5,6-FUSED-BICYCLIC COMPOUNDS AND COMPOSITIONS FOR

THE TREATMENT OF PARASITIC DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

The appliation claims the benefit of U.S. provisional application serial no. 62/520,277 filed June 15, 2017, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds, the use thereof for inhibiting the chymotrypsin-like activity of the proteasomes (proteasomes are proteolytic enzymes) of the kinetoplastid parasites and methods of using such compounds to treat or prevent leishmaniasis, and Chagas disease.

BACKGROUND OF THE INVENTION

Leishmaniasis is a disease caused by protozoan parasites that belong to the genus

Leishmania and is transmitted by the bite of certain species of sand fly. There are four main forms of Leishmaniasis. Cutaneous leishmaniasis is the most common form of leishmaniasis. Visceral leishmaniasis, also called kala-azar, is the most serious form in which the parasites migrate to the vital organs. Visceral leishmaniasis is caused by the parasite Leishmania donovani, and is potentially fatal if untreated.

Leishmaniasis is mostly a disease of the developing world, and is rarely known in the developed world outside a small number of cases, mostly in instances where troops are stationed away from their home countries. Leishmaniasis is endemic in many tropical and subtropical countries, and is found in parts of about 88 countries. Approximately 350 million people live in these areas. The settings in which leishmaniasis is found range from rainforests in Central and South America to deserts in West Asia and the Middle East. It affects as many as 12 million people worldwide, with 1 .5-2 million new cases each year. The visceral form of leishmaniasis has an estimated incidence of 500,000 new cases and 60,000 deaths each year. More than 90 percent of the world's cases of visceral leishmaniasis are in India, Bangladesh, Nepal, Sudan, and Brazil. Kabul is estimated as the largest center of cutaneous leishmaniasis in the world, with approximately 67,500 cases as of 2004.

The two main therapies for visceral leishmaniasis are the antimony derivatives sodium stibogluconate (PENTOSTAM®) and meglumine antimoniate (GLUCANTIM®). Sodium stibogluconate has been used for about 70 years and resistance to this drug is a growing problem. In addition, the treatment is relatively long and painful, and can cause undesirable side effects. Amphotericin (AmBisome®) is now the treatment of choice. Miltefosine

(IMPAVIDO®) and paromomycin are the other treatment alternatives. These drugs are known to produce a definitive cure in >90% of patients. Amphotericin is expansive and administered intravenously, and is not affordable for most patients. Paromomycin, although affordable, requires intramuscular injections for 3 weeks; compliance is a major issue. Miltefosine is an oral drug and has shown to be more effective and better tolerated than other drugs. However, there are problems associated with the use of miltefosine that arise from its teratogenicity and pharmacokinetics. Miltefosine was eliminated much slower from the body and was still detectable five months after the end of treatment. The presence of subtherapeutic miltefosine concentrations in the blood beyond five months after treatment might contribute to the selection of resistant parasites; moreover, measures for preventing the teratogenic risks of miltefosine must be reconsidered. This led to some reluctance to taking Miltefosine by affected populations. Currently, no vaccines are in routine use.

Chagas disease, also called American trypanosomiasis, is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi. T. cruzi is commonly transmitted to humans and other mammals by the blood-sucking "kissing bugs" of the subfamily Triatominae (family Reduviidae).

Chagas disease is contracted primarily in the Americas. It is endemic in twenty one Central and Latin American countries; particularly in poor, rural areas of Mexico, Central America, and South America. Large-scale population movements from rural to urban areas of Latin America and to other regions of the world have increased the geographic distribution of Chagas disease, and cases have been noted in many countries, particularly in Europe. Although there are triatomine bugs in the U.S., only very rarely vector borne cases of Chagas disease have been documented.

Each year, an estimated 10 to 15 million people across the world are infected with Chagas disease, most of whom do not know they are infected. Every year, 14,000 people die from the disease. In Central and South America, Chagas kills more people than any other parasite- borne disease, including malaria. By applying published seroprevalence figures to immigrant populations, CDC estimates that more than 300,000 persons with Trypanosoma cruzi infection live in the United States. Most people with Chagas disease in the United States acquired their infections in endemic countries.

The symptoms of Chagas disease vary over the course of an infection. In the early, acute stage, symptoms are mild and usually produce no more than local swelling at the site of infection. The initial acute phase is responsive to antiparasitic treatments, with 60-90% cure rates. After 4-8 weeks, individuals with active infections enter the chronic phase of Chagas disease that is asymptomatic for 60-80% of chronically infected individuals through their lifetime. However, the remaining 20 - 40% of infected people will develop debilitating and sometimes life-threatening medical problems over the course of their lives.

There is no vaccine against Chagas disease. Treatment for Chagas disease focuses on killing the parasite and managing signs and symptoms. During the acute phase of Chagas disease, the drugs currently available for treatment are benznidazole and nifurtimox. Once Chagas disease reaches the chronic phase, medications are not effective for curing the disease. Instead, treatment depends on the specific signs and symptoms. However, problems with these current therapies include their diverse side effects, the length of treatment, and the requirement for medical supervision during treatment. Resistance to the two frontline drugs has already occurred. The antifungal agent Amphotericin b has been proposed as a second-line drug, but this drug is costly and relatively toxic.

There remains a need for new and better treatments and therapies for leishmaniasis and Chagas disease. The present invention meets that needs.

The present invention relates to compounds having antiparasitic activity against kinatoplastid protozoa. It relates particularly to compounds that inhibit growth of kinatoplastid parasite cells through inhibition of the parasitic proteasome, and thereof useful as a therapy for leishmaniasis and Chagas disease.

SUMMARY OF THE INVENTION

Within certain aspects, provided herein is a compound of Formula (I) or subformulae thereof:

or a pharmaceutically acceptable salt or stereoisomer thereof; wherein :

X is N or CR°;

Y is C or N;

L is a bond, -0-(CR ⁵ R ⁶ ) _P or -(CR ⁵ R ⁶ ) _q -;

Ar is phenyl, which is unsubstituted or substituted with 1 -5 deuterium or 1 -2 R ⁴ ; wherein R ⁴ is independently halo, Ci- ₄ alkyl, halo-substituted Ci- ₄ alkoxyl, -C(0)OR ⁷ , cyano, or C ₃ -6cycloalkyl;

R° is Ci-4alkyl or cyano;

R ¹ , R ² and R ⁷ are Ci- ₄ alkyl;

R ³ are independently Ci-4alkyl or halo; wherein R ³ is attached to any carbon atom in the ring or two R ³ are attached to the same carbon atom in the ring;

R ⁵ and R ⁶ are independently H, deuterium or Ci-4alkyl;

m is 0-2; and

p and q are independently 1 -4.

In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I) or subformulae thereof, or a pharmaceutically acceptable salt or stereoisomer thereof; and one or more pharmaceutically acceptable carriers.

In yet another aspect, the invention provides a combination, in particular a pharmaceutical combination, comprising a therapeutically effective amount of a compound of Formula (I) or subformulae thereof, or a pharmaceutically acceptable salt or stereoisomer thereof; and one or more therapeutically active agent(s).

In another aspect, the present invention provides a compound of Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, for use in treating, preventing, inhibiting, ameliorating, or eradicating the pathology and/or symptomology of a disease caused by a kinetoplastid parasite.

Unless specified otherwise, the term "compounds of the present invention" refers to compounds of Fomula I and subformulae thereof (e.g., Formula (II)), salts of the compound, hydrates or solvates of the compounds; as well as all stereoisomers (including diastereoisomers and enantiomers), tautomers, isotopically labeled compounds (including deuterium

substitutions), and inherently formed moieties. Compounds of the present invention further comprise polymorphs of compounds of Formula (I) (or subformulae thereof) and salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural, unless otherwise indicated herein or clearly contradicted by the context.

As used herein, the term "Ci- ₄ alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to four carbon atoms, and which is attached to the rest of the molecule by a single bond. The term "Ci-4alkyl" is to be construed accordingly. Examples of Ci-4alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1 -methylethyl (/so-propyl), n-butyl, and 1 ,1 -dimethylethyl (t- butyl).

As used herein, the term "Ci- ₄ alkoxy" refers to a radical of the formula -OR _a where R _a is a Ci- ₄ alkyl radical as generally defined above. Examples of Ci- ₄ alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy and isobutoxy.

As used herein, the term "cyano" means the radical * ^_ C≡N

The term "C ₃ -6cycloalkyl" refers to a stable monocyclic saturated hydrocarbon radical consisting solely of carbon and hydrogen atoms, having from three to six carbon atoms, and which is attached to the rest of the molecule by a single bond. Examples of C3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

"Halogen" or "halo" refers to bromo, chloro, fluoro or iodo; preferably fluoro, chloro or bromo.

As referred to herein, the term "substituted" means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that normal valencies are maintained and that the substitution results in a stable compound. When a substituent is oxo (i.e., =0), then 2 hydrogens on the atom are replaced. In cases wherein there are nitrogen atoms (e.g., amines) present in compounds of the present invention, these may be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxides) to afford other compounds of the invention. When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3 R groups, then said group may be unsubstituted or substituted with up to three R groups, and at each occurrence R is selected independently from the definition of R.

The term "EC50", as used herein, refers to the molar concentration of a modulator that produces 50% efficacy.

As used herein, the term "pharmaceutical composition" refers to a compound of the invention , or a pharmaceutically acceptable salt thereof, together with at least one

pharmaceutically acceptable carrier, in a form suitable for topical or parenteral administration.

As used herein, the term "pharmaceutically acceptable carrier" refers to a substance useful in the preparation or use of a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22 ^nd Ed. Pharmaceutical Press, 2013, pp. 1 049-1 070).

As used herein, the term "inhibit", "inhibition" or "inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

As used herein, the term "prevent", "preventing" or "prevention" of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

As used herein, the term "subject" refers to primates (e.g., humans, male or female, dogs, rabbits, guinea pigs, pigs, rats and mice). In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

The term "a therapeutically effective amount" of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a subject, is effective to (1 ) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease caused by the proliferation of a kinetoplatid parasite; or (2) reduce or inhibit the proliferation of a kinetoplatid parasite.

As used herein, the term "treat", "treating" or "treatment" of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.

Embodiments of the Invention

The present invention relates to compounds having antiparasitic activitiy against kinatoplastid protozoa. It relates particularly to compounds that inhibit growth of kinatoplastid parasite cells through inhibition of the parasitic proteasome, and thereof useful as a therapy for leishmaniasis and Chagas disease.

Various enumerated embodiments of the invention are described herein. Features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.

Embodiment 1 . A compound of Formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, as described in the Summary of the Invention.

Embodiment 2. The compound of Formula (I) according to Embodiment 1 , or a

pharmaceutically acceptable salt or stereoisomer thereof, wherein X is N.

Embodiment 3. The compound of Formula (I) according to Embodiment 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R°, R ¹ , R ² and R ³ are methyl.

Embodiment 4. The compound of Formula (I) according to Embodiment 1 , or a

pharmaceutically acceptable salt or stereoisomer thereof, wherein said compound is of Formula

(II):

Embodiment 5. The compound of Formula (I) according to any one of Embodiments 1 - 4, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y is C.

Embodiment 6. The compound of Formula (I) according to any one of Embodiments 1 -5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein L is a bond, -0-,

(CH ₂ )i-3-, ^* -OCH ₂ -, ^* -OCH ₂ CH ₂ -, ^* - CH ₂ 0-, ^* -OCH(CH ₃ )- or ^* -OCD ₂ -; wherein * represents the point of attachment of L to Y.

Embodiment 7. The compound of Formula (I) according to Embodiment 6, or a

pharmaceutically acceptable salt or stereoisomer thereof, wherein L is a bond, -CH ₂ CH ₂ - or ^* -OCH ₂ -.

Embodiment 8. The compound of Formula (I) according to any one of Embodiments 1 - 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ⁴ is chloro, fluoro, CN, -C(0)0-CH ₂ CH ₃ , methyl, isopropyl, t-butyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy or cyclopropyl.

Embodiment 9. The compound of Formula (I) according to Embodiment 1 , or a pharmaceutically acceptable salt or stereoisomer thereof, wherein said compound is selected from Examples 1 -80 in Table 1 .

Embodiment 10. The compound of Formula (I) according to Embodiment 1 , or a pharmaceutically acceptable salt or stereoisomer thereof, wherein said compound is selected from :

Embodiment 11 . A pharmaceutical composition comprising a compound according to any one of Embodiments 1 -10 or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more pharmaceutically acceptable carriers

Embodiment 12. A combination comprising a compound according to any one of

Embodiments 1 -10, or a pharmaceutically acceptable salt or stereoisomer or stereoisomer thereof, and one or more therapeutically active agents.

Embodiment 13. A method of inhibiting growth and proliferation of a kinetoplastid parasite in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of the compound according to any one of Embodiments 1 -10, or a pharmaceutically acceptable salt or stereoisomer thereof.

Embodiment 14. A method for treating the pathology and/or symptomology of a disease caused by a parasite, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of Embodiments 1 -10, or a

pharmaceutically acceptable salt or stereoisomer thereof, and optionally in combination with a second agent.

Embodiment 14A. The method according to Embodiment 14, wherein said disease is caused by a kinetoplastid parasite.

Embodiment 15. The method according to Embodiment 14 or 14A, wherein the disease is leishmaniasis or Chagas disease.

Embodiment 16. The method according to Embodiment 15, wherein the disease is leishmaniasis.

