fold dilutions were prepared in 0.85% sodium chloride solution. Then, 10- 100 pL of each dilution (as appropriate) were spread on the surface of Mueller-Hinton agar plates and incubated at 37°C for 24 hours. Plate count technique was used to determine the number of viable cells for each time point. The experiments were performed in triplicate. Meropenem and ciprofloxacin were used as quality control antibiotics.

Examination of bacterial cell walls by atomic force microscope (AFM): Compound lo was tested against the bacterial strain MRS A- 1. Exponentially growing bacterial cells were prepared in Mueller-Hinton broth at a concentration of approximately 10 ⁸ CFU/mL by adjusting the turbidity to 0.5 with a McFarland™ densitometer (DEN-1B, Grant-Bio, UK). Three tubes with a volume of 4 mL of the bacterial suspension were prepared: one tube served as a negative control (growth control) containing the bacterial suspension without any treatment, one tube was used as a positive control with the antibiotic meropenem (a carbapenem which acts by inhibiting cell wall synthesis) at a final concentration of 40 pg/mL, and the third tube contained the test compound lo at a final concentration of 25 m g/mL. The three tubes were incubated at 37°C for 4 hours. Bacterial cells were harvested from 2 mL broth culture by centrifugation at 3,000 x g for 5 minutes. Pelleted bacterial cells were washed with 1 mL of sterile molecular biology grade water (Sigma, US) twice, and then pelleted again by centrifugation at 3,000 x g for 5 minutes. Pelleted bacterial cells were finally suspended in 50 pF molecular biology grade water. Then, a 5 pL of concentrated bacterial suspension was spread on the surface of clean glass slide coated with poly-L-lysine (Knittel-Glass, Germany) and allowed to dry in air for 15 minutes. The slide was washed with sterile water to remove unattached bacterial cells and allowed to dry for 15 minutes. The slides were examined by atomic force microscopy (Nano Science Nanosurf Easy Scan 2 Flex™ AFM, Switzerland). Height, amplitude, and phase images were simultaneously acquired using tapping mode in air. Farge scans (50 pm by 50 pm) were performed on randomly chosen sections of the glass slides to check the morphology of the bacterial population. High- magnification images (both 10 pm by 10 pm and 5 pm by 5 pm) were then taken for visualizing the bacterial cell wall.

Cytotoxicity: The effect of the compounds on the survival of normal human fibroblast cells (F180) at different concentrations (5 and 2 pM) was tested by sulforhodamine B (SRB) assay. The anticancer drug doxorubicin was used as positive control and cells treated with DMSO served as a negative control. The antibiotics ciprofloxacin, amikacin, and meropenem were also included in the assay as controls. The surviving fraction of each compound was then calculated in reference to the negative control (DMSO).