RU2818168C2 - Methods of treating protozoal infections - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to a pharmaceutical composition for treating or preventing an infection or colonization caused by a protozoal agent in a subject, wherein the pharmaceutical composition contains a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof selected from a group consisting of: NCL178, NCL179, NCL180, NCL181, NCL182, NCL183, NCL184, NCL185, NCL187, NCL189, NCL193, NCL194, NCL195, NCL196, NCL197, NCL198, NCL199, NCL200, NCL201, NCL202, NCL203, NCL204, NCL205, NCL206, NCL207, NCL208, NCL209, NCL210, NCL211, NCL212, NCL213, NCL214, NCL218, NCL220, NCL221, NCL222, NCL223, NCL230, NCL238, NCL239, NCL240, NCL241, NCL242, NCL243, NCL244, NCL245, NCL246, NCL247, NCL255, NCL256, NCL262, NCL264, also relates to a medical device for treating or preventing colonization or infection caused by a protozoal agent in a subject, where said medical device contains a pharmaceutical composition and is presented in the form, selected from a group comprising a plaster, bandage, dressing or implant, and also refers to a compound selected from a list of compounds or pharmaceutically acceptable salts thereof, consisting of: 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL240); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,5-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-4-hydroxyphenolate (NCL242); 4,6-bis(2-((E)-3,4-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-amine (NCL243); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalen-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidine-2-amine (NCL246); 4,6-bis(2-((E)-3,4,5-trihydroxybenzylidene)hydrazinyl)pyrimidine-2-amine (NCL247); 4,6-bis(2-((E)-[1,1'-biphenyl]-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL255); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidine-2-amine (NCL262); 4,6-bis(2-((E)-naphthalen-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264).

EFFECT: group of inventions provides a wider range of products in treating or preventing infection or colonization caused by a protozoal agent.

26 cl, 9 tbl, 14 ex, 13 dwg

Description

TECHNICAL FIELD

The present invention relates to compounds of Formula I and their use in methods of treating or preventing a protozoal infection in a subject using a compound of Formula I, to the use of a compound of Formula I in the manufacture of a medicament for treating a protozoal infection in a subject, and to medical devices used in the method of treating or preventing protozoal infection in the subject.

BACKGROUND OF THE ART

Giardiasis, amoebiasis and cryptosporidiosis are the most common protozoal parasitic diseases of the human intestinal tract and are also a common cause of intestinal diseases in animals. Various medications are used to treat these diseases, but many have a significant degree of unreliability in eliminating parasites from the intestines, and many are associated with frequent unwanted side effects that often lead to contraindications for use in some circumstances. There is a need in the art for an improved treatment that will require one or a very small number of doses, will provide a very high level of clearance of the target protozoal agent, will destroy intestinal cysts (to reduce environmental contamination and source of re-infection), will not cause or will cause only mild and well-tolerated side effects, will not have pre-existing resistance, will be safe for use in pregnant and lactating women and other animal species, and/or will be suitable for use as a prophylactic drug.

Many existing antiprotozoal agents, especially for the treatment of giardiasis, have suboptimal actual efficacy. For metronidazole, therapeutic rates against giardia can be high, but very low rates of 60% (measured by protozoan clearance) have also been described in adults and pediatric patients when administered for 5-10 days. Metronidazole and tinidazole are reported to have an average efficacy of approximately 89%. A more recent anti-Guardia agent, nitazoxanide, showed an overall response rate of 75% (measured by protozoan clearance), ranging from 64 to 94%.

To improve compliance, it is highly desirable that the dosing schedule be simple and short-term. As described above, the duration of metronidazole dosing is most often 5-10 days, which allows for the possibility of missed treatment, poor adherence, and reduced effectiveness or treatment failure.

Acquired resistance of giardia to available treatments is widespread and is an increasingly common cause of treatment failure. This is not surprising since most current recommended antiprotozoal agents have been in use for many decades. In a review of antiprotozoal agents approved by the FDA between 1987 and 2013. (Kesselheim, A. S. and J. J. Darrow (2014). "Drug Development and FDA Approval, 1938–2013." New England Journal of Medicine 370(26): e39) only 7 new agents are described for all protozoal diseases (mefloquin in 1989, eflornithine in 1990, halofantrine and atovaquone in 1992, nitazoxanide in 2002, tinidazole in 2004, and the combination of artemether and lumefantrine in 2009). Among these agents, only nitazoxanide and tinidazole are used to treat giardia, and both drugs have an average efficacy of less than 90%.

Often, the adverse effect profile limits the use of antiprotozoal agents. There are many side effects of currently available antiprotozoal agents. For example, side effects for the commonly used metronidazole include metallic taste, nausea, headache, dizziness, leukopenia, insomnia and irritability. CNS cytotoxicity has been reported less frequently, especially at high doses, and alcohol consumption is not recommended when taking metronidazole due to the potential for a disulfiram-like reaction. Metronidazole has been shown to be mutagenic in bacteria; Based on animal studies, the drug is carcinogenic when taken in high doses and over an extended period. Adverse effects of quinacrine include headache, nausea, vomiting, and bitter taste, leading to poor compliance. After administration, yellowing of the skin, urine and sclera may also occur. Blue or black pigmentation of the nails, urticarial rash, and scaly dermatitis may also occur. Other side effects reported are hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, toxic psychosis and exacerbation of psoriasis. Quinacrine, along with other antiprotozoal agents, can cross the placenta and reach the fetus and, as a result, its use is contraindicated during pregnancy due to a possible association with congenital malformations. Other severe side effects reported include psychiatric disorders.

For the treatment of cryptosporidium and amebiasis caused by the amoeba Entamoeba histolytica, available treatments are even more limited than those for giardia, with no consistently effective and specific treatment for cryptosporidium available (Cabada, M. M. and A. C. White, Jr. (2010). “Treatment of cryptosporidiosis: do we know what we think we know?" Curr Opin Infect Dis 23(5): 494-499).

There is an unmet clinical need for antiprotozoal agents with a novel mechanism of action to complement and/or replace currently available antiprotozoal agents whose effectiveness is continually declining due to antiprotozoal resistance mechanisms. Additionally, there remains a need for alternative antiprotozoal agents to treat infections caused by multidrug-resistant protozoa. However, as described in the Pharmaceutical Research and Manufacturers of America's 2013 report “Medicines in Development for Infectious Diseases,” no new antiprotozoal agents have been developed and no expansion of the pipeline has been observed to provide promising results compared with existing treatments.

The object of the present invention is to overcome at least one of the disadvantages inherent in the prior art.

The description of the prior art presented above is intended only to facilitate understanding of the present invention. The description provided does not constitute an acknowledgment or acceptance that any material mentioned is or was part of the public knowledge as of the priority date of this application.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a method of treating or preventing protozoal colonization or infection in a subject, comprising the step of administering a therapeutically effective amount of a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof to the subject, wherein the protozoal infection is caused by a protozoal agent.

In one embodiment of the present invention, the compound of Formula I is:

In another embodiment of the invention, R ₁ represents H, cycloalkyl, Formula II or Formula III;

In another embodiment of the invention, R ₃ is selected from the group consisting of: H; _NH2 ; NHNH _2; O-CH ₂ -CH ₃ ; NH-C(O)-phenyl; N-CH-chlorophenyl; NH-chlorophenyl; NH-CH ₂ -chlorophenyl; NH-N-CH-cycloalkyl; N and R ₄ represents CH-N-CH-CCl- and R ₄ is linked to R ₃ ; Formula IV; Formula V; and Formula VI;

In another embodiment, A ₀ is N, C, CH, or A ₀ is C and A ₀ is linked to R ₄ to form a triazole ring.

In another embodiment, R ₄ is CH-N-CH-CCl-. Preferably, _R4 is linked to _R3 if _R3 is N.

In another embodiment, A ₁ is N, C, NH, -(CH) ₂ -N-, -(C ₆ H ₅ )C-CH-N- or Formula VII;

In another embodiment, A ₂ is N, C, NH, NC(O)-phenyl, or Formula VII, or (CH ₂ ) _n , where n is an integer from 1 to 3, inclusive.

In another embodiment of the invention, A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₈ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ , A ₁₅ , A _16, A ₁₇ , A ₁₈ , A ₁₉ , A ₂₀ , A ₂₁ A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ independently represent C, O, N, NH, S.

In another embodiment of the invention, A ₉ represents C, O, N, NH, NC(O)-O-CH ₂ -CH ₃ , NC(O)-O-CH(CH ₃ ) ₂ , NC(O)-NH -CH ₂ -CH ₃ , NC(O)-NH-CH ₂ -phenyl, NC(O)-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₃ , NC(O)-CH ₂ - furan-2-yl.

In another embodiment, A ₁₀ is C, NH, -N-(CH) ₂ -, -N-CH-C(C ₆ H ₅ )-.

In another embodiment, A ₂₂ is -CH(CH ₃ )-, -N-CH-, -NC(CH ₃ )-, NC(CH ₂ OH)-.

In another embodiment, R ₂ is H, COOH, CH ₂ NH ₂ , CH ₂ OH, CH ₂ NHNH ₂ , methyl, ethyl, propyl, butyl, pentyl, heptyl, cyclopentyl, CH ₂ (cyclohexyl), (CH ₂ ) ₂ (cyclopentyl) or Formula VII, and R ₂ and R ₄ are linked together to form a pyrimidine, pyrazine or triazine ring, or R ₂ and R ₉ are linked together to form a pyrrolidinyl oxindole ring.

In another embodiment, R ₄ is H ₂ , N, NH, O, S, or R ₄ and A ₀ are linked together to form a triazole ring, or R ₄ is N and R ₄ and R ₂ are linked together to form a pyrimidine ring rings.

In another embodiment of the invention, R ₇ represents H, Cl, Br, F, OH, CH ₃ , OCH ₃ , SCH ₃ , CN, CCH, CF ₃ , OCF ₃ , SCF ₃ , NO ₂ , butyl, tert-butyl, dimethylamino, phenyl, n-propyl, iso-propyl, -NH-C(O)-CH ₃ , -(CH) ₂ -COOH, piperazin-1-yl or R ₇ and R ₈ are linked together to form a substituted or unsubstituted, a saturated or unsaturated aliphatic ring, a heterocyclic ring or a benzene ring.

In another embodiment, R ₆ , R ₈ , R ₁₄ , R ₁₆ , R ₂₅ and R ₂₇ are independently H, OH, Cl, F, Br, CH ₃ , CN, OCH ₃ , COOH, NO ₂ , CF ₃ , R ₈ and R ₇ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, R ₁₄ and R ₁₅ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring , R ₈ and R ₉ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or R ₁₄ and R ₁₃ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring rings.

In another embodiment, R ₅ , R ₉ , R ₁₇ , R ₂₄ and R ₂₈ are independently H, O, OH, Cl, F, Br, NH ₂ , CH ₃ , CF ₃ , OCH ₃ , CN, NO ₂ , phenyl, -NH-CH(OH)-CH ₃ , -NH-C(O)-CH ₃ , or R ₉ and R ₈ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring, or benzene ring , or R ₁₃ and R ₁₄ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring.

In another embodiment, R ₁₀ , R ₁₁ , R ₁₉ , R ₂₀ , R ₂₂ and R ₂₃ are independently H, Cl or Br, or R ₁₀ and R ₁₁ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring , heterocyclic ring or benzene ring, or R ₁₉ and R ₂₀ are linked together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or R ₂₂ and R ₂₃ are linked together to form a substituted or unsubstituted, saturated or unsaturated an aliphatic ring, a heterocyclic ring or a benzene ring.

In another embodiment, R _12, R ₁₈ and R ₂₁ are independently H, COOH, CH ₂ NH ₂ , CH ₂ OH, methyl, ethyl, propyl, butyl, cyclopentyl, or R ₁₂ and R ₁₃ are linked together to form a pyrrolidinyl oxindole rings.

In another embodiment, R ₁₅ and R ₂₆ independently represent H, Cl, Br, F, OH, CH ₃ , OCH ₃ , SCH ₃ , CN, CF ₃ , OCF ₃ , SCF ₃ , NO ₂ , CCH, n- butyl, tert-butyl, dimethylamino, phenyl, n-propyl, iso-propyl, -NH-C(O)-CH ₃ , -(CH) ₂ -COOH, piperazin-1-yl, or R ₁₅ and R ₁₄ are linked together with the formation of a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, and

In another embodiment, "----" represents a double bond or a single bond.

In another embodiment of the invention, the compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, is selected from the compounds presented in FIG. 1.

In another embodiment, a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof contains the following features:

where A ₀ represents C;

where A ₁ represents N; or Formula VII;

where A ₂ represents N; or NH;

where A _3, A ₄ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₄ , A ₁₅ represent N; or C;

where A ₅ , A ₁₃ , A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C;

where A ₈ and A ₂₁ represent S;

where A ₉ represents NH;

where A ₁₀ represents N;

where A ₂₂ represents –N-CH-; -NC(CH ₃ )-; or –NC(CH ₂ OH)-;

where R ₁ represents H; Formula II; Formula III; cycloalkyl;

where R ₂ represents H; methyl; ethyl; CH ₂ NHNH ₂ ; _CH2OH ; butyl; cyclopentyl; or Formula VII, and R ₂ is linked to R ₄ to form a pyrimidine ring;

where R ₃ represents NH ₂ ; Formula IV; Formula V; Formula V; Formula VI; NH ₂ , NH-N-CH-cycloalkyl; or O-CH ₂ -CH ₃ ;

where R ₄ represents NH; O; S; or R ₄ is N and R ₄ and R ₂ are linked together to form a pyrimidine ring;

where R ₇ represents H; F; Cl; CF ₃ ; methyl; R ₇ and R ₈ are linked together to form an unsubstituted benzene ring; OH; tert-butyl; phenyl; dimethylamino; iso-propyl; n-propyl; CN; CCH; n-butyl; SCH ₃ ; R ₇ and R ₈ are linked together to form an unsubstituted, unsaturated heterocyclic ring; OCH ₃ ; Br; OCF ₃ ; piperazin-1-yl; or SCF ₃ ;

where R ₆ , R ₈ , R ₁₄ and R ₁₆ independently represent H; OH; F; OCH ₃ ; CF ₃ ; methyl; Cl; CN; Br; R ₈ and R ₇ are linked together to form an unsubstituted benzene ring; R ₈ and R ₇ are linked together to form an unsubstituted, unsaturated heterocyclic ring; R ₁₄ and R ₁₅ are linked together to form an unsubstituted benzene ring; or R ₁₄ and R ₁₅ are linked together to form an unsubstituted, unsaturated heterocyclic ring;

where R ₅ , R ₉ , R ₁₃ and R ₁₇ independently represent H; OH; _NH2 ; Cl; F; OCH ₃ ; OH; -NH-CH(OH)-CH ₃ ;

where R ₁₂ represents H; methyl; ethyl; _CH2OH ; or cyclopentyl;

where R ₁₅ represents H; F; Cl; CF ₃ ; methyl; R ₇ and R ₈ are linked together to form an unsubstituted benzene ring; OH; tert-butyl; phenyl; dimethylamino; iso-propyl; n-propyl; CN; CCH; n-butyl; SCH ₃ ; R ₁₅ and R ₁₄ are linked together to form an unsubstituted, unsaturated heterocyclic ring; OCH ₃ ; Br; OCF ₃ ; piperazin-1-yl; or SCF ₃ ;

where R ₂₄ and R ₂₈ independently represent H; OH; or Cl;

where R ₂₅ and R ₂₇ independently represent H; or OH;

where R ₂₆ represents H; _CH3 ; Br; Cl; OH; dimethylamino; -OP(O)(OEt) ₂ ; CF ₃ ; or F; And

where "----" independently represents a single or double bond.

In another embodiment of the invention, the compound is selected from the group consisting of: NCL008; NCL009; NCL023; NCL025; NCL026; NCL028; NCL029; NCL036; NCL037; NCL039; NCL040; NCL050; NCL061; NCL062; NCL064; NCL065; NCL068; NCL075; NCL076; NCL078; NCL079; NCL080; NCL081; NCL084; NCL085; NCL086; NCL088; NCL089; NCL090; NCL092; NCL094; NCL095; NCL097; NCL098; NCL099; NCL101; NCL104; NCL105; NCL106; NCL108; NCL111; NCL112; NCL114; NCL115; NCL116; NCL118; NCL119; NCL121; NCL122; NCL123; NCL124; NCL125; NCL126; NCL130; NCL131; NCL132; NCL133; NCL135; NCL136; NCL137; NCL138; NCL139; NCL140; NCL141; NCL144; NCL145; NCL146; NCL147; NCL148; NCL150; NCL152; NCL153; NCL154; NCL156; NCL157; NCL158; NCL159; NCL161; NCL162; NCL164; NCL165; NCL166; NCL167; NCL168; NCL169; NCL170; NCL171; NCL172; NCL173; NCL174; NCL176; NCL177; NCL178; NCL179; NCL180; NCL181; NCL183; NCL184; NCL185; NCL186; NCL187; NCL188; NCL189; NCL190; NCL193; NCL194; NCL195; NCL196; NCL197; NCL198; NCL199; NCL200; NCL201; NCL202; NCL203; NCL204; NCL205; NCL206; NCL207; NCL208; NCL209; NCL210; NCL211; NCL212; NCL213; NCL215; NCL216; NCL217; NCL218; NCL219; NCL220; NCL221; NCL222; NCL223; NCL223; NCL224; NCL225; NCL226; NCL227; NCL228; NCL229; NCL230; NCL231; NCL232; NCL233; NCL234; NCL235; NCL236; NCL237; NCL238; NCL239; NCL240; NCL241; NCL242; NCL243; NCL244; NCL245; NCL246; NCL247; NCL248; NCL249; NCL250; NCL251; NCL252; NCL253; NCL254; NCL255; NCL256; NCL257; NCL258; NCL259; NCL260; NCL261; NCL262; NCL263; NCL264; NCL265; NCL266; NCL267; NCL268; NCL269; NCL270; NCL271; NCL272; NCL273; NCL274; and NCL275.

In another embodiment of the invention, the compound is selected from the group consisting of: NCL028; NCL040; NCL062; NCL078; NCL079; NCL080; NCL081; NCL084; NCL088; NCL089; NCL097; NCL099; NCL123; NCL146; NCL157; NCL158; NCL177; NCL179; NCL188; NCL193; NCL195; NCL196; NCL197; NCL199; NCL202; NCL204; NCL205; NCL215; NCL216; NCL217; NCL219; NCL221; NCL245 and NCL246.

In a preferred embodiment of the invention, the compound is selected from the group consisting of: NCL062; NCL078; NCL079; NCL080; NCL081; NCL084; NCL089; NCL097; NCL099; NCL157; NCL158; NCL179; NCL188; NCL193; NCL195; NCL196; NCL199; NCL204; NCL216; NCL217; NCL219; NCL221; NCL245 and NCL246.

In a more preferred embodiment of the invention, the compound is selected from the group consisting of: NCL062; NCL089; NCL097; NCL099; NCL157; NCL179; NCL188; NCL193; NCL195; NCL196; NCL216; NCL219; and NCL221.

In a more preferred embodiment of the invention, the compound is selected from the group consisting of: NCL062; NCL097; NCL099; NCL157; NCL179; NCL188; NCL195; and NCL196.

In another embodiment of the invention, the compound is a chloride salt.

In another embodiment of the invention, the compound is a compound selected from the group consisting of: NCL812 and NCL062. In another embodiment of the invention, the compound is not selected from the group consisting of: NCL812 and NCL062. For example, the compound is NCL099.

In another embodiment of the invention, the compound is a compound selected from the group consisting of: NCL020, NCL021, NCL024, NCL035, NCL072, NCL077, NCL107, NCL109, NCL134, NCL143, NCL143, NCL151, NCL155 and NCL160. In another embodiment of the invention, the compound is not a compound selected from the group consisting of: NCL020, NCL021, NCL024, NCL035, NCL072, NCL077, NCL107, NCL109, NCL134, NCL143, NCL143, NCL151, NCL155 and NCL160. For example, the compound is NCL099.

In another embodiment, the subject is an animal, most preferably selected from the group consisting of: human, canid, feline, bovine, ovine, goat, porcine, avian, fish and equine species.

In another embodiment, the compound is administered to a subject in a dosage range of 0.1 mg/kg to 250 mg/kg body weight.

In another embodiment of the invention, the protozoan agent is selected from the group of the protozoan family containing: Acanthamoeba, Babesia, Balamuthia, Balantidium, Besnoitia, Blastocystis, Chilomastix, Cochlosoma, Cryptosporidium, Cyclospora, Cystoisospora, Cytauxzoon, Dientamoeba, Eimeria, Endolimax, Entamoeba, Giardia, moproteus , Hammondia Hartmannella, Hepatozoon, Hexamita, Histomonas, Isospora, Leishmania (including subgenus Viannia), Leucocytozoon, Naegleria, Neospora, Pentatrichomonas, Plasmodium (including subgenus Plasmodium (Bennettinia) (birds); Plasmodium (Giovannolaia) (birds); Plasmodium (Haemamoeba ) (birds); Plasmodium (Huffia) (birds); Plasmodium (Plasmodium) (higher primates, including Plasmodium brasilianum, cynomolgi, inui, knowlesi, malariae]; Plasmodium (Sauramoeba) (lizards); Plasmodium (Vinckeia)), Sappinia, Sarcocystis, Tetratrichomonas, Theileria, Toxoplasma, Trichomonas, Tritrichomonas, Trypanosoma (subgenera Duttonella, Herpetosoma, Nannomonas, Schizotrypanum, Trypanozoon), Tyzzeria and Wenyonella.

In another embodiment of the invention, the protozoal agent is selected from the group of protozoa listed in Table 1 (see below).

In another embodiment of the present invention, the protozoal agent is a resistant protozoan.

In another embodiment of the invention, the infection or colonization is caused by a mixture of at least two protozoal agents.

In another embodiment, the protozoal agent is resistant to one or more compounds selected from the group presented in Table 2 (see below).

In another embodiment of the invention, the protozoal agent is resistant to the specified compound when the protozoal agent is exposed to the compound in a concentration range selected from the following: 0.001 μg/ml - 10,000 μg/ml; 0.01 µg/ml - 1000 µg/ml; 0.10 µg/ml - 100 µg/ml; and 1 µg/ml - 50 µg/ml.

In another embodiment, the protozoal infection or colonization in the subject is a zoonosis.

In another embodiment of the invention, protozoal infection or colonization in a subject essentially causes an indication selected from the group consisting of: trypanosomiasis (caused by the order Kinestoplastorida of the family Trypanosamidae); amebiasis (caused by the Sarcodina subtype of the Amoebidorida order of the Endamoebidae family); babesiosis (caused by the Piroplasmida class of the Babesiidae family); balantidiosis (caused by the Ciliphora type of the order Trichostomatorida family Balantidiidae); Chagas disease (caused by the order Kinestoplastorida family Trypanosomatidae); cryptosporidiosis (caused by the Coccidea subclass of the family Cryptosporidiidae); giardiasis (caused by the class Zoomastigophora of the order Diplomonadida of the family Diplomonadida, including Giardia lamblia); leishmaniasis (caused by the class Zoomastigophora of the Trypanosomatidae family of the genus Leishmania); Malaria (caused by the genus Plasmodium, including P. falciparum, P. vivax, P. ovale and P. malariae), sarcocystosis (caused by the subclass Coccidia of the order Eucoccidiorida of the family Sarcocystidae); toxoplasmosis (caused by the subclass Coccidia of the order Eucoccidiorida of the family Sarcocystidae, including Toxoplasma gondii); cyclosporiasis (caused by the species Cyclospora cayetanensis); infections caused by free-living amoebae (caused by Naegleria, Acanthamoeba and/or Balamuthia); malaria in non-human primates (caused by the genus Plasmodium); microsporidiosis (caused by Enterocytozzon bieneusi, Encephalitizoon intestinalis and/or E. cuniculi); atypical human trypanosomiasis (T. lewisi, T. evansi, T. brucei, T. vivax, and/or T. congolense); trichomoniasis (caused by Trichomonas vaginalis); amoebic dysentery (caused by Entamoeba histolyticum); and acanthamoebiasis (caused by Acanthamoeba).

In another embodiment, a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof is administered to a subject via oral administration.

In another embodiment, a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof is administered to a subject together with a second antimicrobial agent selected from the group consisting of the compounds listed in Table 2 (see below).

In another embodiment, a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof is administered to a subject via parenteral administration.

In another embodiment, a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof is administered to a subject via topical administration.

In a further aspect, the present invention includes an antiprotozoal pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof and optionally a pharmaceutically acceptable excipient or carrier.

In a further aspect, the present invention includes an antiprotozoal veterinary composition comprising a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof and optionally a veterinarily acceptable excipient or carrier.

In another embodiment of the invention, the specified composition contains an additional antimicrobial agent selected from the group presented in table 2 (see below).

In another embodiment of the invention, the composition is adapted for oral administration.

In another embodiment of the invention, the composition is adapted for parenteral administration.

In another embodiment of the invention, the composition is adapted for topical administration.

In a further aspect, the present invention includes the use of a compound of Formula I or a therapeutically acceptable salt thereof in the manufacture of a medicament for the treatment of protozoal colonization or infection in a subject. In another embodiment of the present invention, use comprises administering to a subject a therapeutically effective amount of a compound of Formula I or a therapeutically acceptable salt thereof. In another embodiment, a compound of Formula I is administered to a subject in a dosage range of 0.1 mg/kg to 250 mg/kg body weight. In one preferred embodiment, the compound is administered to a subject at a dose ranging from the group consisting of: 0.1 mg/kg to 100 mg/kg body weight; from 0.1 mg/kg to 50 mg/kg body weight; from 1 mg/kg to 20 mg/kg body weight; from 1 mg/kg to 10 mg/kg body weight; and from 2 mg/kg to 5 mg/kg body weight. In one preferred embodiment, the compound is administered to a subject at a dose that, after 1 hour, achieves a plasma concentration of the compound in a range selected from the group consisting of: 0.01 μg/ml - 100 μg/ml; 0.1 µg/ml - 10 µg/ml; 1 µg/ml - 8 µg/ml. Preferably, after 1 hour, the dose achieves a plasma concentration of the compound greater than 8 μg/ml. Preferably, after 7 hours, the dose achieves a plasma concentration of the compound greater than 8 μg/ml. Preferably, after 24 hours, the dose achieves a plasma concentration of the compound greater than 8 μg/ml.

In one preferred embodiment, the compound is administered to a subject at a frequency selected from the group consisting of: every 30 minutes; each hour; every 2 hours; every 3 hours; every 4 hours; every 5 hours; every 6 hours; every 8 hours; every 12 hours; once a day; once every 2 days; once a week; two times a week; once every 2 weeks; once a month; once every 2 months; once every 3 months; once every 4 months; once every 5 months; once every 6 months; once a year; once every 2 years; and once.

In another embodiment of the invention, the drug is administered to a subject via oral administration. In another embodiment of the invention, the drug is administered to a subject via parenteral administration. In another embodiment of the invention, the drug is administered to a subject via topical administration.

In a further aspect, the present invention includes a medical device used in a method of treating or preventing protozoal colonization or infection in a subject, the medical device comprising a composition according to the present invention. In another embodiment, the medical device is in a form selected from the group consisting of: a patch, bandage, bandage, or implant applied to the site of protozoal colonization or infection in a subject.

In a further aspect, the present invention includes a method for killing protozoa, comprising the step of contacting the protozoan with a compound of Formula I or a therapeutically acceptable salt thereof.

In a further aspect, the present invention includes the use of a compound of Formula I or a therapeutically acceptable salt thereof to kill protozoa, and said use includes the step of contacting the protozoan with a compound of Formula I or a therapeutically acceptable salt thereof.

In a further aspect, the present invention includes substantially such method, composition, apparatus or use as described herein with reference to the accompanying examples and figures.