Embodiment 16A. The method according to Embodiment 16, wherein the disease is visceral leishmaniasis or cutaneous leishmaniasis.

Embodiment 17. The method according to any one of Embodiments 16 or 16A, further comprising administering said second agent selected from stibogluconate, meglumine antimoniate, amphotericin, miltefosine, and paromomycin.

Embodiment 18. The method according to Embodiment 15, wherein the disease is Chagas diease.

Embodiment 18A. The method according to Embodiment 18A, further comprising administering said second agent selected from benznidazole, nifurtimox and amphotericin.

Embodiment 1 9. A compound according to any one of Embodiments 1 -10, or a

pharmaceutically acceptable salt thereof, for use in the treatment of leishmaniasis or Chagas disease.

Embodiment 20. Use of a compound according to any one of Embodiments 1 -10, or a pharmaceutically acceptable salt or stereoisomer thereof, in the manufacture of a medicament for the treatment of leishmaniasis or Chagas disease.

Embodiment 21 . A compound of Formula (I) or subformulae thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of Embodiments 1 -10 for use in therapy.

Embodiment 22. A compound of Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of Embodiments 1 -10, for use as a medicament.

Embodiment 23. Use of a compound of the Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, according to any one of Embodiment 1 -1 0, in the manufacture of a medicament for the treatment of a disease selected from leishmaniasis, and Chagas diseases.

Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible stereoisomers or as mixtures thereof, for example as pure optical isomers, or as stereoisomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. The present invention is meant to include all such possible stereoisomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms. Optically active (R)- and (S)- stereoisomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.

As used herein, the terms "salt" or "salts" refers to an acid addition or base addition salt of a compound of the invention. "Salts" include in particular "pharmaceutical acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of am ino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,

toluenesulfonic acid, sulfosalicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

In another aspect, the present invention provides compounds of Formula (I) or subformulae thereof, in acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, subsalicylate, sulfate, tartrate, tosylate trifenatate,

trifluoroacetate or xinafoate salt form.

In another aspect, the present invention provides compounds of Formula (I) or subformulae thereof, in sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, copper, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine or tromethamine salt form.

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen.

Further, incorporation of certain isotopes, particularly deuterium (i.e., ² H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index or tolerability. It is understood that deuterium in this context is regarded as a substituent of a compound of the formula (I). The concentration of deuterium, may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted as being deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). It should be understood that the term "isotopic enrichment factor" can be applied to any isotope in the same manner as described for deuterium.

Other examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F ³¹ P, ³² P, ³⁵ S, ³⁶ CI, ¹²³ l, ¹²⁴ l, ¹²⁵ l respectively. Accordingly it should be understood that the invention includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as ³ H and ¹⁴ C, or those into which non-radioactive isotopes, such as ² H and ¹³ C are present. Such isotopically labelled compounds are useful in metabolic studies (with ¹⁴ C), reaction kinetic studies (with, for example ² H or ³ H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ¹⁸ F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically- labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

The compounds of the present invention are either obtained in the free form, as a salt thereof. As used herein, the terms "salt" or "salts" refers to an acid addition or base addition salt of a compound of the invention. "Salts" include in particular "pharmaceutical acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,

toluenesulfonic acid, sulfosalicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium , sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

In another aspect, the present invention provides compounds of the present invention in acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide/hydrobromide,

bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, subsalicylate, sulfate, tartrate, tosylate trifenatate,

trifluoroacetate or xinafoate salt form thereof, and a pharmaceutically acceptable carrier.

Pharmacology and Utility

In one aspect, the invention relates to small molecules compounds of Formula (I) that target the proteasome of kinoplastid parasites and are useful as a therapy; particularly, for preventing and treating diseases and conditions that are caused by the infection of kinoplastid parasites. The activity of a compound according to the present invention can be assessed by the in vitro and in vivo methods described in the Biological Assay section infra. It is understood that the assays illustrate the invention without in any way limiting the scope of the invention.

Thus, as a further aspect, the present invention provides for the use of a compound of Formula (I) or subformulae thereof, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as a therapy for a disease or condition that can benefit from inhibition of growth and proliferation of kinetoplastid parasites; and for the manufacture of a medicament, such as for the treatment of a disease caused by the growth and proliferation of a kinetoplastid parasite. In one embodiment, the disease being treated is selected from leishmaniasis, and Chagas disease. In another embodiment, the disease being treated is Leishmaniasis caused by the parasite including, but is not limited to, Leishmania donovani, Leishmania infantum, Leishmania braziliensis, Leishmania panamensis, Leishmania guayanensis, Leishmania amazonensis, Leishmania mexicana, Leishmania tropica, Leishmania major, In one embodiment, the disease being treated is visceral leishmaniasis. In another embodiment, the disease being treated is cutaneous leishmaniasis. In another embodiment, the disease being treated is Chagas disease caused by the parasites Trypanosoma cruzi.

In accordance with the foregoing, the present invention further provides a method for preventing or treating leishmaniasis, or Chagas disease in a subject in need of such treatment, comprising administering to said subject a therapeutically effective amount of a compound selected from Formula (I) or subformulae thereof, a pharmaceutically acceptable salt or stereoisomer thereof. The required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.

Pharmaceutical Compositions, Dosage and Administration

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration (e.g. by injection, infusion, transdermal or topical administration), and rectal administration. Topical administration may also pertain to inhalation or intranasal application. The pharmaceutical compositions of the present invention can be made up in a solid form (including, without limitation, capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including, without limitation, solutions, suspensions or emulsions). Tablets may be either film coated or enteric coated according to methods known in the art. Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of:

a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol ; for tablets also

c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired

d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and

e) absorbents, colorants, flavors and sweeteners.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 1 -1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1 - 500 mg or about 1 -250 mg or about 1 -150 mg or about 0.5-100 mg, or about 1 -50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The compounds of the present invention can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either enterally, parenterally,

advantageously intravenously, e.g., as a suspension or in aqueous solution. The dosage in vitro may range between about 10 ³ molar and 10 ⁹ molar concentrations. A therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1 -500 mg/kg, or between about 1 -100 mg/kg.

Compounds of the invention can be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). The compound of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The compound of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents. A therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is

therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the invention.

Products provided as a combined preparation include a composition comprising the compound of Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of Formula (I) and the other therapeutic agent(s) in separate form, e.g. in the form of a kit. In one embodiment, the invention provides a pharmaceutical composition comprising a compound of Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and another therapeutic agent(s). Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, as described above. In another embodiment, the invention provides a product comprising a compound of Formula (I) and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy.

In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of Formula (I) or subformulae thereof, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like. The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of the invention and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the invention and the other therapeutic agent.

Accordingly, the invention provides the use of a compound of Formula (I) for treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the medicament is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the medicament is administered with a compound of Formula (I).

The invention also provides a compound of Formula (I) for use in a method of treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the compound of Formula (I) is prepared for administration with another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the other therapeutic agent is prepared for administration with a compound of Formula (I). The invention also provides a compound of Formula (I) for use in a method of treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the compound of Formula (I) is administered with another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the other therapeutic agent is administered with a compound of Formula (I).

The invention also provides the use of a compound of Formula (I) for treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition caused by the growth and proliferation of a kinetoplastid parasite, wherein the patient has previously (e.g. within 24 hours) been treated with a compound of Formula (I).

In one embodiment, for the treatment of Leishmaniasis, the other therapeutic agent is selected from tstibogluconate, meglumine antimoniate, Amphotericin, Miltefosine and paromomycin. In one embodiment, for the treatment of Chagas disease, the other therapeutic agent is selected from benznidazole, nifurtimox and/or Amphotericin.

Where the compounds of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co- drug employed, on the specific drug employed, on the condition being treated and so forth.

Processes for Making Compounds of the Invention

The compounds of the present invention can be prepared in a number of ways known to one skilled in the art of organic synthesis in view of the methods, reaction schemes and examples provided herein. For illustrative purposes, the reaction schemes depicted below provide potential routes for synthesizing the compounds of the present invention as well as key intermediates. For a more detailed description of the individual reaction steps, see the

Examples section below. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the inventive compounds. Although specific starting materials and reagents are depicted in the schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art. All methods described herein can be performed in any suitable order, unless otherwise indicated or otherwise clearly contradicted by context.

Compounds of Formula (I) can be prepared as generally illustrated in Scheme 1 , wherein R ¹ , ² , R ³ , X, Y, L, Ar and m are as defined in the Summary of the Invention.

Scheme 1

Intermediate lA can be prepared from generally commercially available agents, followed by acid catalyzed cyclization with 5-membered amino- heterocycle to afford fused bicyclic ring.

Examples

The Examples herein merely illuminate the invention and does not limit the scope of the invention otherwise claimed. Temperatures are given in degrees Celsius. If not mentioned otherwise, all evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20- 133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR.

All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume 21 ). Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples. Where desired, conventional protecting groups are used to protect reactive functional groups in accordance with standard practice, for example, see T.W. Greene and RG.M. Wuts in

"Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991 .

Unless specified otherwise, the starting materials are generally available from non-excluding commercial sources such as Aldrich Chemicals (Milwaukee, Wis.), TCI Fine Chemicals (Japan), Shanghai Chemhere Co., Ltd. (Shanghai, China), Aurora Fine Chemicals LLC (San Diego, CA), FCH Group (Ukraine), Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn, N.J.), Maybridge Chemical Company, Ltd. (Cornwall, England), Tyger Scientific (Princeton, N.J.), AstraZeneca Pharmaceuticals (London, England), Chembridge Corporation (USA), Matrix Scientific (USA), Conier Chem & Pharm Co., Ltd (China), Enamine Ltd (Ukraine), Combi-Blocks, Inc. (San Diego, USA), Oakwood Products, Inc. (USA), Apollo Scientific Ltd. (UK), Allichem LLC. (USA) and Ukrorgsyntez Ltd (Latvia); or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1 -19, Wiley, New York (1967-1999 ed.), Larock, R.C., Comprehensive Organic Transformations, 2 ^nd -ed., Wiley-VCH Weinheim, Germany (1999), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer- Verlag, Berlin, including supplements (also available via the Beilstein online database).

Abbreviations

Abbreviations as used herein, are defined as follows: "1 x" for once, "2x" for twice, "3x" for thrice, " ^S C" for degrees Celsius, "aq" for aqueous, "Col" for column, "eq" for equivalent or equivalents, "g" for gram or grams, "mg" for milligram or milligrams, "L" for liter or liters, "mL" or "ml" for milliliter or milliliters, "μί" or "μΙ" for microliter or microliters, "N" for normal, "M" for molar, "μΜ" for micromolar, "nM" for nanomolar, "mol" for mole or moles, "mmol" for millimole or millimoles, "min" for minute or minutes, "h" or "hrs" for hour or hours, "RT" for room temperature, "ON" for overnight, "atm" for atmosphere, "psi" for pounds per square inch, "cone." for concentrate, "aq" for aqueous, "sat" or "sat'd" for saturated, "MW" for molecular weight, "mw" or '^wave" for microwave, "mp" for melting point, "Wt" for weight, "MS" or "Mass Spec" for mass spectrometry, "ESI" for electrospray ionization mass spectroscopy, "HR" for high resolution, "HRMS" for high resolution mass spectrometry, "LCMS" for liquid

chromatography mass spectrometry, "HPLC" for high pressure liquid chromatography, "RP HPLC" for reverse phase HPLC, "TLC" or "tic" for thin layer chromatography, "NMR" for nuclear magnetic resonance spectroscopy, "nOe" for nuclear Overhauser effect spectroscopy, "1 H" for proton, "δ " for delta, "s" for singlet, "d" for doublet, "t" for triplet, "q" for quartet, "m" for multiplet, "br" for broad, "Hz" for hertz, "ee" for "enantiomeric excess" and "α", "β", "R","r", "S", "s", "E", and "Z" are stereochemical designations familiar to one skilled in the art.

The following abbreviations used herein below have the corresponding meanings:

AIBN azobisisobutyronitrile

Bn benzyl

Boc fert-butoxy carbonyl

Boc ₂ 0 di-fert-butyl dicarbonate

Bu butyl

CS2CO3 cesium carbonate anhydrous

CHCI3 chloroform

DAST diethylaminosulfurtrifluoride

DBU 2,3,4,6,7,8,9,10-octahydropyrimido[1 ,2-a]azepine

DCM dichloromethane

DMAP 4-dimethylaminopyridine

DMF dimethylformamide

DMSO dimethylsulfoxide

DPPA diphenylphosphoryl azide

EA ethyl acetate

Equiv. equivalence

Et ethyl

Et ₂ 0 diethyl ether

EtOH ethanol

EtOAc ethyl acetate

HATU 2-(7-Aza-1 H-benzotriazole-1 -yl)-1 ,1 ,3,3-tetramethyluronium

hexafluorophosphate

HCI hydrochloric acid

/ ^' -Bu isobutyl

/ ^' -Pr isopropyl

KOAc potassium acetate

UAIH4 lithium aluminium hydride

Me methyl

mCPBA 3-chloroperoxybenzoic acid MeCN acetonitrile

Mn0 ₂ manganese dioxide

N ₂ nitrogen

NaBH ₄ sodium borohydride

NaHC0 ₃ sodium bicarbonate

Na ₂ S0 ₄ sodium sulfate

NBS /V-Bromosuccinimide

Ph phenyl

PPh ₃ triphenylphosphine

Ph ₃ P=0 triphenylphosphine oxide

Rt retention factor

rt or RT room temperature ( °C)

f-Bu or Bu' fert-butyl

T3P® Propane phosphonic acid anhydride

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

Instrumentation

Method 1 : The system consists of:

- LEAP PAL Autosampler

- Waters Acquity Binary Sovent Manager (UPB)

- Waters Acq u ity P D A Detecto r ( U P D)

- Waters Acquity ELS Detector (UPE)

- Waters ZQ mass spectrometer

- Waters Mass Lynx Software

The system flows at 1 mL/min with each sample being screened through a 1 .7um 2.1 x30mm Waters Acquity BEH C18 column. Mobile phase A is Water + 0.05% formic acid and mobile phase B is Acetonitrile + 0.05% formic acid.