In a preferred aspect of the invention, the compound of Formula I is robenidine (also referred to herein as NCL812 and also known as 1,3-bis[(E)-(4-chlorophenyl)methyleneamino]guanidine), which has the following structure:

In one preferred embodiment, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents O; R ₈ and R ₁₄ are CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL101):

In another preferred embodiment, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N and A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₇ and R ₈ represent H; R ₄ represents NH; R ₉ represents Cl; and "----" in Formula I between A ₀ and A ₁ and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL015):

In another preferred embodiment, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are F; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL021):

In another preferred embodiment, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₈ and R ₁₄ represent F; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL023):

In another preferred embodiment, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents Formula IV A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₂ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₉ and R ₁₃ are OCH ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL028):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₈ and R ₁₄ are OCH ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL029):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₈ and R ₉ represent H; R ₄ represents NH; R ₇ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL030):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL035):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are methyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL038):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₈ , R ₁₂ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₉ and R ₁₃ are methyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL039):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₈ and R ₁₄ are methyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL040):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₄ represents NH; R ₇ represents CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL041):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₇ , and R ₉ are H; R ₄ represents NH; R ₈ represents CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL043):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₄ represents NH; R ₇ represents methyl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL044):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₇ , and R ₉ are H; R ₄ represents NH; R ₈ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL052):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₈ and R ₁₄ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL054):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ , R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL061):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL062):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NHNH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ represents methyl, R ₅ , R ₆ , R ₈ and R ₉ represent H; R ₄ represents NH; R ₇ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL069):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , and R ₁₇ are H; R ₄ represents S; R ₈ and R ₁₄ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL070):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ , R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₄ represents NH; R ₇ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL072):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NHNH ₂ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₂ represents methyl; R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₄ represents NH; R ₇ represents CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example of a connection according to this embodiment includes (NCL073):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R _7, R ₁₅ and R ₁₇ are Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL074):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₅ is CF ₃ ; R ₁₇ represents F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL078):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₅ represents F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL079):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₂ represents methyl; R ₁₅ is CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL080):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; R ₁₂ represents methyl; and "----" in Formula IA ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL081):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₇ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL082):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; R ₁₇ represents F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL084):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₅ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₄ represents CN; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL086):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , and R ₁₆ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₇ represents F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL088):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ represents Cl; R ₁₅ is CF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL089):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₉ , R ₁₂ , R ₁₃ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₈ are linked together to form an unsubstituted benzene ring; R ₁₄ and R ₁₅ are linked together to form an unsubstituted benzene ring; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL093):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; A ₁ represents N; A ₂ represents NH; R ₁ represents cyclohexyl; R ₃ represents NH-N-CH-cyclohexyl; R ₄ represents NH; R ₂ represents H; and "----" in Formula I between A ₀ and A ₁ represents a double bond. An example connection according to this embodiment includes (NCL094):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₉ , R ₁₂ , R ₁₃ and R ₁₇ are H; R ₄ represents NH; R ₆ , R ₇ , R ₈ , R ₁₄ , R ₁₅ , and R ₁₆ are OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL097):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are tert-butyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL099):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₇ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are H; R ₄ represents NH; R _5, R ₆ , R ₁₆ , and R ₁₇ are OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL101):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₈ , R ₁₂ , R ₁₄ , and R ₁₇ are H; R ₄ represents NH; R _6, R ₇ , R ₉ , R ₁₃ , R ₁₅ , and R ₁₆ are OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL104):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₁₂ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R _7, R ₈ , R ₉ , R ₁₃ , R ₁₄ , and R ₁₅ are OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL097):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , and R ₁₆ are H; R ₄ represents NH; R ₅ and R ₁₇ represent OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL107):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , and R ₁₇ are H; R ₄ represents NH; R ₆ and R ₁₆ represent OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL108):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₇ are H; R ₄ represents NH; R _6, R ₇ , R ₁₅ , and R ₁₆ are OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL111):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are phenyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL112):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are dimethylamino; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL113):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₇ are H; R ₄ represents NH; R ₆ and R ₁₆ are OCH ₃ ; R ₇ and R ₁₅ represent OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL117):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are isopropyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL120):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are n-propyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL121):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₇ are H; R ₄ represents NH; R ₆ , R ₇ , R ₁₅ , and R ₁₆ are F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL123):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CCH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL126):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , and R ₁₇ are H; R ₄ represents NH; R ₆ and R ₁₆ are Br; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL131):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are butyl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL136):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ is -C(C ₆ H ₅ )-CH-N- and A ₁₀ is -N-CH-C(C ₆ H ₅ )-; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL138):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CH ₃ S; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL140):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula III; R ₃ is Formula VI; A ₀ represents C; R ₂ and R ₂₁ represent H; A ₁ and A ₂₀ represent N; A ₂ and A ₁₉ represent NH; A ₈ and A ₂₁ represent S; R ₄ represents NH; R ₁₀ and R ₁₁ are linked together to form a substituted benzene ring; R ₂₂ and R ₂₃ are linked together to form a substituted benzene ring; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula III and Formula VI represent double bonds. An example of a connection according to this embodiment includes (NCL141):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL143):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₉ , R ₁₂ , R ₁₃ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₈ are linked together to form an unsubstituted heterocyclic ring; R ₁₄ and R ₁₅ are linked together to form an unsubstituted, unsaturated heterocyclic ring; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL146):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ is -(CH) ₂ -N- and A ₁₀ is –N-(CH) ₂ -; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are OCH ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL150):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ represent OH; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL151):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are ethyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL153):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are Br; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL155):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₁₂ , R ₁₄ , R ₁₆ , and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; R ₉ and R ₁₃ are NH ₂ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL157):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are ethyl; R ₅ and R ₁₇ represent OH; R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL158):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are cyclopentyl; R ₅ and R ₁₇ represent OH; R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example of a connection according to this embodiment includes (NCL159):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are OCF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL160):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are piperazin-1-yl; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL161):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents O-CH ₂ -CH ₃ ; A ₁ represents N; A ₂ represents NH; A ₀ , A ₃ , A _{4 ,} A ₅ , A ₆ and A ₇ represent C; R ₂ represents methyl; R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₄ represents NH; R ₇ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL162):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are SCF ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL166):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; R ₅ and R ₁₇ are -NH-CH(OH)-CH ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL168):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ represents Cl. An example of a connection according to this embodiment includes (NCL179):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ represents NH ₂ ; A ₁ represents N; A ₂ and R ₄ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , and A ₇ represent C; R ₅ , R ₆ , R ₈ , and R ₉ are H; R ₂ represents butyl; R ₇ represents Cl; and "----" in Formula I between A ₀ and A ₁ , and all "----" in Formula II represent double bonds. An example connection according to this embodiment includes (NCL188):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ is CH ₃ . An example connection according to this embodiment includes (NCL195):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ represents OH. An example connection according to this embodiment includes (NCL196):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ is Br. An example connection according to this embodiment includes (NCL193):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₆ , R ₂₇ and R ₂₈ are H; and A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ are C. An example of a compound according to this embodiment includes (NCL199):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –NC(CH ₃ )-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ represents Cl. An example connection according to this embodiment includes (NCL204):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ , R ₆ , R ₈ , R ₉ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₆ are H; R ₄ represents NH; R ₇ and R ₁₅ represent Cl; R ₅ and R ₁₇ are F; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL216):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CH ₃ ; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL217):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ is Formula II; R ₃ is Formula IV; A ₁ and A ₁₀ represent N; A ₂ and A ₉ represent NH; A ₀ , A ₃ , A ₄ , A ₅ , A ₆ , A ₇ , A ₁₁ , A ₁₂ , A ₁₃ , A ₁₄ and A ₁₅ represent C; R ₂ and R ₁₂ are methyl; R ₅ , R ₆ , R ₈ , R ₉ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are H; R ₄ represents NH; R ₇ and R ₁₅ are CH3; and "----" in Formula I between A ₀ and A ₁ , all "----" in Formula II and Formula IV represent double bonds. An example connection according to this embodiment includes (NCL219):

In another preferred embodiment of the present invention, the compound is a compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein A ₀ represents C; R ₁ represents H; A ₂ and R ₄ represent N; R ₃ represents NH ₂ ; A ₁ represents Formula VII; R ₂ is Formula VII and R ₂ is linked to R ₄ to form a pyrimidine ring; "----" in Formula I between R ₂ and A ₀ , and between A ₁ and A ₂ represent double bonds; A ₂₂ represents –N-CH-; R ₂₄ , R ₂₅ , R ₂₇ and R ₂₈ represent H; A ₂₃ , A ₂₄ , A ₂₅ , A ₂₆ and A ₂₇ represent C; and R ₂₆ is CF ₃ . An example connection according to this embodiment includes (NCL221):

In another aspect, the present invention includes a compound selected from the list of compounds presented in FIG. 1. In one preferred embodiment, the connection is a connection selected from the following connections: NCL231 - NCL275. In a further aspect, the present invention includes a composition containing a compound of the present invention. In a further aspect, the present invention includes a pharmaceutical composition containing a compound of the present invention together with a pharmaceutically acceptable excipient and/or carrier. In a further aspect, the present invention includes a veterinary composition comprising a compound of the present invention together with a suitable excipient and/or carrier.

Terms used herein have their usual meaning known in the art unless otherwise noted. As used herein, the term robenidine, NCL812 (also known as 1,3-bis[(E)-(4-chlorophenyl)methyleneamino]guanidine) refers to a compound having the following chemical structure:

Preferably, the protozoal agent is selected from the group of genera, subgenera and species listed in Table 1.

Table 1

Acanthamoeba (scientific classification: Eukaryota; Amoebozoa; Discosea; Longamoebia; Centramoebida; Acanthamoebidae), including Acanthamoeba astronyxis, Acanthamoeba castellanii, Acanthamoeba comandoni, Acanthamoeba culbertsoni, Acanthamoeba divionensis, Acanthamoeba echinulata, Acanthamoeba griffini, ba hatchetti, Acanthamoeba healyi, Acanthamoeba jacobsi, Acanthamoeba lenticulata, Acanthamoeba lugdunensis, Acanthamoeba mauritaniensis, Acanthamoeba palestinensis, Acanthamoeba paradivionensis, Acanthamoeba pearcei, Acanthamoeba polyphaga, Acanthamoeba pustulosa, Acanthamoeba quina, Acanthamoeba rhysodes, Acanthamoeba royreba, Acanthamoeba stevensoni, hamoeba terricola, Acanthamoeba triangularis, Acanthamoeba tubiashi, Acanthamoeba sp. and unclassified Acanthamoeba. Babesia (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Piroplasmida; Babesiidae), including Babesia bennetti, Babesia bicornis, Babesia bigemina, Babesia cf. bigemina, Babesia bovis, Babesia cf. bovis, Babesia caballi, Babesia canis (and subspecies Babesia canis canis, Babesia canis rossi and Babesia canis vogeli), Babesia capreoli, Babesia conradae, Babesia crassa, Babesia cf. crassa GU184, Babesia divergens, Babesia cf. divergens, Babesia cf. divergens AdL5, Babesia duncani, Babesia equi, Babesia felis, Babesia cf. felis, Babesia gibsoni, Babesia hongkongensis, Babesia kiwiensis, Babesia lengau, Babesia leo, Babesia major, Babesia microti, Babesia cf. microti, Babesia cf. microti MES-2012, Babesia motasi, Babesia muratovi, Babesia occultans, Babesia odocoilei, Babesia cf. odocoilei, Babesia orientalis, Babesia cf. orientalis, Babesia ovata, Babesia ovis, Babesia poelea, Babesia rodhaini, Babesia vesperuginis, Babesia vitalii, Babesia sp. "venatorum" and unclassified Babesia. Balamuthia (scientific classification: Eukaryota; Amoebozoa; Discosea; Longamoebia; Centramoebida; Balamuthiidae), including Balamuthia mandrillaris Balantidium (scientific classification: Eukaryota; Alveolata; Ciliophora; Intramacronucleata; Litostomatea; Trichostomatia; Vestibuliferida; Balantidiidae), including Balantidium cf. coli, Balantidium ctenopharyngodoni and Balantidium entozoon. Besnoitia (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Besnoitia akodoni, Besnoitia bennetti, Besnoitia besnoiti, Besnoitia caprae, Besnoitia darlingi, Besnoitia jellisoni, Besnoitia neo tomofelis, Besnoitia oryctofelisi and Besnoitia tarandi. Blastocystis (scientific classification: Eukaryota; Stramenopiles), including Blastocystis cycluri, Blastocystis geocheloni, Blastocystis hominis, Blastocystis lapemi, Blastocystis pythoni, Blastocystis ratti and Blastocystis sp. subtypes. Chilomastix (scientific classification: Eukaryota; Fornicata; Retortamonadidae), including Chilomastix caulleryi, Chilomastix mesnili and Chilomastix wenrichi. Cochlosoma (scientific classification: Eukaryota; Parabasalia; Trichomonadida; Trichomonadidae), including Cochlosoma anatis. Cryptosporidium (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Cryptosporidiidae), including Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium cf. bovis G2, Cryptosporidium cf. bovis G3, Cryptosporidium cf. bovis G4, Cryptosporidium cf. bovis G7, Cryptosporidium cf. molnari, Cryptosporidium cf. parvum, Cryptosporidium cf. suis, Cryptosporidium cuniculus, Cryptosporidium erinacei, Cryptosporidium fayeri, Cryptosporidium felis, Cryptosporidium fragile, Cryptosporidium galli, Cryptosporidium hominis, Cryptosporidium macropodum, Cryptosporidium meleagridis, Cryptosporidium molnari, muris, Cryptosporidium parvum, mouse genotype of Cryptosporidium parvum, Cryptosporidium ryanae, Cryptosporidium saurophilum, Cryptosporidium scrofarum, Cryptosporidium serpentis, Cryptosporidium struthionis, Cryptosporidium suis, Cryptosporidium tyzzeri, Cryptosporidium ubiquitum, Cryptosporidium viatorum, Cryptosporidium wrairi, Cryptosporidium xiaoi and unclassified Cryptosporidium. Cyclospora (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Eimeriidae), including Cyclospora cayetanensis, Cyclospora cercopitheci, Cyclospora colobi, Cyclospora papionis and Cyclospora sp. Cystoisospora (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Cystoisospora belli, Cystoisospora felis, Cystoisospora ohioensis, Cystoisospora cf. ohioensis, Cystoisospora rivolta, Cystoisospora suis and Cystoisospora timoni. Cytauxzoon (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Piroplasmida; Theileriidae), including Cytauxzoon felis, Cytauxzoon manul and unclassified Cytauxzoon. Dientamoeba (scientific classification: Eukaryota; Parabasalia; Tritrichomonadida; Dientamoebidae), including Dientamoeba fragilis. Eimeria (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Eimeriidae), including Eimeria acervulina, Eimeria adenoeides, Eimeria ahsata, Eimeria alabamensis, Eimeria albigulae, Eimeria alorani, Eimeria anguillae, Eimeria anseris, antrozoi, Eimeria apionodes, Eimeria arizonensis, Eimeria arloingi, Eimeria arnyi, Eimeria auburnensis, Eimeria auritusi, Eimeria banffensis, Eimeria bovis, Eimeria brunetti, Eimeria bukidnonensis, Eimeria burdai, Eimeria cahirinensis, Eimeria callospermophili, Eimeria canadensis, Eimeria catron ensis, Eimeria caviae, Eimeria chaetodipi , Eimeria chinchillae, Eimeria chobotari, Eimeria coecicola, Eimeria crandallis, Eimeria cylindrica, Eimeria cylindrospora, Eimeria daviesae, Eimeria dipodomysis, Eimeria dispersa, Eimeria ellipsoidalis, Eimeria exigua, Eimeria falciformis, Eimeria faurei, Eimeria flavescens, Eimeria furonis, Eimeria gallopavonis, Eimeria gruis, Eimeria hermani, Eimeria hessei, Eimeria intestinalis, Eimeria irresidua, Eimeria krijgsmanni, Eimeria lamae, Eimeria lancasterensis, Eimeria langebarteli, Eimeria leucisci, Eimeria leucopi, Eimeria macropodis, Eimeria macusaniensis, Eimeria magna, Eimeria maxima, Eimeria media E, imeria meleagrimitis, Eimeria mitis, Eimeria mivati, Eimeria cf. mivati, Eimeria myoxi, Eimeria nafuko, Eimeria necatrix, Eimeria nemethi, Eimeria nieschulzi, Eimeria ontarioensis, Eimeria onychomysis, Eimeria ovinoidalis, Eimeria papillata, Eimeria pavonina, Eimeria percae, Eimeria perforans, Eimeria peromysci, Eimeria phalacrocoraxae, Eimeria pilarensis , Eimeria piriformis, Eimeria polita, Eimeria porci, Eimeria praecox, Eimeria pragensis, Eimeria quokka, Eimeria ranae, Eimeria reedi, Eimeria reichenowi, Eimeria rioarribaensis, Eimeria rutili, Eimeria scabra, Eimeria scholtysecki, Eimeria separata, Eimeria setonicis, Eimeria sevilletensis, Eimeria siliculiformis , Eimeria sinensis , Eimeria stiedai, Eimeria subepithelialis, Eimeria subspherica, Eimeria telekii, Eimeria tenella, Eimeria cf. tenggilingi L12_Ros, Eimeria trichosuri, Eimeria tropidura, Eimeria variabilis, Eimeria vejdovskyi, Eimeria vermiformis, Eimeria vilasi, Eimeria weybridgensis, Eimeria wyomingensis, Eimeria zuernii and unclassified Eimeria. Endolimax (scientific classification: Eukaryota; Amoebozoa; Archamoebae; Entamoebidae), including Endolimax nana and Endolimax piscium. Entamoeba (scientific classification: Eukaryota; Amoebozoa; Archamoebae; Entamoebidae), including Entamoeba bangladeshi, Entamoeba bovis, Entamoeba chattoni, Entamoeba coli, Entamoeba dispar, Entamoeba ecuadoriensis, Entamoeba equi, Entamoeba gingivalis, Entamoeba hartmanni, Entamoeba histolytica, insolita, Entamoeba invadens , Entamoeba moshkovskii, Entamoeba muris, Entamoeba nuttalli, Entamoeba polecki, Entamoeba ranarum, Entamoeba struthionis, Entamoeba suis and Entamoeba terrapinae. Giardia (scientific classification: Eukaryota; Fornicata; Diplomonadida; Hexamitidae; Giardiinae), including Giardia ardeae, Giardia intestinalis (synonyms Giardia duodenalis and Giardia lamblia) (and various various communities), Giardia microti, Giardia muris, Giardia psittaci and unclassified unclassified Giardia . Haemoproteus (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Haemosporida), including Haemoproteus balmorali, Haemoproteus belopolskyi, Haemoproteus chelodinae, Haemoproteus coatneyi, Haemoproteus columbae, Haemoproteus danilewskyii, Haemoproteus elani, moproteus enucleator, Haemoproteus fringillae, Haemoproteus hirundinis, Haemoproteus homobelopolskyi, Haemoproteus iwa, Haemoproteus jenniae, Haemoproteus kopki, Haemoproteus lanii, Haemoproteus magnus, Haemoproteus majoris, Haemoproteus mesnili, Haemoproteus micronuclearis, Haemoproteus minutus, Haemoproteus motacillae, Haemoproteus multipigmentatus, Haemoproteus nucleofascialis, Haemoproteus pallidulus, Haemoproteus pallidus, Haemoproteus parabelopolskyi, Haemoproteus paranucleophilus, Haemoproteus passeris , Haemoproteus pastoris, Haemoproteus payevskyi, Haemoproteus picae, Haemoproteus ptyodactylii, Haemoproteus sacharovi, Haemoproteus sanguinis, Haemoproteus sylvae, Haemoproteus syrnii, Haemoproteus tartakovskyi, Haemoproteus turtur, Haemoproteus vacuola tus and unclassified Haemoproteus. Hammondia (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Hammondia hammondi, Hammondia heydorni, Hammondia triffittae and Hammondia sp. Fox-2000. Hartmannella (scientific classification: Eukaryota; Amoebozoa; Tubulinea; Euamoebida; Tubulinida; Hartmannellidae), including Hartmannella abertawensis, Hartmannella cantabrigiensis, Hartmannella cf. vermiformis [currently known as Vermamoeba vermiformis] and Hartmanella sp. Hepatozoon (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Adeleorina; Hepatozoidae), including Hepatozoon americanum, Hepatozoon ayorgbor, Hepatozoon canis, Hepatozoon catesbianae, Hepatozoon cf. catesbianae, Hepatozoon clamatae, Hepatozoon cf. clamatae, Hepatozoon erhardovae, Hepatozoon felis, Hepatozoon seychellensis, Hepatozoon sipedon, Hepatozoon tuatarae, Hepatozoon ursi and unclassified Hepatozoon. Hexamita (scientific classification: Eukaryota; Fornicata; Diplomonadida; Hexamitidae; Hexamitinae), including Hexamita inflata, Hexamita nelsoni and Hexamita sp. Histomonas (scientific classification: Eukaryota; Parabasalia; Tritrichomonadida; Dientamoebidae), including Histomonas meleagridis and Histomonas sp. Isospora (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Eimeriidae), including Isospora anthochaerae, Isospora gryphoni, Isospora hypoleucae, Isospora insularius, Isospora lesouefi, Isospora orlovi, Isospora peromysis and spora robini. Leishmania (scientific classification: Eukaryota; Euglenozoa; Kinetoplastida; Trypanosomatidae; Leishmaniinae), including the subgenus Leishmania (including the Leishmania aethiopica species complex, Leishmania aristidesi, Leishmania deanei, Leishmania donovani species complex, Leishmania hertigi, Leishmania major species complex, Leishmania major x donovani, Leishmania mexicana species complex, Leishmania tropica species complex, lizard Leishmania (Leishmania adleri, Leishmania gymnodactyli, Leishmania hoogstraali, Leishmania tarentolae, Leishmania sp. NC29/Iran/2007), subgenus Viannia (including Leishmania braziliensis species complex, Leishmania garnhami, Leishman species complex ia guyanensis, Leishmania lainsoni species complex, Leishmania lindenbergi, Leishmania naiffi species complex, Leishmania utingensis and unclassified Leishmania). Leucocytozoon (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Haemosporida), including Leucocytozoon buteonis, Leucocytozoon caulleryi, Leucocytozoon dubreuili, Leucocytozoon fringillinarum, Leucocytozoon gentili, Leucocytozoon lovati, Leucocytozoon macle ani, Leucocytozoon majoris, Leucocytozoon quynzae, Leucocytozoon sabrazesi, Leucocytozoon schoutedeni, Leucocytozoon simondi, Leucocytozoon squamatus, Leucocytozoon toddi, Leucocytozoon ziemanni, Leucocytozoon sp. and unclassified Leucocytozoon. Naegleria (scientific classification: Eukaryota; Heterolobosea; Schizopyrenida; Vahlkampfiidae), including Naegleria americana, Naegleria andersoni, Naegleria angularis, Naegleria antarctica, Naegleria arctica, Naegleria australiensis, Naegleria cf. australiensis, Naegleria canariensis, Naegleria carteri, Naegleria chilensis, Naegleria clarki, Naegleria dobsoni, Naegleria dunnebackei, Naegleria endoi, Naegleria fowleri, Naegleria fultoni, Naegleria galeacystis, Naegleria gallica, Naegleria gruberi, Naegleria cf. gruberi, Naegleria indonesiensis, Naegleria italica, Naegleria jadini, Naegleria jamiesoni, Naegleria laresi, Naegleria lovaniensis, Naegleria mexicana, Naegleria minor, Naegleria morganensis, Naegleria neoantarctica, Naegleria neochilensis, Naegleria neodobsoni, Naegleria neopolaris, Naegleria pagei, Naegleria paradobsoni, Naegleria peruana, Naegleria philippinensis, Naegleria polaris, Naegleria pringsheimi, Naegleria pussardi, Naegleria robinsoni, Naegleria schusteri, Naegleria spitzbergeniensis, Naegleria tenerifensis, Naegleria tihangensis, Naegleria sp. and unclassified Naegleria. Neospora (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Neospora caninum, Neospora hughesi and Neospora sp. Pentatrichomonas (scientific classification: Eukaryota; Parabasalia; Trichomonadida; Trichomonadidae), including Pentatrichomonas hominis. Plasmodium (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Haemosporida), including Plasmodium ovale, Plasmodium simiovale, Plasmodium simium, Plasmodium vivax and subgenus Plasmodium (Bennettinia) (birds); Plasmodium (Giovannolaia) (birds); Plasmodium (Haemamoeba) (birds); Plasmodium (Huffia) (birds); Plasmodium (Laverania) (greater primates, includes Plasmodium falciparum); Plasmodium (Novyella) (birds); Plasmodium (Paraplasmodium) (lizards); Plasmodium (Plasmodium) (greater primates, includes Plasmodium brasilianum, Plasmodium cynomolgi, Plasmodium inui, Plasmodium knowlesi, Plasmodium malariae]); Plasmodium (Sauramoeba) (lizards); and Plasmodium (Vinckeia). Sappinia (scientific classification: Eukaryota; Amoebozoa; Discosea; Longamoebia; Thecamoebida), including Sappinia diploidea, Sappinia pedata and Sappinia sp. Sarcocystis (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Sarcocystis albifronsi, Sarcocystis alces, Sarcocystis alceslatrans, Sarcocystis anasi, Sarcocystis arctica, Sarcocystis arieticanis, Sarcocystis ather idis, Sarcocystis aucheniae, Sarcocystis buffalonis, Sarcocystis calchasi, Sarcocystis campestris, Sarcocystis canis, Sarcocystis capracanis, Sarcocystis capreolicanis, Sarcocystis cf. clethrionomyelaphis JJH-2013, Sarcocystis columbae, Sarcocystis cornixi, Sarcocystis cruzi, Sarcocystis cf. cruzi, Sarcocystis dasypi, Sarcocystis dispersa, Sarcocystis elongata, Sarcocystis falcatula, Sarcocystis cf. falcatula, Sarcocystis fayeri, Sarcocystis felis, Sarcocystis cf. felis WACF-2013, Sarcocystis fusiformis, Sarcocystis gallotiae, Sarcocystis gigantea, Sarcocystis gracilis, Sarcocystis grueneri, Sarcocystis hardangeri, Sarcocystis hirsuta, Sarcocystis hjorti, Sarcocystis hominis, Sarcocystis lacertae, Sarcocystis lamacanis, lindsayi, Sarcocystis miescheriana, Sarcocystis moulei, Sarcocystis mucosa, Sarcocystis muris, Sarcocystis nesbitti, Sarcocystis neurona [equine protozoal myeloencephalitis (EPM) agent], Sarcocystis cf. neurona, Sarcocystis neurona-like protozoan, Sarcocystis ovalis, Sarcocystis oviformis, Sarcocystis ramphastosi, Sarcocystis rangi, Sarcocystis rangiferi, Sarcocystis rileyi, Sarcocystis rodentifelis, Sarcocystis scandinavica, Sarcocystis silva, Sarcocystis sinensis, Sarcocystis singaporens is, Sarcocystis suihominis, Sarcocystis taeniata, Sarcocystis tarandi, Sarcocystis tarandivulpes, Sarcocystis tenella, Sarcocystis truncata, Sarcocystis turdusi, Sarcocystis wobeseri, Sarcocystis zamani, Sarcocystis zuoi and unclassified Sarcocystis. Tetratrichomonas (scientific classification: Eukaryota; Parabasalia; Trichomonadida; Trichomonadidae), including Tetratrichomonas brumpti, Tetratrichomonas buttreyi, Tetratrichomonas gallinarum, Tetratrichomonas limacis, Tetratrichomonas prowazeki, Tetratrichomonas undula and unclassified Tetratrichomonas. Theileria (scientific classification: Eukaryota; Alveolata; Apicomplexa; Aconoidasida; Piroplasmida; Theileriidae), including Theileria annae, Theileria annulata, Theileria annulata strain Ankara, Theileria bicornis, Theileria brachyuri, Theileria buffeli, Theileria cf. buffeli A MEC-2013, Theileria cf. buffeli B, Theileria cf. buffeli C, Theileria cf. buffeli MC-2012, Theileria capreoli, Theileria cervi, Theileria equi, Theileria fuliginosus, Theileria lestoquardi, Theileria cf. lestoquardi (Atbara), Theileria cf. lestoquardi G4, Theileria cf. lestoquardi G6, Theileria luwenshuni, Theileria mutants, Theileria cf. mutants 3, Theileria cf. mutants A MEC-2013, Theileria cf. mutants B MEC-2013, Theileria cf. mutants C MEC-2013, Theileria orientalis, Theileria orientalis complex isolate 9172, Theileria orientalis complex isolate 9196, Theileria orientalis strain Shintoku, Theileria ovis, Theileria cf. ovis G4, Theileria cf. ovis G6, Theileria parva, Theileria parva bovis, Theileria parva lawrencei, Theileria parva parva, Theileria parva strain Muguga, Theileria penicillata, Theileria separata, Theileria sergenti, Theileria sinensis, Theileria cf. sinensis, Theileria cf. sinensis MC-2012, Theileria taurotragi, Theileria uilenbergi, Theileria velifera, Theileria cf. velifera A, Theileria cf. velifera A MEC-2013, Theileria cf. velifera B, Theileria cf. velifera B MEC-2013, Theileria cf. velifera (Syncerus caffer), Theileria youngi, Theileria sp. and unclassified Theileria. Toxoplasma (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Sarcocystidae), including Toxoplasma gondii. Trichomonas (scientific classification: Eukaryota; Parabasalia; Trichomonadida; Trichomonadidae), including Trichomonas canistomae, Trichomonas equibuccalis, Trichomonas gallinae, Trichomonas stableri, Trichomonas tenax and Trichomonas vaginalis. Tritrichomonas (scientific classification: Eukaryota; Parabasalia; Tritrichomonasida; Tritrichomonadae), including Tritrichomonas augusta, Tritrichomonas foetus, Tritrichomonas mobilensis, Tritrichomonas muris, Tritrichomonas nonconforma and Tritrichomonas suis. Trypanosoma (scientific classification: Eukaryota; Euglenozoa; Kinetoplastida; Trypanosomatidae), including subgenus Duttonella (Trypanosoma vivax and Trypanosoma sp. T78), subgenus Herpetosoma (Trypanosoma blanchardi, Trypanosoma lewisi, Trypanosoma rabinowitschae, Trypanosoma rangeli, Trypanosoma sp. SJP-2011), subgenus Nannomonas (Trypanosoma congolense, Trypanosoma simiae, Trypanosoma simiae Tsavo, Trypanosoma sp. Fly9), subgenus Schizotrypanum (Trypanosoma cruzi, Trypanosoma dionisii, Trypanosoma erneyi, Trypanosoma vespertilionis), Trypanosoma indeterminate (Trypanosoma avium, Trypanosoma cf. avium TRM-2012, Trypanosoma bennetti, Trypanosoma binneyi, Trypanosoma boissoni, Trypanosoma caninum, Trypanosoma carassii, Trypanosoma cascavelli, Trypanosoma cervi, Trypanosoma chattoni, Trypanosoma chelodinae, Trypanosoma cobitis, Trypanosoma conorhini, Trypanosoma copemani, Trypanosoma corvi, Trypanosoma culicavium, Trypanosoma cyclops, Trypanosoma danilewskyi, Trypanosoma desterrensis , Trypanosoma everetti, Trypanosoma evotomys, Trypanosoma fallisi, Trypanosoma gallinarum, Trypanosoma godfreyi, Trypanosoma granulosum, Trypanosoma grayi, Trypanosoma grosi, Trypanosoma hastatus, Trypanosoma irwini, Trypanosoma kuseli, Trypanosoma leeuwenhoeki, Trypanosoma livingstonei, Trypanosoma mega, Trypanosoma melophagium, Trypanosoma microti, Trypanosoma minasense, Trypanosoma murmanensis, Trypanosoma musculi, Trypanosoma nabiasi, Trypanosoma neveulemairei, Trypanosoma nudigobii, Trypanosoma ophiocephali, Trypanosoma otospermophili, Trypanosoma percae, Trypanosoma pestanai, Trypanosoma pleuronectidium, Trypanosoma ralphi, Trypanosoma ranarum, Trypanosoma rot atorium, Trypanosoma scelopori, Trypanosoma siniperca, Trypanosoma terrestris, Trypanosoma theileri, Trypanosoma therezieni, Trypanosoma triglae, Trypanosoma varani), subgenus Trypanozoon (Trypanosoma brucei, Trypanosoma cf. brucei Msubugwe, Trypanosoma equiperdum, Trypanosoma evansi and unclassified Trypanosoma). Tyzzeria (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Eimeriidae) species. Wenyonella (scientific classification: Eukaryota; Alveolata; Apicomplexa; Conoidasida; Coccidia; Eucoccidiorida; Eimeriorina; Eimeriidae) species.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Additional features of the present invention are described in more detail in the following description of certain non-limiting embodiments. The present description is included solely for purposes of illustration of the present invention. It should not be construed as limiting the broad scope of the invention, disclosure, or description of the invention set forth above. The description presented is made with reference to the accompanying graphic materials, in which:

In FIG. 1 shows the chemical name and chemical structure of compounds NCL001 to NCL230;

In FIG. 2 is a graph illustrating the activity of NCL099 against Giardia duodenalis;

In FIG. 3 is a diagram illustrating the effect of NCL812 and metronidazole on the adhesion of Giardia duodenalis trophozoites;

In FIG. 4 is a graph illustrating the activity of NCL812 against Giardia duodenalis;

In FIG. 5 is a graph illustrating the activity of NCL062 against Giardia duodenalis;

In FIG. 6 is a diagram illustrating the activity of metronidazole against Giardia duodenalis;

In FIG. 7 is a graph showing erythrocyte lysis based on n-fold minimum inhibitory concentration;

In FIG. Figure 8 shows photographs demonstrating changes in the ultrastructure of giardia trophozoites under the influence of NCL812. A-B: control, 0.1% DMSO, C: metronidazole control, D-G: trophozoites treated with NCL812 (1 hour);

In FIG. 9 is a graph showing the cumulative release of NCL812 and NCL099 from Dosage Form B according to Example 7;

In FIG. 10 is a graph showing the activity of NCL compounds at a concentration of 10 μM against T. brucei (black) and L. donovani (gray);

In FIG. 11 is a table illustrating the physicochemical and metabolic characteristics of nine NCL compounds;

In FIG. 12 is a graph illustrating plasma concentration-time profiles for NCL026, NCL195, NCL259 and NCL812; And

In FIG. 13 is a graph illustrating plasma concentrations of NCL195 in male Swiss mouse mice following intraperitoneal (IP) administration of a mean dose of 43 mg/kg.

DESCRIPTION OF IMPLEMENTATION OPTIONS OF THE INVENTION

general description

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific illustrative methods or compositions described herein. It should also be understood that the terminology used herein is intended only to describe specific embodiments of the invention and is not intended to be limiting.

All publications cited herein, including patents or patent applications, are incorporated by reference in their entirety. However, the applications that are mentioned herein are included only to describe and disclose the methods, protocols and reagents mentioned in the publication that can be used in connection with the present invention. The citation of publications mentioned herein should not be construed as an admission that the present invention is not entitled to date such description back by virtue of the earlier invention.

In addition, the practice of the present invention, unless otherwise indicated, utilizes conventional microbiology techniques that are well within the art. Such traditional technologies are known to experienced specialists.

As used herein and in the appended claims, the singular forms include the plural unless the context clearly indicates otherwise.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although any materials and methods similar or equivalent to those described herein can be used to practice the present invention, preferred materials and methods are described herein.

The invention described herein may include one or more ranges of values (eg, size, concentration, dose, etc.). A range of values should be understood to include all values within the range, including the values that define the range, as well as values adjacent to the specified range that produce the same or substantially similar result as the values immediately adjacent to the value. which defines the limit of the range.

The pharmaceutical or veterinary compositions of the present invention may be administered in various unit dosages depending on the route of administration, the target site, the physiological condition of the patient, and the administration of other drugs. For example, unit dosage form suitable for oral administration includes solid dosage forms such as powders, tablets, pills and capsules, and liquid dosage forms such as elixirs, syrups, solutions and suspensions. The active ingredients can also be administered parenterally in the form of sterile liquid dosage forms. Gelatin capsules may contain the active ingredient and inactive ingredients such as powdered carriers, glucose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate and the like.

The expression "therapeutically effective amount" as used herein refers to an amount sufficient to inhibit protozoal growth associated with protozoal infection or colonization. Thus, reference to administering a therapeutically effective amount of a compound of Formula I in accordance with the methods or compositions of the present invention refers to a therapeutic effect in which significant protozoacid or protozoastatic activity causes significant inhibition of protozoal infection. The term "therapeutically effective amount" as used herein refers to a sufficient amount of the composition to provide the desired biological, therapeutic and/or prophylactic result. Desired results include elimination of protozoal infection or colonization, or reduction and/or alleviation of signs, symptoms or causes of disease, or any other desired change in the biological system. The effective amount in any individual case can be determined by one skilled in the art through routine experimentation. In relation to a pharmaceutical or veterinary composition, effective amounts may be doses that are recommended to modulate the disease state or its signs or symptoms. Effective amounts vary with the composition used and the route of administration used. Effective amounts are typically optimized based on pharmacokinetic and pharmacodynamic characteristics as well as various patient-specific factors such as age, weight, sex, etc., and the area affected by the disease or disease-causing microbes.

As used herein, the terms “treat” or “cure” refer to the complete or partial elimination of the symptoms and signs of a pathological condition. For example, when treating a protozoal infection or colonization, treatment provides complete or partial elimination of signs of infection. Preferably, when treating an infection, the treatment reduces or eliminates infecting protozoal pathogens, resulting in microbiological cure.

In this context, the term "protozoa" refers to members of the large domain of eukaryotic single-celled microorganisms. Typically a few micrometers in length, protozoa have a variety of shapes, from spheres to rods and spirals, and may be present as single cells or may be present in linear chains or clusters of varying numbers and shapes. Preferably, the terms "protozoa" and its adjectives "protozoan", "protozoans" refer to protozoa. These terms may refer to a strain susceptible to the action of antiprotozoal agents or to a strain resistant to the action of antiprotozoal agents.

As used herein, the term “resistant protozoa” refers to a protozoal isolate that exhibits resistance to any of the following antimicrobial agents listed in Table 2.

Table 2: Antimicrobial agents

Chemical class Examples 4-aminoquinoline Amodiaquine, chloroquine, hydroxychloroquine, piperaquine (bis-4-aminoquinoline) 8-aminoquinoline Bulaquin, pamaquin, primaquine, tafenoquine Acetamide Thiolutin Acridine dye Acriflavine, mepacrine (quinacrine) Alkylphosphocholine Miltefosine Allylamine Terbinafine Aminoglycosides Paromomycin Aminophenanthridium Homidium, isometamidium chloride Aminopyridine antimalarials MMV390048 Pentavalent antimony preparations Sodium stibogluconate, meglumine antimonate Arsenic preparations (trivalent and pentavalent) Acetarsol (5+), arstinol (3+), carbarsone (5+), difetarsone (5+), glycobiarsol (5+), melarsomin (3+), melarsoprol (3+), nitarsone (5+), oxofenarsine ( 3+), roxarsone (5+), triparsamide (5+) Arylamino alcohol Halofantrine, lumefantrine, quinine/quinidine Azonaphthalene dyes Trypan blue, trypan red Azoles (triazoles and imidazoles) Albaconazole, itraconazole, ketoconazole, posaconazole, ravuconazole Benzamide Zoxamide Benzenediol Resveratrol Benzimidazoles and probenzimidazoles Albendazole, fenbendazole, febantel, mebendazole, omeprazole Bicyclohexylammonium Fumagillin Carbamate Disulfiram Cinnamamidoadenosine Puromycin Coumarin Flocumafen Diamidines Amicarbalide, diminazene diaceturate, imidocarb dipropionate, pafuramidine, pentamidine isethionate, fenamidine isethionate, propamidine, stilbamidine Dichloroacetamide Clefamide, etofamide, teclosan Dichloroacetamide Diloxanide furoate Difluoromethylornithine Eflornithine Dihydrofolate reductase/thymidylate synthase inhibitors Diaveridine, ormethoprim, pyrimethamine, trimethoprim Dihydroorotate dehydrogenase (DHODH) inhibitors Dinitroaniline Trifluralin, oryzalin Dinitrocarbanilide + pyrimidinol Nicarbazine Dithiocarbamate Tiram Ethoxybenzoic acid Etopabat Fluoroquinolones Ciprofloxacin, enrofloxacin, marbofloxacin Guanidines Chlorproguanil, cycloguanil, lauroguanide, proguanil, robenidine Halogenated 8-hydroxyquinoline Iodoquinol, chloroquinaldone, tilbroquinol, broxyquinoline, diiodohydroxyquinoline, clioquinol Hydroxyoxo-cyclohexenecarbaldehydeoxime Sethoxydim, tralkoxydim, alloxydim, clethodim and cycloxydim Hydroxyquinolones Buquinolate, decoquinate, nequinate Imidazolopiperazine Kaf156 Isoquinoline Emetine/dehydroemetine Lincosamides Clindamycin, lincomycin Macrolides Azithromycin, clarithromycin, erythromycin, roxithromycin, spiramycin Methylquinolinium Quinapyramine, quinuronium sulfate Miscellaneous Pyridabene Naphthoquinones Atovaquone, buparvaquon, parvaquon Naphthyridine Pyronaridine Nitrobenzamides Aclomid, dinitolmid Nitrofurans Nifurtimox, furaltodone, furazolidone, nifuratel, nifuroxime, nifurzol Nitroimidazoles Azanidazole, benznidazole, carnidazole, dimetridazole, fexnidazole, ipronidazole, metronidazole, nimorazole, ornidazole, propenidazole, ronidazole, satranidazole, secnidazole, ternidazole, tinidazole Nitrothiazoles Nitazoxanide, aminotrozole (nithiamide), forminitrazole, niridazole, tenonitrozole Organometallic antiprotozoal agents Auranofin, Ferroquine Oxaborole (including benzoxaboroles) SCYX-7158 Phenoxyphenol Triclosan phenylsulfamide Tolylfluanid Phosphonic acid derivative Fosmidomycin Phosphonomethylglycine Glyphosate Phosphoramidothionic acid Amiprofos-methyl Polyene Amphotericin B, mepartricin, hahimycin, gamycin Polyether ionophores Laidlomycin, lasalocid, maduramicin, monensin, narasin, salinomycin, semduramycin Polypeptide Bacitracin (zinc, methylene disalicylate), cecropins, cyclosporins, dermaseptin, magainins, tachyplesin, thiostrepton Polysulfonated naphthylamine Suramin Propylphosphonic acid Fosmidomycin Purinamine Arprinocid Pyrazolopyran Pyrazolopyrimidine Allopurinol Pyridinols Clopidol Pyrimidine Fenarimol Pyrrolidinediol Anisomycin Quinazolinone Febrifugin, halofuginone Quinoline Mefloquine, nequinate (methylbenzoquat), quinfamide, tiliquinol Quinoxaline Carbadox Rifamycin Rifaximin Spiroindolon KAE609 (formerly NITD609), cypargamine Strobilurin Fluacripyrim, azoxystrobin, trifloxystrobin, dimoxystrobin Sulfonamides Sulfadiazine, sulfadimethoxine, sulfadoxine, sulfaguanidine, sulfamethazine (sulfadimidine), sulfamethoxazole, sulfanitran, sulfaquinoxaline, sulfamethoxypyrazine, cyazofamide Sulfone Dapsone Tetracyclines Chlortetracycline, doxycycline, oxytetracycline, tetracycline, tigecycline Tetraoxanes Thiamine analogues Amprolius Thiophenone Thiolactomycin Translation elongation factor 2 (eEF2) inhibitor DDD107498 Triazine Clazuril, diclazuril, ponazuril, toltrazuril Triazole Bittertanol Trioxane (sesquiterpene lactones, artemisinins) Artemether, artesunate, dihydroartemisinin, artemotil, artemisinin, artitere, artemisone Trioxolane (including ozonides) Arterolan, OZ277, OZ439

Pharmaceutically and veterinarily acceptable salts include salts that retain the biological effectiveness and properties of the compounds described herein and that are not biologically or otherwise undesirable. In many cases, the compounds described herein can form salts with acids and/or bases due to the presence of amino groups and/or carboxyl groups or the like. Suitable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, including, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines such as, by way of example only, alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl )amines, alkenylamines, dialkenylamines, trialkenylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines ines, cycloalkenylamines , di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines us, mixed di- and triamines, in wherein at least two substituents on the amine are different and selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic moiety, and the like. Also included are amines in which two or three substituents, together with the nitrogen atom of the amino group, form a heterocyclic or heteroaryl group.

Pharmaceutically and veterinarily acceptable acid addition salts can be prepared from inorganic and organic acids. Inorganic acids that can be used include, by way of example only, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Organic acids that may be used include, by way of example only, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid , benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

Pharmaceutically or veterinarily acceptable salts of the compounds useful herein can be synthesized from a parent compound that contains a basic or acidic moiety using conventional chemical methods. Typically, such salts can be prepared by reacting the compounds in free acid or base forms with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof; in general, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred. A list of suitable salts is provided in Remington, Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, Pennsylvania, (1985), p. 1418, the description of which is incorporated herein by reference. Examples of such acceptable salts are iodide, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methyl benzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, γ-hydroxybutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexine-1,4-dioate, hexine-1,6-dioate, caproate, caprylate, chloride, cinnamate, citrate, decanoate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, propanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartrate and etc.

The pharmaceutical or veterinary compositions of the present invention may be formulated in the usual manner, optionally together with other pharmaceutically acceptable excipients, in forms suitable for oral, parenteral or topical administration. Routes of administration may include parenteral, such as intramuscular, subcutaneous, and intravenous, oral, topical, and direct site administration, such as intraocular, intraauricular, intrauterine, intranasal, intramammary, intraperitoneal, intralesional, etc.

The pharmaceutical or veterinary compositions of the present invention may be formulated for oral administration. Traditional inactive ingredients may be added to provide desired color, flavor, stability, buffering capacity, dispersion and other known desired characteristics. Examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white dyes, etc. Standard diluents can be used to obtain compressed tablets. Tablets and capsules can be formulated as sustained release formulations to release the drug continuously over a period of time. Compressed tablets may be in the form of sugar-coated or film-coated tablets, or as enteric-coated tablets for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and/or flavoring agents to improve patient compliance with therapy. For example, an oral dosage form containing the compounds of the present invention may be a tablet containing any or a combination of the following excipients: anhydrous calcium phosphate, microcrystalline cellulose, lactose, hydroxypropyl methylcellulose and talc.

The compositions described herein may be presented in liquid dosage form. Examples of preferred liquid compositions include solutions, emulsions, injections, and capsule solutions. The liquid dosage form may contain a solution that contains the therapeutic agent dissolved in a solvent. In general, any solvent that has the desired effect, in which the therapeutic agent is dissolved and which can be administered to the subject, can be used. In general, any concentration of therapeutic agent that has the desired effect can be used. In some embodiments, the dosage form is a solution that is an unsaturated, saturated, or supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components. In some embodiments, the resulting solution is an in situ gel forming dosage form. Solvents and types of solutions that can be used are known to those skilled in the art of drug delivery technology.

The compositions described herein may be presented in the form of a liquid suspension. Liquid suspensions can be prepared by standard methods known in the art. Examples of liquid suspensions include microemulsions, complexing compounds, and stabilizing suspensions. The liquid suspension can be in undiluted or concentrated form. Liquid suspensions for oral use may contain suitable preservatives, antioxidants and other excipients which are known in the art to act as one or more of dispersing agents, suspending agents, thickening agents, emulsifiers, humectants, solubilizing agents, stabilizing agents, flavorings and sweeteners, coloring agents, etc. The liquid suspension may contain glycerin and water.

The composition described herein may be in the form of a paste for oral administration. The oral paste can be prepared by standard methods known in the art.

The composition described herein may be presented in a liquid dosage form for injection, such as intramuscular injection, and may be prepared by methods known in the art. For example, the liquid dosage form may contain polyvinylpyrrolidone K30 and water.

The composition described herein may be in the form of topical preparations. The topical preparation may be in the form of a lotion or cream prepared by methods known in the art. For example, the lotion may be formulated with an aqueous or oily base and may contain one or more excipients known in the art to act as thickeners, emulsifiers, flavoring or fragrance agents, preservatives, chelating agents, pH modifiers, antioxidants and so on. For example, a topical dosage form containing one or more compounds of the present invention may be a gel containing any of the following excipients or a combination thereof: PEG 8000, PEG 4000, PEG 200, glycerin, propylene glycol. Compound NCL812 can be further formulated as a solid dispersion using SoluPlus (BASF, www.soluplus.com) and can be formulated with any or a combination of the following excipients: PEG 8000, PEG 4000, PEG 200, glycerin and propylene glycol.

For administration as an aerosol, the composition of the present invention is provided in a finely divided form along with a non-toxic surfactant and a nebulizer. The surfactant is preferably soluble in the spray agent. Such surfactants may include esters or partial esters of fatty acids.

Alternatively, the compositions of the present invention may be formulated for delivery by injection. For example, the compound is delivered by injection using one of the following routes: intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous.

Alternatively, the compositions of the present invention may be formulated using nanotechnology-based drug delivery methods known in the art. Nanotechnology-based drug delivery systems have the advantage of improving bioavailability, patient compliance, and reducing side effects.

The dosage form of the composition according to the present invention includes a preparation of nanoparticles in the form of nanosuspensions or nanoemulsions, based on the solubility of the compound. Nanosuspensions are dispersions of nanosized drug particles obtained by bottom-up or top-down technology and stabilized by suitable excipients. This approach can be applied to the compounds of the present invention, which may have poor water and fat solubility, to improve saturation solubility and dissolution characteristics. An example of such technology is described in the publication of Sharma and Garg (2010) (Pure drug and polymer-based nanotechnologies for the improved solubility, stability, bioavailability, and targeting of anti-HIV drugs. Advanced Drug Delivery Reviews, 62: pp. 491-502) . Saturation solubility is understood as a compound-specific constant that depends on temperature, properties of the dissolution medium and particle size (less than 1-2 μm).

The composition according to the present invention may be presented in the form of a nanosuspension. For nanosuspensions, increasing surface area can lead to increased solubility at saturation. Nanosuspensions are colloidal drug delivery systems consisting of particles less than 1 μm in size. The compositions of the present invention may be in the form of nanosuspensions, including nanocrystalline suspensions, solid lipid nanoparticles (SLNs), polymer nanoparticles, nanocapsules, polymer micelles and dendrimers. Nanosuspensions can be prepared using a top-down approach in which larger particles can be reduced to nanometer size through various techniques known in the art, including wet grinding and high-pressure homogenization. Alternatively, nanosuspensions can be produced using bottom-up technology, in which controlled precipitation of particles from solution can be accomplished.

The composition according to the present invention may be presented in the form of a nanoemulsion. Nanoemulsions, as a rule, are transparent systems of the “oil in water” or “water in oil” type, with droplet sizes of 100-500 nm, in which the compounds in question are present in the hydrophobic phase. The preparation of nanoemulsions may provide improved solubility of the compounds of the present invention described herein, resulting in improved bioavailability. Nanosized suspensions may also contain agents for electrostatic or steric stabilization, such as polymers and surfactants. Compositions in the form of SLN may contain biodegradable lipids such as triglycerides, steroids, waxes, and emulsifiers such as soy lecithin, egg lecithin and poloxamers. Preparation of the SLN formulation may involve dissolving/dispersing the drug in a molten lipid followed by hot or cold homogenization. When using hot homogenization, the molten lipid phase can be dispersed in the aqueous phase to form an emulsion. It can be solidified by cooling to form SLN. Using cold homogenization, the lipid phase can be solidified in liquid nitrogen and reduced to micron size. The resulting powder can be homogenized under high pressure in an aqueous surfactant solution.

The compounds of Formula I described herein can be dissolved in oils/liquid lipids and stabilized to form an emulsion dosage form. Nanoemulsions can be produced using high-energy and low-energy droplet size reduction technologies. High energy methods may include high pressure homogenization, sonication and microfluidization. When using a low-energy method, solvent diffusion and phase inversion lead to the spontaneous formation of a nanoemulsion. The lipids used in nanoemulsions may be selected from the group consisting of triglycerides, soybean oil, safflower oil and sesame oil. Other components such as emulsifiers, antioxidants, pH modifiers and preservatives may also be added.

The composition may be in the form of a controlled release drug and may contain a degradable or nondegradable polymer, hydrogel, organogel, or other physical construct that modifies the release of the compound. It is understood that such dosage forms may contain additional inactive ingredients that are added to provide desired color, stability, buffering capacity, dispersion, or other known desired characteristics. Such dosage forms may further comprise liposomes such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use in the present invention can be prepared from standard vesicle-forming lipids, typically including neutral and negatively charged phospholipids and a sterol such as cholesterol.

Dosage forms according to the present invention may have the advantage of increased solubility and/or stability of the compounds, in particular for dosage forms obtained using nanotechnological methods. Such increased stability and/or stability of compounds of Formula I may improve bioavailability and enhance drug exposure for oral and/or parenteral dosage forms.

As used herein, unless the context otherwise requires, the term “comprises” or variations thereof such as “comprise” or “comprising” are to be understood to include the specified integer or group of integers, but not to exclude any other integer or groups of integers.

EXAMPLES

EXAMPLE 1: Obtaining analogues of NCL812.

Materials and methods

NCL812

Analytical grade NCL812 was obtained with a determined efficiency of 960 mg/g (i.e. 96%). The powder was stored in a closed sample container away from direct sunlight and at room temperature at the research facility. Aliquots (1 mL) of the stock solution (containing 25.6 mg/mL NCL812 in DMSO) were prepared and stored at −80 °C and thawed immediately before use.

Synthesis and testing of NCL812 analogues

Analogues of NCL001-NCL275 shown in Fig. 1 was synthesized by standard methods known in the art. For example, to obtain compounds NCL097; NCL157; NCL179; NCL188; NCL195; and NCL196 used the following methods:

NCL 097 (2,2'-bis[(3,4,5-trihydroxyphenyl)methylene]carbonimido-dihydrazide hydrochloride)

A suspension of 3,4,5-trihydroxybenzaldehyde (412.0 mg, 2.673 mmol, 2.21 eq.) and N,N′-diaminoguanidine hydrochloride (152.0 mg, 1.211 mmol) in EtOH (5 ml) was treated with microwave radiation ( 150 W) at 100 °C for 10 minutes. The reaction mixture was then allowed to cool to ambient temperature. The resulting precipitate was collected and washed with ice-cold EtOH (5 mL) and Et ₂ O (5 mL) to give carbonimido dihydrazide (369.0 mg, 77%) as a pale brown solid. Tpl. 292 °C (decomposed). ^1H NMR (300 MHz, DMSO-d6) δ 9.06 (w s, 6H), 8.25 – 8.01 (m, 4H), 6.83 (s, 4H). ¹³ C NMR (75 MHz, DMSO-d6) δ 152.2, 149.7, 146.2, 136.5, 123.7, 107.4. MS-NR (IER ⁺ ): 361.95 [M + 1] ⁺ .

NCL157 (2,2'-bis[(2-amino-4-chlorophenyl)methylene]carbonimido-dihydrazide hydrochloride)

Synthesis of 2-amino-4-chloro-N-methoxy-N-methylbenzamide. To a solution of 2-amino-4-chlorobenzoic acid (5.6691 g, 33.041 mmol, N,O-dimethylhydroxylamine hydrochloride (5.7504 g, 58.954 mmol, 1.78 eq.), N-(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (7.7925 g, 40.649 mmol, 1.23 eq.) and N-hydroxybenzotriazole hydrate (5.2371 g, 38.793 mmol) (anhydrous), 1.17 eq.) in DMF ( 100 mL), diisopropylethylamine (18.0 mL, 13.4 g, 104 mmol, 3.15 eq.) was added and the brown solution was stirred at room temperature for 7 hours. The reaction mixture was then concentrated in vacuo, then diluted with 1M NaOH (100 ml) and extracted with CH ₂ Cl ₂ (3 x 100 ml). The combined organic extracts were washed with 1M HCl (100 ml), then dried over MgSO ₄ and concentrated in vacuo to give a brown syrup. The resulting oily substance was then further dried at 60 °C under high vacuum to obtain crude Weinreb amide (7.021 g, 99%) as a brown syrupy substance that crystallized on standing. The crude material was used without further purification. ^1H NMR (400 MHz, _CDCl3 ) δ 7.24 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 18 Hz, 1H), 6.54 (dd, J = 8 ,4, 1.9 Hz, 1H), 4.75 (s, 2H), 3.48 (s, 3H), 3.24 (s, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 169.2, 148.4, 137.1, 130.6, 116.6, 116.1, 115.0, 61.1, 34.0.

Synthesis of 2-amino-4-chlorobenzaldehyde. Crude 2-amino-4-chloro-N-methoxy-N-methylbenzamide (751.1 mg, 3.532 mmol) was divided into batches of approximately 120 mg and each was dissolved in THF (10 ml) and cooled to 0 °C, then each add LiAlH ₄ (2M in THF, 0.5 ml) and stir the solutions for 16 hours, allowing the reaction mixtures to reach room temperature. The reaction mixtures were quenched with saturated NH ₄ Cl (1 ml), then combined, diluted with saturated NaHCO ₃ (160 ml) and extracted with CHCl ₃ (2 x 150 ml, 1 x 75 ml). The combined organic layers were dried over MgSO ₄ and concentrated in vacuo to give crude benzaldehyde (463.3 mg, 85%) as yellow-orange crystals. The material was used without additional purification. ¹ H (400 MHz, CD ₃ OD) 9.77 (d, J = 0.7 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 6.83 – 6.71 ( m, 1H), 6.63 (dd, J = 8.4, 1.9 Hz, 1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 194.6, 153.0, 142.5, 138.4, 118.3, 116.8, 116.1.

Synthesis of 2,2'-bis[(2-amino-4-chlorophenyl)methylene]carbonimido-dihydrazide hydrochloride. A suspension of 2-amino-4-chlorobenzaldehyde (128.0 mg, 0.823 mmol, 1.78 eq.) and N,N′-diaminoguanidine hydrochloride (58.0 mg, 0.462 mmol) in EtOH (2 ml) was treated with microwave radiation ( 100 W) at 60 °C for 5 minutes. Most of the solvent was then removed in vacuo, EtOH (1 ml) was added and the flask was transferred to the freezer to allow crystallization. The resulting precipitate was collected and washed with EtOH (1 mL) to give carbonimido dihydrazide (21.0 mg, 13%) as a pale yellow solid. ^1H NMR (400 MHz, DMSO- _d6 ) δ 11.71 (w s, 2H), 8.40 (s, 2H), 8.37 (s, 2H), 7.29 (d, J = 8 .4 Hz, 2H), 6.87 (d, J = 2.0 Hz, 2H), 6.73 (w s, 4H), 6.59 (dd, J = 8.3, 2.0 Hz, 2H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 152.1, 151.5, 148.9, 136.0, 134.7, 115.1, 114.5, 112.8.