The DAD acquires data between 214 nm and 400 nm at 2.5 Hz; 214 nm and 254 nm are extracted during data processing. The ZQ acquires typically between 180 am u and 800 amu. If the sample mass lies outside of the 180 amu range another MS method that scans over a broader range will, be used.

Method 2:

Quantitative QC analysis is conducted on the "Pacer"LC/MS/CLND system, which consists of:

- Waters Acquity Sampler Manager

- Waters Acquity Binary pump

- Waters Acquity Photodiode Array Detector (PDA)

- Antek 8060-R Chemiluminescent Nitrogen Detector (CLND)

- Waters 3100 Mass Detector

- Leap Technologies HTC PAL autosampler

UPLC Pump Method:

Mobile Phases:

A. 95% H20/5% MeOH/IPA (75/25) + 0.05% formic acid

B. MeOH/IPA (75/25) + 0.035% formic acid

Column: Thermo Syncronis 2.1 x30mm, 1 .7u particle C18

MS Method"Scan range is 150-1000amu

UV Scan: 200-400 nm

(1A) Methyl 3-oxo-2-(4-phenethylpiperidin-1 -yl)butanoate (1A)

To a stirred solution of 4-phenethylpiperidine (7.0g, 37 mmol) and /V-ethyl-/V- isopropylpropan-2-amine (DIEA, 10.51 g, 14.17ml_, 0.742g/ml) in DMF (15 ml_) was added methyl 2-chloro-3-oxobutanoate slowly (8.35g, 55.5 mmol) and the reaction was stirred at room temperature for 24 h. LC-MS indicated that 4-phenethylpiperidine (M/Z = 190.1 ) still was not consumed. Methyl 2-chloro-3-oxobutanoate was added every two hours for three times (3 x 2.78g, 18.5 mmol) and stirred 21 h. The reaction was monitored with LCMS at this time, which showed that the reaction was complete with the desired compound as a major product. The reaction mixture was diluted with ethyl acetate (250 ml) and washed with saturated aqueous Na2C03 (100 ml) and water (4 x 120 ml). The combined organic layers were dried and evaporated to offer crude mixture, which was dried overnight under vacuum at 45 °C to give 1 A as a liquid with yellow, brown like color (-11 .3 g). M/Z (M+H) = 304.2.

2,5-dimethyl-6-(4-phenethylpiperidin-1 -yl)-H ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)-one (Step B) A mixture of methyl 3-oxo-2-(4-phenethylpiperidin-1 -yl)butanoate (1 A) (11 g, 36.3 mmol), 3- methyl-1 H-1 ,2,4-triazol-5-amine (8.89 g, 91 mmol) and 4-methylbenzenesulfonic acid (6.24g,

36.3 mmol) was heated from 140°C to 160 °C with vigorous stirring using a stir bar. The reaction mixture changed from solid to liquid, which then turned into a solid again. The crude mixture was dissolved in MeOH (150 ml_) and divided into three portions. Each portion was purified using a 330 grams combiflash column eluting with 0.1 to 3% MeOH in DCM to give (1 ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.83 (s, 1 H), 7.32 - 7.25 (m, 2H), 7.24 - 7.20 (m, 2H), 7.20 - 7.13 (m, 1 H), 3.40 (t, J = 10.4 Hz, 2H), 2.68 - 2.56 (m, 4H), 2.22 (s, 3H), 2.19 (s, 3H), 1 .75 - 1 .64 (m, 2H), 1 .61 - 1 .46 (m, 2H), 1 .30 - 1 .13 (m, 3H). Method 2: RT = 3.37 min.; M/Z (M+H) = 352.3.

Examples 2-25 infra were synthesized according to the protocol described for Example 1 , using the appropriate amine analog.

Example 2: 2,5-dimethyl-6-(2-methyl-4-(4-(trifluoromethoxy)phenyl)piper azin-1-yl)- Γ1 ,2,41triazolori ,5-alpyrimidi -7(4H)-one

¹ H NMR (400 MHz, Chloroform-d) δ 7.13 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 3.85 (dt, J =6.7, 3.5 Hz, 1 H), 3.42 - 3.06 (m, 4H), 2.83 (d, J = 10.9 Hz, 1 H), 2.63 (d, J = 3.4 Hz, 1 H), 2.54 (s, 3H), 2.07 (s, 3H), 1 .13 (d, J = 6.5 Hz, 3H). Method 1 : RT = 1 .07 min.; M/Z (M+H) = 423.2. Example 3: 2,5-dimethyl-6-(4-(p-tolyl)piperazin-1-yl)-ri ,2,4ltriazolori ,5-alpyrimidin-7(4H)- one

¹ H NMR (400 MHz, Methanol-d4) δ 7.13 - 7.01 (m, 2H), 6.97 - 6.81 (m, 2H), 4.00

2H), 3.44 (d, J = 49.0 Hz, 2H), 3.02 - 2.64 (m, 4H), 2.45 (s, 3H), 2.39 (s, 3H), 2.26 (s,

Method 1 : RT = 0.67 min.; M/Z (M+H) = 339.2.

Example 4: 6-(3-(4-chlorophenyl)pyrrolidin-1-yl)-2,5-dimethyl-[1 ,2,4]triazolo[1 ,5- a]pyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δδ 7.39-7.37, (m, 4H), 3.57 - 3.50 (m, 2H), 3.44 - 3.40 (m, 2H), 3.28 - 3.24 (m, 2H), 3.11 - 3.07 (m, 1 H), 2.39 (s, 3H), 2.34 (s, 3H). Method 1 : RT = 1 .00 min.; M/Z (M+H) = 344.1 .

Example 5: 2,5-dimethyl-6-(3-methyl-4-(4-(trifluoromethoxy)phenyl)piper azin-1-yl)- H ,2,41triazolori ,5-alpyrimidi -7(4H)-one

¹ H NMR (400 MHz, Methanol-cW) δ 7.87 (d, J = 8.8 Hz, 2H), 7.69 - 7.51 (m, 2H), 4.14 (t, J = 12.6 Hz, 2H), 4.02 - 3.84 (m, 2H), 3.75 (d, J = 11 .8 Hz, 1 H), 3.28 - 3.18 (m, 2H),3.17 - 3.08 (m, 2H), 2.60 (s, 3H), 2.45 (s, 3H), 1 .10 (d, J = 6.3 Hz, 3H). Method 1 : RT = 0.97 min.; M/Z (M+H) = 423.2.

Example 6: 6-(4-(4-isopropylphenyl)-3-methylpiperazin-1-yl)-2,5-dimethy l-

A mixture of tert-butyl 3-methylpiperazine-1 -carboxylate (50 mg, 0.25 mmol), 1 -bromo-4- isopropylbenzene (50 mg, 0.25 mmol), BINAP (47 mg, 0.075 mmol), NaOtBu (72 mg, 0.753 mmol), and Pd2dba3 (23 mg, 0.025 mmol), in dioxane at r/t in a 20ml_ vial was degassed under nitrogen. Stirred at 140 in a sealed 5ml_ microwave vial in the Biotage microwave for 30min. After the reaction was complete as shown by LCMS, the crude reaction was filtered and the solvent evacuated. The crude product was purified by normal phase silica gel column via ISCO chromatography using EtOAC/Hex (10-100%) to give tert-butyl 4-(4-isopropylphenyl)-3- methylpiperazine-1 -carboxylate (6A). M/Z (M+H) = 319.5.

Ethyl 2-(4-(4-isopropylphenyl)-3-methylpiperazin-1 -yl)-3-oxobutanoate (6B) and 6-(4-(4- isopropylphenyl)-3-methylpiperazin-1 -yl)-2,5-dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one (6) were synthesized following a protocol similar to compounds (1A) and (1 ) from Example 1 , respectively.

Example 7: (R)-6-(3-(4-chlorophenyl)pyrrolidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolon ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, CD30D) δδ 7.39 - 7.35, (m, 2H), 7.31 - 7.28 (m, 2H), 3.60 - 3.48 (m, 2H), 3.43 - 3.35 (m, 2H), 3.24 - 3.19 (m, 1 H), 2.49 (s, 3H), 2.43 (s, 3H), 2.38 - 2.35 (m, 1 H), 2.17 - 2.10 (m, 1 H). Method 1 : RT = 1 .00 min.; M/Z (M+H) = 344.1 .

Example 8: (S)-6-(3-(4-chlorophenyl)pyrrolidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolon ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, CD ₃ OD) δδ 7.38 - 7.35, (m, 2H), 7.31 - 7.29 (m, 2H), 3.61 - 3.50 (m, 2H), 3.41 - 3.35 (m, 2H), 3.24 - 3.20 (m, 1 H), 2.49 (s, 3H), 2.42 (s, 3H), 2.39 - 2.35 (m, 1 H), 2.16 - 2.10 (m, 1 H). Method 1 : RT = 1 .01 min.; M/Z (M+H) = 344.1 .

Example 9: 2.5-dimethyl-6-((3R.4R¾-3-methyl-4-(4-(trifluoromethoxy¾ph envnpiperidin-1-vn- H ,2,41triazolori ,5-alpyrimidin-7(4H)-one

Example 9 was prepared following the same protocol as described on Example 1 using the appropriate amino analog, (cis)-3-methyl-4-(4-(trifluoromethoxy)phenyl)piperidine, which was prepared as described in WO2008012622. ¹ H NMR (400 MHz, DMSO-cfe) δ 7.39 (dd, J = 1 1 .7, 8.7 Hz, 2H), 7.34 - 7.28 (m, 2H), 3.87 - 3.72 (m, 1 H), 3.01 - 2.81 (m, 2H), 2.39 (s, 3H), 2.31 (s, 3H), 2.10 - 1 .88 (m, 2H), 1 .86 - 1 .50 (m, 4H), 1 .36 - 1.08 (m, 2H). Method V. RT = 1 .17 min.; M/Z (M+H) = 422.2.

Example 10: 6-(4-(4-(tert-butyl)phenyl)-3-methylpiperazin-1 -yl)-2,5-dimethyl-

¹ H NMR (400 MHz, DMSO-d6) d 11 .10 (s, 1 H), 7.68 (d, J = 38.3 Hz, 2H), 7.26 (d, J = 15.0 Hz, 1 H), 6.90 (d, J = 23.5 Hz, 1 H), 3.39 - 2.57 (m, 4H), 2.44 (s, 2H), 2.35 (s, 3H), 1 .39 - 1 .25 (m, 9H), 1 .12 (s, 1 H), 0.93 (s, 2H). Method 1 : RT = 0.77 min.; M/Z (M+H) = 395.2.

Example 11 : Ethyl 4-(4-(2.5-dimethyl-7-oxo-4.7-dihvdro-ri.2.41triazolon .5-alPyrimidin-6- yl)piperazin-1-yl)benzoate

¹ H NMR (400 MHz, Chloroform-d) δ 7.92 - 7.77 (m, 2H), 6.92 (d, J = 7.5 Hz, 2H), 4.27 (q, J = 6.8 Hz, 2H), 3.94 - 3.43 (m, 4H), 3.12 - 2.68 (m, 4H), 2.58 (s, 3H), 2.49 (s, 3H), 1 .31 (td, J = 7.1 , 3.3 Hz, 3H). Method 1 : RT = 0.92 min.; M/Z (M+H) = 397.2.

Example 12: 4-(4-(2.5-dimethyl-7-oxo-4.7-dihvdro-H .2.41.riazoloH .5-alPyrimidin-6- yl)piperazin-1-yl)benzonitrile

Method 1 : RT = 0.88 min.; M/Z (M+H) = 350.2.

Example 13: 6-(4-(3,4-dichlorophenyl)piperazin-1-yl)-2,5-dimethyl-ri,2,4 ltriazolori,5- alpyrimidin-7(4H)-one

Method 1 : RT = 1.07 min.; M/Z (M+H) = 393.1.

Example 14: 2,5-dimethyl-6-(4-(3-(trifluoromethyl)phenethyl)piperidin-1- yl)-

¹ H NMR (400 MHz, DMSO-cfe) δ 7.58 (d, J= 2.2 Hz, 1H), 7.54 (dd, J= 3.1, 1.9 Hz, 3H), 3.30 (s, 2H), 2.77-2.64 (m, 4H), 2.35 (s, 3H), 2.32 (s, 3H), 1.74 (d, J= 10.9 Hz, 2H), 1.56 (s, 2H), 1.33- 1.16 (m,3H). Method 1: RT = 1.19 min.; M/Z (M+H) = 420.2.