NCL179 (4,6-bis(2-(4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine)

A suspension of 2-amino-4,6-dihydrazinylpyrimidine (67.3 mg, 0.434 mmol) and 4-chlorobenzaldehyde (198.8 mg, 1.414 mmol, 3.26 eq.) in EtOH (25 ml) was refluxed for 16 hours. At this time, the condenser was removed and the solution was concentrated to a volume of about 1 ml, and the resulting precipitate was hot filtered and washed with Et ₂ O (10 ml) to obtain aminopyrimidine (42.8 mg, 25%) as an off-white amorphous powder. Tpl. 275 °C (decomposed). ^1H NMR (400 MHz, DMSO) δ 10.70 (s, 2H), 8.02 (s, 2H), 7.67 (d, J = 8.4 Hz, 4H), 7.52 (d, J = 8.4 Hz, 4H), 6.28 (s, 1H), 5.85 (s, 2H). ¹³ C NMR (101 MHz, DMSO) δ 162.8, 162.6, 138.8, 134.1, 133.1, 128.9, 127.6, 73.5.

NCL188 ((E)-2-(1-(4-chlorophenyl)pentylidene)hydrazine-1-carboximidamide hydrochloride)

A suspension of 1-(4-chlorophenyl)pentanone (1.8319 g, 9.3146 mmol, 1.95 eq.) and aminoguanidine hydrochloride (527.6 mg, 4.773 mmol) in EtOH (15 ml) was heated at 65 °C in within 16 hours. The crude reaction mixture was cooled to room temperature, then diluted with Et ₂ O (60 ml) and cooled to 0 °C to precipitate unreacted aminoguanidine hydrochloride (174.5 mg). The mother liquors were then concentrated in vacuo and the residue was dissolved in Et ₂ O (20 ml). The solution was then boiled and hexane (10 ml) was added to give carboximidamide as a cream-colored solid. ^1H NMR (400 MHz, DMSO) δ 11.54 (s, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.90 (s, 3H), 7.47 (d, J = 8.6 Hz, 2H), 2.91 – 2.82 (m, 2H), 1.48 – 1.32 (m, 4H), 0.89 – 0.84 (m, 3H). ¹³ C NMR (101 MHz, DMSO) δ 156.2, 153.8, 134.8, 134.4, 128.7, 128.4, 28.1, 26.6, 22.0, 13.8

NCL195 (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine)

A suspension of 2-amino-4,6-dihydrazinopyrimidine (58.9 mg, 0.380 mmol) and 4-methylbenzaldehyde (0.10 ml, 100 mg, 0.832 mmol, 2.19 eq.) in EtOH (4 ml) was heated at reflux refrigerator for 16 hours. The reaction mixture was cooled to room temperature, then the granular precipitate was collected by washing with Et ₂ O (20 ml). The "pellets" were then broken up and the solid was further washed with Et ₂ O (10 ml) to obtain pyrimidine (85.8 mg, 63%) as a white "loose" powder. Tpl. 274-276 °C. ^1H NMR (400 MHz, DMSO) δ 10.51 (s, 2H), 8.00 (s, 2H), 7.54 (d, J = 8.0 Hz, 4H), 7.26 (d, J = 7.9 Hz, 4H), 6.26 (s, 1H), 5.77 (s, 2H), 2.34 (s, 6H). ¹³ C NMR (101 MHz, DMSO) δ 162.8, 162.6, 140.1, 138.4, 132.5, 129.4, 126.0, 73.3, 21.0.

NCL196 (4',4'-((1E,1'E)-((2-aminopyridin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol)

A suspension of 2-amino-4,6-dihydrazinopyrimidine (70.4 mg, 0.454 mmol) and 4-hydroxybenzaldehyde (140.3 mg, 1.149 mmol, 2.53 eq.) in EtOH (3 ml) was refluxed for 16 hours. The reaction mixture was cooled to room temperature and the precipitate was collected by washing with Et ₂ O (25 mL) to give pyrimidine (91.4 mg, 55%) as an off-white powder. Tpl. 298 °C (decomposed). ^1H NMR (400 MHz, DMSO) δ 10.31 (s, 2H), 9.74 (s, 2H), 7.94 (s, 2H), 7.48 (d, J = 8.6 Hz, 4H), 6.83 (d, J = 8.6 Hz, 4H), 6.20 (s, 1H), 5.70 (s, 2H). ¹³ C (101 MHz, DMSO) δ 162.7, 162.5, 158.3, 140.5, 127.7, 126.3, 115.7, 73.0.

EXAMPLE 2: Giardia adhesion assay

Target. The purpose of this study was to determine the anti-giardial activity of NCL099 and NCL812.

Ways. Giardia strain WB was grown to confluence (grown and maintained in TYI-S-33 medium with 10% fetal bovine serum). For analysis, the confluent culture medium was replaced with fresh TYI-S-33 medium. The cultures were then cold-shocked (on ice) for 40 minutes to release the trophozoites. The cell density of the cultures was adjusted to 1 x 10 ⁶ cells/ml, and 1 ml was added to each well of a 24-well plate already containing 1 ml of diluted compounds (see below). A cover glass was placed at the bottom of each well and the analytical mixture was incubated under anaerobic conditions (in a candle bottle) for 2.5 hours at 37 °C. After incubation, the coverslips were removed, air dried, and stained with Diff-Quick dye (a variant of Romanovsky staining). Coverslips were mounted onto glass slides using Polymount mounting medium. Images were taken at 10x magnification at 5 random positions of each coverslip. Free Dotcount software was used to count the number of cells per image and the resulting data were analyzed using GraphPad Prism version 6. Compound stock solutions were prepared in DMSO at 25.6 mg/mL. Compounds were diluted 1:100 in TYI-S-33 medium. Then serial dilutions 1:2 were carried out in TYI-S-33 medium in 24-well plates.

Results. The results of the study are presented in Fig. 2-3. In FIG. Figure 2 shows the in vitro activity of NCL099 against Giardia duodenalis. A significant decrease in the number of adherent cells was observed at NCL099 concentrations of 11 μg/ml (P=0.0099), 38 μg/ml (P=0.001), and 128 μg/ml (P=<0.0001) compared with the control sample (Fig. .2). As the concentration of NCL099 increased, an increase in activity was observed. In FIG. Figure 3 shows the in vitro activity of NCL812 and metronidazole against Giardia duodenalis. Significant reductions in the number of adhesive trophozoites were observed for metronidazole (P=0.0002) and NCL812 (P=<0.0001). The samples were kept for 5 hours rather than 2.5 hours.

Conclusion. This study shows that NCL099 and NCL812 inhibit the ability of Giardia cells to adhere to surfaces, thereby limiting the ability of this pathogen to cause disease (as adhesion is required for disease initiation).

EXAMPLE 3: Resazurin Recovery Assay

Target. Determine the activity of NCL812 and NCL062 against Giardia duodenalis in vitro

Ways. Giardia trophozoites were grown to confluence. The medium was replaced with fresh medium and cold shocked for 40 minutes (as described above). Cells were diluted to a concentration of approximately 500,000 cells/mL and 100 μL was added to each well of the assay plate (except for the well containing only the media control). The analytical mixture was incubated under anaerobic conditions (in a candle bottle) for 42 hours at 37 °C. Alamarblue™ was added at a concentration of 10% and the samples were incubated under anaerobic conditions for another 6 hours. After incubation, the absorbance of each sample was read at 570 and 630 nm. The percentage recovery of resazurin (Alamarblue™) was calculated and data analyzed using GraphPad Prism version 6 software. Experimental conditions: The assay was performed in 96-well plates in a total volume of 200 μl. 100 μl of a stock solution of NCL812 or NCL062 (concentration 25.6 mg/ml in DMSO) or metronidazole (concentration 5 mg/ml in DMSO) was added to 9.9 ml of TYI-S-33 medium, then serially diluted 1:2 in that same environment. Cells were added as described above.

Results. The results are presented in Fig. 4-6. All three compounds tested (NCL812, NCL062 and metronidazole) demonstrated a decrease in the metabolic activity of trophozoites (indicated by a decrease in the percentage of resazurin recovery). Both NCL compounds showed superior activity compared to metronidazole.

Conclusion. Both compounds NCL812 and NCL062 demonstrate inhibitory activity against Giardia duodenalis trophozoites in vitro.

EXAMPLE 4: Red Blood Cell Hemolysis Assay

Target. The purpose of this study was to determine the in vitro toxicity of NCL812, NCL099 and NCL062 against mammalian cells.

Ways. Whole sheep blood was purchased from Thermofisher scientific. Red blood cells were separated from other blood components by centrifugation at 1500 g for 10 minutes, and washed with saline 3 times. Red blood cells were diluted to a concentration of approximately 1 × ¹⁰ cells/ml in saline. 50 μl of NCL compounds diluted in DMSO (see below) was added to 1.5 ml Eppendorf tubes, and 500 μl of diluted red blood cells was added to each tube. Red blood cells were incubated with the compounds for 30 minutes at 37°C with gentle shaking (75 rpm). After incubation, the cells were placed on ice for 5 minutes and then centrifuged at 1500 rpm. for 10 minutes to obtain a cell pellet. The supernatant was removed and serially diluted in saline. The absorbance of the supernatant and dilutions was read at 550 nm. Sample lysis was compared to cells lysed 100% in distilled water and analyzed using GraphPad Prism version 6 software.

Results. The results of this study are presented in Fig. 7 (NCL062 tested from 1 to 128 μg/ml, NCL812 tested from 2 to 256 μg/ml, NCL099 tested from 8 to 124 μg/ml). In FIG. Figure 7 shows erythrocyte lysis as a percentage of the positive control (100% lysed blood cells in water) at various concentrations.

Conclusion. This study shows that the concentration required for each compound to cause toxic effects in mammalian cells (red blood cells) is much higher than the concentration required to cause effects in protozoal cells (giardia). This study also shows that NCL099 has higher selectivity for bacterial and protozoal cells than for mammalian cells compared to NCL812 and NCL062.

EXAMPLE 5: Transmission Electron Microscopy Study

Target. To determine the effect that NCL812 has on the ultrastructure of Giardia duodenalis.

Ways. Giardia strain WB was grown to confluence (as described above) and the old medium was replaced with new medium containing 6 μg/ml NCL812, 25 μg/ml metronidazole, or 0.1% DMSO (control). Samples were incubated at 37°C for 1 hour (NCL-treated) or 4 hours (metronidazole-treated and DMSO controls), then cold-shocked to release trophozoites. Treated samples were centrifuged at 900 x g for 10 minutes and washed twice with cold saline. The resulting cell pellet was resuspended in pre-cooled fixative (1.25% gluteraldehyde, 4% paraformaldehyde in PBS with 4% sucrose, pH 7.2) and left overnight. The samples were subsequently fixed in 2% osmium tetroxide solution for 1 hour and dehydrated using varying series of ethanol (70-100%). The samples were embedded in epoxy resin and stained with uranyl acetate and lead citrate, then examined under a FEI Tecnai G2 Spirit transmission electron microscope.

Results. The results of the study are presented in Fig. 8. This figure shows a significant change in the ultrastructure of trophozoites treated with NCL (D-G) compared to control samples (A-C). The development of vacuoles and destruction of the cytoplasmic membrane were observed.

Conclusion. NCL812 causes significant changes in the ultrastructure of Giardia duodenalis.

EXAMPLE 6: Dosage forms of compounds

The following dosage forms were prepared using methods standard in the art.

Dosage Form A – Dosage form for topical use - PEG-based gel with compounds according to the invention

4.0 g PEG 4000;

3.5 g PEG 200;

0.6 g propylene glycol;

1.9 g water; And

0.204 g Compound (e.g. NCL099)

PEG 4000, PEG 200 and propylene glycol were mixed and heated to 150 °C and until all solid crystals dissolved. The compounds were added to water and sonicated for 30 minutes until completely suspended. The compound solution and gel solutions were mixed and left to cool and solidify. Dosage Form A is likely to exhibit acceptable viscosity, ease of application to the skin, uniform suspension, and a uniform and fine texture.

Dosage form B – Dosage form for topical use - PEG-based gel with compounds according to the invention

3.0 g PEG 4000;

1.0 g PEG 8000;

3.0 g PEG 200;

1.0 g propylene glycol;

1.9 g water; And

0.202 g Compound (e.g. NCL099)

PEG 4000, PEG 8000, PEG 200 and propylene glycol were mixed and heated to 150 °C and until all solid crystals dissolved. A compound (eg NCL099) was added to water and sonicated for 30 minutes until completely suspended. The compound solution and gel solutions were mixed and left to cool and solidify. Dosage Form B exhibits acceptable viscosity, ease of application to the skin, uniform suspension and a uniform and fine texture.

Dosage form C – Dosage form for topical use - PEG-based gel with Compound-Soluplus

2.5 g PEG 4000;

4.0 g PEG 200;

2.5 g propylene glycol;

1.0 g water; And

1.8 g solid dispersion of Compound-Soluplus.

Soluplus was purchased from BASF (www.soluplus.com). Compound-Soluplus was prepared by methods standard in the art. PEG 4000, PEG 200, Compound-Soluplus and propylene glycol were mixed and heated to 150 °C and until all solid crystals dissolved. Water was added and then the solution was treated with ultrasound. The solution was left to cool and solidify. Dosage Form C exhibits acceptable viscosity, ease of application to the skin, uniform suspension, and a uniform and fine texture.

Dosage form D - Dosage form tablet

30 mg anhydrous calcium hydrogen phosphate;

80 mg microcrystalline cellulose;

50 mg lactose;

8 mg hydroxypropyl methylcellulose

1.5 mg talc

10 mg Compound (eg NCL099)

The excipients were weighed and mixed for 5 minutes. The mixture was loaded into the hopper of a tablet press and the machine was operated in a manner standard in the art. Dosage Form D exhibits acceptable tablet hardness, disintegration and friability.

Dosage form E – oral suspension

2.0 ml glycerin;

1.5 ml absolute ethanol;

600 mg NCL812; And

Up to 60 ml of carrier (Ora Sweet and Ora Plus, 1:1).

NCL 812 powder was sieved through a 75 µm sieve. 600 mg of sieved NCL 812 was mixed with 2.0 ml of glycerol and 1.5 ml of absolute ethanol. The mixture was placed in a mortar and manually crushed until the NCL 812 was evenly suspended. The suspension was sonicated for 30 minutes. Then the carrier (55 ml of a mixture of Ora Sweet and Ora Plus) was added to the suspension and crushed for another 10 minutes. The volume was adjusted to 60 ml using a mixture of Ora plus and Ora sweet by transferring the mixture into a graduated cylinder.

Dosage Form E demonstrates acceptable suspension and acceptable short-term stability.

Dosage form F – intramuscular injection

20 mg/ml Polyvinylpyrrolidone K30 (PVPK30);

0.09 mg/ml NCL812; And

50 ml water.

A two percent w/v solution of PVP K30 was prepared by adding 1.0 g of PVP K30 to 50 mL of MilliQ water. The solution was then placed in an ultrasonic dispersant for 30 minutes to equilibrate and 4.5 mg NCL 812 was added to the PVP solution and placed in a shaker incubator at a maximum speed of 10 rpm. for 24 hours at a controlled temperature of 25±1°C. The solution was transferred into 5 ml tubes and tested for clarity, appearance, pH and short-term stability. The pH of the solution was 7.25.

Dosage Form F demonstrates acceptable clarity and short-term stability.

EXAMPLE 7: Release of NCL812 and NCL099 from Dosage Form B.

Purpose: The purpose of this study was to measure the release of NCL812 and NCL099 from Dosage Form B prepared in Example 6.

Methodology: Franz diffusion cells were used to quantify the release rates of NCL 812 and NCL099 from their topical dosage forms. The receiving chamber was loaded with five milliliters of absolute ethanol, which was chosen as the desired release medium. The temperature of the receiving fluid was kept constant at 32±1°C using a water jacket. Cellulose acetate membranes with a pore size of 0.45 μm (Pall Corporation) were selected and placed between the donor and receptor chambers. Then a number of test samples (dosage form B) were loaded into the donor chamber. One milliliter of receptor fluid was collected at regular time intervals of 0.25, 0.50, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, and 24 hours through a sampling port. One milliliter of fresh absolute alcohol was immediately returned to the receptor chamber. UV-HPLC was used to analyze the contents of the collected receptor fluids.

Results and conclusions: In Fig. 9 shows the cumulative release of NCL812 and NCL099 over time. This study demonstrates that Formulation B provides an acceptable release profile for NCL812 and NCL099.

EXAMPLE 8: List of NMR spectroscopy data for compounds NCL812, NCL001-NCL275

NMR spectroscopy was performed on compounds NCL812, NCL001-NCL275 using methods standard in the art. Lists of NMR spectroscopy data are presented in Table 3.

Table 3: List of NMR spectroscopy data for compounds NCL812, NCL001-NCL275

NCL Code NMR NCL812 1H NMR (400 MHz, DMSO) δ 12.04 (w s, 2H), 8.48 (w s, 1H), 8.37 (w s, 2H), 7.97 (d, J = 8.6 Hz, 4H), 7.57 (d, J = 8.6 Hz, 4H) NCL001 1H NMR (400 MHz, DMSO) δ 10.84 (w s, 2H), 8.17 (w s, 2H), 7.77 (d, J = 8.2 Hz, 4H), 7.50 (d , J = 8.6 Hz, 4H) NCL002 1H NMR (400 MHz, DMSO) δ 11.06 (s, 2H), 8.58 (w s, 2H), 8.17 (w s, 2H), 7.50 - 7.52 (m, 2H) , 7.41 - 7.45 (m, 4H) NCL003 1H NMR (400 MHz, DMSO) δ 10.71 (s, 2H), 8.17 (w s, 2H), 7.73 - 7.88 (m, 4H), 7.28 (t, J=8 ,8 Hz, 4H) NCL004 1H NMR (400 MHz, DMSO) δ 10.89 (w s, 2H), 8.19 (w s, 2H), 7.65 (w s, 2H), 7.43 - 7.56 (m, 4H ), 7.19 - 7.27 (m, 2H) NCL005 1H NMR (400 MHz, DMSO) δ 10.94 (w s, 2H), 8.43 (w s, 2H), 8.10 (w s, 2H), 7.39 - 7.52 (m, 2H ), 7.21 - 7.35 (m, 4H) NCL006 1H NMR (400 MHz, DMSO) δ 10.50 (s, 2H), 8.11 (w s, 2H), 7.68 (d, J = 8.6 Hz, 4H), 6.99 (d, J = 8.6 Hz, 4H), 3.80 (s, 6H) NCL007 1H NMR (400 MHz, DMSO) δ 11.10 (w s, 2H), 8.24 (w s, 2H), 7.81 - 8.03 (m, 8H) NCL008 1H NMR (400 MHz, DMSO) δ 11.24 (w s, 2H), 8.51 (w s, 2H), 8.18 - 8.29 (m, 2H), 7.90 (d, J= 7.4 Hz, 2H), 7.80 (t, J = 7.6 Hz, 2H), 7.59 (t, J = 7.0 Hz, 2H) NCL009 1H NMR (400 MHz, DMSO) δ 11.02 (s, 2H), 8.26 (w s, 4H), 8.07 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.65 (t, J = 7.8 Hz, 2H) NCL010 1H NMR (400 MHz, DMSO) δ 10.74 (b s, 2H), 8.15 (b s, 2H), 7.25 - 7.39 (m, 6H), 6.94-7.01 ( m, 2H), 3.82 (s, 6H) NCL011 1H NMR (400 MHz, DMSO) δ 11.02 (s, 2H), 8.28 (w s, 2H), 8.13 (s, 2H), 8.04 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 7.68 (t, J = 8.0 Hz, 2H) NCL012 1H NMR (400 MHz, DMSO) δ 11.04 (w s, 2H), 8.27 (w s, 2H), 7.97 (d, J = 7.8 Hz, 4H), 7.80 (d , J = 8.2 Hz, 4H) NCL013 1H NMR (400 MHz, DMSO) δ 11.22 (w s, 2H), 8.55 (w s, 2H), 8.35 (d, J = 7.0 Hz, 2H), 7.73 - 7 .82 (m, 4H), 7.57 - 7.65 (m, 2H) NCL014 1H NMR (400 MHz, DMSO) δ 10.30 (s, 1H), 7.81 (s, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 6.53 (w s, 2H) NCL015 1H NMR (400 MHz, DMSO) δ 12.02 (w s, 1H), 8.55 (s, 1H), 8.27-8.33 (m, 1H), 7.79 (w s, 3H) , 7.51 - 7.56 (m, 1H), 7.39 - 7.51 (m, 2H) NCL016 1H NMR (400 MHz, DMSO) δ 11.98 (w s, 1H), 8.39 (s, 1H), 8.19-8.26 (m, 1H), 7.80 (w s, 3H) , 7.46 - 7.58 (m, 1H), 7.20 - 7.38 (m, 2H) NCL017 1H NMR (400 MHz, DMSO) δ 11.79 (w s, 1H), 8.17 (s, 1H), 7.87 (d, J = 9.8 Hz, 1H), 7.71 (w s , 3H), 7.62 (d, J = 7.4 Hz, 1H), 7.45 - 7.54 (m, 1H), 7.25-7.32 (m, 1H) NCL018 1H NMR (400 MHz, DMSO) δ 10.66 (s, 2H), 8.47 (w s, 2H), 7.91-8.00 (m, 2H), 7.19 - 7.32 (m , 6H), 2.42 (s, 6H) NCL019 1H NMR (400 MHz, DMSO) δ 10.68 (w s, 2H), 8.15 (w s, 2H), 7.57 (s, 2H), 7.52 (d, J = 7.4 Hz , 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.21 (d, J = 7.4 Hz, 2H), 2.36 (s, 6H) NCL020 1H NMR (400 MHz, DMSO) δ 12.37 (w s, 2H), 8.83 (w s, 2H), 8.63 (w s, 2H), 8.39 - 8.44 (m, 2H ), 7.55 - 7.60 (m, 2H), 7.44 - 7.55 (m, 4H) NCL021 1H NMR (400 MHz, DMSO) δ 12.11 (w s, 1H), 8.52 (w s, 2H), 8.40 (w s, 2H), 8.02 (t, J = 8.6 , 4H), 7.35 (t, J = 8.8 Hz, 4H) NCL022 1H NMR (400 MHz, DMSO) δ 12.19 (w s, 2H), 8.65 (w s, 2H), 8.58 (w s, 1H), 8.34 (t, J = 7.6 Hz, 2H), 7.51 - 7.60 (m, 2H), 7.34 (t, J = 8.2 Hz 4H) NCL023 1H NMR (400 MHz, DMSO) δ 12.08 (w s, 2H), 8.38 (w s, 2H), 7.92-8.00 (m, 2H), 7.65-7.71 ( m, 2H), 7.50 - 7.58 (m, 2H), 7.29 - 7.37 (m, 2H) NCL024 1H NMR (400 MHz, DMSO) δ 12.32 (w s, 2H), 8.67 (w s, 2H), 8.44 (w s, 2H), 8.15 (d, J = 8.6 Hz, 4H), 7.98 (d, J = 8.6 Hz, 4H) NCL025 1H NMR (400 MHz, DMSO) δ 8.75 (w s, 2H), 8.50 (d, J = 8.2 Hz, 2H), 7.97 (d, J = 7.4 Hz, 2H) , 7.85 (t, J = 7.6 Hz, 2H), 7.68 (t, J = 7.6 Hz, 2H) NCL026 1H NMR (400 MHz, DMSO) δ 12.26 (ws, 1H), 8.66 (ws, 1H), 8.55 (s, 2H), 8.43 (ws, 2H), 8. 21 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 7.8 Hz, 2H), 7.71 (t, J = 7.8 Hz, 2H) NCL027 1H NMR (400 MHz, DMSO) δ 11.78 (w s, 2H), 8.31 (w s, 3H), 7.87 (d, J = 8.6 Hz, 4H), 7.04 (d , J = 8.6 Hz, 4H), 3.83 (s, 6H) NCL028 1H NMR (400 MHz, DMSO) δ 12.00 (w s, 2H), 8.75 (w s, 2H), 8.39 (w s, 2H), 8.22 (d, J = 6.7 Hz, 2H), 7.44 - 7.52 (m, 2H), 7.14 (d, J = 8.2 Hz, 2H), 7.05 (t, J = 7.6 Hz, 2H), 3.89 (s, 6H) NCL029 1H NMR (400 MHz, DMSO) δ 11.98 (b s, 2H), 8.48 (b s, 2H), 8.36 (b s, 2H), 7.56 (b s, 2H), 7. 35 - 7.49 (m, 4H), 7.04-7.10 (m, 2H), 3.84 (s, 6H) NCL030 1H NMR (400 MHz, DMSO) δ 11.83 (w s, 1H), 8.16 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.75 (w s , 1H), 7.53 (d, J = 8.2 Hz, 2H) NCL031 1H NMR (400 MHz, DMSO) δ 11.91 (w s, 1H), 8.22 (s, 1H), 8.09 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H) NCL032 1H NMR (400 MHz, DMSO) δ 12.12 (s, 1H), 8.48 (s, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.86 (w s, 2H), 7.80 (t, J = 7.8 Hz, 2H), 7.64 (t, J = 7.6 Hz, 1H) NCL033 1H NMR (400 MHz, DMSO) δ 11.93 (s, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H ), 7.90 (d, J = 7.8 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H) NCL034 1H NMR (400 MHz, DMSO) δ 11.87 (w s, 1H), 8.48 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.70 (w s , 2H), 7.38 - 7.49 (m, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 3 .86 (s, 3H) NCL035 1H NMR (400 MHz, DMSO) δ 12.32 (w s, 2H), 8.69 (w s, 2H), 8.49 (w s, 2H), 8.18 (d, J = 7.8 Hz, 4H), 7.86 (d, J = 8.2 Hz, 4H) NCL036 1H NMR (400 MHz, DMSO) δ 12.51 (w s, 1H), 8.80 (w s, 2H), 8.72 (w s, 1H), 8.59 (d, J = 7.8 Hz, 2H), 7.78 - 7.91 (m, 4H), 7.71 (t, J = 8.0 Hz, 2H) NCL037 1H NMR (400 MHz, DMSO) δ 12.28 (b s, 2H), 8.70 (b s, 2H), 8.50 (b s, 2H), 8.38 (b s, 2H), 8. 22 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H) NCL038 1H NMR (400 MHz, DMSO) δ 11.92 (w s, 2H), 8.41 (w s, 2H), 8.36 (w s, 2H), 7.83 (d, J = 8.2 Hz, 4H), 7.31 (d, J = 7.8 Hz, 4H), 2.37 (s, 6H) NCL039 1H NMR (400 MHz, DMSO) δ 11.99 (w s, 2H), 8.73 (w s, 2H), 8.41 (w s, 2H), 8.19 (d, J = 7.8 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H), 7.30 (t, J = 7.8 Hz, 4H), 2.46 (s, 6H) NCL040 1H NMR (400 MHz, DMSO) δ 11.97 (b s, 2H), 8.44 (b s, 2H), 8.37 (b s, 2H), 7.76 (b s, 2H), 7. 71 (d, J = 7.8 Hz, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 2.38 ( s, 6H) NCL041 1H NMR (400 MHz, DMSO) δ 11.94 (s, 1H), 8.25 (s, 1H), 8.11 (d, J = 7.8 Hz, 2H), 7.71 - 7.91 (m, 4H) NCL042 1H NMR (400 MHz, DMSO) δ 12.04 (s, 1H), 8.46 - 8.56 (m, 2H), 7.70 - 7.93 (m, 5H), 7.66 (t, J = 7.8 Hz, 1H) NCL043 1H NMR (400 MHz, DMSO) δ 11.88 (s, 1H), 8.33 (s, 1H), 8.26 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H ), 7.80 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H) NCL044 1H NMR (400 MHz, DMSO) δ 11.71 (w s, 1H), 8.13 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 2.35 (s, 3H) NCL045 1H NMR (400 MHz, DMSO) δ 11.69 (w s, 1H), 8.45 (s, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.67 (w s , 2H), 7.30 - 7.39 (m, 1H), 7.20 - 7.29 (m, 2H), 2.42 (s, 3H) NCL046 1H NMR (400 MHz, DMSO) δ 11.64 (w s, 1H), 8.12 (s, 1H), 7.53 - 7.77 (m, 4H), 7.34 (t, J=7 .8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 2.35 (s, 3H) NCL047 1H NMR (400 MHz, DMSO) δ 10.47 (s, 1H), 8.23 (s, 1H), 8.16 - 8.21 (m, 1H), 7.42 - 7.50 (m, 1H), 7.30 - 7.40 (m, 2H), 6.57 (w s, 2H) NCL048 1H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 8.08 - 8.15 (m, 1H), 8.05 (s, 1H), 7.34 - 7.44 (m, 1H), 7.17 - 7.28 (m, 2H), 6.54 (w s, 2H) NCL049 1H NMR (400 MHz, DMSO) δ 10.51 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 6.63 (w s, 2H) NCL050 1H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.11 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 6.60 (w s, 2H) NCL051 1H NMR (400 MHz, DMSO) δ 10.43 (s, 1H), 8.37 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H ), 7.77 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H) NCL052 1H NMR (400 MHz, DMSO) δ 11.95 (s, 1H), 8.16 (s, 1H), 8.10 (s, 1H), 7.80 (w s, 2H), 7.76 ( d, J = 7.0 Hz, 1H), 7.40 - 7.55 (m, 2H) NCL053 1H NMR (400 MHz, DMSO) δ 11.88 (s, 1H), 8.17 (s, 1H), 7.95 (dd, J = 8.8, 5.7 Hz, 2H), 7.76 (w s, 1H), 7.30 (t, J = 9.0 Hz, 2H) NCL054 1H NMR (400 MHz, DMSO) δ 12.17 (b s, 2H), 8.61 (b s, 2H), 8.39 (b s, 2H), 8.16 (b s, 2H), 7. 83 (d, J = 7.0 Hz, 2H), 7.45 - 7.61 (m, 4H) NCL055 1H NMR (400 MHz, DMSO) δ 10.34 (s, 1H), 7.93 (s, 1H), 7.80 (s, 1H), 7.54 - 7.67 (m, 1H), 7 .32 - 7.46 (m, 2H), 6.58 (w s, 1H) NCL056 1H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.82 (s, 1H), 7.78 (dd, J = 8.8, 5.7 Hz, 2H), 7.21 (t, J = 8.8 Hz, 1H), 6.49 (w s, 2H) NCL057 1H NMR (400 MHz, DMSO) δ 10.33 (s, 1H), 7.82 (s, 1H), 7.71 (d, J = 9.8 Hz, 1H), 7.35 - 7.53 (m, 2H), 7.10 - 7.23 (m, 1H), 6.57 (w s, 1H) NCL058 1H NMR (400 MHz, DMSO) δ 10.08 (s, 1H), 7.78 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.40 (w s, 2H), 3.78 (s, 3H) NCL059 1H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.80 (s, 1H), 7.34 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H ), 7.21 (d, J = 7.8 Hz, 1H), 6.91 (dd, J = 7.8, 2.0 Hz, 1H), 6.51 (w s, 2H), 3, 79 (s, 3H) NCL060 1H NMR (400 MHz, DMSO) δ 10.56 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.19 (w s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 6.62 (w s, 2H ) NCL061 1H NMR (400 MHz, DMSO) δ 11.71 (w s, 2H), 8.91 (w s, 1H), 8.28 (d, J = 7.8 Hz, 4H), 7.82 (d , J = 8.2 Hz, 4H), 2.49 (w s, 6H) NCL062 1H NMR (400 MHz, DMSO) δ 11.68 (w s, 2H), 8.78 (w s, 1H), 8.10 (d, J = 8.6 Hz, 4H), 7.52 (d , J = 8.6 Hz, 4H), 2.43 (s, 6H) NCL063 1H NMR (400 MHz, DMSO) δ 7.99 (w s, 4H), 7.83 (d, J = 7.8 Hz, 6H) NCL064 1H NMR (400 MHz, DMSO) δ 7.94 (d, J = 7.8 Hz, 4H), 7.84 (t, J = 7.6 Hz, 4H), 7.64 (t, J = 7 .6 Hz, 4H) NCL065 1H NMR (400 MHz, DMSO) δ 12.20 (w s, 1H), 11.86 (w s, 1H), 8.65 (w s, 1H), 8.50 (w s, 1H), 8 .03-8.27 (m, 3H), 7.90 (d, J = 7.8 Hz, 2H), 7.68 (t, J = 7.6 Hz, 2H) NCL066 1H NMR (400 MHz, DMSO) δ 7.43 - 7.67 (m, 7H), 7.23 - 7.34 (m, 2H) NCL067 1H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 8.16 (s, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H ), 7.67 (d, J = 7.3 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H) NCL068 1H NMR (400 MHz, DMSO) δ 8.47 (w s, 1H), 8.18 (d, J = 7.8 Hz, 3H), 8.07 (d, J = 8.6 Hz, 2H) , 7.85 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 2.40 (s, 3H) NCL069 1H NMR (400 MHz, DMSO) δ 9.44 (w s, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.94 (w s, 2H), 7.48 (d , J = 8.6 Hz, 2H), 2.31 (s, 3H) NCL070 1H NMR (400 MHz, DMSO) δ 8.12 (w s, 1H), 7.71 (w s, 3H), 7.49 (w s, 6H) NCL071 1H NMR (400 MHz, DMSO) δ 11.43 (s, 1H), 8.40 (s, 1H), 8.16 (w s, 1H), 8.10 (d, J = 7.4 Hz, 1H), 7.95 (w s, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.95 (t , J = 7.4 Hz, 1H), 3.82 (s, 3H) NCL072 1H NMR (400 MHz, DMSO) δ 7.91 (w d, J = 8.2 Hz, 3H), 7.52 (d, J = 8.6 Hz, 2H) NCL073 1H NMR (400 MHz, DMSO) δ 9.51 (w s, 1H), 8.22 (d, J = 8.3 Hz, 2H), 8.01 (w s, 2H), 7.77 (d , J = 8.3 Hz, 2H), 2.36 (s, 3H) NCL074 1H NMR (400 MHz, DMSO) δ 12.46 (w s, 1H), 8.79 (w s, 1H), 8.66 (w s, 2H), 8.46 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 7.0 Hz, 2H), 7.77 (w s, 1H), 7.58 (d, J = 6.7 Hz, 3H) NCL075 1H NMR (400 MHz, DMSO) δ 8.48 (w s, 1H), 8.26 (d, J = 7.4 Hz, 2H), 8.19 (d, J = 7.4 Hz, 2H) , 7.75-7.93 (m, 4H), 2.46 (s, 3H) NCL076 1H NMR (400 MHz, DMSO) δ 8.53 (w s, 2H), 8.29 - 8.46 (m, 2H), 7.99 (d, J = 7.8 Hz, 2H), 7. 83 (d, J = 7.8 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 2.37 ( s, 3H) NCL077 1H NMR (400 MHz, DMSO) δ 12.28 (w s, 1H), 8.67 (w s, 1H), 8.49 (w s, 1H), 8.42 (w s, 1H), 8 .18 (d, J = 7.8 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H) NCL078 1H NMR (400 MHz, DMSO) δ 12.33 (w s, 1H), 8.74 (w s, 2H), 8.58 (t, J = 7.6 Hz, 1H), 8.42 (w s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.87 (d, J = 10.2 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H) NCL079 1H NMR (400 MHz, DMSO) δ 12.08 (w s, 1H), 8.54 (w s, 1H), 8.39 (w s, 2H), 7.93 - 8.09 (m, 4H ), 7.57 (d, J = 8.2 Hz, 2H), 7.35 (t, J = 8.6 Hz, 2H) NCL080 1H NMR (400 MHz, DMSO) δ 12.04 (w s, 1H), 11.36 (w s, 1H), 8.40 (w s, 1H), 8.25 (d, J = 8.2 Hz, 2H), 8.00 (d, J = 7.0 Hz, 2H), 7.82 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 6.7 Hz, 2H), 2.44 (s, 3H) NCL081 1H NMR (400 MHz, DMSO) δ 8.39 (w s, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H) , 7.57 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 2.39 (s, 3H) NCL082 1H NMR (400 MHz, DMSO) δ 12.40 (w s, 1H), 8.84 (w s, 1H), 8.63 (w s, 2H), 8.42 (d, J = 7.8 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.2 Hz, 3H), 7.43 - 7.54 (m, 2H) NCL083 1H NMR (400 MHz, DMSO) δ 12.30 (b s, 1H), 8.64 (b s, 2H), 8.41 (b s, 2H), 8.17 (b s, 1H), 7. 99 (d, J = 8.6 Hz, 2H), 7.82 (d, J = 6.7 Hz, 1H), 7.45 - 7.64 (m, 4H) NCL084 1H NMR (400 MHz, DMSO) δ 12.38 (w s, 1H), 8.64 (w s, 2H), 8.30 - 8.50 (m, 2H), 7.99 (d, J= 8.6 Hz, 2H), 7.62 (d, J = 10.4, 1.4 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.6 Hz, 1H) NCL085 1H NMR (400 MHz, DMSO) δ 8.77 (w s, 1H), 8.68 (w s, 2H), 8.52 (d, J = 8.2 Hz, 1H), 8.46 (w s, 1H), 7.92 - 8.06 (m, 3H), 7.84 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H) NCL086 1H NMR (400 MHz, DMSO) δ 12.39 (b s, 1H), 8.68 (b s, 2H), 8.57 (b s, 1H), 8.43 (b s, 2H), 8. 21 (d, J = 7.4 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.94 (d, J = 7.0 Hz, 1H), 7.70 ( t, J = 7.2 Hz, 1H), 7.58 (d, J = 7.8 Hz, 2H) NCL087 1H NMR (400 MHz, DMSO) δ 8.71 (w s, 2H), 8.48 (w s, 1H), 8.43 (w s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.99 (t, J = 8.0 Hz, 4H), 7.58 (d, J = 8.6 Hz, 2H) NCL088 1H NMR (400 MHz, DMSO) δ 12.33 (w s, 1H), 8.68 (w s, 1H), 8.61 (w s, 2H), 8.42 (w s, 1H), 8 .35 (t, J = 7.4 Hz, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.49 - 7.64 (m, 3H), 7.27 - 7, 41 (m, 2H) NCL089 1H NMR (400 MHz, DMSO) δ 11.45 (w s, 1H), 8.76 - 8.94 (m, 1H), 8.11 (d, J = 8.0 Hz, 4H), 7, 53 (d, J = 8.0 Hz, 4H), 2.42 (w s, 6H) NCL090 1H NMR (400 MHz, DMSO) δ 10.60 (w s, 1H), 8.68 (s, 1H), 8.09 (w s, 1H), 7.95 (w s, 4H), 7. 32 - 7.71 (m, 10H) NCL091 1H NMR (400 MHz, DMSO) δ 9.61 (w s, 1H), 8.19 (d, J = 6.3 Hz, 2H), 7.90 (w s, 2H), 7.56 - 7 .73 (m, 2H), 7.26 - 7.40 (m, 2H) NCL092 1H NMR (400 MHz, DMSO) δ 12.38 (w s, 1H), 7.82 - 7.94 (m, 2H), 7.18 - 7.63 (m, 7H), 4.39 (w s, 2H) NCL 093 1H NMR (300 MHz, DMSO) δ 8.64 (s, 4H), 8.35 – 8.24 (m, 4H), 8.06 – 7.93 (m, 6H), 7.64 – 7, 54 (m, 4H), NCL 094 1H NMR (300 MHz, CDCl3) δ 7.16 (d, J = 4.7 Hz, 2H), 6.10 (w s, 3H), 2.27 – 2.14 (m, 2H), 1, 84 – 1.61 (m, 10H), 1.37 – 1.13 (m, 10H). NCL 095 1H NMR (300 MHz, DMSO) δ 8.39 (s, 2H), 8.36 – 8.11 (m, 4H), 7.78 (s, 2H), 7.12 (d, J = 1, 4 Hz, 2H). NCL 096 1H NMR (300 MHz, MeOD) δ 8.08 (d, J = 8.2 Hz, 2H), 7.61 – 7.53 (m, 4H), 7.45 – 7.30 (m, 6H) , 7.17 – 6.97 (m, 4H). NCL 097 1H NMR (300 MHz, DMSO) δ 9.06 (w s, 6H), 8.25 – 8.01 (m, 4H), 6.83 (s, 4H). 13C NMR (75 MHz, DMSO) δ 152.2, 149.7, 146.2, 136.5, 123.7, 107.4. NCL 098 1H NMR (300 MHz, DMSO) δ 8.65 (s, 2H), 8.53 (s, 2H), 8.40 (s, 2H), 8.24 (d, J = 7.3 Hz, 2H ), 8.09 – 7.98 (m, 2H), 7.62 (t, J = 7.7 Hz, 2H). NCL 099 1H NMR (300 MHz, DMSO) δ 8.56 – 8.32 (m, 4H), 7.85 (d, J = 8.3 Hz, 4H), 7.49 (d, J = 8.3 Hz , 4H), 1.31 (s, 18H). 13C NMR (75 MHz, DMSO) δ 153.7, 152.7, 148.8, 130.7, 127.8, 125.6, 34.7, 31.0. NCL 100 1H NMR (300 MHz, DMSO) δ 12.39 (w s, 2H), 8.55 (s, 2H), 8.46 (s, 2H), 8.01 – 7.88 (m, 4H), 7.55 – 7.41 (m, 6H). NCL 101 1H NMR (300 MHz, DMSO) δ 12.06 (b s, 2H), 9.71 (b s, 2H), 9.21 (b s, 2H), 8.70 (b s, 2H), 8. 30 (s, 2H), 7.50 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.7 Hz, 2H), 6.70 (t, J = 7.7 Hz, 2H). NCL 102 1H NMR (300 MHz, DMSO) δ 12.86 (w s, 2H), 8.89 (s, 2H), 8.77 (s, 2H), 8.52 (d, J = 7.9 Hz, 2H), 8.11 (d, J = 8.1 Hz, 2H), 7.91 – 7.78 (m, 2H), 7.77 – 7.65 (m, 2H). NCL 103 1H NMR (300 MHz, DMSO) δ 11.81 (w s, 2H), 10.32 – 9.85 (m, 4H), 8.52 (s, 2H), 8.12 (s, 2H), 7.85 (d, J = 8.4 Hz, 2H), 6.43 (s, 2H), 6.33 (d, J = 8.5 Hz, 2H). NCL 104 1H NMR (300 MHz, DMSO) δ 11.72 (b s, 2H), 9.78 (b s, 2H), 9.45 (s, 2H), 8.48 (s, 2H), 8.34 (w s, 2H), 8.04 (s, 2H), 7.33 (s, 2H), 6.44 (s, 2H). NCL 105 1H NMR (300 MHz, DMSO) δ 11.75 (w s, 2H), 9.71 (w s, 2H), 9.15 (w s, 2H), 8.86 – 8.40 (m, 4H ), 8.13 (s, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 8.6 Hz, 2H). NCL 106 1H NMR (300 MHz, DMSO) δ 9.12 (w s, 4H), 8.29 (s, 2H), 8.20 (s, 2H), 7.10 (s, 2H), 6.93 ( s, 2H), 3.84 (s, 6H). NCL 107 1H NMR (300 MHz, DMSO) δ 12.19 (s, 2H), 10.25 (s, 2H), 8.70 (s, 2H), 8.34 (s, 2H), 8.06 (d , J = 7.8 Hz, 2H), 7.35 – 7.23 (m, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.87 (t, J = 7, 5 Hz, 2H). NCL 108 1H NMR (300 MHz, DMSO) δ 8.00 (s, 2H), 7.26 – 7.08 (m, 6H), 6.98 – 6.43 (m, 4H). NCL 109 1H NMR (300 MHz, DMSO) δ 8.60 (d, J = 1.2 Hz, 2H), 8.33 – 8.19 (m, 4H), 8.15 (d, J = 8.0 Hz , 2H), 7.74 – 7.61 (m, 2H), 7.12 (s, 2H). NCL 110 1H NMR (300 MHz, DMSO) δ 8.85 (s, 2H), 8.58 (s, 2H), 8.31 (d, J = 8.7 Hz, 4H), 8.23 (d, J = 8.9 Hz, 4H). NCL 111 1H NMR (300 MHz, DMSO) δ 11.80 (w s, 2H), 10.30 – 9.80 (m, 4H), 8.52 (s, 2H), 8.12 (s, 2H), 7.84 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 1.8 Hz, 2H), 6.33 (d, J = 8.6 Hz, 2H). NCL 112 1H NMR (300 MHz, DMSO) δ 12.48 (w s, 2H), 8.62 (s, 2H), 8.51 (s, 2H), 8.04 (d, J = 7.5 Hz, 4H), 7.85 – 7.69 (m, 8H), 7.54 – 7.36 (m, 6H). NCL 113
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1H NMR (400 MHz, DMSO) δ 11.92 (w s, 2H), 8.24 (s, 2H), 8.16 (s, 2H), 7.71 (d, J = 8.9 Hz, 4H), 6.74 (d, J = 8.9 Hz, 4H), 2.98 (s, 12H). NCL 114 1H NMR (400 MHz, DMSO) δ 12.67 (w s, 2H), 8.81 (s, 2H), 8.40 (s, 2H), 8.06 (d, J = 1.8 Hz, 4H), 7.68 (t, J = 1.8 Hz, 2H). NCL 115 1H NMR (400 MHz, DMSO) δ 12.09 (w s, 2H), 8.44 (s, 2H), 8.34 (s, 2H), 7.63 (d, J = 1.4 Hz, 2H), 7.33 (dd, J = 8.3, 1.6 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 3.86 (s, 6H), 3, 81 (s, 6H). NCL 116 1H NMR (400 MHz, DMSO) δ 12.27 (w s, 2H), 8.49 (s, 2H), 8.44 – 8.27 (m, 4H), 7.57 – 7.44 (m , 10H), 7.41 – 7.33 (m, 6H). NCL 117 1H NMR (400 MHz, DMSO) δ 9.77 (w s, 2H), 8.36 (s, 2H), 8.29 (s, 2H), 7.58 (d, J = 1.5 Hz, 2H), 7.23 (dd, J = 8.1, 1.2 Hz, 2H), 6.87 (d, J = 8.1 Hz, 2H), 3.86 (s, 6H). NCL 118 1H NMR (400 MHz, DMSO) δ 12.83 (w s, 2H), 8.76 (s, 2H), 8.69 (s, 2H), 8.31 – 8.21 (m, 2H), 7.45 – 7.31 (m, 4H). NCL 119 1H NMR (400 MHz, DMSO) δ 12.13 (w s, 2H), 10.35 (s, 2H), 8.39 (s, 2H), 8.35 (s, 2H), 7.85 ( d, J = 8.7 Hz, 4H), 7.72 (d, J = 8.6 Hz, 4H), 2.08 (s, 6H). NCL 120 1H NMR (400 MHz, DMSO) δ 8.62 – 8.29 (m, 4H), 7.85 (d, J = 8.2 Hz, 4H), 7.33 (d, J = 8.2 Hz , 4H), 2.98 – 2.87 (m, 2H), 1.21 (d, J = 6.9 Hz, 12H). NCL 121 1H NMR (400 MHz, DMSO) δ 8.60 – 8.30 (m, 4H), 7.84 (d, J = 8.1 Hz, 4H), 7.28 (d, J = 8.1 Hz , 4H), 2.59 (t, J = 7.5 Hz, 4H), 1.64 – 1.54 (m, 4H), 0.88 (t, J = 7.3 Hz, 6H). NCL 122 1H NMR (400 MHz, DMSO) δ 8.56 (s, 2H), 8.42 (d, J = 2.0 Hz, 2H), 8.38 (s, 2H), 8.09 (dd, J = 8.7, 2.0 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H). NCL 123 1H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 8.43 (s, 2H), 8.28 – 8.16 (m, 2H), 7.77 – 7.64 (m, 2H), 7.58 – 7.46 (m, 2H). 13C NMR (101 MHz, DMSO) δ 153.0, 150.8 (dd, J = 250.6, 13.0 Hz), 149.9 (dd, J = 245.9, 13.2 Hz), 146 .6, 131.2 (dd, J = 6.4, 3.4 Hz), 126.0 (dd, J = 6.4, 2.8 Hz), 117.8 (d, J = 17.7 Hz), 115.7 (d, J = 18.5 Hz). NCL 124 1H NMR (400 MHz, DMSO) δ 10.84 (w s, 2H), 9.29 (s, 2H), 8.80 (d, J = 8.6 Hz, 2H), 8.43 (s, 2H), 7.94 (d, J = 9.0 Hz, 2H), 7.87 (d, J = 7.9 Hz, 2H), 7.64 – 7.56 (m, 2H), 7. 43 – 7.38 (m, 2H), 7.34 (d, J = 8.9 Hz, 2H). NCL 125 1H NMR (400 MHz, DMSO) δ 9.21 (w s, 2H), 8.42 – 8.17 (m, 4H), 7.43 (d, J = 1.9 Hz, 2H), 7, 25 (dd, J = 8.4, 1.9 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 3.83 (s, 6H). NCL 126 1H NMR (400 MHz, DMSO) δ 12.27 (w s, 2H), 8.58 (s, 2H), 8.42 (s, 2H), 7.96 (d, J = 8.3 Hz, 4H), 7.58 (d, J = 8.2 Hz, 4H), 4.39 (s, 2H). NCL 127 1H NMR (400 MHz, DMSO) δ 12.84 (w s, 2H), 8.84 (s, 2H), 8.74 (s, 2H), 8.46 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 1.6 Hz, 2H), 7.54 (dd, J = 8.6, 1.2 Hz, 2H). NCL 128 1H NMR (400 MHz, DMSO) δ 8.64 (s, 2H), 8.36 (s, 2H). NCL 129 1H NMR (400 MHz, DMSO) δ 8.84 (s, 2H), 8.69 (s, 2H), 8.41 (dd, J = 7.8, 1.7 Hz, 2H), 7.70 (dd, J = 8.0, 1.0 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H)*, 7.44 – 7.37 (m, 2H)*. NCL 130 1H NMR (400 MHz, DMSO) δ 8.66 (s, 2H), 8.37 (s, 2H), 7.79 (d, J = 1.6 Hz, 2H), 7.68 – 7.62 (m, 2H), 3.92 (s, 6H), 3.78 (s, 6H). NCL 131 1H NMR (400 MHz, DMSO) δ 12.45 (w s, 2H), 8.68 (s, 2H), 8.41 (s, 2H), 8.29 (s, 2H), 7.87 ( d, J = 7.8 Hz, 2H), 7.70 – 7.63 (m, 2H), 7.44 (t, J = 7.9 Hz, 2H). NCL 132 1H NMR (400 MHz, DMSO) δ 12.69 (w s, 2H), 8.61 (s, 4H), 7.34 (dd, J = 9.0, 2.8 Hz, 2H), 7. 22 (td, J = 8.6, 2.9 Hz, 2H), 7.03 (dd, J = 8.8, 4.6 Hz, 2H), 5.29 (s, 4H). NCL 133 1H NMR (400 MHz, DMSO) δ 12.51 (w s, 2H), 8.49 – 8.28 (m, 4H), 8.16 (s, 2H), 7.35 (s, 2H). NCL 134 1H NMR (400 MHz, DMSO) δ 12.42 (w s, 2H), 8.60 (s, 2H), 8.42 (s, 2H), 7.91 (d, J = 8.5 Hz, 4H), 7.69 (d, J = 8.5 Hz, 4H). NCL 135 1H NMR (400 MHz, DMSO) δ 12.24 (w s, 2H), 8.66 (s, 2H), 8.49 (w s, 2H), 7.79 (s, 2H), 7.24 (s, 2H), 3.88 (s, 6H), 3.85 (s, 6H). NCL 136 1H NMR (400 MHz, DMSO) δ 12.21 (w s, 2H), 8.44 (s, 2H), 8.39 (s, 2H), 7.83 (d, J = 8.2 Hz, 4H), 7.30 (d, J = 8.2 Hz, 4H), 2.63 (t, J = 7.7 Hz, 4H), 1.61 – 1.52 (m, 4H), 1, 36 – 1.26 (m, 4H), 0.90 (t, J = 7.3 Hz, 6H). NCL 137 1H NMR (400 MHz, DMSO) δ 12.94 (w s, 2H), 8.68 (s, 2H), 8.33 (s, 2H), 7.60 (d, J = 7.9 Hz, 4H)*, 7.49 (dd, J = 8.7, 7.4 Hz, 2H)*. NCL 138
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1H NMR (400 MHz, DMSO) δ 12.12 (w s, 2H), 8.21 (s, 2H), 7.97 (s, 2H), 7.54 – 7.47 (m, 6H), 7.42 – 7.36 (m, 6H), 7.31 – 7.21 (m, 8H), 6.84 (d, J = 9.8 Hz, 2H). NCL 139 1H NMR (400 MHz, DMSO) δ 12.65 (w s, 2H), 9.63 (d, J = 1.8 Hz, 2H), 8.88 – 8.58 (m, 6H), 8, 13 – 8.01 (m, 4H), 7.88 – 7.79 (m, 2H), 7.68 (t, J = 7.4 Hz, 2H). NCL 140 1H NMR (400 MHz, DMSO) δ 12.17 (w s, 2H), 8.46 (s, 2H), 8.37 (s, 2H), 7.86 (d, J = 8.2 Hz, 4H), 7.34 (d, J = 8.2 Hz, 4H), 2.53 (s, 6H). NCL 141 1H NMR (400 MHz, DMSO) δ 8.66 (s, 2H), 8.60 (d, J = 1.9 Hz, 2H), 8.52 (w s, 2H), 8.49 (s, 2H), 8.09 (d, J = 8.6 Hz, 2H), 7.50 (dd, J = 8.6, 2.0 Hz, 2H). NCL 142 1H NMR (400 MHz, DMSO) δ 8.80 (s, 2H), 7.41 – 7.20 (m, 12H), 5.56 (s, 2H), 3.79 (d, J = 3, 4 Hz, 4H). NCL 143 1H NMR (400 MHz, DMSO) δ 11.91 (w s, 2H), 8.66 (w s, 2H), 8.10 – 8.00 (m, 4H), 7.51 – 7.41 ( m, 6H), 2.45 (s, 6H). NCL 144 1H NMR (400 MHz, DMSO) δ 12.36 (w s, 2H), 8.38 – 8.23 (m, 4H), 7.16 (d, J = 3.5 Hz, 2H), 6, 82 (d, J = 3.5 Hz, 2H). NCL 145 1H NMR (400 MHz, DMSO) δ 12.41 (w s, 2H), 8.41 – 8.21 (m, 4H), 7.20 (d, J = 3.5 Hz, 2H), 6, 73 (d, J = 3.5 Hz, 2H). NCL 146 1H NMR (400 MHz, DMSO) δ 11.97 (w s, 2H), 11.45 (s, 2H), 8.47 (s, 2H), 8.30 (s, 2H), 8.02 ( s, 2H), 7.80 (dd, J = 8.6, 0.9 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.45 – 7.40 (m , 2H), 6.53 (s, 2H). 13C NMR (101 MHz, DMSO) δ 152.4, 150.6, 137.4, 127.6, 126.7, 124.5, 121.8, 120.3, 111.9, 102.0. NCL 147 1H NMR (400 MHz, DMSO) δ 13.09 (w s, 2H), 9.97 (s, 2H), 9.01 (s, 2H), 8.73 (s, 2H), 8.19 – 8.09 (m, 4H), 7.95 – 7.84 (m, 4H). NCL 148 1H NMR (400 MHz, DMSO) δ 12.44 (s, 4H), 8.60 (s, 2H), 8.44 (s, 2H), 7.99 (d, J = 8.3 Hz, 4H ), 7.81 (d, J = 8.3 Hz, 4H), 7.63 (d, J = 16.0 Hz, 2H), 6.66 (d, J = 16.0 Hz, 2H). NCL 149 1H NMR (400 MHz, DMSO) δ 8.75 (d, J = 6.1 Hz, 4H), 8.68 (s, 2H), 8.47 (s, 2H), 8.07 (d, J = 6.1 Hz, 4H). NCL 150 1H NMR (400 MHz, DMSO) δ 12.10 (w s, 2H), 8.23 – 8.07 (m, 4H), 7.55 (d, J = 8.7 Hz, 4H), 7, 13 (d, J = 16.0 Hz, 2H), 6.99 (d, J = 8.7 Hz, 4H), 6.81 (dd, J = 16.0, 9.4 Hz, 2H), 3.79 (s, 6H). NCL 151 1H NMR (400 MHz, DMSO) δ 11.92 (w s, 2H), 10.13 (w s, 2H), 8.28 (s, 4H), 7.75 (d, J = 8.5 Hz , 4H), 6.86 (d, J = 8.5 Hz, 4H). NCL 152 1H NMR (400 MHz, DMSO) δ 12.08 (w s, 2H), 9.35 (s, 2H), 8.66 (s, 2H), 8.48 (s, 2H), 7.67 ( d, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 6.88 (t, J = 7.6 Hz, 2H), 2.23 (s, 6H). NCL 153 1H NMR (400 MHz, DMSO) δ 11.68 (s, 2H), 8.78 (s, 2H), 8.09 (d, J = 8.4 Hz, 4H), 7.52 (d, J = 8.6 Hz, 4H), 2.92 (k, J = 7.5 Hz, 4H), 1.12 (t, J = 7.4 Hz, 6H). NCL 154 1H NMR (400 MHz, DMSO) δ 12.09 (s, 2H), 8.69 (s, 2H), 8.07 (d, J = 8.6 Hz, 4H), 7.50 (d, J = 8.6 Hz, 4H), 3.01 – 2.88 (m, 4H), 1.49 – 1.39 (m, 8H), 0.88 (t, J = 6.6 Hz, 6H) . NCL 155 1H NMR (400 MHz, DMSO) δ 11.63 (s, 2H), 8.76 (s, 2H), 8.01 (d, J = 8.6 Hz, 4H), 7.65 (d, J = 8.6 Hz, 4H), 2.41 (s, 6H). NCL 156 1H NMR (400 MHz, DMSO) δ 12.18 (s, 2H), 8.71 (s, 2H), 8.08 (d, J = 8.0 Hz, 4H), 7.50 (d, J = 8.3 Hz, 4H), 3.01 – 2.87 (m, 4H), 1.58 – 1.46 (m, 4H), 1.01 (t, J = 7.1 Hz, 6H) . NCL 157 1H NMR (400 MHz, DMSO) δ 11.71 (w s, 2H), 8.40 (s, 2H), 8.37 (s, 2H), 7.29 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 2.0 Hz, 2H), 6.73 (w s, 4H), 6.59 (dd, J = 8.3, 2.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 152.1, 151.5, 148.9, 136.0, 134.7, 115.1, 114.5, 112.8. NCL 158 1H NMR (400 MHz, DMSO) δ 13.03 (w s, 1H), 10.49 (w s, 1H), 9.35 (w s, 1H), 7.24 (d, J = 7.6 Hz, 2H), 7.11 – 6.61 (m, 6H), 2.5 (contains CH2 groups identified by COSY, but they are covered by the solvent signal), 0.93 (s, 6H). 13C NMR (101 MHz, DMSO) δ 157.8, 155.4, 152.9, 134.4, 130.1, 118.7, 115.6, 29.9, 10.8. *Correlation spectroscopy (COSY) was used to determine that the signal due to methylene protons occurred under the DMSO signal. Line broadening is evident in the 13C-NMR spectrum (due to tautomerization effects), making carbon assignment difficult. NCL 159 1H NMR (400 MHz, DMSO) δ 12.69 (b s, 1H), 10.44 (b s, 1H), 9.15 (b s, 1H), 7.21 (b s, 2H), 7. 09 – 6.60 (m, 6H), 2.98 (p, J = 7.7 Hz, 2H), 1.84 – 1.01 (m, 16H). NCL 160 1H NMR (400 MHz, DMSO) δ 12.49 (w s, 2H), 8.65 (s, 2H), 8.49 (s, 2H), 8.10 (d, J = 8.7 Hz, 4H)*, 7.47 (d, J = 8.3 Hz, 4H)*. NCL 161
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1H NMR (400 MHz, DMSO) δ 7.73 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 3.22 – 3.13 (m , 4H), 2.99 – 2.86 (m, 4H), 2.24 (s, 3H). NCL162 1H NMR (400 MHz, CDCl3) δ 7.76 – 7.70 (m, 2H), 7.35 – 7.30 (m, 2H), 4.99 (s, 2H), 4.34 (k, J = 7.1 Hz, 2H), 2.39 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H). NCL 163 1H NMR (400 MHz, DMSO) δ 13.88 (s, 1H), 13.21 (s, 1H), 11.50 (s, 2H), 9.70 (s, 2H), 7.70 (s , 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.14 (t, J = 7.5 Hz, 2H), 7.00 (d, J = 7.8 Hz, 2H ). NCL164 1H NMR (400 MHz, DMSO) δ 10.85 (s, 2H), 9.38 (s, 2H), 8.64 (s, 3H), 7.90 (s, 3H), 7.34 (d , J = 8.5 Hz, 2H), 7.19 (s, 3H), 6.83 (d, J = 2.1 Hz, 2H), 6.57 (dd, J = 8.5, 2, 1 Hz, 2H). NCL165 1H NMR (400 MHz, DMSO) δ 10.87 (w s, 1H), 9.53 (w s, 1H), 8.77 (w s, 3H), 7.61-7.44 (m, 8H ), 7.40 (w s, 3H), 7.27 (w s, 3H), 4.07 (w s, 2H). NCL166 1H NMR (400 MHz, DMSO) δ 12.70 (w s, 2H), 8.75 (w s, 2H), 8.55 (w s, 2H), 8.13 (d, J = 8.2 Hz, 4H), 7.81 (d, J = 8.1 Hz, 4H). NCL167 1H NMR (400 MHz, DMSO) δ 14.09 (s, 1H), 8.12 (d, J = 6.9 Hz, 2H), 7.97 (dd, J = 6.4, 3.0 Hz , 2H), 7.56 (ddd, J = 8.2, 6.7, 2.9 Hz, 7H). NCL168 1H NMR (400 MHz, DMSO) δ 11.86 (w s, 2H), 8.42 (w s, 4H), 7.35 (d, J = 6.8 Hz, 4H), 6.75 (s , 2H), 6.67 (d, J = 7.9 Hz, 2H), 3.44 (d, J = 6.9 Hz, 2H)*, 1.24 (t, J = 5.3 Hz, 6H). *Signal partially covered by H2O signal in DMSO NCL170 1H NMR (400 MHz, DMSO) δ 12.29 (s, 2H), 10.29 (s, 2H), 8.55 (s, 2H), 8.45 (s, 2H), 8.13 (d , J = 8.5 Hz, 2H), 7.71 (s, 2H), 7.35 (dd, J = 8.5, 1.8 Hz, 2H), 2.12 (s, 6H). NCL171 1H NMR (400 MHz, DMSO) δ 11.63 (s, 2H), 9.84 (s, 2H), 8.46 (s, 2H), 8.02 (s, 2H), 7.74 (d , J = 8.8 Hz, 2H), 6.30 (d, J = 7.4 Hz*, 2H), 6.17 (s, 2H), 2.94 (s, 12H). *A poorly separated doublet causes a reduced binding constant. NCL172 1H NMR (400 MHz, DMSO) δ 12.00 (s, 2H), 8.92 (s, 2H), 8.64 (dd, J = 4.7, 0.6 Hz, 2H), 8.57 (d, J = 8.1 Hz, 2H), 7.89 (td, J = 8.0, 1.6 Hz, 2H), 7.50 – 7.41 (m, 2H), 2.52 ( s, 6H). NCL 173 ^1H NMR (400 MHz, DMSO) δ 11.49 (w s, 2H), 10.62 (w s, 2H), 8.55 (w s, 2H), 7.59 (d, J = 8, 3 Hz, 2H), 6.98 (d, J = 1.9 Hz, 2H), 6.92 (dd, J = 8.4, 2.0 Hz, 2H), 2.37 (s, 6H) . NCL174 1H NMR (400 MHz, DMSO) δ 12.02 (s, 2H), 10.81 (s, 2H), 8.63 (s, 2H), 8.38 (s, 2H), 8.12 (d , J = 8.3 Hz, 2H), 7.13 – 6.84 (m, 4H). NCL 175 1H NMR (400 MHz, DMSO) δ 12.63 (w s, 2H), 8.90 (d, J = 2.1 Hz, 2H), 8.74 (s, 2H), 8.56 – 8, 42 (m, 4H), 7.66 (d, J = 8.4 Hz, 2H). NCL 176 1H NMR (400 MHz, DMSO) δ 11.94 (w s, 2H), 8.44 (s, 2H), 8.36 (s, 2H), 8.07 (dd, J = 4.8, 1 .7 Hz, 2H), 7.72 (dd, J = 7.6, 1.4 Hz, 2H), 7.19 (s, 4H), 6.67 (dd, J = 7.5, 4, 9 Hz, 2H). NCL 177 1H NMR (400 MHz, DMSO) δ 8.67 (s, 2H), 7.97 (s, 4H), 7.50 (d, J = 8.6 Hz, 4H), 4.81 (s, 4H ). NCL178 1H NMR (400 MHz, DMSO) δ 10.17 (s, 2H), 8.24 (s, 1H), 7.83 (d, J = 8.6 Hz, 4H), 7.50 (d, J = 8.6 Hz, 4H), 6.97 (s, 1H), 2.32 (s, 6H). NCL179 1H NMR (400 MHz, DMSO) δ 10.70 (s, 2H), 8.02 (s, 2H), 7.67 (d, J = 8.4 Hz, 4H), 7.52 (d, J = 8.4 Hz, 4H), 6.28 (s, 1H), 5.85 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.8, 162.6, 138.8, 134.1, 133.1, 128.9, 127.6, 73.5. NCL180 1H NMR (400 MHz, DMSO) δ 10.62 (s, 2H), 8.22 (d, J = 0.9 Hz, 1H), 7.82 – 7.74 (m, 4H), 7.53 – 7.47 (m, 4H), 6.93 (d, J = 0.6 Hz, 1H), 5.85 (t, J = 5.3 Hz, 2H), 4.74 (d, J = 5.2 Hz, 4H). NCL181 1H NMR (400 MHz, DMSO) δ 11.20 (s, 2H), 8.17 (s, 1H), 8.09 (s, 2H), 7.72 (d, J = 7.4 Hz, 4H ), 7.54 (d, J = 7.6 Hz, 4H), 6.83 (s, 1H). NCL182 1H NMR (400 MHz, CDCl3) δ 7.44 – 7.19 (m, 6H), 5.67 (s, 1H), 5.42 (s, 1H), 5.06 (s, 2H), 4 .95 – 4.63 (m, 1H), 1.52 (d, J = 6.8 Hz, 3H). NCL183 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.43 – 7.13 (m, 10H), 6.19 (s, 1H), 5.94 (s, 1H), 4 .95 – 4.48 (m, 2H), 1.56 (d, J = 6.8 Hz, 4H), 1.50 (d, J = 6.5 Hz, 2H). NCL184 1H NMR (400 MHz, MeOD) δ 7.44 – 7.04 (m, 10H), 5.26 – 4.53 (m, 7H), 1.51 – 1.35 (m, 6H). NCL188
76
1H NMR (400 MHz, DMSO) δ 11.54 (s, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.90 (s, 3H), 7.47 (d, J = 8.6 Hz, 2H), 2.91 – 2.82 (m, 2H), 1.48 – 1.32 (m, 4H), 0.89 – 0.84 (m, 3H). 13C NMR (101 MHz, DMSO) δ 156.2, 153.8, 134.8, 134.4, 128.7, 128.4, 28.1, 26.6, 22.0, 13.8. NCL190 1H NMR (400 MHz, DMSO) δ 11.42 (s, 1H), 8.06 (d, J = 8.7 Hz, 3H), 8.01 – 7.71 (m, 5H), 7.53 (d, J = 8.7 Hz, 3H), 4.90 (s, 2H), 3.69 (w s, 3H). NCL191 1H NMR (400 MHz, DMSO) δ 11.51 (s, 1H), 8.85 (s, 3H), 7.99 (d, J = 8.6 Hz, 7H), 7.46 (d, J = 8.6 Hz, 4H), 2.35 (s, 3H). NCL192 1H NMR (400 MHz, DMSO) δ 11.65 (s, 1H), 8.21 (s, 4H), 7.82 (dd, J = 7.6, 1.9 Hz, 2H), 7.53 – 7.39 (m, 3H). NCL193 1H NMR (400 MHz, DMSO) δ 10.71 (s, 2H), 8.00 (s, 2H), 7.66 (d, J = 8.5 Hz, 4H), 7.60 (d, J = 8.6 Hz, 4H), 6.27 (s, 1H), 5.86 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.7, 162.6, 138.8, 134.5, 131.8, 127.9, 121.7, 73.5. NCL194 1H NMR (400 MHz, DMSO) δ 12.01 (w s, 1H), 10.84 (s, 2H), 9.98 (w s, 1H), 9.79 (w s, 1H), 7. 96 (w s, 3H), 7.79 (w s, 2H), 7.67 – 7.37 (m, 6H), 5.37 (w s, 1H). NCL195 1H NMR (400 MHz, DMSO) δ 10.51 (s, 2H), 8.00 (s, 2H), 7.54 (d, J = 8.0 Hz, 4H), 7.26 (d, J = 7.9 Hz, 4H), 6.26 (s, 1H), 5.77 (s, 2H), 2.34 (s, 6H). 13C NMR (101 MHz, DMSO) δ 162.8, 162.6, 140.1, 138.4, 132.5, 129.4, 126.0, 73.3, 21.0. NCL196 1H NMR (400 MHz, CDCl3) δ 10.31 (s, 2H), 9.74 (s, 2H), 7.94 (s, 2H), 7.48 (d, J = 8.6 Hz, 4H ), 6.83 (d, J = 8.6 Hz, 4H), 6.20 (s, 1H), 5.70 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 162.7, 162.5, 158.3, 140.5, 127.7, 126.3, 115.7, 73.0. NCL197 1H NMR (400 MHz, DMSO) δ 10.51 (s, 2H), 9.55 (s, 2H), 7.95 (s, 2H), 7.22 (t, J = 7.9 Hz, 2H ), 7.11 – 7.04 (m, 4H), 6.76 (d, J = 8.4 Hz, 2H), 6.23 (s, 1H), 5.80 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.8, 162.6, 157.7, 140.4, 136.4, 129.9, 117.4, 116.1, 112.4, 73.3. NCL199 1H NMR (400 MHz, DMSO) δ 10.60 (s, 2H), 8.04 (s, 2H), 7.66 (d, J = 7.5 Hz, 4H), 7.45 (t, J = 7.1 Hz, 4H), 7.40 – 7.34 (m, 2H), 6.30 (s, 1H), 5.82 (s, 2H). 13C NMR (101 MHz, DMSO) δ 163.3, 163.1, 140.5, 135.7, 129.3, 129.2, 126.5, 73.9. NCL200 1H NMR (400 MHz, DMSO) δ 10.51 (s, 2H), 8.02 (s, 2H), 7.58 (d, J = 8.3 Hz, 4H), 7.47 (d, J = 8.3 Hz, 4H), 6.25 (s, 1H), 5.77 (s, 2H), 1.31 (s, 18H). NCL201 1H NMR (400 MHz, DMSO) δ 10.19 (s, 2H), 7.91 (s, 2H), 7.47 (d, J = 8.8 Hz, 4H), 6.77 (d, J = 8.9 Hz, 4H), 6.16 (s, 1H), 5.63 (s, 2H). NCL202 1H NMR (600 MHz, DMSO) δ 13.43 (s, 1H), 11.45 (s, 2H), 10.28 (w s, 1H), 9.47 (s, 1H), 8.38 ( s, 2H), 7.71 (dd, J = 7.7, 1.3 Hz, 1H), 7.67 (wd, J = 7.0 Hz, 2H), 7.53 – 7.48 ( m, 1H), 7.28 – 7.23 (m, 2H), 7.03 – 6.98 (m, 2H), 6.96 – 6.90 (m, 4H), 6.56 (w s , 1H). *Due to tautomers and rotamers due to the proximity of phenol to hydrazone, >17 protons were observed. 13C NMR (151 MHz, DMSO) δ 167.7, 163.2, 162.6, 156.7*, 141.4*, 135.3, 133.9, 131.0, 130.5, 120.7 *, 119.95, 119.8, 118.9, 117.6, 116.7, 116.6*. *Line broadening due to tautomers and rotamers makes signal assignment difficult. NCL203 1H NMR (400 MHz, DMSO) δ 12.60 – 10.94 (m, 3H), 7.79 – 7.39 (m, 3H), 2.25 (s, 2H), 1.82 – 1, 59 (m, 10H), 1.35 – 1.15 (m, 10H). NCL204 1H NMR (400 MHz, DMSO) δ 9.58 (d, J = 7.6 Hz, 2H), 7.82 (s, 4H), 7.50 (s, 4H), 6.45 (d, J = 9.6 Hz, 1H), 5.75 (s, 2H), 2.28 (d, J = 9.3 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 163.48, 162.11, 143.34, 137.68, 132.88, 128.34, 127.16, 75.15, 13.01. NCL205 1H NMR (600 MHz, DMSO) δ 10.92 (s, 2H), 8.42 (s, 2H), 7.98 (d, J = 7.5 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 6.35 (s, 1H), 5.95 (s, 2H). 13C NMR (151 MHz, DMSO) δ 162.75, 162.68, 136.0, 132.4, 132.0, 130.1, 129.9, 127.6, 126.2, 73.7. NCL206 1H NMR (400 MHz, DMSO) δ 10.75 (s, 2H), 8.09 (s, 2H), 7.52 (d, J = 8.1 Hz, 4H), 7.24 (d, J = 8.0 Hz, 4H), 6.71 (s, 1H), 2.33 (s, 6H). NCL207
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1H NMR (400 MHz, DMSO) δ 11.43 (s, 2H), 11.05 (s, 2H), 8.31 (s, 2H), 7.42 (s, 2H), 7.29 – 7 .18 (m, 2H), 6.89 (t, J = 7.7 Hz, 4H), 6.74 (s, 2H). NCL208 1H NMR (400 MHz, DMSO) δ 9.63 (s, 2H), 7.82 (d, J = 8.5 Hz, 4H), 7.46 (d, J = 8.5 Hz, 4H), 6.73 (s, 2H), 2.29 (s, 6H). NCL209 1H NMR (400 MHz, DMSO) δ 10.75 (s, 2H), 9.55 (s, 2H), 8.04 (s, 2H), 7.21 (t, J = 7.8 Hz, 2H ), 7.06 (s, 2H), 7.02 (d, J = 7.6 Hz, 2H), 6.84 – 6.57 (m, 4H). NCL210 1H NMR (400 MHz, DMSO) δ 11.16 (s, 2H), 8.22 (s, 2H), 7.85 (d, J = 8.3 Hz, 4H), 7.80 (d, J = 8.5 Hz, 4H), 6.92 (s, 2H). NCL211 1H NMR (400 MHz, DMSO) δ 10.56 (s, 2H), 9.78 (s, 2H), 8.02 (s, 2H), 7.45 (d, J = 8.6 Hz, 4H ), 6.80 (d, J = 8.6 Hz, 4H), 6.62 (s, 2H). NCL212 1H NMR (400 MHz, DMSO) δ 10.94 (s, 2H), 8.10 (s, 2H), 7.67 – 7.60 (m, 4H), 7.61 – 7.54 (m, 4H), 6.80 (s, 2H). NCL213 1H NMR (400 MHz, DMSO) δ 10.23 (w s, 2H), 7.27 (s, 2H), 2.27 – 1.98 (m, 2H), 1.78 – 1.42 (m , 10H), 1.33 – 1.00 (m, 10H). NCL214 1H NMR (400 MHz, DMSO) δ 10.84 (s, 2H), 8.14 (s, 2H), 7.63 (d, J = 7.4 Hz, 4H), 7.46 – 7.33 (m, 3H), 6.76 (s, 2H). NCL215 1H NMR (600 MHz, DMSO) δ 12.06 (s, 2H), 8.72 (s, 2H), 7.97 (t, J = 8.4 Hz, 2H), 7.56 (dd, J = 11.1, 1.9 Hz, 2H), 7.38 (dd, J = 8.5, 1.9 Hz, 2H), 2.42 (d, J = 2.9 Hz, 6H). NCL216 1H NMR (400 MHz, DMSO) δ 12.43 (w s, 2H), 8.66 (w s, 2H), 8.62 (w s, 2H), 8.38 (t, J = 8.3 Hz, 2H), 7.61 (dd, J = 10.5, 1.9 Hz, 2H), 7.45 (dd, J = 8.6, 1.6 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 160.7 (d, J = 254.5 Hz), 152.8*, 140.8*, 136.3 (d, J = 10.8 Hz), 128.5 , 125.3, 120.2 (d, J = 10.0 Hz), 116.7 (d, J = 24.7 Hz).
*Wide signals NCL217 1H NMR (400 MHz, DMSO) δ 11.66 (s, 2H), 8.61 (s, 2H), 7.94 (d, J = 8.2 Hz, 4H), 7.25 (d, J = 8.1 Hz, 4H), 2.41 (s, 6H), 2.35 (s, 6H). 13C NMR (101 MHz, DMSO) δ 154.0, 153.3, 139.7, 133.9, 129.0, 127.0, 21.0, 14.9. NCL218 1H NMR (400 MHz, DMSO) δ 10.73 (s, 2H), 8.05 (s, 2H), 7.70 (d, J = 8.58 Hz, 4H), 7.31 – 7.27 (m, 4H), 6.24 (s, 1H), 5.92 (s, 2H), 4.17 (dk, J = 7.06, 8.70 Hz, 8H), 1.28 (td, J = 1.01, 7.05 Hz, 12H). NCL219 1H NMR (400 MHz, DMSO) δ 11.74 (s, 2H), 8.60 (s, 2H), 7.95 (d, J = 8.6 Hz, 4H), 7.45 (d, J = 8.6 Hz, 4H), 2.42 (s, 6H), 1.31 (s, 18H). 13C NMR (101 MHz, DMSO) δ 154.1, 153.3, 152.6, 134.0, 126.8, 125.0, 34.5, 31.0, 14.9. NCL220 1H NMR (400 MHz, DMSO) δ 10.61 (s, 2H), 8.03 (s, 2H), 7.70 (dd, J = 8.7, 5.6 Hz, 4H), 7.29 (t*, J = 8.9 Hz, 4H), 6.27 (s, 1H), 5.82 (s, 2H). NCL221 1H NMR (600 MHz, DMSO) δ 10.89 (s, 2H), 8.11 (s, 2H), 7.86 (d, J = 8.2 Hz, 4H), 7.81 (d, J = 8.4 Hz, 4H), 6.34 (s, 1H), 5.94 (s, 2H). NCL222 1H NMR (400 MHz, DMSO) δ 10.76 (s, 2H), 7.99 (s, 2H), 7.70 (td, J = 1.7, 9.2 Hz, 2H), 7.54 – 7.41 (m, 4H), 6.30 (s, 1H), 5.87 (s, 2H). NCL223 1H NMR (400 MHz, DMSO) δ 10.48 (s, 2H), 10.07 (s, 2H), 7.98 (s, 2H), 7.65 (d, J = 8.7 Hz, 4H ), 7.58 (d, J = 8.7 Hz, 4H), 6.24 (s, 1H), 5.76 (s, 2H), 2.07 (s, 6H). NCL224 1H NMR (400 MHz, DMSO) δ 8.36 (s, 1H), 7.96 – 7.90 (m, 2H), 7.86 (s, 1H), 7.79 – 7.73 (m, 2H), 7.54 – 7.47 (m, 6H), 4.26 (k, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H). NCL225 1H NMR (400 MHz, DMSO) δ 8.35 (s, 1H), 7.98 – 7.91 (m, 2H), 7.87 (s, 1H), 7.79 – 7.74 (m, 2H), 7.51 (dd, J = 2.8, 8.5 Hz, 6H), 3.99 (d, J = 6.5 Hz, 2H), 1.95 (hept, J = 6.7 Hz, 1H), 0.93 (d, J = 6.7 Hz, 6H). NCL226 1H NMR (400 MHz, DMSO) δ 10.95 (s, 1H), 9.18 (t, J = 5.6 Hz, 1H), 8.38 (d, J = 10.1 Hz, 2H), 8.32 (s, 1H), 7.99 – 7.92 (m, 2H), 7.83 (d, J = 8.5 Hz, 2H), 7.57 – 7.46 (m, 5H) , 3.30 – 3.20 (m, 2H), 1.14 (t, J = 7.2 Hz, 3H). NCL227 1H NMR (400 MHz, DMSO) δ 11.00 (s, 1H), 9.68 (s, 1H), 8.52 (s, 1H), 8.41 (s, 1H), 8.32 (s , 1H), 8.01 – 7.80 (m, 4H), 7.56 – 7.46 (m, 4H), 7.36 (d, J = 6.00 Hz, 4H), 7.30 – 7.21 (m, 1H), 4.56 – 4.33 (m, 2H). NCL228 1H NMR (400 MHz, DMSO) δ 10.95 (s, 1H), 9.22 (t, J = 5.6 Hz, 1H), 8.40 (s, 1H), 8.34 (s, 1H ), 8.32 (s, 1H), 7.98 – 7.90 (m, 2H), 7.87 – 7.80 (m, 2H), 7.55 – 7.48 (m, 4H), 3.22 (k, J = 6.6 Hz, 2H), 1.56 – 1.46 (m, 2H), 1.39 – 1.25 (m, 6H), 0.88 (t, J = 6.6 Hz, 3H). NCL229
78
1H NMR (400 MHz, DMSO) δ 10.98 (s, 1H), 9.60 (s, 1H), 8.54 (s, 1H), 8.40 (s, 1H), 8.30 (s , 1H), 8.01 – 7.89 (m, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.64 – 7.59 (m, 1H), 7.54 – 7.47 (m, 4H), 6.42 (dd, J = 1.8, 3.3 Hz, 1H), 6.31 (m, 1H), 4.44 (d, J = 5.5 Hz , 2H).