Example 15: 6-(4-((3,4-difluorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-r i,2,4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.35-6.99 (m, 3H), 4.57 (s, 2H), 3.81 (s, 1H), 3.49 (dt, J = 3.3, 1.7 Hz, 2H), 2.82 (s, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.09 (s, 2H), 1.63 (s, 2H). Method 1 : RT = 0.97 min.; M/Z (M+H) = 390.2. Example 16: 6-(4-((3,5-difluorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-r i,2,4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 6.99 (d, J= 7.1 Hz, 2H), 6.92-6.70 (m, 1H), 4.61 (s, 2H), 3.92-3.70 (m, 1H), 3.56-3.44 (m, 2H), 2.82 (s, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.10 (d, J = 7.3 Hz, 2H), 1.64 (d, J= 13.3 Hz, 2H). Method 1: RT = 1.00 min.; M/Z (M+H) = 390.2.

Example 17: 2,5-dimethyl-6-(4-phenylpiperidin-1-yl)-ri,2,4ltriazolori,5- alpyrimidin-7(4H)- one

¹ H NMR (400 MHz, DMSO-de) d 12.87 (s, 1H), 7.31 (d, J = 5.6 Hz, 4H), 7.20 (dqd, J = 7.2, 4.9, 2.4 Hz, 1H), 3.49 (t, J = 11.3 Hz, 2H), 2.80 (d, J = 11.2 Hz, 2H), 2.60 (tt, J = 11.5, 3.8 Hz, 1 H), 2.41 (s, 3H), 2.34 (s, 3H), 1.85 - 1.66 (m, 4H). Method 1 : RT = 0.98 min.; M/Z (M+H) = 324.2.

Method 1 : RT = 1.02 min.; M/Z (M+H) = 337.2.

Example 19: 6-(4-((2,6-difluorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-r i,2,4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.27 (s, 1 H), 7.22 - 7.14 (m, 2H), 4.69 (s, 2H), 3.94 3.66 (m, 1H), 3.50 (s, 2H), 2.82 (s, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.11 (d, J= 16.6 Hz, 2H), 1.63 (s,2H). Method 1: RT = 0.97 min.; M/Z (M+H) = 390.2.

Example 20: 6-(4- 2.3-difluorobenzvnoxy¾piperidin-1-vn-2.5-dimethyl-ri.2.4ltr iazolori.5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.23 (s, 1 H), 7.11 - 6.96 (m, 2H), 4.65 (s, 2H), 3.96 - 3.63 (m, 1H), 3.61 -3.46 (m, 2H), 2.83 (s, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.10 (s, 2H), 1.64 (s, 2H). Method 1: RT = 0.97 min.; M/Z (M+H) = 390.2.

Example 21: 6-(4-((2.5-difluorobenzvnoxy¾piperidin-1-vn-2.5-dimethyl-ri .2.4ltriazolon.5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.40 (tt, J= 8.4, 6.5 Hz, 1H), 7.00 (t, J= 7.9 Hz, 2H), 4.68 (s, 2H), 3.88 - 3.63 (m, 1 H), 3.59 - 3.44 (m, 2H), 2.81 (s, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.17-1.95 (m, 2H), 1.59 (s, 2H). Method 1 : RT = 0.93 min.; M/Z (M+H) = 390.2.

Example 22: 6-(4-((3-fluorophenoxy)methyl)piperidin-1-yl)-2,5-dimethyl-r i,2,4ltriazolori,5- alpyrimidin-7(4H)-one

Compound 22 was made in a similar manner as Example 1 using 4-((3- fluorophenoxy)methyl)piperidine hydrochloride in place of 4-phenethylpiperidine. ¹ H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H), 7.35 - 7.27 (m, 1H), 6.86 - 6.72 (m, 3H), 3.91 - 3.85 (m, 2H), 3.42 - 3.35 (m, 2H), 2.74 (d, J = 9.0 Hz, 2H), 2.36 (s, 3H), 2.33 (s, 3H), 1.84 - 1.74 (m, 3H), 1.41 -1.28(m,2H). Method 1: RT = 1.04 min.; M/Z (M+H) = 372.2. Example 23: 6-(4-((3-fluorobenzyl)oxy)-3-methylpiperidin-1-yl)-2,5-■&l t;dimethyl-

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.36 (tdd, J = 8.2, 5.9, 2.9 Hz, 1H), 7.16 (dd, J= 22.6, 8.9 Hz, 2H), 7.01 (d, J= 7.2 Hz, 1H), 4.61 (s, 2H), 3.96-3.63 (m, 1H), 3.62-3.43 (m, 3H), 2.81 (s, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.10 (s, 2H), 1.64 (s,2H), 1.23(s, 3H). Method 1: RT = 1.03 min.; M/Z (M+H) = 386.2.

Example 24: 6-(4-((2-chlorophenoxy)methyl)piperidin-1-yl)-2,5-dimethyl-r i ,2,41triazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δδ 7.44-7.42 (m, 1H), 7.32 - 7.27 (m, 1H), 7.18-7.13 (m, 1H), 6.97-6.91 (m, 1H), 3.65-3.61 (m, 2H), 2.81 -2.71 (m, 2H), 2.37 (s, 3H), 2.33 (s, 3H), 1.91 - 1.82 (m, 3H), 1.41 - 1.38 (m, 2H). Method 1 : RT = 1.07 min.; M/Z (M+H) = 388.2.

Example 25: 2,5-dimethyl-6-(4-phenethoxypiperidin-1-yl)-ri,2,4ltriazolor i,5-alpyrimidin- 7(4H¾-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.31 - 7.25 (m, 4H), 7.22-7.18 (m, 1H), 3.66-3.56 (m, 5H), 2.83 - 2.80 (m, 2H), 2.70 - 2.68 (m, 2H), 2.35 (s, 3H), 2.52 (s, 3H), 1.97 - 1.94 (m, 2H), 1.45- 1.40 (m,2H). Method 1 : RT = 0.97 min.; M/Z (M+H) = 368.2.

Example 26: 6-(4-((2.4-difluorobenzyl)oxy)piperidin-1-yl)-2.5-dimethyl-r i.2.4ltriazolori.5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.51 (s, 1 H), 7.04 - 6.87 (m, 2H), 4.62 (s, 2H), 3.90 - 3.59 (m, 1 H), 3.57 - 3.43 (m, 2H), 2.82 (s, 2H), 2.48 (s, 3H), 2.41 (s, 3H), 2.08 (s, 2H), 1 .62 (s, 2H). Method 1 : RT = 0.97 min.; M/Z (M+H) = 390.2.

Example 27: 6-(4-((4-chlorobenzyl)oxy)piperidin-1 -yl)-2,5-dimethyl-ri ,2,41triazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.48 - 7.22 (m, 4H), 4.60 (s, 2H), 3.57 - 3.41 (m, 3H), 2.88 - 2.72 (m, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.15 - 2.04 (m, 2H), 1.75 - 1 .53 (m, 2H). Method 1 : RT = 1 .03 min.; M/Z (M+H) = 388.1 .

Example 28: 2- (1-(2,5-dimethyl-7-oxo-4,7-dihvdro-ri,2,4ltriazolon ,5-alpyrimidin-6-

¹ H NMR (400 MHz, Methanol-d4) δ 7.75 (d, J = 7.7 Hz, 1 H), 7.74 - 7.57 (m, 2H), 7.53 - 7.37 (m, 1 H), 4.76 (s, 2H), 3.66 - 3.40 (m, 3H), 2.92 - 2.74 (m, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.20 - 2.08 (m, 2H), 1 .77 - 1 .55 (m, 2H). Method 1 : RT = 0.87 min. ; M/Z (M+H) = 379.1 .

Example 29: 6-(4-((3-chlorobenzyl)oxy)piperidin-1 -yl)-2,5-dimethyl-ri ,2,41triazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.36 (s, 3H), 7.35 (dd, J = 4.5, 1 .4 Hz, 1 H), 4.59 (s, 2H), 3.54 - 3.45 (m, 3H), 2.88 - 2.76 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.15 - 2.02 (m, 2H), 1 .73 - 1 .51 (m, 2H). Method 1 : RT = 1 .03 min.; M/Z (M+H) = 388.1 .

Example 30:3-(((1-(2,5-dimethyl-7-oxo-4,7-dihvdro-ri ,2,4ltriazolori,5-alpyrimidin-6-

1 H NMR (400 MHz, Methanol-d4) δ 7.75 (s, 1 H), 7.69 (s, 1 H), 7.66 (dd, J = 7.8, 1 .4 Hz, 1 H),

7.55 (t, J = 7.7 Hz, 1 H), 4.66 (s, 2H), 3.58 - 3.41 (m, 3H), 2.89 - 2.73 (m, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.17 - 2.05 (m, 2H), 1 .72 - 1 .53 (m, 2H). Method 1 : RT = 0.87 min.; M/Z (M+H) = 379.1 .

Example 31 : 6-(4-((2-chlorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.64 - 7.44 (m, 1 H), 7.39 (d, J = 7.7 Hz, 1 H), 7.36 - 7.24 (m, 2H), 4.69 (s, 2H), 3.61 - 3.42 (m, 3H), 2.91 - 2.74 (m, 2H), 2.49 (s, 3H), 2.42 (s, 3H), 2.18 - 2.03 (m, 2H), 1 .75 - 1 .54 (m, 2H). Method 1 : RT = 1.02 min. ; M/Z (M+H) = 388.1 .

Example 32: 4-(((1-(2,5-dimethyl-7-oxo-4,7-dihvdro-ri ,2,4ltriazolori ,5-alpyrimidin-6-

¹ H NMR (400 MHz, Methanol-d4) δ 7.73 (d, J = 8.3 Hz, 2H), 7.58 (s, 2H), 4.69 (s, 2H), 3.56 - 3.50 (m, 3H), 2.89 - 2.76 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.16 - 2.06 (m, 2H), 1 .70 - 1 .57 (m, 2H). Method 1 : RT = 0.87 min.; M/Z (M+H) = 379.1 .

Example 33: 2,5-dimethyl-6-(4-((3-methylbenzyl)oxy)piperidin-1 -yl)-H ,2,41triazolon ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.26 - 7.19 (m, 2H), 7.16 (d, J = 7.2 Hz, 1 H), 7.11 (d, J = 7.3 Hz, 1 H), 4.57 (s, 2H), 3.57 - 3.40 (m, 3H), 2.90 - 2.74 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.35 (s, 3H), 2.16 - 2.02 (m, 2H), 1 .72 - 1 .53 (m, 2H). Method 1 : RT = 1 .00 min.; M/Z (M+H) = 368.2.

Example 34: 2,5-dimethyl-6-(3-(4-(trifluoromethoxy)phenoxy)pyrrolidin-1- yl)- H ,2,41triazolori ,5-alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-cfe) δ 7.28 (dd, J = 9.2, 1 .0 Hz, 2H), 7.04 (d, J = 9.1 Hz, 2H), 5.05 (tt, J = 6.4, 3.1 Hz, 1 H), 3.57 (dd, J = 9.8, 5.9 Hz, 1 H), 3.27 (q, J = 7.7 Hz, 2H), 3.20 - 3.10 (m, 2H), 2.46 - 2.35 (m, 1 H), 2.33 (s, 3H), 2.29 (s, 3H). Method 1 : RT = 1 .06 min. ; M/Z (M+H) = 410.2.

Example 53: 6-(4-((4-fluorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.49 - 7.33 (m, 2H), 7.13 - 6.95 (m, 2H), 4.58 (s, 2H), 3.57 - 3.40 (m, 3H), 2.89 - 2.73 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.14 - 2.01 (m, 2H), 1 .71 - 1 .54 (m, 2H). Method 1 : RT = 0.94 min.; M/Z (M+H) = 372.1 .

Example 36: 2,5-dimethyl-6-(4-((2-methylbenzyl)oxy)piperidin-1 -yl)-H ,2,41triazolon ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.36 - 7.27 (m, 1 H), 7.25 - 7.12 (m, 3H), 4.61 (s, 2H), 3.60 - 3.41 (m, 3H), 2.92 - 2.72 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.38 (s, 3H), 2.19 - 2.05 (m, 2H), 1.69 - 1.52 (m, 2H). Method 1 : RT = 0.99 min.; M/Z (M+H) = 368.2.

Example 37: 6-(4-(3,5-difluorophenethyl)piperidin-1-yl)-2,5-dimethyl-ri ,2,41triazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δδ 7.05 - 6.96 (m, 3H), 3.30 -3.29 (m, 2H), 2.68 - 2.55 (m, 4H), 2.34 (s, 3H), 2.29 (s, 3H), 1.73- 1.71 (m, 2H), 1.62 - 1.52 (m, 2H), 1.32 - 1.19 (m, 3H). Method 1 : RT = 1.15 min.; M/Z (M +H) = 388.2.

Example 38: 6-(4-((2-fluorobenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-ri,2, 4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.48 (s, 1H), 7.38-7.25 (m, 1H), 7.18 (td, J =7.5, 1.0 Hz, 1H), 7.13 -6.97 (m, 1H), 4.66 (s, 2H), 3.49 (d, J= 1.6 Hz, 3H), 2.82 (s, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.09, (s, 2H), 1.63 (s, 2H). Method 1 : RT = 0.94 min.; M/Z (M+H) = 372.2.

Example 39: 6-(4-((4-methoxybenzyl)oxy)piperidin-1-yl)-2,5-dimethyl-ri,2 ,4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.20-7.15 (m, 2H), 6.82 (d, J=2.1 Hz, 1H), 6.81 (d, J = 2.2 Hz, 1H), 5.15 (s, 2H), 3.67 (s, 3H), 3.63-3.31 (m, 2H), 2.68 (s, 2H), 2.40 (s, 3H), 2.34 (s, 3H), 1.82 (dd, J= 12.2, 4.0 Hz, 2H), 1.52 (d, J= 11.9 Hz, 2H). Method 1: RT= 0.80 min.; M/Z (M +H) = 384.20. Example 40: 6-(4-(benzyloxy)piperidin-1 -yl)-2,5-dimethyl-ri ,2,41triazolori ,5-alpyrimidin- 7(4H¾-one

1H NMR (400 MHz, DMSO-cfe) δ 12.84 (s, 1H), 7.36 (d, J= 4.8 Hz, 4H), 7.28 (ddd, J= 8.6,

5.0, 3.7 Hz, 1H), 4.54 (s, 2H), 3.57 (d, J= 1.0 Hz, 1H), 3.34 (s, 3H), 2.71 (d, J= 10.4 Hz, 1H), 2.36 (s, 3H), 2.32 (bro„ 2H), 1.94 (d, J = 60.2 Hz, 2H), 1.48 (s, 1 H). Method 1 : RT = 0.92 min.; M/Z (M+H) = 354.2.

Example 41: 6-(4-((3-fluorobenzvnoxy¾piperidin-1-vn-2.5-dimethyl-ri.2.4 ltriazolon.5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) 7.36 (td, J= 7.9, 5.9 Hz, 1H), 7.16 (dd, J=21.8, 8.5 Hz, 2H), 7.00 (td, J= 8.5, 2.3 Hz, 1H), 4.61 (s, 2H), 3.58 - 3.38 (m, 3H), 2.94 - 2.67 (m, 2H), 2.49 (s, 3H), 2.42 (s,3H), 2.09 (s, 2H), 1.63 (d, J = 8.2 Hz, 2H). Method 1 : RT = 0.96 min.; M/Z (M+H) = 372.2.

Example 42: 2,5-dimethyl-6-(4-(3-phenylpropyl)piperidin-1-yl)-ri,2,4ltri azolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-cfe) δ 7.31 -7.24 (m, 2H), 7.23-7.14 (m, 3H), 3.29 (d, J= 13.4 Hz, 2H), 2.71 - 2.61 (m, 2H), 2.58 (t, J = 7.6 Hz, 2H), 2.33 (s, 3H), 2.32 (s, 3H), 1.62 (ddd, J = 15.3, 11.4, 8.9 Hz, 4H), 1.38 - 1.03 (m, 5H). Method 1 : RT = 1.18 min.; M/Z (M+H) = 366.2.

Example 43: 6-(4-(2-fluorophenethyl)piperidin-1-yl)-2,5-dimethyl-ri,2,4l triazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) ¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 7.29 - 7.15 (m, 2H), 7.09 (td, J= 7.4, 1.3 Hz, 1H), 7.02 (ddd, J= 10.4, 8.1, 1.3 Hz, 1H), 3.43 (s, 2H), 2.87-2.63 (m, 4H), 2.47 (s, 3H), 2.42 (s, 3H), 1.81 (d, J = 9.6 Hz, 2H), 1.59 (s, 2H), 1.36 (s, 3H). Method 1 : RT= 1.12 min.; M/Z(M+H) = 370.20.

Example 44: 6-(4-(3-fluorophenethyl)piperidin-1-yl)-2,5-dimethyl-ri,2,4l triazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-cfe) δ 7.32 (td, J= 7.5, 6.0 Hz, 1H), 7.09-7.04 (m, 2H), 7.03- 6.96 (m, 1H), 3.27 (d, J= 10.3 Hz, 2H), 2.73 -2.61 (m, 4H), 2.35 (s, 3H), 2.32 (s, 3H), 1.82- 1.66 (m, 2H), 1.55 (q, J = 7.8, 7.2 Hz, 2H), 1.25 (m, 3H). RT= 1.609 min.; M/Z (M+H) = 370.15.

Example 45: 6-(4-(benzyloxy)-3,3-dimethylpiperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 4.3 Hz, 4H), 7.34 - 7.23 (m, 1H), 4.64 (d, J = 12.1 Hz, 1H), 4.52 -4.30 (m, 1 H), 3.70 - 3.39 (m, 1 H), 3.28 - 3.00 (m, 2H), 2.74 (d, J = 9.5 Hz, 1 H), 2.35 (d, J = 22.3 Hz, 6H), 2.27 (s, 1 H), 1.90 (d, J = 10.3 Hz, 1 H), 1.09 (s, 3H), 0.88 (s, 3H) Method 1 : RT = 1.09 min.; M/Z (M+H) = 382.2.

Example 46: 2,5-dimethyl-6-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl )-

H ,2,41triazolori ,5-alpyrimidin-7(4H)-one

(46A)

Methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenyl)pipehdin-1 -vObutanoate (46A)

A mixture of methyl 2-chloro-3-oxobutanoate (134 mg, 0.887 mmol), 4-(4- (trifluoromethoxy)phenyl)piperidine (250 mg, 0.887 mmol) and potassium carbonate (123 mg, 0.887 mmol) in acetonitrile (2 mL) was stirred at rt for 3 days. Then reaction mixture was diluted with 20 mL of water and extracted with EtOAc (50 mL). Organic layer was dried over Na2S04 and concentrated to give a crude product that was used without purification.

2,5-dimethyl-6-(4-(4-(trifluoromethoxy)phenyl)piperidin-1 -yl)-[1 ,2,4ltriazolo[1 ,5-alpyrimidin- 7(4H -one (2)

A mixture of methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenyl)piperidin-1 -yl)butanoate (46A) (300 mg, 0.835 mmol) and 3-methyl-1 H-1 ,2,4-triazol-5-am ine (123 mg, 1 .252 mmol) in acetic acid (10 mL) was heated at 130 °C for 2 h . Then reaction mixture was diluted with water (20 mL), adjusted to pH=6 by addition of saturated Na2C03 solution and extracted with EtOAc twice. Organic layers were combined, dried over Na ₂ S0 ₄ and concentrated to give a crude product, which was purified by ISCO with 0-1 00% EtOAc:EtOH (3:1 ) in hexanes to give two peaks of same mass. Peak two was the desired product based on NMR. ¹ H NMR (400 MHz, DMSO-d6) δ 7.48 - 7.40 (m , 2H), 7.30 (dt, J = 7.8, 1 .1 Hz, 2H) , 3.49 (t, J = 11 .4 Hz, 2H), 2.87 - 2.74 (m , 2H), 2.74 - 2.60 (m , 1 H), 2.41 (s, 3H), 2.34 (s, 3H), 1 .87 - 1 .63 (m , 4H). Method 1 : RT = 1 .12 min. ; M/Z (M+H) = 408.1 .

Examples 47-53 were synthesized following the general procedure in Example 46, using the appropriate amine analog.

Example 47: 6-(4-(4-chlorophenyl)piperazin-1 -yl)-2,5-dimethyl-ri,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.29 - 7.18 (m , 2H), 7.02 - 6.92 (m , 2H), 3.81 - 3.40 (m , 4H), 3.02 - 2.57 (m , 4H), 2.40 (s, 3H), 2.34 (s, 3H). Method 1 : RT = 0.92 min. ; M/Z (M+H) = 359.1 .

Example 48: 6-(4-(4-fluorophenyl)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori,5- alpyrimidin-7(4H¾-one

¹ H NMR (400 MHz, DMSO-d6) δ 7.39 - 7.30 (m, 2H), 7.17 - 7.07 (m, 2H), 3.53 - 3.43 (m, 2H), 2.86 -2.73 (m, 2H), 2.66 - 2.57 (m, 1 H), 2.40 (s, 3H), 2.33 (s, 3H), 1 .84 - 1 .63 (m, 4H). Method 1 : RT = 1 .00 min.; M/Z (M+H) = 342.2.

Example 49: 6-(4-(4-chlorophenyl)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 1 .07 min. ; M/Z (M+H) = 358.1 .

Example 50: 6-(4-(4-methoxyphenyl)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

M/Z (M+H) = 354.2.

Example 51 : 6-(4-(4-fluorophenoxy)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 0.95 min. ; M/Z (M+H) = 358.1 .

Example 52: 6-(4-(4-chlorophenyl)piperazin-1-yl)-2,5-dimethyl-7-oxo-4,7- dihvdropyrazolori ,5-alpyrimidine-3-carbonitrile

¹ H NMR (400 MHz, DMSO-d6) δ 7.29 - 7.20 (m, 2H), 7.07 - 6.95 (m, 2H), 3.56 (s, 4H), 3.06 - 2.55 (m, 4H), 2.42 (s, 3H), 2.37 (s, 3H). Method 1 : RT = 1 .02 min.; M/Z (M+H) = 383.1 .

Example 53: 6-(4-(4-chlorophenyl)piperazin-1-yl)-2,3,5-trimethylpyrazolo ri ,5-alpyrimidin- 7(4H)-one

¹ H NMR (400 MHz, DMSO-d6) δ 11 .56 (s, 1 H), 7.29 - 7.20 (m, 2H), 7.03 - 6.94 (m, 2H), 3.63 (s, 4H), 2.86 - 2.62 (m, 4H), 2.40 (s, 3H), 2.20 (s, 3H), 2.04 (s, 3H). M/Z (M+H) = 372.1 .

Example 54: 2,5-dimethyl-6-(4-(2-methyl-4-(trifluoromethoxy)phenyl)piper azin-1-yl)- H ,2,41triazolori ,5-alpyrimidin-7(4H)-one

Benzyl 4-(2,5-dimethyl-7-oxo-4,7-dihydro-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperazine-1 - carboxylate (54A) was made by the method similar to Example 46 ¹ H NMR (400 MHz, Methanol-d4) δ 7.44 - 7.21 (m, 5H), 5.16 (s, 2H), 4.19 - 3.98 (m, 2H), 3.61 - 3.39 (m, 2H), 3.23 ^■ 2.97 (m, 2H), 2.85 - 2.58 (m, 2H), 2.50 (s, 3H), 2.42 (s, 3H). M/Z (M+H) = 383.2.

2,5-dimethyl-6-(piperazin-1 -yl)-[1 ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)-one (54B)

A mixture of benzyl 4-(2,5-dimethyl-7-oxo-4,7-dihydro-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-6- yl)piperazine-1 -carboxylate (250 mg, 0.654 mmol) and Pd-C (69.6 mg, 0.654 mmol) in MeOH (10 mL) was charged with H ₂ balloon and stirred at rt for 1 h. The reaction mixture was filtered through a pad of CELITE® and washed with a mixture of MeOH and dichloromethane, then water. The filtrate was collected and concentrated to give the desired product, which was used without purification. M/Z (M+H) = 249.2.

2,5-dimethyl-6-(4-(2-methyl-4-(trif luoromethoxy)phenyl)piperazin-1 -yl)-H ,2,4ltriazolo[1 ,5- alpyrimidin-7(4H)-one (54)

A mixture of 1 -bromo-2-methyl-4-(trifluoromethoxy)benzene (15 mg, 0.059 mmol), 2,5- dimethyl-6-(piperazin-1 -yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one (14.60 mg, 0.059 mmol), BINAP (10.99 mg, 0.018 mmol), Pd ₂ (dba) ₃ (5.39 mg, 5.88 μη οΙ) and NaOtBu (6.78 mg, 0.071 mmol) in toluene (5 ml_) was degassed for a couple of times and refilled with N ₂ . The mixture was heated at 110 °C for 8 h; and the solvent removed and the residue purified by HPLC with 0- 90% ACN in water. ¹ H NMR (400 MHz, Methanol-d4) δ 7.21 - 7.16 (m, 1 H), 7.13 - 7.03 (m, 2H), 3.85 (s, 2H), 3.13 - 2.90 (m, 4H), 2.80 (s, 2H), 2.54 (s, 3H), 2.44 (s, 3H), 2.39 (s, 3H). Method 1 : RT = 1 .13 min., M/Z (M+H) = 423.1 .

Examples 55-67 described infra were synthesized according to the protocol described for Eaxmple 54, using the appropriate aryl analog.

Example 55: 6-(4-(2-cvclopropyl-4-fluorophenyl)piperazin-1-yl)-2,5-dimet hyl- H ,2,41triazolori ,5-alpyrimidin- -one

¹ H NMR (400 MHz, DMSO-d6) δ 7.09 (dd, J = 8.8, 5.5 Hz, 1 H), 6.91 (td, J = 8.5, 3.0 Hz, 1 H), 6.58 (dd, J = 10.6, 3.0 Hz, 1 H), 3.76 - 3.59 (m, 2H), 3.15 - 3.05 (m, 2H), 2.90 - 2.78 (m,

2H), 2.77 - 2.69 (m, 2H), 2.69 - 2.67 (m, 1 H), 2.42 (s, 3H), 2.34 (s, 3H), 1 .11 - 0.98 (m, 2H), 0.79 - 0.68 (m, 2H). Method 1 : RT = 1 .04 min.; M/Z (M+H) = 383.0.

Example 56: 6-(4-(4-chloro-2-methylphenyl)piperazin-1-yl)-2,5-dimethyl-r i ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 1 .07 min. ; M/Z = 373 Example 57: 6-(4-(4-fluorophenyl)piperazin-1-yl)-2,5-dimethyl-H ,2,41triazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 0.74 min. ; M/Z (M+H) = 343.2.

Example 58: 2,5-dimethyl-6-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl )- H ,2,41triazolori ,5-alpyrimidin-7 -one

Method 1 : RT = 1 .06 min. ; M/Z (M+H) = 409.1 .

Example 59: 6-(4-(4-f luoro-2-methylphenvhpiperazin-1 -vn-2.5-dimethyl-n .2.41triazolon .5- alpyrimidin-7(4H)-one

Method 1 : RT = 0.96 min. ; M/Z (M+H) = 357.2.