NCL230 1H NMR (400 MHz, DMSO) δ 10.41 (s, 2H), 7.98 (s, 2H), 7.59 (d, J = 8.9 Hz, 4H), 7.02 (d, J = 8.9 Hz, 4H), 6.23 (s, 1H), 5.73 (s, 2H), 3.80 (s, 6H). NCL231 1H NMR (400 MHz, DMSO) δ 7.71 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 6.07 (s, 2H), 5.78 (s, 2H), 2.22 (s, 3H), 1.28 (s, 9H). 13C NMR (101 MHz, DMSO) δ 159.03, 149.99, 147.94, 137.04, 125.31, 124.70, 34.22, 31.09, 13.59. NCL232 1H NMR (400 MHz, DMSO) δ 7.96 (s, 1H), 7.57 (d, J = 7.2 Hz, 2H), 7.34 (d, J = 7.3 Hz, 2H), 5.91 (s, 2H), 5.53 (s, 2H), 1.28 (s, 9H). 13C NMR (101 MHz, DMSO) δ 160.25, 150.33, 143.29, 134.17, 126.01, 125.10, 34.35, 31.08. NCL233 1H NMR (400 MHz, DMSO) δ 11.52 (s, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.89 (s, 4H), 7.46 (d, J = 8.7 Hz, 2H), 2.90 – 2.78 (m, 2H), 1.53 – 1.37 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H) . 13C NMR (101 MHz, DMSO) δ 156.12, 153.60, 134.76, 134.39, 128.69, 128.38, 28.39, 19.40, 13.60. NCL234 1H NMR (400 MHz, DMSO) δ 11.42 (s, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.86 (s, 4H), 7.48 (d, J = 8.7 Hz, 2H), 2.85 (k, J = 7.6 Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 156.08, 154.70, 134.44, 134.38, 128.64, 128.42, 20.07, 10.69. NCL235 1H NMR (400 MHz, DMSO) δ 8.25 (s, 1H), 8.22 (t, J = 8.5 Hz, 1H), 7.50 (d, J = 10.5 Hz, 1H), 7.42 (s, 4H), 7.34 (d, J = 8.4 Hz, 1H). OR 1H NMR (400 MHz, DMSO) δ 8.31 – 8.15 (m, 2H), 7.73 – 7.14 (m, 6H). 13C NMR (101 MHz, DMSO) δ 160.16 (d, J = 253.3 Hz), 157.07, 136.99 (d, J = 4.2 Hz), 134.83 (d, J = 10 .7 Hz), 128.07 (d, J = 3.6 Hz), 125.08 (d, J = 3.2 Hz), 121.22 (d, J = 10.0 Hz), 116.45 (d, J = 24.8 Hz). NCL236 1H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 7.95 – 7.64 (m, 5H), 7.53 (d, J = 10.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO) δ 159.90 (d, J = 252.9 Hz), 156.21, 148.64, 134.88 (d, J = 10.8 Hz), 131.48 (d , J = 3.9 Hz), 125.07 (d, J = 11.4 Hz), 124.74 (d, J = 3.3 Hz), 116.79 (d, J = 26.3 Hz) , 17.80 (d, J = 6.1 Hz). NCL237 1H NMR (400 MHz, DMSO) δ 14.38 (s, 1H), 7.30 (d, J = 8.6 Hz, 1H), 6.73 – 6.66 (m, 2H), 6.39 (s, 2H), 6.27 (s, 2H), 2.57 (d, J = 7.3 Hz, 2H), 1.63 – 1.46 (m, 5H), 1.32 (s, 1H), 1.13 – 0.97 (m, 3H), 0.89 – 0.77 (m, 2H). 13C NMR (101 MHz, DMSO) δ 160.62, 158.15, 152.94, 134.16, 130.52, 124.27, 119.53, 116.22, 44.10, 36.13, 32 .45, 25.98, 25.57. NCL238 1H NMR (400 MHz, DMSO) δ 10.92 (s, 2H), 8.61 (d, J = 1.7 Hz, 2H), 8.14 (dd, J = 8.3, 2.0 Hz , 2H), 8.05 (s, 2H), 7.59 (d, J = 8.3 Hz, 2H), 6.30 (s, 1H), 5.93 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.67, 162.59, 149.65, 147.85, 135.86, 130.63, 124.61, 73.64. NCL239 1H NMR (400 MHz, DMSO) δ 10.82 (s, 2H), 8.80 (d, J = 1.6 Hz, 2H), 8.54 (dd, J = 4.7, 1.5 Hz , 2H), 8.11 – 8.03 (m, 4H), 7.46 (dd, J = 7.9, 4.8 Hz, 2H), 6.33 (s, 1H), 5.91 ( s, 2H). 13C NMR (101 MHz, DMSO) δ 162.74, 162.62, 149.41, 147.69, 137.19, 132.57, 131.02, 123.96, 73.55. NCL240 1H NMR (600 MHz, DMSO) δ 10.91 (s, 2H), 8.60 – 8.53 (m, 2H), 8.10 (s, 2H), 7.94 (d, J = 7, 7 Hz, 2H), 7.87 (t, J = 7.3 Hz, 2H), 7.38 – 7.30 (m, 2H), 6.35 (s, 1H), 5.94 (w s , 2H). 13C NMR (151 MHz, DMSO) δ 162.7*, 154.0, 149.4, 140.8, 136.7, 123.2, 119.1, 73.7*
*2D NMR analysis suggests that signals from the two quaternary carbons of the pyrimidine core occur at 162.7 ppm. NCL241 1H NMR (400 MHz, DMSO) δ 11.03 (s, 2H), 8.62 (d, J = 3.3 Hz, 4H), 8.01 (s, 2H), 7.60 (d, J = 3.2 Hz, 4H), 6.38 (s, 1H), 6.00 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.71, 162.69, 150.18, 142.21, 137.53, 120.15, 73.96. NCL242 1H NMR (400 MHz, DMSO) δ 10.61 (s, 2H), 10.42 (s, 1H), 9.78 (s, 2H), 9.08 (s, 1H), 8.87 (s , 1H), 8.84 (s, 2H), 8.20 (s, 2H), 7.08 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 2.5 Hz , 2H), 6.89 – 6.76 (m, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.64 (dd, J = 8.7, 2.7 Hz, 2H), 5.91 (s, 1H), 5.84 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.66, 162.18, 151.58, 149.94, 149.88, 149.08, 140.17, 120.97, 120.39, 118.32, 117 .43, 117.23, 116.73, 115.04, 112.60, 72.69. NCL243 1H NMR (400 MHz, DMSO) δ 10.25 (s, 2H), 9.18 (s, 4H), 7.87 (s, 2H), 7.11 (d, J = 1.4 Hz, 2H ), 6.90 (dd, J = 8.1, 1.4 Hz, 2H), 6.78 (d, J = 8.1 Hz, 2H), 6.14 (s, 1H), 5.69 (c, 2H). 13C NMR (101 MHz, DMSO) δ 163.16, 162.89, 147.24, 146.03, 141.55, 127.12, 119.30, 116.24, 113.09, 73.36. NCL244 1H NMR (400 MHz, DMSO) δ 10.71 (s, 3H), 9.95 (s, 3H), 9.30 (s, 3H), 8.96 (s, 1H), 8.26 (s , 2H), 7.13 (d, J = 7.7 Hz, 1H), 7.01 (d, J = 7.4 Hz, 2H), 6.95 (d, J = 7.6 Hz, 1H ), 6.86 – 6.74 (m, 3H), 6.71 (t, J = 7.7 Hz, 2H), 5.95 (s, 1H), 5.85 (s, 2H). 13C NMR (101 MHz, DMSO) δ 163.39, 162.70, 162.18, 147.40, 145.67, 145.47, 144.80, 140.68, 121.27, 120.58, 119 .43, 119.18, 118.88, 118.44, 117.96, 116.00, 72.75. NCL245 1H NMR (400 MHz, DMSO) δ 10.75 (s, 2H), 8.83 – 8.78 (m, 2H), 8.74 (s, 2H), 8.05 – 8.00 (m, 2H), 7.98 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 7.0 Hz, 2H), 7.67 – 7.53 (m, 6H), 6, 51 (s, 1H), 5.92 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.94, 162.74, 139.74, 133.69, 130.51, 129.90, 129.25, 128.84, 126.93, 126.11, 125 .99, 125.60, 123.98, 73.46. NCL246 1H NMR (400 MHz, DMSO) δ 10.55 (s, 2H), 7.87 (d, J = 9.2 Hz, 2H), 7.59 (d, J = 7.4 Hz, 4H), 7.37 (t, J = 7.5 Hz, 4H), 7.29 (t, J = 7.3 Hz, 2H), 7.01 (dd, J = 16.1, 9.3 Hz, 2H ), 6.84 (d, J = 16.0 Hz, 2H), 6.13 (s, 1H), 5.78 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.52, 142.73, 136.42, 135.24, 128.76, 128.19, 126.69, 126.04, 73.35. NCL247 1H NMR (400 MHz, DMSO) δ 10.20 (s, 2H), 9.00 (s, 4H), 8.43 (s, 2H), 7.80 (s, 2H), 6.59 (s , 4H), 6.07 (s, 1H), 5.67 (s, 2H). NCL248 1H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 8.66 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.60 – 7.44 (m, 3H), 5.95 (s , 2H), 5.58 (s, 2H). 13C NMR (101 MHz, DMSO) δ 160.68, 142.16, 133.59, 132.18, 130.24, 128.52, 127.94, 126.48, 125.77, 125.58, 125 .02, 124.11. NCL249
80
1H NMR (400 MHz, DMSO) δ 7.82 (d, J = 9.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7 .6 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 6.95 (dd, J = 16.0, 9.5 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 4.38 (s, 4H). NCL250 1H NMR (400 MHz, DMSO) δ 8.34 (s, 2H), 7.78 – 7.65 (m, 4H), 7.54 – 7.43 (m, 4H), 3.87 (s, 4H). 13C NMR (101 MHz, DMSO) δ 160.85, 135.20, 134.84, 129.44, 128.77, 60.70. NCL251 1H NMR (400 MHz, DMSO) δ 8.35 (s, 2H), 7.79 – 7.70 (m, 4H), 7.55 – 7.45 (m, 4H), 3.60 (d, J = 0.9 Hz, 4H), 1.67 (t, J = 2.7 Hz, 4H). 13C NMR (101 MHz, DMSO) δ 159.49, 135.06, 134.98, 129.42, 128.74, 60.20, 28.25. NCL252 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 2H), 7.71 – 7.60 (m, 4H), 7.42 – 7.33 (m, 4H), 3.71 (td, J = 6.8, 1.1 Hz, 4H), 2.10 (p, J = 6.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.00, 136.54, 134.74, 129.25, 128.90, 59.16, 31.90. NCL253 1H NMR (400 MHz, DMSO) δ 7.89 (s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 5.71 (s, 2H), 5.25 (s, 2H), 2.91 (s, 6H). NCL254 1H NMR (400 MHz, DMSO) δ 11.30 (s, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.80 (s, 4H), 7.47 (d, J = 8.7 Hz, 2H), 2.99 – 2.68 (m, 2H), 1.68 – 1.13 (m, 6H), 0.84 (t, J = 7.2 Hz, 3H) . 13C NMR (101 MHz, DMSO) δ 156.06, 153.87, 134.79, 134.40, 128.68, 128.41, 30.95, 26.63, 25.60, 21.95, 13 ,86. NCL255 1H NMR (400 MHz, DMSO) δ 10.69 (s, 2H), 8.09 (s, 2H), 7.77 (s, 8H), 7.71 (d, J = 7.5 Hz, 4H ), 7.47 (t, J = 7.6 Hz, 4H), 7.37 (t, J = 7.3 Hz, 2H), 6.42 (s, 1H), 5.87 (s, 2H ). 13C NMR (101 MHz, DMSO) δ 162.80, 162.64, 140.22, 139.71, 139.48, 134.31, 128.97, 127.64, 127.05, 126.66, 126 .55, 73.49. NCL256 1H NMR (400 MHz, DMSO) δ 10.47 (s, 4H), 9.81 (s, 2H), 8.14 (s, 2H), 7.29 (d, J = 8.4 Hz, 2H ), 6.40 – 6.24 (m, 4H), 5.78 (s, 3H). NCL257 1H NMR (400 MHz, DMSO) δ 11.73 (s, 1H, isomer A), 10.65 (s, 1H, isomer A), 8.99 (s, 1H, isomer A), 8.75 (d , J = 8.6 Hz, 1H, isomer A), 7.92 (d, J = 8.9 Hz, 1H, isomer A masks the signal of isomer B), 7.85 (d, J = 7.7 Hz, 1H, isomer A), 7.73 – 7.50 (m, 5H, isomers A & B), 7.39 (t, J = 7.6 Hz, 1H, isomer A masks isomer B signal), 7.24 (d, J = 8.9 Hz, 1H, isomer A). 1H NMR (400 MHz, DMSO) δ 12.88 (s, 1H, isomer B), 9.99 (s, 1H, isomer B), 8.65 (d, J = 8.8 Hz, 1H, isomer B ), 8.07 – 8.01 (m, 2H, isomer B), 7.95 (d, J = 9.0 Hz, 1H, isomer B), 7.47 – 7.42 (m, 1H), 7.30 (d, J = 8.8 Hz, 1H, isomer B), 7.29 (d, J = 9.0 Hz, 1H, isomer B). Masked signals identified in NMR of isomer A. 13C NMR (101 MHz, DMSO) δ 157.22, 154.74, 146.77, 133.32, 131.19, 128.61, 128.20, 128.07, 124 ,17, 123.58, 118.12, 109.69. NCL258 1H NMR (400 MHz, DMSO) δ 7.33 (s, 8H), 3.64 (s, 4H), 2.42 (s, 4H), 1.42 (s, 4H). 13C NMR (101 MHz, DMSO) δ 140.20, 130.86, 129.64, 127.95, 52.14, 48.53, 27.32. NCL259 1H NMR (400 MHz, DMSO) δ 11.37 (s, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.84 (s, 4H), 7.46 (d, J = 8.6 Hz, 2H), 2.83 (d, J = 7.1 Hz, 2H), 1.69 – 1.43 (m, 6H), 1.08 (s, 5H). 13C NMR (101 MHz, DMSO) δ 156.02, 153.02, 135.35, 134.32, 128.84, 128.33, 35.79, 33.41, 31.97, 25.65. NCL260 1H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 8.3 Hz, 4H), 7.21 (d, J = 8.4 Hz, 4H), 3.70 (s, 4H), 2.70 (s, 4H). NCL261
81
1H NMR (400 MHz, DMSO) δ 12.09 (s, 1H), 8.34 (s, 1H), 8.24 – 8.17 (m, 2H), 7.96 (dd, J = 8, 8, 4.6 Hz, 3H), 7.83 (s, 4H), 7.61 – 7.54 (m, 2H). 13C NMR (101 MHz, DMSO) δ 155.42, 146.87, 133.90, 132.70, 131.23, 129.46, 128.35, 128.31, 127.81, 127.35, 126 .82, 123.04. NCL262 1H NMR (400 MHz, DMSO) δ 9.98 (s, 2H), 7.42 – 7.07 (m, 12H), 5.97 (d, J = 25.2 Hz, 1H), 5.59 (s, 2H), 2.79 (t, J = 7.6 Hz, 4H), 2.57 – 2.44 (m, 4H). 13C NMR (101 MHz, DMSO) δ 162.74, 162.47, 143.11, 141.16, 128.40, 128.27, 125.86, 72.62, 33.61, 32.43. NCL263 1H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.47 (t, J = 7.6 Hz, 1H), 8.40 (s, 1H), 7.90 (s, 4H ), 7.81 (d, J = 10.4 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H). NCL264 1H NMR (400 MHz, DMSO) δ 10.78 (s, 2H), 8.23 (s, 2H), 8.10 – 7.87 (m, 10H), 7.56 (s, 4H), 6 .46 (s, 1H), 5.88 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.83, 162.67, 140.09, 133.19, 133.12, 133.00, 128.45, 128.08, 127.76, 126.96, 126 .67, 126.53, 122.37, 73.63. NCL265 1H NMR (400 MHz, DMSO) δ 7.78 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 5.85 (s, 2H), 5.45 (s, 2H), 2.90 – 2.74 (m, 2H), 1.49 – 1.33 (m, 2H), 1.33 – 1.11 (m, 8H), 0. 84 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 160.12, 149.95, 138.11, 131.48, 127.92, 127.10, 31.31, 29.43, 28.69, 26.44, 25 .86, 22.07, 13.96. NCL266 1H NMR (400 MHz, DMSO) δ 7.77 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 5.85 (s, 2H), 5.43 (s, 2H), 2.90 – 2.77 (m, 2H), 1.82 – 1.65 (m, 3H), 1.62 – 1.28 (m, 6H), 1, 21 – 1.00 (m, 2H). 13C NMR (101 MHz, DMSO) δ 160.07, 150.06, 138.09, 131.49, 127.96, 127.08, 39.95, 32.79, 32.22, 25.17, 24 ,86. NCL267 1H NMR (400 MHz, DMSO) δ 11.62 (s, 1H), 8.58 (s, 1H), 8.06 (d, J = 4.7 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 152.2, 145.5, 141.3, 133.8, 133.4, 132.6, 128.8, 128.2. NCL268 1H NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 8.54 (s, 1H), 8.03 (s, 2H), 7.62 (d, J = 7.6 Hz, 2H ), 7.24 (d, J = 7.4 Hz, 2H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO) δ 152.4, 145.5, 142.9, 139.2, 132.1, 131.7, 129.4, 128.7, 126.6, 21.0. NCL269 1H NMR (400 MHz, DMSO) δ 11.66 (s, 1H), 10.89 (w s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.51 (w s, 3H), 7.42 (d, J = 8.0 Hz, 1H), 7.22 – 7.14 (m, 1H), 7 .14 – 7.06 (m, 1H), 2.39 (s, 3H). 13C NMR (101 MHz, DMSO) δ 155.6, 151.9, 137.2, 129.5, 124.2, 122.8, 122.3, 120.6, 113.8, 111.7, 15 ,9.