Example 60: 6-(4-(2,4-difluorophenyl)piperazin-1-yl)-2,5-dimethyl-ri,2,4 ltriazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 0.92 min. ; M/Z (M+H) = 361 .1 . Example 61 : 6-(4-(4-chloro-3-fluorophenyl)piperazin-1-yl)-2,5-dimethyl-r i,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

Method 1 : RT = 1 .02 min. ; M/Z (M+H) = 377.1 .

Example 62: 6-(4-(4-methoxyphenyl¾piperazin-1-vn-2.5-dimethyl-ri.2.4ltr iazolon .5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d6) δ 7.27 - 7.20 (m, 1 H), 6.99 (d, J = 8.2 Hz, 1 H), 6.88 (s, 2H), 3.71 (s, 3H), 3.55 (s, 4H), 3.01 - 2.70 (m, 4H), 2.41 (s, 3H), 2.34 (s, 3H). Method 1 : RT = 0.56 min.; M/Z (M+H) = 355.2.

Example 63: 6-(4-(4-(difluoromethoxy)phenyl)piperazin-1-yl)-2,5-dimethyl - H ,2,41triazolori ,5-alpyrimid -7(4H)-one

¹ H NMR (400 MHz, DMSO-d6) δ 7.29 - 6.83 (m, 5H), 3.72 - 3.57 (m, 4H), 2.98 - 2.68 (m, 4H), 2.40 (s, 3H), 2.34 (s, 3H). Method 1 : RT = 0.85 min. ; M/Z (M+H) = 391 .1 .

Example 64: 6-(4-(4-chloro-2-methoxyphenyl)piperazin-1-yl)-2,5-dimethyl- H ,2,41triazolori ,5-alpyrimidin- -one

¹ H NMR (400 MHz, DMSO-d6) δ 7.01 (t, J = 1 .3 Hz, 1 H), 6.94 (d, J = 1 .3 Hz, 2H), 3.84 (s, 3H), 3.78 - 3.72 (m, 4H), 3.35 - 3.22 (m, 2H), 2.83 - 2.60 (m, 2H), 2.40 (s, 3H), 2.34 (s, 3H). Method 1 : RT = 0.85 min.; M/Z (M+H) = 389.1 .

Example 65: 6-(4-(2,4-dichlorophenyl)piperazin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H¾-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.63 (s, 1 H), 7.46 - 7.43 (m, 1 H), 7.39 - 7.27 (m, 1 H), 3.75- 3.56 (m, 2H), 3.29 - 3.23 (m, 2H), 2.90 - 2.61 (m, 4H), 2.47 (s, 3H), 2.40 (s, 3H). Method 1 : RT = 1 .09 min.; M/Z (M+H) = 393.1 .

Example 66: 2,5-dimethyl-6-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl) -ri ,2,4ltriazolori ,5- alpyrimidin-7(4H¾-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δδ 7.58 (d, J = 0.02 Hz, 1 H), 7.16 (d, J = 0.03 Hz, 2H), 3.89 (broad, 2H), 3.29 - 3.23 (m, 2H), 2.95-2.80 (m, 4H), 2.47 (s, 3H), 2.40 (s, 3H). Method 1 : RT = 1 .03 min.; M/Z (M+H) = 393.2.

Example 67: 6-(4-(3-chlorophenyl)piperazin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d ₆ ) δδ 7.18 - 7.14 (m, 1 H), 6.92 - 6.92 (m, 1 H), 6.89 - 6.86 (m, 1 H), 6.74 - 6.71 (m, 1 H), 3.65-3.47 (m, 4H), 2.83 - 2.60 (m, 4H), 2.32 (s, 3H), 2.26 (s, 3H). Method 1 : RT = 0.96 min.; M/Z (M+H) = 359.1 .

Example 68: 2,5-dimethyl-6-(4-(4-(trifluoromethoxy)benzyl)piperazin-1-yl )- H ,2,41triazolori ,5-alpyrimidin-7(4H)-one

A mixture of 1 -(bromomethyl)-4-(trifluoromethoxy)benzene (10 mg, 0.039 mmol), 2,5- dimethyl-6-(piperazin-1 -yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one (54B) (9.74 mg, 0.039 mmol), Na ₂ C0 ₃ (12.47 mg, 0.118 mmol) in DMF (1 mL) was heated at 100 °C for 1 h. The reaction mixture was purified by HPLC with 0-90% acetonitrile in water to give the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1 H), 7.76 - 7.61 (m, 2H), 7.52 (d, J = 8.1 Hz, 2H), 4.42 (d, J = 5.2 Hz, 2H), 3.70 (t, J = 12.7 Hz, 2H), 3.50 - 3.45 (m, 2H), 3.22 - 3.11 (m, 2H), 2.95 - 2.79 (m, 2H), 2.39 (s, 3H), 2.34 (s, 3H). RT=0.64min; M/Z (M+H) = 423.1 .

Example 69: 2,5-dimethyl-6-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl )- Γ1 ,2,41triazolori ,5-alpyrimidin-7(4H)-one

Tert-butyl 4-(4-(trif luoromethoxy)phenyl)piperazine-1 -carboxylate (69A)

A mixture of 1 -bromo-4-(trifluoromethoxy)benzene (4000 mg, 16.60 mmol), tert-butyl piperazine-1 -carboxylate (3400 mg, 18.26 mmol), BINAP (3100 mg, 4.98 mmol), Pd ₂ (dba) ₃ (1520 mg, 1 .660 mmol) and NaOtBu (1914 mg, 19.92 mmol) in toluene (5 mL) was degassed for a couple of times and refilled with N ₂ . Then the mixture was heated at 110 °C for 8 h, followed by aqueous work up and extracted with EtOAc. Organic layer was dried over Na ₂ S0 ₄ and concentrated to give a crude, which was purified by ISCO with 0-50% EtOAc in hexanes to give the desired product. M/Z (M+H) = 347.1 . Methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenyl)piperazin-1 -vDbutanoate (69B)

A mixture of tert-butyl 4-(4-(trifluoromethoxy)phenyl)piperazine-1 -carboxylate (69A) (1 g, 2.89 mmol) in 25 mL of HCI (4N in dioxane) was stirred at rt for 1 h. Then solvent was removed and residue was dissolved in acetonitrile (50 mL) and methyl 2-chloro-3-oxobutanoate (0.522 g, 3.46 mmol), K2CO3 (1 .197 g, 8.66 mmol) were added and stirred at rt for 2 days. The reaction mixture was subject to standard aqueous workup and extracted with EtOAc. Organic layer was dried over Na2S04 and concentrated to give a crude product that was purified by ISCO with 0- 50% EtOAc in hexanes. M/Z (M+H) = 361 .1 .

2,5-dimethyl-6-(4-(4-(trifluoromethoxy)phenyl)piperazin-1 -yl)-[1 ,2,4ltriazolo[1 ,5-alpyrimidin- 7(4H)-one (69)

A mixture of methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenyl)piperazin-1 -yl)butanoate (12B) (450 mg, 1 .249 mmol) and 3-methyl-1 H-1 ,2,4-triazol-5-amine (184 mg, 1 .873 mmol) in Acetic Acid (10 mL) was heated at 130 °C for 2 h. Then reaction mixtre was diluted with water (25 mL) and adjusted to pH=6 by addition of Na ₂ C0 ₃ solution. The resluting mixture was extracted with EtOAc twice. Organic layers were dried over Na2S04 and concentrated to give a crude product that was purified by ISCO with 0-100% EtOAc:EtOH (3:1 ) in hexanes to give two peaks of same mass. Peak two was the desired one based on NMR and LCMS. ¹ H NMR (400 MHz, Methanol- d ₄ ) δ 7.44 - 7.21 (m, 5H), 5.16 (s, 2H), 4.19 - 3.98 (m, 2H), 3.61 - 3.39 (m, 2H), 3.23 - 2.97 (m, 2H), 2.85 - 2.58 (m, 2H), 2.50 (s, 3H), 2.42 (s, 3H). M/Z (M+H) = 409.1 .

Example 70: 2,5 dimethyl-6-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-yl)- -alpyrimidin-7(4H)-one

(70A)

Methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1 -vDbutanoate (70A)

A mixture of 4-(4-(trifluoromethoxy)phenoxy)piperidine (750 mg, 2.87 mmol), methyl 2- chloro-3-oxobutanoate (519 mg, 3.45 mmol), K2CO3 (1190 mg, 8.61 mmol) in acetonitrile (5 mL) was stirred at rt for 8 h. Water (25 mL) was added, and extracted with EtOAc. Organic layers were combined and dried over Na ₂ S0 ₄ , and concentrated to give a crude product that was used without further purification.

2,5-dimethyl-6-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1 -yl)-i1 ,2,4ltriazolo[1 ,5-alpyrimidin- 7(4H)-one (70)

A mixture of methyl 3-oxo-2-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1 -yl)butanoate (70A) (40 mg, 0.107 mmol),Ts-OH (3.04 mg, 0.016 mmol) and 3-methyl-1H-1,2,4-triazol-5-amine (15.68 mg, 0.160 mmol) in toluene (1 mL) was heated at 120 °C for overnight. The reaction mixture was cooled and solvent was removed to give a residue, which was purified by HPLC to give desired product. ¹ H NMR (400 MHz, Methanol-d4) δ 7.24 - 7.15 (m, 2H), 7.12 - 6.95 (m, 2H), 4.39 (s, 1 H), 3.96 - 3.76 (m, 1 H), 3.74 - 3.53 (m, 2H), 2.98 - 2.77 (m, 1 H), 2.51 (s, 3H), 2.43 (s, 3H), 2.24- 2.06 (m, 2H), 2.06- 1.89 (m, 1H), 1.85- 1.60 (m, 1H). Method 1: RT= 1.11 min.; M/Z(M+H) = 424.1.

Examples 71-74 were prepared following the protocol in Example 70, using the appropriate amine reagent.

Example 71 : 6-(4-(3-fluorophenyl)piperidin-1-yl)-2,5-dimethyl-ri,2,4ltri azolori,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.36 - 7.24 (m, 1H), 7.11 (dt, J = 7.6, 1.2 Hz, 1H), 7.03 (dt, J = 10.5, 2.1 Hz, 1H), 6.95 - 6.86 (m, 1H), 3.70 - 3.53 (m, 2H), 2.97 - 2.82 (m, 2H), 2.74 - 2.61 (m, 1 H), 2.53 (s, 3H), 2.43 (s, 3H), 2.02 - 1.71 (m, 4H). Method 1 : RT = 1.00 min.; M/Z (M+H) = 342.1.

Example 72: 6-(4-(4-chlorophenoxy¾piperidin-1-vn-2.5-dimethyl-ri.2.4ltr iazolon.5- alpyrimidin-7(4H¾-one

¹ H NMR (400 MHz, Methanol-d4) δ 7.33 - 7.17 (m, 2H), 7.08 - 6.85 (m, 2H), 4.44 - 4.27 (m, 1H), 3.95 - 3.73 (m, 1H), 3.72 - 3.54 (m, 2H), 2.95 - 2.76 (m, 1H), 2.50 (s, 3H), 2.43 (s, 3H), 2.22 - 2.05 (m, 2H), 2.06 - 1.91 (m, 1 H), 1.83 - 1.61 (m, 1 H). Method 1 : RT = 1.05 min.; M/Z (M+H) = 374.1.

Example 73: 6-(4-(4-fluorobenzyl)piperidin-1-yl)-2,5-dimethyl-ri,2,4ltri azolori,5- alpyrimidin-7(4H)-one ¹ H NMR (400 MHz, DMSO-d6) δ 7.26 - 7.18 (m, 2H), 7.14 - 7.05 (m, 2H), 3.36 - 3.13 (m, 2H), 2.72 - 2.58 (m, 2H), 2.57 - 2.52 (m, 2H), 2.34 (s, 3H), 2.31 (s, 3H), 1.66 - 1.42 (m, 3H), 1.32 -1.08(m,2H). Method 1: RT = 1.05 min.; M/Z (M+H) = 356.1.

Example 74: 2,5-dimethyl-6-(4-((4-methylbenzyl)oxy)piperidin-1 -yl)-H ,2,41triazolori ,5- alpyrimidin-7(4H)-one

¹ H NMR (400 MHz, DMSO-d6) δ 7.24 (d, J = 7.4 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 4.49 (s, 2H), 2.36 (s, 3H), 2.32 (s, 3H), 2.30 (s, 3H). Method 1 : RT = 1.00 min.; M/Z (M+H) = 368.2.

Example 75: 6-(4-(benzyloxy)-3,3-difluoropiperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori,5-

tert-butyl 3,3-difluoro-4-hvdroxypiperidine-1-carboxylate (75A)

To a suspension of 3,3-difluoropiperidin-4-ol (1g; 5.76mmol) in THF (5ml_) was added triethylamine (0.86 mL; 6.34 mmol) followed by Boc anhydride (1.32g; 6.05mmol), and the mixture was stirred at RT for 16h. The reaction mixture was then diluted with water (100 mL) and extraxted with EtOAc (3x 50 mL), the organic layer was washed with 1N HCI (2 x 30 mL), brine, dried over anhydrous Na2S04, and concentrated to provide the desired product as a white solid . ¹ H NMR (400 MHz, DMSO-cfe) δ 5.73 (d, J= 5.4 Hz, 1H), 3.91 - 3.63 (m, 2H), 3.61 -3.41 (m, 2H), 3.29 (d, J=4.8Hz, 1H), 1.75 (ddt, J= 15.3, 7.7, 3.8 Hz, 1H), 1.56 (dtt, J= 11.0, 7.0, 3.1 Hz, 1 H), 1 .40 (s, 9H). M/Z (M+H) = 182.1 .

tert-butyl 4-(benzyloxy)-3,3-difluoropiperidine-1 -carboxylate (75B)

To as solution of tert-butyl 3,3-difluoro-4-hydroxypiperidine-1 -carboxylate (1 g; 4.22 mmol) in THF (10 mL) and cooled in an ice bath was added sodium hydride (0.34 g; 8.43 mmo) and the mixture was stirred for 1 h. Benzyl bromide (0.61 mL; 5.06 mmol) was added dropwise and the mixture was stirred at RT for 48h. The reaction mixture was diluted with water (200mL) and extracted with EtOAC (3x100 mL). The organic layer was washed with water (2x100mL), brine, dried over anhydrous Na ₂ S0 ₄ , and concentrated to provide the desired product as an amber thick oil. Ή NMR (400 MHz, DMSO-cfe) δ 7.45 - 7.25 (m, 5H), 4.74 - 4.57 (m, 2H), 3.97 - 3.83 (m, 1 H), 3.83 - 3.69 (m, 1 H), 3.66 - 3.52 (m, 1 H), 3.54 - 3.42 (m, 1 H), 1 .93 - 1 .80 (m, 1 H), 1 .78 - 1 .61 (m, 1 H), 1 .40 (s, 9H). M/Z (M+H+Na) = 350.1.

4-(benzyloxy)-3,3-difluoropiperidine trifluoro acetic salt (75C)

The above oil (0.745g, 4.22 mmol) was diluted in dichloromethane (10 mL) and treated with trifluoroacetic acid (5mL; 65 mmol), and the mixture was stirred at RT for 1 h. The reaction mixture was concentrated and the residue tirurated with dichloromethane to provide the title compound as a white semi-solid. ¹ H NMR (400 MHz, DMSO-cfe) δ 9.21 (s, 2H), 7.39 - 7.15 (m, 5H), 4.72 - 4.49 (m, 2H), 4.01 - 3.85 (m, 1 H), 3.63 - 3.42 (m, 2H), 3.14 - 2.91 (m, 2H), 2.07 - 1 .94 (m, 1 H), 1 .94 - 1 .78 (m, 1 H). M/Z (M+H) = 228.1 .

Methyl 2-(4-(benzyloxy)piperidin-1 -yl)-3-oxobutanoate (75D)

The compound was prepared using 4-(benzyloxy)-3,3-difluoropiperidine trifluoro acetic salt (0.25g, 0.733 mmol) following the same procedure described for compound 1 A. M/Z (M+H) = 342.0.

6-(4-(benzyloxy)-3,3-difluoropiperidin-1 -yl)-2,5-dimethyl-[1 ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)-one (75)

The compound was prepared from methyl 2-(4-(benzyloxy)piperidin-1 -yl)-3-oxobutanoate

(31 mg; 0.045mmol) using the same procedure as described for Example 1. ¹ H NMR (400 MHz, DMSO-cfe) δ 7.50 - 7.10 (m, 6H), 4.70 (s, 2H), 3.84 (s, 2H), 3.53 (d, J = 17.8 Hz, 1 H), 3.10 (q, J = 7.3 Hz, 2H), 2.35 (s, 3H), 2.33 (s, 3H), 2.04 (d, J = 10.6 Hz, 1 H), 1 .91 - 1 .57 (m, 1 H). Method 1 : RT = 0.96 min.; M/Z (M+H) = 390.1 .

Example 76: 6-(4-(1-(3-fluorophenyl)ethoxy)piperidin-1-yl)-2,5-dimethyl- ri ,2,4ltriazolori ,5- alpyrimidin-7(4H)-one

3-methyl-1 H-1 ,2,4-triazol-5-amine

Benzyl 4-(1 -(3-fluorophenyl)ethoxy)piperidine-1 -carboxylate (76A)

To a mixture of 1 -(3-fluorophenyl)ethanol (3000 mg, 21 .40 mmol) in THF (75 mL) was added triethylamine (3.28 mL, 23.55 mmol) and followed by addition of chlorotrimethylsilane (2.84 mL, 22.48 mmol) slowly at 0 °C. The resulting mixture was stirred at the same temperature for 30min, subsequently diluted with 100 mL of hexanes and filtered. The filtrate was collected and concentrated to give an oil, which was dissolved in dichloromethane (100 mL). The solution was cooled down to -78 °C and followed by addition of benzyl 4-oxopiperidine-1 -carboxylate (4993 mg, 21 .40 mmol), triethylsilane (3.76 mL, 23.55 mmol) and trimethylsilyl

trifluoromethanesulfonate (1.937 mL, 10.70 mmol) and stirred at -78 °C for 30 min. The reaction mixture was warmed to rt and stirred for 30 min,, then 50 mL of water was added and extracted with dicholoromethane. The organic layers were combined and dried over Na2S04,

concentrated to give a crude product that was purified by ISCO with 0-40 % EtOAc in hexanes to yield the desired product. ¹ H NMR (400 MHz, Methanol-d4) δ 7.32 - 7.17 (m, 6H), 7.07 (d, J = 7.7 Hz, 1 H), 7.00 (dt, J = 10.0, 2.0 Hz, 1 H), 6.93 - 6.85 (m, 1 H), 5.02 (s, 2H), 4.58 (q, J = 6.4 Hz, 1 H), 3.82 - 3.59 (m, 2H), 3.46 - 3.33 (m, 1 H), 3.18 - 2.98 (m, 2H), 1.86 - 1 .74 (m, 1 H), 1 .66 - 1 .54 (m, 1 H), 1 .51 - 1 .34 (m, 2H), 1 .31 - 1 .27 (m, 3H). M/Z (M+H) = 358.1.

4-(1 -(3-fluorophenyl)ethoxy)piperidine (76B)

To a mixture of benzyl 4-(1 -(3-fluorophenyl)ethoxy)piperidine-1 -carboxylate (76A) (3940 mg, 11 .02 mmol) in MeOH (25 mL) was added Pd-C (1173 mg, 11.02 mmol) and charged with H ₂ balloon. The mixture was stirred at rt for overnight. The solid was filtered off through a pad of CELITE®. The filtrate was collected and concentrated to give an oil. M/Z (M+H) = 224.2.

6-(4-(1 -(3-fluorophenyl)ethoxy)piperidin-1 -yl)-2,5-dimethyl-[1 ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)- one (76)

Example 76 was made by the method similar to Example 70. ¹ H NMR (400 MHz, Methanol- d4) δ 7.36 (td, J = 7.9, 5.9 Hz, 1 H), 7.18 (d, J = 7.7 Hz, 1 H), 7.11 (d, J = 9.8 Hz, 1 H), 7.05 - 6.92 (m, 1 H), 4.77 - 4.58 (m, 1 H), 3.42 - 3.32 (m, 2H), 2.85 - 2.63 (m, 2H), 2.47 (s, 3H), 2.41 (s, 3H), 2.20 - 2.05 (m, 1 H), 1 .85 - 1 .67 (m, 2H), 1 .68 - 1 .51 (m, 2H), 1 .41 (d, J = 6.7 Hz, 3H). Method 1 : RT = 1 .00 min.; M/Z (M+H) = 386.1 .

Example 77: 6-(4-(3-fluorophenethoxy)piperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5- alPyrimidin-7(4H¾-one

tert-butyl 4-(3-fluorophenethoxy)piperidine-1 -carboxylate (77A)

Potassium (1 -Boc-4-piperidinyloxy)methyl trifluoroborate (656 mg, 2.041 mmol), potassium carbonate (434 mg, 3.14 mmol), PEPPSI™ - iPr catalyst (53.4 mg, 0.079 mmol), toluene (8 mL), Water (0.421 mL) and 1 -(chloromethyl)-3-fluorobenzene (0.190 mL, 1 .570 mmol) were combined to give a suspension. Nitrogen was bubbled through the reaction mixture for 2 min. The reaction vial was sealed and heated to 120 for 23 hrs. The reaction was filtered through CELITE® and the filtrate was adsorbed onto CELITE® and purified by silica gel

chromatography eluting with 0-40% EtOAc in hexanes to give a clear oil. ¹ H NMR (400 MHz, DMSO-d6) δ 7.32 (td, J = 8.0, 6.4 Hz, 1 H), 7.13 - 7.07 (m, 2H), 7.06 - 6.98 (m, 1 H), 3.63 (t, J = 6.8 Hz, 2H), 3.59 - 3.50 (m, 2H), 3.50 - 3.41 (m, 1 H), 3.08 - 2.95 (m, 2H), 2.82 (t, J = 6.8 Hz, 2H), 1 .80 - 1 .67 (m, 2H), 1 .38 (s, 9H), 1 .35 - 1 .23 (m, 2H).

4-(3-fluorophenethoxy)piperidine (77B)

TFA (0.5 mL) was added to a solution of tert-butyl 4-(3-fluorophenethoxy)piperidine-1 - carboxylate (365 mg, 1 .129 mmol) in dichloromethane (2 mL) and the reaction was stirred for 2 hr. The reaction was concentrated and the crude mixutre was partitioned between 2M aq. Na ₂ C0 ₃ and EtOAc. The aqueous layer was extracted twice with EtOAc, and the combined organic layers were dried over Na2S04, filtered and concentrated to give a clear oil. M/Z (M+H) = 224.2.

6-(4-(3-fluorophenethoxy)piperidin-1 -yl)-2,5-dimethyl-[1 ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)-one iZZl

Example 77 was made in a similar manner as Example 1 using in 4-(3- fluorophenethoxy)piperidine in place of 4-phenethylpiperidine. ¹ H NMR (400 MHz, DMSO-d6) δ 7.39 - 7.28 (m, 1 H), 7.12 (d, J = 9.4 Hz, 2H), 7.07 - 6.97 (m, 1 H), 3.66 (t, J = 6.8 Hz, 2H), 3.32 - 3.30 (m, 1 H), 2.84 (t, J = 6.5 Hz, 2H), 2.78 - 2.60 (m, 2H), 2.35 (s, 3H), 2.32 (s, 3H), 2.21 - 2.12 (m, 1 H), 2.12 - 2.03 (m, 1 H), 2.02 - 1.86 (m, 2H), 1 .48 - 1 .26 (m, 2H). NH proton is not present. Method 1 : RT = 0.99 min.; M/Z (M+H) = 386.2.

Example 78: 2.5-dimethyl-6-(4-((phenyl-d5¾methoxy-d2¾piperidin-1-vn-n .2.41triazoloH .5- alpyrimidin-7(4H)-one

e

Tert-butyl 4-((phenyl-ds)methoxy-d?)pipehdine-1 -carboxylate (78A)

To a suspension of NaH (1 .011 g, 42.1 mmol) in THF (15 mL) was slowly added tert-butyl 4- hydroxypiperidine-1 -carboxylate (5.65 g, 28.1 mmol) and stirred at rt for 20 min. Then 1 - (bromomethyl)-3-fluorobenzene (5 g, 28.1 mmol) was added and heated at 80 °C for 1 h. The reaction was complete as shown by LCMS, and water (50 mL) was added and extracted with EtOAc. Organic layers were combined and dried over Na ₂ S0 ₄ , concentrated to give a crude product that was purified by ISCO with 0-30% EtOAc in hexanes to give the desired product. ¹ H NMR (400 MHz, Methanol-d ₄ ) δ 3.80 - 3.69 (m, 2H), 3.68 - 3.56 (m, 1 H), 3.22 - 3.03 (m, 2H), 1 .94 - 1 .81 (m, 2H), 1 .58 - 1 .47 (m, 2H), 1 .46 (s, 9H). M/Z (M+Na) = 321 .0.

4-((phenyl-ds)methoxy-d?)pipehdine (78B)

Tert-butyl 4-((phenyl-d5)methoxy-d2)piperidine-1 -carboxylate (78A) (3240 mg, 10.86 mmol) was added into 20 mL of 4N HCI solution in dioxane and stirred at rt for 1 h. The solvent was removed to give the desired product, which was used without further purification. M/Z = 199.1 (M+1 ).

Example 78 was made by the method similar to Example 1 . ¹ H NMR (400 MHz, Methanol- d ₄ ) δ 3.59 - 3.38 (m, 3H), 2.92 - 2.71 (m, 2H), 2.48 (s, 3H), 2.43 - 2.39 (m, 3H), 2.18 - 2.00 (m, 2H), 1 .70 - 1 .49 (m, 2H). Method 1 : RT = 0.92 min.; M/Z (M+H) = 361 .1 . Example 79: 6-(4-((3-fluorobenzyl)oxy)-3,3-dimethylpiperidin-1-yl)-2,5-d imethyl-

Tert-butyl 4-((2-fluorobenzyl)oxy)-3,3-dimethylpiperidine-1 -carboxylate (79A) was made by method similar to compound 78A. M/Z (M+Na) = 360.2.