NCL270 1H NMR (400 MHz, DMSO) δ 11.71 (s, 2H), 11.33 (w s, 2H), 8.33 (d, J = 7.8 Hz, 2H), 8.05 (d, J = 2.7 Hz, 2H), 7.83 (w s, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.24 – 7.18 (m, 2H), 7 ,18 – 7.12 (m, 2H), 2.50 (s, 6H, hidden by the DMSO-d6 signal). 13C NMR (101 MHz, DMSO) d 153.2, 153.0, 137.2, 129.8, 124.2, 122.8, 122.4, 120.6, 113.7, 111.8, 15 ,9. NCL271 1H NMR (400 MHz, DMSO) δ 12.23 (w s, 2H), 8.68 (w s, 2H), 8.06 (d, J = 8.7 Hz, 4H), 7.50 (d , J = 8.7 Hz, 4H), 3.03 – 2.86 (m, 4H), 1.52 – 1.36 (m, 8H), 1.34 – 1.23 (m, 4H), 0.84 (t, J = 7.3 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 155.7, 154.4, 134.6, 134.6, 128.9, 128.4, 30.9, 27.2, 25.8, 22.0, 13 ,9.

NCL272 1H NMR (400 MHz, DMSO) δ 12.28 (w s, 2H), 8.68 (w s, 2H), 8.06 (d, J = 8.5 Hz, 4H), 7.50 (d , J = 8.5 Hz, 4H), 3.04 – 2.86 (m, 4H), 1.53 – 1.35 (m, 8H), 1.32 – 1.14 (m, 12H), 0.83 (t, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 155.6, 154.3, 134.6 (2 × C)*, 128.9, 128.4, 31.2, 28.6, 28.5, 27.1 , 26.1, 22.0, 13.9.
*Determined by 2D NMR analysis. NCL273 1H NMR (400 MHz, acetone) δ 13.20 (w s, 2H), 8.19 (w s, 2H), 7.96 (d, J = 8.6 Hz, 4H), 7.46 (d , J = 8.7 Hz, 4H), 3.14 – 3.05 (m, 4H), 2.15 (dt, J = 15.5, 7.8 Hz, 2H), 1.88 – 1, 78 (m, 4H), 1.67 – 1.46 (m, 12H), 1.31 – 1.17 (m, 4H). 13C NMR (101 MHz, acetone) δ 157.7, 156.6, 136.2, 136.1, 129.5, 129.4, 40.9, 33.3, 33.1, 28.4, 25 ,8. NCL274 1H NMR (400 MHz, DMSO) δ 11.99 (s, 2H), 11.64 (s, 2H), 8.50 (w s, 4H), 8.10 (s, 2H), 8.02 ( s, 2H), 7.44 (d, J = 7.3 Hz, 2H), 7.36 (d, J = 7.0 Hz, 2H). NCL275 1H NMR (400 MHz, DMSO) δ 11.51 (w s, 2H), 8.77 (w s, 2H), 8.10 – 7.90 (m, 4H), 7.54 (d, J= 8.3 Hz, 4H), 4.81 (w s, 4H), 3.59 (k, J = 6.8 Hz, 4H), 1.20 – 1.03 (m, 6H).

EXAMPLE 9: Antiguardiase activity of NCL analogues 231 – 247 and NCL219.

Objective: The objective of this study was to determine the in vitro activity of NCL219 and NCL231–NCL247 against Giardia duodenalis using a resazurin reduction assay.

Methods: Giardia trophozoites were grown to confluence. The medium was replaced with fresh medium and cold shocked for 40 minutes. Compounds were prepared in DMSO and serially diluted 2-fold in DMSO, starting with a 1/100 dilution of the stock solution (e.g., an initial dilution of a 128 mg/mL stock solution would be 128 μg/mL). 2 μl of dilutions were added to the appropriate wells and 198 μl of TYI-S-33 medium was added to each well. Cells were diluted to a concentration of approximately 500,000 cells/mL and 100 μL was added to each well of the assay plate (except for the well containing only the media control). The analytical mixture was incubated under anaerobic conditions (in a candle bottle) for 5 hours at 37 °C. The medium was removed and replaced with 100 µl of warm PBS, then Alamarblue™ was added to a concentration of 10%. Samples were incubated under anaerobic conditions until color appeared. After incubation, the absorbance of each sample was read at 570 and 630 nm. Percent recovery of resazurin (Alamarblue™) was calculated (using the formula below) and data analyzed using GraphPad Prism version 6 software. Percent recovery of Alamarblue was calculated using the formula: ((E _oxi 630 × A ₅₇₀ ) – (E _oxi 570 × A ₆₃₀ ))/((Ered ₅₇₀ × C ₆₃₀ )-(E _red × C ₅₇₀ )) × 100, where: E _oxi 630 = 34798, E _oxi 570 = 80586, A ₅₇₀ = absorbance at 570 nm, A ₆₃₀ = absorbance at 630 nm, E _red 570 = 155677, E _red 630 = 5494, C ₆₃₀ = negative control well absorbance at 630 nm and C ₅₇₀ = negative control well absorbance at 570 nm.

Results and Conclusions: Three of the compounds tested in this assay demonstrated excellent inhibitory activity against Giardia duodenalis in vitro, NCL245 (9.6 µM), NCL246 (5.4 µM) and NCL219 (1.03 µM). The results are presented in Table 4.

Table 4

NCL code [Stock solution] (mg/ml) S. aureus ATCC 29213
(µg/ml) Giardia W.B.
(µg/ml) Giardia
W.B.
(µM) NCL231 25.6 32.00 9.6 41.24 NCL232 12.8 64.32 20 93.48 NCL233 25.6 64.32 68 247.3 NCL234 25.6 64.00 More than 64* 0 NCL235 25.6 128 More than 64* NC NCL236 25.6 16 More than 64* NC NCL237 25.6 4.8 22.8^ NC NCL238 6.4 More than 64 More than 64* NC NCL239 12.8 32 111 334 NCL240 25.6 More than 128 Approximately 30 Approximately 91.73 NCL241 25.6 More than 128 Approximately 30 Approximately 91.29 NCL242 25.6 More than 128 32 Approximately 60.33 NCL243 25.6 More than 128 639 1754 NCL244 25.6 16 More than 64* NC NCL245 25.6 8 4 9,591 NCL246 25.6 8 2 5,446 NCL247 25.6 More than 128 40 92.87 NCL219 10.0 More than 100 0.4 1,030

*IC50 could not be calculated

^only 1 repetition

IC50 after 5 hours, MIC after 24 hours, NC – does not converge

EXAMPLE 10: Activity of NCL analogues against trypanosomatids

Background: Trypanosomatids cause significant morbidity and mortality in humans, with an estimated 1.3 million new cases per year resulting in approximately 30,000 deaths due to Leishmania sp. alone. In addition, trypanosomatids such as Trypanosoma brucei (endemic to Africa) cause significant morbidity and mortality in humans (up to 66 million people are affected), as well as significant losses in the livestock industry (the disease is known as nagana). Currently, chemotherapy available for these organisms is limited and causes unwanted toxic side effects. This study examined the in vitro efficacy of 20 chemical analogues from the NCL series (details shown in Table 5) against the procyclic stage of T. brucei and the promastigote stage of Leishmania donovani. Analogues that demonstrated promising in vitro activity against any of these parasites were tested in vitro for selectivity against a mouse macrophage cell line (ATCC RAW 264.7).

Purpose: The purpose of this study was: (1) to evaluate the in vitro antiparasitic activity of 20 structurally related aminoguanidines (from the NCL series) against T. brucei and L. donovani; and (2) determine the selectivity of these compounds against parasites compared to mammalian cells.

Methods: Antimicrobial agents (Table 5) were dissolved in DMSO to a final concentration of 10 mM. Pentamidine (Sigma) was used as a positive control and was prepared in the same way as NCL compounds.

Table 5.

Screening for L. donovani. Procyclic promastigotes from exponentially grown cultures maintained in DME-L+ supplemented with Bob were used for all analyses. Compounds were initially tested for activity at 10 μM. Compounds were diluted in culture medium to a final volume of 10 μM in 96-well plates. Promastigotes were diluted to a density of approximately 8 × 10 ⁵ cells/ml, then added to the assay plate to produce a final cell density of 4 × 10 ⁵ cells/ml in 96-well plates. Cells were incubated for 96 hours at 27°C, then Alamarblue (Thermofisher) was added. Fluorescence was read at excitation 530 nm and emission 590 nm. Compounds that showed inhibitory activity at 10 μM were further examined to determine IC ₅₀ values. Compounds were serially diluted in triplicate in a 96-well plate in cell growth medium such that concentrations ranged from 0.005 to 10 μM, and promastigotes were added to a final concentration of 1 x 10 ⁶ cells/ml. Cells were incubated at 27°C for 72 hours, then alamar blue was added. Fluorescence was measured as described above.

Screening for T. brucei. Procyclic promastigotes from exponentially grown cells maintained in SDM-79 medium were used for all analyses. Compounds were initially tested for activity at 10 μM. Compounds were diluted in culture medium to a concentration of 20 μM and added to a 96-well plate; after the addition of promastigotes (final concentration 4 × 10 ⁵ cells/ml), the concentration of the compounds was 10 μM. Cells were incubated for 48 hours at 27°C, then alamar blue was added and fluorescence was measured as described above. Compounds that showed inhibitory activity at 10 μM were further examined to determine IC ₅₀ values. Compounds were serially diluted in triplicate in culture medium to obtain final concentrations ranging from 0.004 to 10 μM. Promastigotes were added at a final concentration of 4 × ¹⁰⁵ cells/ml. Cells were incubated at 27°C for 48 hours, then alamar blue was added. In addition, promastigotes at a concentration of 8 × 10 ⁵ were treated with NCL026 for 1.5 hours, then the drug was removed by centrifugation at 5000 rpm. for 7 minutes and resuspended the cells in culture medium. Cells were incubated for 96 hours at 27°C and metabolic activity (Alamar blue assay) and morphological changes were observed daily. The control culture was treated with DMSO instead of NCL026.

Cell toxicity assay. Mouse macrophages (RAW 264.7) were grown in RPMI1640 medium supplemented with L-glutamine and 10% fetal bovine serum. Cells were trypsinized at 80% confluence and subcultured every 3–4 days. For cytotoxicity assays, cells were diluted to a final cell concentration of 2 × ¹⁰⁴ cells/mL and 198 μL was added to each well of a 96-well plate. Cells were incubated in a humidified incubator for 2 hours at 37°C with 5% CO ₂ , then NCL compounds were added (2 μl/well, pre-diluted in DMSO). Only campothecin, Triton X and DMSO were used as controls. Cells were treated with compounds for 24 hours. Metabolic activity of cells was determined using the WST-1 analytical system (Roche Life Science). The supernatant was removed, and 100 μl of PBS with 10% WST-1 was added to each well and incubated for 1 hour, then the absorbance was read at 450 nm. The selectivity coefficient of compounds was determined by dividing the IC ₅₀ of macrophages by the IC ₅₀ against parasites. IC ₅₀ was determined using Graphpad Prism software.

Results: The activity of robenidine and 19 structural analogues against the procyclic promastigote stage of L. donovani and T. brucei was tested at 10 µM. Among the compounds tested, 70% showed a ≥90% reduction in metabolic activity in L. donovani, with 15% showing a similar reduction in metabolic activity in T. brucei (see Figure 10). The procyclic stage of T. brucei and the promastigote stage of L. donovani were treated with the indicated compounds for 48 or 96 hours, respectively, and then the effect was measured using resazurin dye. Analyzes were performed in triplicate. P = pentamidine. Accuracy ± SD.

Among the compounds active against L. donovani, NCL026, NCL028, NCL041, NCL042, NCL062, NCL099, NCL195, NCL201, NCL219 and NCL246 had the highest activity, inhibiting parasites by 100%. NCL026, NCL062 and NCL246 had the greatest inhibitory effect against T. brucei. NCL026, NCL062 and NCL246 were highly effective against both parasite species. A further study was conducted to select compounds with anti-parasite activity to determine IC ₅₀ values. IC ₅₀ values were determined for NCL028, NCL099, NCL166, NCL201, NCL245, NCL246 and NCL812 against L. donovani. IC ₅₀ ranged from 0.37 μM (NCL028) to 6.48 μM (NCL245 and NCL246). The IC ₅₀ value against T. brucei was determined for the 6 most effective analogues NCL024, NCL026, NCL062, NCL171, NCL195 and NCL246. Among these analogs, the most effective were NCL026, NCL171 and NCL195 with IC ₅₀ values of 1.7, 1.4 and 1.5 μM, respectively. The highest IC ₅₀ value determined was 4.2 μM for NCL246. A recovery assay was performed to determine the ability of T. brucei to recover from short-term exposure to NCL026. After 1.5 hours of exposure, GI ₅₀ or IC ₅₀ was determined. The selectivity index (SI) was determined by dividing the GI ₅₀ of macrophages by the IC ₅₀ of parasites. When SI ≥10, the compound was considered selective for the parasite. Analyzes were performed in triplicate. Accuracy ± SD. Based on this analysis, the selectivities of the compounds ranged from 0.57 to 27.9. In general, a selectivity factor less than 10 is considered to indicate relative lack of selectivity, while a selectivity factor greater than 10 is considered to indicate selectivity for the parasite. Based on this rule, only compound (NCL171) can be considered relatively selective against T. brucei, and 4 compounds are highly selective against L. donovani in vitro (NCL028, NCL 099, NCL113 and NCL219).

Table 6

Compound GI ₅₀ macrophage (µM) T. brucei L. donovani IC ₅₀ (µM) S.I. IC ₅₀ (µM) S.I. NCL 024 12.74±2.79 3.35±0.11 3.79 - - NCL 026 9.60±0.99 1.68±0.63 5.71 2.5±0.13 3.84 NCL 028 8.10±2.76 - - 0.29±0.05 27.9 NCL 062 7.15±1.27 4.04±0.83 1.75 - - NCL 099 7.66±0.56 - - 0.37±0.04 20.7 NCL 113 12.90±3.49 - - 0.92±0.06 14 NCL 166 9.46±0.05 - - 3.23±1.00 2.78 NCL 171 12.50±3.61 1.37±0.03 9.14 - - NCL 195 5.78±0.33 1.46±0.76 3.79 - - NCL 201 12.28±2.06 - - 2.92±0.22 4.23 NCL 219 19.26±3.77 - - 0.80±0.10 24.08 NCL 245 3.7±0.71 - - 7.17±2.05 0.57 NCL 246 13.83±0.07 4.18±0.37 3.31 6.72±2.99 2.06 NCL 812 14.85±1.34 - - 2.9±0.24 5.12

Conclusions. This study shows that some of the tested compounds showed high inhibitory activity against L. donovani or T. brucei in vitro. Based on in vitro selectivity coefficient, NCL171 is the most promising against T. brucei, and NCL028, NCL099 and NCL219 are the most promising against L. donovani.

EXAMPLE 11: Physicochemical and metabolic properties of NCL026, NCL028, NCL099, NCL171, NCL177, NCL195, NCL217, NCL259 and NCL812.

Objective: The objective of this study was to determine the physicochemical and metabolic properties of NCL026, NCL028, NCL099, NCL171, NCL177, NCL195, NCL217, NCL259 and NCL812.

The physicochemical and metabolic characteristics of nine compounds were evaluated using a combination of in silico and experimental techniques, and the results are summarized in Fig. eleven.

The calculated physicochemical parameters of each compound were generally within the range typically associated with compounds exhibiting “drug” properties. However, the polar surface area values of NCL026 and NCL171 were close to the upper limit recommended for good membrane permeability, reflecting the relatively high number of heteroatoms in the two structures. All compounds showed low kinetic solubility under neutral pH conditions, with the exception of NCL259, which had more moderate solubility. Most compounds showed high solubility under acidic conditions (pH 2), suggesting increased ionization at low pH. Measured partition coefficient values were relatively high at pH 7.4, with LogD7.4 values ranging from 3.6 to greater than 5.3. LogD values were lower under acidic conditions (pH 3) but could still be considered moderate to high (2.8 to 4.9). The observed pH-dependent solubility and partition coefficient results are consistent with the basic characteristics of the compounds predicted from their structures. The metabolic stability of nine compounds was assessed in human and mouse liver microsomes. Five of these compounds, NCL026, NCL177, NCL195, NCL259 and NCL812, showed low degradation rates in both types of liver microsomes (EH values less than 0.3). NCL099, NCL171, and NCL217 exhibited intermediate to high degradation rates (EH values between 0.49 and 0.88), with degradation rates of each compound being largely comparable between species. NCL028 showed a low rate of degradation in human liver microsomes and a high rate of degradation in mouse liver microsomes, which may indicate significant differences in the metabolism of this compound between species. No measurable degradation of any of the compounds was observed in the control (no cofactor) incubation mixtures in either species, suggesting that there is no major cofactor-independent metabolism contributing to the overall metabolic rate.

Experimental methods

Physicochemical parameters calculated using ChemAxon JChem software

Theoretical physicochemical values for each compound were calculated using the ChemAxon chemical cartridge in JChem for Excel software. The calculated parameters and a brief explanation of their meaning are presented below.

Molecular Weight (MW): Ideally, MW should be less than 500 for good membrane permeability.

Polar Surface Area (PSA): Calculated using a simplified 2D modeling approach and validated by comparison with a more complex 3D modeling strategy. The value was calculated at pH 7.4, which takes into account the ionization of the molecule. It is generally accepted that PSA values of less than about 120 Å ² provide acceptable oral drug absorption and membrane permeability.

Freely Rotating Bonds: The number of single bonds that are not arranged in a ring or sterically hindered system and are not bonded to a hydrogen atom. For good membrane permeability, the number of freely rotating bonds (FRBs) must be less than or exactly 10 (see D. Veber et al, J. Med. Chem. 2002, 45, 2615-2623).

Hydrogen bond donors/acceptors: The number of hydrogen bond donors and acceptors provides an indicator of a molecule's hydrogen bonding ability, which is inversely related to membrane permeability. Ideally, the number of hydrogen bond donors should be less than 5 and the number of hydrogen bond acceptors should be less than 10.

pKa: The basic physicochemical measure of the acidity of a compound. In the context of drug development, these values themselves indicate only the significance of ionization under physiological conditions.

Assessing solubility using nephelometry

Compounds in DMSO were added to either phosphate buffer pH 6.5 or 0.01 M HCl (pH about 2.0) with a final concentration of 1% in DMSO. The samples were then analyzed using nephelometry to determine the solubility range. (See C. D. Bevan and R. S. Lloyd, Anal. Chem. 2000, 72, 1781-1787).

LogD measurement

The distribution coefficient (LogD) values of the test compounds were estimated by correlating their chromatographic retention properties and the characteristics of a number of standard compounds with known distribution coefficient values. The method used was an HPLC modification of the method developed by Lombardo (see F. Lombardo et al, J. Med. Chem. 2001, 44, 2490-2497).

Microsomal stability

Incubation Methods: Metabolic stability assays were performed by incubating each test compound (at 1 μM) with human and mouse liver microsomes (Xenotech, batch no. 1210057 and 1310211, respectively) at 37 °C and at a protein concentration of 0.4 mg/ml. The metabolic reaction was initiated by the addition of an NADPH-regenerating system (i.e., NADPH is a cofactor required for CYP450-mediated metabolism) and quenched at various time points during the 60-minute incubation period by adding acetonitrile containing diazepam as an internal standard. Control samples (containing no NADPH) were included (and quenched after 2, 30 and 60 minutes) to control for possible degradation in the absence of cofactor. Analytical conditions: Instrument: Waters Micromass Xevo G2 QTOF connected to a Waters Acquity UHPLC instrument; Detection: Positive electrospray ionization mode with MSE; cone voltage 30 V; column: Ascentis Express amide column (50 × 2.1 mm, 2.7 µm); LC conditions: Gradient cycle time: 4 minutes; injection volume: 5 µl; flow rate: 0.4 ml/min; mobile phase: acetonitrile-water gradient with 0.05% formic acid; metabolite; Definition: Metabolite screening was not included in this study, however, since the data were read in MSE mode, which allows for simultaneous acquisition of low and high collision energy MS spectra, the results can be used to later search for a metabolite if warranted.

Calculations: Test compound concentration versus time data were fitted to an exponential decay function to determine the first order rate constant for substrate depletion. If an obvious deviation from first order kinetics was observed, then only the original linear part of the profile was used to determine the decomposition rate constant (k). Then, using methods standard in the art, the depletion rate constant of each substrate was used to calculate: [1] the degradation half-life, [2] the intrinsic in vitro clearance value (CLint, in vitro); [3] predicted value of intrinsic clearance in the liver in vivo (CLint); [4] predicted in vivo blood clearance (CLblood); and [5] predicted in vivo liver clearance rate (EH). In the calculations described above, the following scaling parameters were taken into account (Table 7).

Table 7

Kinds Liver weight (g liver/kg body weight) Microsomal protein (mg/g liver weight) Hepatic blood flow (Q) (ml/min./kg body weight) Person ^a 25.7 32 20.7 Mouse ^a 54.9 47 120

^a Ring et al. (2011) Journal of Pharmaceutical Sciences, 100:4090-4110.

Prediction of in vivo liver clearance rates: Predicted liver clearance (EH) rates from microsomal data, derived from relative in vitro degradation rates of test compounds, were used to classify compounds into low (less than 0.3), intermediate (0.3) – 0.7), high (0.7 – 0.95) or very high (more than 0.95) clearance.

Results: Physicochemical and metabolic characteristics of nine compounds were evaluated using a combination of in silico and experimental techniques, and the results are summarized in FIG. eleven.

EXAMPLE 12: Effect of NCL026, NCL195, NCL259 and NCL812 on male Swiss non-linear mice after IV administration

Objective: The objective of this study was to evaluate the systemic effects of NCL026, NCL195, NCL259 and NCL812 in male Swiss nonlinear mice after IV administration at a dose of 5 mg/kg.

Methods: Systemic exposure to NCL026, NCL195, NCL259 and NCL812 was studied in fasted male Swiss nonlinear mice weighing 26.2 - 32.1 g. Animals were given food and water ad libitum during the pre- and post-dose sampling period. Each compound was administered IV via tail vein bolus (vehicle 20% (v/v) DMSO in PEG 400, dose volume 1 mL/kg, n=8 mice per compound). Post-dose blood samples were collected at 5, 15, 30, 120, 240 and 480 minutes post-dose (n=2 per time point for each compound). A maximum of two samples were collected from each mouse, with samples collected either by submandibular puncture (∼120 μl; sample collected from conscious animals) or terminal cardiac puncture (0.6 ml; with mice anesthetized using isoflurane inhalation). No urine samples were collected because mice were kept in cages with bedding during the study. Blood was collected directly into Eppendorf polypropylene tubes containing heparin as an anticoagulant and a stabilizing cocktail (containing Complete® (EDTA protease inhibitor cocktail) and potassium fluoride) to minimize possible degradation of test compounds ex vivo in blood/plasma samples. After collection, blood samples were immediately centrifuged, supernatant plasma was removed, and stored at −80°C until analyzed by LC-MS using methods known in the art.

Each compound was administered in a vehicle consisting of 20% (v/v) DMSO in PEG 400. Dosage forms were prepared by dissolving the compounds in DMSO before adding PEG 400. The dosage forms were unfiltered before dosing and administered to mice over a 2.5-hour period. hours after receipt. The average measured concentration of each compound in aliquots (n=2) of their respective dosage forms was 4.58, 4.31, 5.26, and 5.25 mg/mL for NCL026, NCL195, NCL259, and NCL812, respectively. The dose administered to each mouse was calculated based on the measured concentration in the IV dosage form, the dose volume, and the individual body weight of the mouse. The plasma concentration dependence was analyzed using non-compartmental methods (WinNonlin version 6.3.0.395). Standard calculations for each pharmacokinetic parameter were performed using standard methods in the art.

One mouse dosed with NCL812 exhibited abnormal behavior (fussy, hyperactive) starting within minutes of dosing; the mouse was euthanized and blood was collected 15 min after dosing. Other animals in this study did not show any adverse reactions or side effects associated with the administered compound. There is evidence of hemolysis observed in plasma samples, however this is likely due to the solvents used in the IV dosage forms (20 (v/v) DMSO in PEG 400), which were necessary due to the limited solubility of the test compounds in the aqueous dosage form carrier .

Results: Plasma concentration-time profiles for NCL026, NCL195, CL259 and NCL812 are presented in FIG. 12. Pharmacokinetic parameters are presented in Table 8. All compounds exhibited moderate to long apparent terminal half-lives.

Table 8

NCL026 NCL195 NCL259 NCL812 Measured dose (mg/kg) 4.8 4.4 5.4 5.3 Apparent t _1/2 (h.) 1.5 2.4 2.9 8.2 ^b Plasma clearance (ml/min./kg) 100.6 16.3 ^56.0a 9.5 ^b V _ss plasma (l/kg) 6.9 2.6 ^{12.8a 5.9b} AUC _0-inf (h*µM) 2.5 12.5 ^5.5a 27.9 ^b

^a Plasma concentration at time zero cannot be determined by log-linear regression of the first two measurements and is therefore taken to be equal to the first observed measurement. Consequently, the AUC from 0 to 5 minutes (and thus the AUC _0-inf ) will be underestimated, and the parameters calculated from the AUC _0-inf are only approximations.

^b The terminal elimination phase is not well defined and the value is only an approximation.

EXAMPLE 13: Effect of NCL195 on male Swiss nonlinear mice after IP administration.

Objective: The objective of this study was to obtain a preliminary indication of the plasma exposure of NCL195 following IP administration at a target dose of 50 mg/kg.

Methods: The dosage form was prepared by dissolving solid NCL195 in DMSO (up to 20% (v/v) of final volume) followed by the addition of PEG 400 to produce a clear yellow solution, which was dosed to mice within 30 minutes of preparation. The measured concentration of NCL195 in the finished dosage form was 21.9 mg/mL, resulting in an average administered dose of 43 mg/kg. Following administration, blood samples were collected for up to 24 hours post-dose (n = 2 mice per time point). A maximum of two samples were collected from each mouse, with samples collected either by submandibular puncture (∼120 μl; sample collected from conscious animals) or terminal cardiac puncture (0.6 ml; with mice anesthetized using isoflurane inhalation). No urine samples were collected because mice were kept in cages with bedding during the study. Blood was collected directly into Eppendorf polypropylene tubes containing heparin as an anticoagulant and a stabilizing cocktail (containing Complete® (protease inhibitor cocktail), potassium fluoride and EDTA) to minimize possible ex vivo degradation of NCL195 in blood/plasma samples. After collection, blood samples were immediately centrifuged, supernatant plasma was removed, and stored at −20°C until analysis by LCMS using methods known in the art.

Results: No adverse reactions or compound-related side effects were observed in any mice following IP administration of NCL195 at a dose of 43 mg/kg. The plasma concentration-time profile (Figure 13) shows that NCL195 is rapidly absorbed after dosing. During the first 7.5 hours post-dose, plasma concentrations remained above 3-4 mcg/mL, but concentrations decreased to 0.2-0.5 mcg/mL between 7.5 and 24 hours post-dose. Assuming that a 2-fold increase in dose would result in a proportionate increase in NCL195 exposure, the data presented suggest that IP administration of 100 mg/kg NCL195 (as a solution dosage form) would result in at least 7.5 hours of exposure with a plasma concentration of more than 8 mcg/ml.

EXAMPLE 14: Activity of NCL analogues against Trypanosoma cruzi

General information: Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). T. cruzi is transmitted by inoculation of infected feces with triatomine vectors through the bite site or through the intact mucosa of a mammalian host. Vector-borne transmission is limited to the regions of North America, Central America and South America. In endemic and non-endemic regions, other routes of infection include transfusion, organ and bone marrow transplantation, and inheritance. Outbreaks attributed to contaminated food or drink have been described in northern South America, with pronounced transmission cycles involving populations of wild vectors and mammalian reservoir hosts. Without effective treatment, the infection becomes lifelong. The most serious consequence of T. cruzi infection is cardiomyopathy, which occurs in 20-30% of infected subjects. The World Health Organization estimates that approximately 6-7 million people were infected worldwide in 2015, mostly in Latin America.

There are only two drugs, nifurtimox, a member of the nitrofurans, and benznidazole, a member of the nitroimidazoles, that are reliably effective against T. cruzi infection. However, each of these agents has significant limitations in effectiveness and safety.

In patients with acute Chagas disease and in patients with congenital Chagas disease, benznidazole and nifurtimox reduce the severity of symptoms, shorten the clinical duration of the disease, and shorten the duration of parasitemia; but the recovery rate in the acute phase is only about 80-90%.

Studies of benznidazole in children with chronic T. cruzi infection found a cure rate of only about 60% based on conversion to negative serological test results 3 to 4 years after treatment.

The use of nifurtimox is associated with gastrointestinal side effects (anorexia, weight loss, nausea and vomiting) in 70% of patients. Neurological toxic effects include irritability, insomnia, disorientation and tremor. Rare but more severe side effects include paraesthesia, polyneuropathy, and peripheral neuritis.

Benznidazole use is often associated with dermatologic adverse effects, usually mild rashes that respond to antihistamines. However, severe or scaly dermatitis, or dermatitis associated with fever and lymphadenopathy, indicates the need for immediate discontinuation of treatment. Dose-dependent peripheral neuropathy that occurs late in the course of therapy requires immediate discontinuation of treatment. Although bone marrow suppression is rare, its occurrence indicates the need for immediate cessation of treatment.

The lack of safe and effective treatments has led to the classification of Chagas disease as a neglected parasitic infection with significant health complications in humans. Global losses from Chagas disease are estimated at more than 7 billion US dollars (Lee, B. Y., K. M. Bacon, M. E. Bottazzi and P. J. Hotez (2013). "Global economic burden of Chagas disease: a computational simulation model." The Lancet Infectious Diseases 13( 4): 342-348), and there remains a critical and unmet need to identify and develop improved treatments.

Purpose and Methods: In an effort to identify new agents for the treatment of Chagas disease, the biological activity of robenidine and 79 analogues against Trypanosoma cruzi was examined in an in vitro screening assay conducted according to the methods described by Keenan et al (Keenan, M., M. J. Abbott, P. W. Alexander, T. Armstrong, W. M. Best, B. Berven, A. Botero, J. H. Chaplin, S. A. Charman, E. Chatelain, T. W. von Geldern, M. Kerfoot, A. Khong, T. Nguyen, J. D. McManus, J. Morizzi, E. Ryan, I. Scandale, R. A. Thompson, S. Z. Wang and K. L. White (2012). "Analogues of fenarimol are potent inhibitors of Trypanosoma cruzi and are effective in a murine model of Chagas disease." Journal of medicinal chemistry 55(9) : 4189-4204), Buckner and colleagues (Buckner, F. S., C. L. Verlinde, A. C. La Flamme and W. C. Van Voorhis (1996). "Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase." Antimicrobial Agents and Chemotherapy 40(11): 2592-2597) and by Van Voorhis and Eisen (1989). "Fl-160. A surface antigen of Trypanosoma cruzi that mimics mammalian nervous tissue." The Journal of Experimental Medicine 169(3): 641-652).

In vitro analysis of T. cruzi to determine IC50.

Tulahuen trypomastigotes expressing the ß-galactosidase gene were used in the T. cruzi assay. Parasites were maintained in vitro with serial passage in L6 cells. Briefly, L6 cells were plated in 96-well flat-bottomed tissue culture plates and incubated at 37°C in 5% CO ₂ for 24 hours to allow cell attachment. T. cruzi trypomastigotes were then added at an MOI of 3 and the plates were incubated for an additional 48 h to allow infection to occur. All stages were carried out in RPMI 1640 medium (without phenol red) supplemented with 10% fetal bovine serum (FBS, Bovogen). Extracellular trypomastigotes were then removed and NCL compounds were added in seven-point serial dilutions done in triplicate. Benznidazole (Epichem Pty Ltd.) was included as a control. After 96 hours of incubation with the compounds, the colorimetric agent, chlorophenol red-fl-D-galactopyranoside (CPRG, Roche) was added at 0.3% v/v. Nonidet P-40. After 4-6 hours, a color change was observed after catabolization of the reagent by viable T. cruzi and absorbance was read at 530 nm using a Dynex microplate reader. % inhibition was calculated using the following equation: % inhibition = 100 - [(T. cruzi with compound - compound only)/(T. cruzi only - medium only)] × 100. For each compound, the % inhibition values were used to construct a calibration curve, according to which _IC50 was calculated. Each analysis was performed at least twice and the average was used.

Results: The IC ₅₀ values of the 80 NCL compounds are presented in Table 9.

Table 9

Activity against Trypanosoma cruzi Compound IC ₅₀
µM NCL026 1.4 NCL089 3.0 NCL042 4.9 NCL080 7.8 NCL036 8.3 NCL039 9.9 NCL024 12 NCL028 13 NCL027 14 NCL041 14 NCL086 14 NCL052 15 NCL075 15 NCL021 16 NCL037 16 NCL038 16 NCL040 16 NCL043 16 NCL083 16 NCL087 17 NCL030 18 NCL023 19 NCL029 19 NCL054 19 NCL076 19 NCL088 19 NCL025 20 NCL035 20 NCL081 20 NCL085 20 NCL015 21 NCL082 22 NCL084 22 NCL016 24 NCL020 24 NCL073 24 NCL045 29 NCL046 32 NCL074 33 NCL002 38 NCL044 39 NCL072 46 NCL022 49 NCL053 49 NCL010 50 NCL034 53 NCL004 56 NCL017 56 NCL032 58 NCL019 61 NCL031 69 NCL033 80 NCL007 82 NCL003 83 NCL077 >10 NCL078 >10 NCL079 >10 NCL812 >11 NCL011 >11 NCL005 >33 NCL018 >33 NCL001 >100 NCL006 >100 NCL008 >100 NCL009 >100 NCL012 >100 NCL013 >100 NCL014 >100 NCL047 >100 NCL048 >100 NCL049 >100 NCL050 >100 NCL051 >100 NCL055 >100 NCL056 >100 NCL057 >100 NCL058 >100 NCL059 >100 NCL060 >100 NCL071 >100

Conclusion: It can be seen that 6 compounds had IC ₅₀ values less than 10 µM, while 30 compounds had IC ₅₀ values less than or exactly 20 µM. The NCL series provides a rich source of agents with activity against Trypanosoma cruzi.

Claims (257)

1. The use of a pharmaceutical composition for the treatment or prevention of colonization or infection caused by a protozoal agent in a subject, where the pharmaceutical composition contains a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); (2Z,2'Z)-2,2'-(pyrimidin-4,6-diylbis(hydrazin-2-yl-1-ylidene))bis(2-(4-chlorophenyl)ethan-1-ol) (NCL180 ); 4,6-bis(2-((E)-4-chlorobenzylidene)hydrazinyl)pyrimidine (NCL181); 6-chloro-N ⁴ -(1-phenylethyl)pyrimidine-2,4-diamine (NCL182); N ⁴ ,N ⁶ -bis(1-phenylethyl)pyrimidine-4,6-diamine (NCL183); N ⁴ ,N ⁶ -bis(1-phenylethyl)pyrimidine-2,4,6-triamine (NCL184); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine hydrochloride (NCL185); 4,6-bis(2-((E)-4-chlorobenzylidene)hydrazinyl)pyrimidine hydrochloride (NCL187); (2Z,2'Z)-2,2'-(pyrimidin-4,6-diylbis(hydrazin-2-yl-1-ylidene))bis(2-(4-chlorophenyl)ethan-1-ol) hydrochloride ( NCL189); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); N',N'''-(2-aminopyrimidin-4,6-diyl)di(benzohydrazide) (NCL194); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL196); 3,3'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL197); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL200); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 2,2'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL202); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL203); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL206); 2,2'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL207); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL208); 3,3'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL209); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,4'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL211); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)-1,3,5-triazin-2-amine (NCL213); 4,6-bis(2-((E)-benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL214); 4-((E)-(2-(2-amino-6-(2-((E)-4-((diethoxyphosphoryl)oxy)benzylidene)hydrazinyl)pyrimidin-4-yl)hydrazono)methyl)phenyl diethyl phosphate (NCL218 ); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); N,N'-(((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(4,1 -phenylene))diacetamide (NCL223); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL240); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,5-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-4-hydroxyphenolate (NCL242); 4,6-bis(2-((E)-3,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL243); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-3,4,5-trihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL247); 4,6-bis(2-((E)-[1,1'-biphenyl]-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL255); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 2. Use according to claim 1, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL196); 3,3'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL197); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 2,2'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL202); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL203); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 2,2'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL207); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4-((E)-(2-(2-amino-6-(2-((E)-4-((diethoxyphosphoryl)oxy)benzylidene)hydrazinyl)pyrimidin-4-yl)hydrazono)methyl)phenyl diethyl phosphate (NCL218 ); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); N,N'-(((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(4,1 -phenylene))diacetamide (NCL223); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 3. Use according to claim 2, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 4. Use according to claim 3, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 5. Application according to any one of paragraphs. 1-4, where the compound is a chloride salt. 6. Application according to any one of paragraphs. 1-5, where the subject is an animal selected from the group consisting of: human, canid, feline, bovine, ovine, goat, pig, bird, fish and equine species. 7. Application according to any one of paragraphs. 1-6, wherein the composition is adapted to administer the compound to a subject in a dosage range of 0.1 to 250 mg/kg body weight. 8. Application according to any one of paragraphs. 1-7, where the protozoan agent is selected from the group of protozoan genus containing: Acanthamoeba, Babesia, Balamuthia, Balantidium, Besnoitia, Blastocystis, Chilomastix, Cochlosoma, Cryptosporidium, Cyclospora, Cystoisospora, Cytauxzoon, Dientamoeba, Eimeria, Endolimax, Entamoeba, Giardia, proteus , Hammondia Hartmannella, Hepatozoon, Hexamita, Histomonas, Isospora, Leishmania, Leucocytozoon, Naegleria, Neospora, Pentatrichomonas, Plasmodium, Sappinia, Sarcocystis, Tetratrichomonas, Theileria, Toxoplasma, Trichomonas, Tritrichomonas, Trypanosoma (subgenera Duttonella, Herpetosoma, nomonas, Schizotrypanum, Trypanozoon ), Tyzzeria and Wenyonella. 9. Application according to any one of paragraphs. 1-8, wherein the infection or colonization is caused by a mixture of at least two protozoal agents. 10. Application according to any one of paragraphs. 1-9, wherein the infection or colonization caused by a protozoal agent in the subject constitutes a zoonosis. 11. Application according to any one of paragraphs. 1-10, where the composition is adapted for oral administration, parenteral administration or topical administration. 12. A pharmaceutical composition for treating or preventing infection or colonization caused by a protozoal agent in a subject, comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); (2Z,2'Z)-2,2'-(pyrimidin-4,6-diylbis(hydrazin-2-yl-1-ylidene))bis(2-(4-chlorophenyl)ethan-1-ol) (NCL180 ); 4,6-bis(2-((E)-4-chlorobenzylidene)hydrazinyl)pyrimidine (NCL181); 6-chloro-N ⁴ -(1-phenylethyl)pyrimidine-2,4-diamine (NCL182); N ⁴ ,N ⁶ -bis(1-phenylethyl)pyrimidine-4,6-diamine (NCL183); N ⁴ ,N ⁶ -bis(1-phenylethyl)pyrimidine-2,4,6-triamine (NCL184); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine hydrochloride (NCL185); 4,6-bis(2-((E)-4-chlorobenzylidene)hydrazinyl)pyrimidine hydrochloride (NCL187); (2Z,2'Z)-2,2'-(pyrimidin-4,6-diylbis(hydrazin-2-yl-1-ylidene))bis(2-(4-chlorophenyl)ethan-1-ol) hydrochloride ( NCL189); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); N',N'''-(2-aminopyrimidin-4,6-diyl)di(benzohydrazide) (NCL194); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL196); 3,3'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL197); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL200); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 2,2'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL202); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL203); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL206); 2,2'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL207); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL208); 3,3'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL209); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,4'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL211); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)-1,3,5-triazin-2-amine (NCL213); 4,6-bis(2-((E)-benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL214); 4-((E)-(2-(2-amino-6-(2-((E)-4-((diethoxyphosphoryl)oxy)benzylidene)hydrazinyl)pyrimidin-4-yl)hydrazono)methyl)phenyl diethyl phosphate (NCL218 ); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); N,N'-(((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(4,1 -phenylene))diacetamide (NCL223); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL240); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,5-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-4-hydroxyphenolate (NCL242); 4,6-bis(2-((E)-3,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL243); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-3,4,5-trihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL247); 4,6-bis(2-((E)-[1,1'-biphenyl]-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL255); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 13. The composition according to claim 12, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL196); 3,3'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL197); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 2,2'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol (NCL202); 4,6-bis(2-((E)-cyclohexylmethylene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL203); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 2,2'-((1E,1'E)-((6-amino-1,3,5-triazin-2,4-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene ))diphenol (NCL207); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4-((E)-(2-(2-amino-6-(2-((E)-4-((diethoxyphosphoryl)oxy)benzylidene)hydrazinyl)pyrimidin-4-yl)hydrazono)methyl)phenyl diethyl phosphate (NCL218 ); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); N,N'-(((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(4,1 -phenylene))diacetamide (NCL223); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 14. The composition according to claim 13, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidine (NCL178); 4,6-bis(2((E)-4-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL179); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL193); 4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine (NCL195); 4,6-bis(2-((E)-4-(tert-butyl)benzylidene)hydrazinyl)pyrimidin-2-amine dihydrochloride (NCL198); 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-amine (NCL199); 4,4'-((1E,1'E)-((2-aminopyrimidin-4,6-diyl)bis(hydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(N,N- dimethylaniline) (NCL201); 4,6-bis(2-((E)-1-(4-chlorophenyl)ethylidene)hydrazinyl)pyrimidin-2-amine (NCL204); 4,6-bis(2-((E)-2-chlorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL205); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL210); 4,6-bis(2-((E)-4-bromobenzylidene)hydrazinyl)-1,3,5-triazin-2-amine (NCL212); 4,6-bis(2-((E)-4-fluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL220); 4,6-bis(2-((E)-4-(trifluoromethyl)benzylidene)hydrazinyl)pyrimidin-2-amine (NCL221); 4,6-bis(2-((E)-3,4-difluorobenzylidene)hydrazinyl)pyrimidin-2-amine (NCL222); 4,6-bis(2-((E)-4-methoxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL230); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 15. The composition according to claim 14, where the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 16. Composition according to any one of paragraphs. 12-15, which is a veterinary composition. 17. Composition according to any one of paragraphs. 12-16, where the compound is a chloride salt. 18. Composition according to any one of paragraphs. 12-17, where the subject is an animal selected from the group consisting of: human, canid, feline, bovine, ovine, goat, pig, bird, fish and equine species. 19. Composition according to any one of paragraphs. 12-18, wherein the composition is adapted to administer the compound to a subject in a dosage range of 0.1 to 250 mg/kg body weight. 20. Composition according to any one of paragraphs. 12-19, where the protozoan agent is selected from the group of the genus of protozoa, containing: Acanthamoeba, Babesia, Balamuthia, Balantidium, Besnoitia, Blastocystis, Chilomastix, Cochlosoma, Cryptosporidium, Cyclospora, Cystoisospora, Cytauxzoon, Dientamoeba, Eimeria, Endolimax, Entamoeba, Giardia, emoproteus , Hammondia Hartmannella, Hepatozoon, Hexamita, Histomonas, Isospora, Leishmania, Leucocytozoon, Naegleria, Neospora, Pentatrichomonas, Plasmodium, Sappinia, Sarcocystis, Tetratrichomonas, Theileria, Toxoplasma, Trichomonas, Tritrichomonas, Trypanosoma (subgenera Duttonella, Herpetosoma, nomonas, Schizotrypanum, Trypanozoon ), Tyzzeria and Wenyonella. 21. Composition according to any one of paragraphs. 12-20, wherein the infection or colonization is caused by a mixture of at least two protozoal agents. 22. Composition according to any one of paragraphs. 12-21, wherein the infection or colonization caused by a protozoal agent in the subject constitutes a zoonosis. 23. Composition according to any one of paragraphs. 12-22, where the composition is adapted for oral administration, parenteral administration or topical administration. 24. A medical device for treating or preventing colonization or infection caused by a protozoal agent in a subject, wherein said medical device contains a pharmaceutical composition according to any one of paragraphs. 12-23 and is presented in a form selected from the group consisting of: patch, bandage, bandage or implant. 25. A compound selected from a list of compounds or pharmaceutically acceptable salts thereof, consisting of: 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL240); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,5-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-4-hydroxyphenolate (NCL242); 4,6-bis(2-((E)-3,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL243); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-3,4,5-trihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL247); 4,6-bis(2-((E)-[1,1'-biphenyl]-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL255); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264). 26. The compound according to claim 25, selected from the list of compounds or their pharmaceutically acceptable salts, consisting of: 4,6-bis(2-((E)-(6-chloropyridin-3-yl)methylene)hydrazinyl)pyrimidin-2-amine (NCL238); 4,6-bis(2-((E)-pyridin-3-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL239); 4,6-bis(2-((E)-pyridin-4-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL241); 4,6-bis(2-((E)-2,3-dihydroxybenzylidene)hydrazinyl)pyrimidine-2-aminium 2-formyl-6-hydroxyphenolate (NCL244); 4,6-bis(2-((E)-naphthalene-1-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL245); 4,6-bis(2-((1E,2E)-3-phenylallylidene)hydrazinyl)pyrimidin-2-amine (NCL246); 4,6-bis(2-((E)-2,4-dihydroxybenzylidene)hydrazinyl)pyrimidin-2-amine (NCL256); 4,6-bis(2-((E)-3-phenylpropylidene)hydrazinyl)pyrimidin-2-amine (NCL262); 4,6-bis(2-((E)-naphthalene-2-ylmethylene)hydrazinyl)pyrimidin-2-amine (NCL264).