Example 79 was made by the method similar to Example 78. ¹ H NMR (400 MHz, DMSO- d6) 5 12.84 (s, 1 H), 7.41 (q, J = 7.8 Hz, 1 H), 7.26 - 7.00 (m, 3H), 4.66 (d, J = 12.6 Hz, 1 H), 4.46 (d, J = 12.7 Hz, 1 H), 3.33 - 3.29 (m, 1 H), 3.19 (d, J = 11.5 Hz, 1 H), 3.11 (dd, J = 11 .4, 4.3 Hz, 1 H), 2.82 - 2.70 (m, 1 H), 2.38 (s, 3H), 2.32 (s, 3H), 2.31 - 2.24 (m, 1 H), 1 .94 - 1 .85 (m, 1 H), 1 .66 - 1 .50 (m, 1 H), 1 .10 (s, 3H), 0.89 (s, 3H). M/Z (M+H) = 400.2.

Example 80: 6-(4-(benzyloxy)-3,5-dimethylpiperidin-1-yl)-2,5-dimethyl-ri ,2,4ltriazolori ,5-

(80A) (80B) < ^80C )

Ethyl 2-(4-(benzyloxy)-3,5-dimethylpiperidin-1 -yl)-3-oxobutanoate (80A)

To a stirred solution of tert-butyl 3,5-dimethyl-4-oxopiperidine-1 -carboxylate (500 mg, 2.2 mmol) in MeOH at 0°C in a 40ml_ vial was added sodium borohydride (166 mg, 4.4 mmol).

Stirred at 25°C for 1 hr. After the reaction was complete as shown by LCMS, the reaction was quenched with a solution of aqueous saturated NaHC03. Methanol was removed in vacuo; EtOAc was added and washed with brine, dried over MgSC , filtered and concentrated in vacuo. M/Z (M+H) = 227.3.

tert-butyl 4-(benzyloxy)-3,5-dimethylpiperidine-1 -carboxylate (80B)

To a stirred solution of ethyl 2-(4-(benzyloxy)-3,5-dimethylpiperidin-1 -yl)-3-oxobutanoate (20 mg, 0.09 mmol) in THF at 25°C in a 40mL vial was added sodium hydride (4 mg, 0.174 mmol). Stirred for 0.5hr. Benzyl bromide (15 mg, 0.09 mmol) was added dropwise and the reaction stirred at 75 °C for 12hrs. When the reaction was complete as shown by LCMS, the solid was filtered under vacuum, washed with EtOAc, and the solvent was evaporated. ¹ H NMR (400 MHz, Chloroform-d) δ 7.45 - 7.30 (m, 8H), 4.68 - 4.57 (m, 2H), 4.53 (s, 1 H), 3.76 (s, 1 H), 2.74 (t, J = 10.0 Hz, 1 H), 1 .75 (dtt, J = 9.0, 4.8, 2.2 Hz, 2H), 1 .49 - 1 .45 (m, 9H), 1 .31 - 1 .23 (m, 1 H), 1 .05 (d, J = 6.5 Hz, 4H), 0.99 (d, J = 6.9 Hz, 3H). M/Z (M+H) = 319.5.

Ethyl 2-(4-(benzyloxy)-3,5-dimethylpiperidin-1 -yl)-3-oxobutanoate (80C)

To neat tert-butyl 4-(benzyloxy)-3,5-dimethylpiperidine-1 -carboxylate (720 mg, 2.25 mmol) at 25°C in a 40ml_ vial was added excess Trifluoroacetic acid and stirred for 1 hr. TFA was removed in vacuo. Diluted the TFA salt in Acetonitrile and added potassium carbonate (935 mg, 6.76 mmol). While stirring the mixture at 25°C, ethyl 2-chloro-3-oxobutanoate was added and the reaction stirred at 25°C for 12hrs. The reaction was filtered under vacuum and washed with EtOAc, and the solvent was evaporated. The crude mixture was diluted in MeOH and purified by reverse phase silica gel via mass triggered HPLC. Solvent was evaporated. M/Z (M+H) = 348.4.

6-(4-(benzyloxy)-3,5-dimethylpiperidin-1 -yl)-2,5-dimethyl-[1 ,2,4ltriazolo[1 ,5-alpyrimidin-7(4H)- one (80)

Compound 80 was synthesized using a protocol similar to Example 1. ¹ H NMR (400 MHz, Methanol-d4) δ 7.43 - 7.33 (m, 4H), 7.30 (dd, J = 8.2, 6.0 Hz, 1 H), 4.64 (s, 2H), 3.38 - 3.33 (m, 1 H), 3.31 (s, 3H), 3.26 (d, J = 11 .4 Hz, 1 H), 2.78 (t, J = 10.0 Hz, 1 H), 2.40 (s, 3H), 2.37 (s, 2H), 1 .98 - 1 .85 (m, 2H), 1 .01 (d, J = 6.5 Hz, 6H). RT = 1 .05 min. ; M/Z (M+H) = 382.2.

BIOLOGICAL ASSAYS

The assays described herein illustrate and does not limit the scope of the invention.

Abbreviations

Abbreviations as used herein below have the corresponding meaning:

CPRG Chlorophenol Red-p-D-galactopyranoside

ATP adenosine triphosphate

BSA bovine serum albumin

DMSO dimethyl sulfoxide

FBS fetal bovine serum

PBS phosphate buffered saline

RPMI 1640 Roswell Park Memorial Institute (RPMI) 1640 medium Assay for inhibition of parasitemia of Leishmania donovani in mouse macrophages

The percentage inhibition of 50%, EC ₅ o, is calculated for each compound using a parasite proliferation assay. The assay measures the increase in the parasite number in the assayed plate well using a DNA intercalating dye, SYBR Green I ^® dye (INVITROGEN) to stain

Leishmania cell nuclei.

L.donovani HU3 strain is propagated by infecting BALB/c mice through tail vein injection with 10 ⁷ Leishmania parasites. Infected mice are allowed to develop infection during 9-11 weeks post-infection. During this time, the parasites accumulate in the infected mouse spleens to large numbers, and the infected mice serve as the source of parasites for the in vitro measurement of compound efficacies. To assay a compound for anti-leishmanial activity, peritoneal macrophages isolated from non-infected BALB/c mice are seeded into 384-well plates at density 2x10 ⁴ macrophages per well in 25 mL of medium (RPMI1640, 10% fetal serum albumin, 10 mM HEPES, 1 mM sodium pyruvate, 1 % Pen/Strep). Subsequently, the seeded plates are placed into an incubator set to maintain 37 °C temperature and atmosphere with 5% C0 ₂ . The next day, Leishmania parasites are isolated from the spleens of mice infected for 9-11 weeks and 4x10 ⁵ isolated parasites in 10 mL of the above media are added to each plate well. Plates are then returned into incubators and infection is allowed to proceed for 24 hours. After the infection of macrophages is completed, 5 mL of compounds of the invention in the above medium, which also contains 5% DMSO, are added to plate wells containing infected macrophages. At the same time control compounds (miltefosine and amphotericin B) and

DMSO are added to plates to serve as the positive and negative controls, respectively. After the compound addition, the plates are returned into incubator and cells infected with parasites are cultured for 5 days. At the end of cultivation, 40 mL of 8% paraformaldehyde is added to plate wells and incubated for 15 min at room temperature. Following the incubation, the

paraformaldehyde from plate wells is aspirated, and 40 mL of PBS containing 0.2% Triton X-100 is added to wells. After 15 min incubation, the solution is aspirated from wells again, and replaced with SYBR® Green Dye solution in PBS (1 :125,000 dilution). Infected cells are imaged with Evotec Opera high-content microscope, and the number of parasites in well is determined by counting parasite nuclei visualized by staining with SYBR® Green dye.

Assay for growth inhibition of kinetoplastid parasite Trypanosoma cruzi

Compounds of the invention can be assayed to measure their capacity to inhibit proliferation of kinetoplastid parasite Trypanosoma cruzi. The screening procedure is for identifying compounds with inhibitor activity against Trypanosoma cruzi amastigotes cultured in 3T3 fibroblast cells. The assay is done using the mammalian stage (amastigotes) of T. cruzi that replicates in the intracellular space of host cells. The host cells are initially infected with the culture-derived trypomastigotes that rapidly invade and then divide as amastigotes. The protocol uses the Tulahuen strain of T. cruzi that has been engineered to express the E. co// ^' beta- galactosidase gene (Lac-Z) (Antimicr. Agents Chemoth. 40:2592, 1996). This allows for a colorimetric readout by using the substrate CPRG and an absorbance plate reader.

3T3 fibroblast cells are re-suspended in RPMI-1640 medium without phenol red medium supplemented with 10% FBS (heat inactivated), 100 μg/ml penicillin, and 100 μg /ml streptomycin. Forty μΙ_ of suspension (1 ,000 cells) is dispensed into 384-well plates and incubated overnight at 37 °C temperature and in atmosphere containing 5% CO2. The following day, 100 nl_ of compounds of the invention in DMSO are added to plate wells containing 3T3 cells. At the same time control compounds (benznidazole and nifurtimox) and DMSO are added to plates to serve as the positive and negative controls, respectively. After that, 10 μΙ_ of media containing 10,000 T. cruzi trypomastigotes are added to each plate well and plates are placed back into incubators. After 6 day incubation, 10 μΙ_ of reagent solution (0.6 mM CPRG, 0.6% NP-40 in PBS) is added to plates and incubated at room temperature for 2 hours. Absorbance is then measured on SpectraMax Gemini fluorimeter to determine relative number of T. cruzi cells present in each plate well. The percentage inhibition of 50%, EC50, is calculated for each compound.

The inhibitory efficacy of the compounds of the invention against proliferation of T cruzi and L. donovani ln mouse peritoneal macrophages are provided in Table 1 . Typically, the compounds analyzed have an EC50 of < 1 μιη. Selected compounds have EC50 less than 50 nM.

Table 1

Mouse

Buckner Macrophage infection

Example infectious

with L.d. No. T. cruzi

Ethiopian ECso (μΜ) HU3

ECso (μΜ)

2,5-dimethyl-6-((3R,4R)-3-methyl-4-(4-

9 (trifluoromethoxy)phenyl)piperidin-1 -yl)- 0.001732 0.0485

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-(tert-butyl)phenyl)-3-methylpiperazin-1 -yl)-

10 0.01584 0.0373 2,5-dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

Ethyl 4-(4-(2,5-dimethyl-7-oxo-4,7-dihydro-

1 1 [1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperazin-1 - 0.1245 0.1435 yl)benzoate

4-(4-(2,5-dimethyl-7-oxo-4,7-dihydro-

12 [1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperazin-1 - >20 ND yl)benzonitrile

6-(4-(3,4-dichlorophenyl)piperazin-1 -yl)-2,5-dimethyl-

13 0.0474 0.2423

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(3-

14 (trifluoromethyl)phenethyl)piperidin-1 -yl)- 0.1357 ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3,4-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

15 0.02171 0.0716 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3,5-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

16 0.02814 0.0521 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-phenylpiperidin-1 -yl)-

17 0.0414 ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,3,5-trimethyl-6-(4-phenylpiperidin-1 -

18 <0.00254 0.00705 yl)pyrazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2,6-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

19 0.0433 ND dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2,3-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

20 0.1081 0.1 105 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2,5-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

21 0.2593 0.2205 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3-fluorophenoxy)methyl)piperidin-1 -yl)-2,5-

22 0.0959 0.677 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3-fluorobenzyl)oxy)-3-methylpiperidin-1 -yl)-2,5-

23 0.0419 0.0408 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2-chlorophenoxy)methyl)piperidin-1 -yl)-2,5-

24 0.324 1 .394 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-phenethoxypiperidin-1 -yl)-

25 0.0703 0.661

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2,4-difluorobenzyl)oxy)piperidin-1 -yl)-2,5-

26 0.01 1 0.203 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((4-chlorobenzyl)oxy)piperidin-1 -yl)-2,5-

27 0.016 0.077 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one Mouse

Buckner Macrophage infection

Example infectious

with L.d. No. T. cruzi

Ethiopian ECso (μΜ) HU3

ECso (μΜ)

2-(((1 -(2,5-dimethyl-7-oxo-4,7-dihydro-

28 [1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperidin-4- 1 .135 1 .778 yl)oxy)methyl)benzonitrile

6-(4-((3-chlorobenzyl)oxy)piperidin-1 -yl)-2,5-

29 0.027 0.0625 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

3-(((1 -(2,5-dimethyl-7-oxo-4,7-dihydro-

30 [1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperidin-4- 0.317 0.967 yl)oxy)methyl)benzonitrile

6-(4-((2-chlorobenzyl)oxy)piperidin-1 -yl)-2,5-

31 0.0437 ND dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

4-(((1 -(2,5-dimethyl-7-oxo-4,7-dihydro-

32 [1 ,2,4]triazolo[1 ,5-a]pyrimidin-6-yl)piperidin-4- 1 .223 1 .444 yl)oxy)methyl)benzonitrile

2,5-dimethyl-6-(4-((3-methylbenzyl)oxy)piperidin-1 -

33 0.0334 0.1354 yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(3-(4-

34 (trifluoromethoxy)phenoxy)pyrrolidin-1 -yl)- 0.13 1 .56

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((4-fluorobenzyl)oxy)piperidin-1 -yl)-2,5-dimethyl-

35 0.021 0.07

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-((2-methylbenzyl)oxy)piperidin-1 -

36 0.02 0.096 yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(3,5-difluorophenethyl)piperidin-1 -yl)-2,5-

37 0.005 0.018 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((2-fluorobenzyl)oxy)piperidin-1 -yl)-2,5-dimethyl-

38 0.081 0.19

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((4-methoxybenzyl)oxy)piperidin-1 -yl)-2,5-

39 1 .16 9.0 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(benzyloxy)piperidin-1 -yl)-2,5-dimethyl-

40 0.135 0.141

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3-fluorobenzyl)oxy)piperidin-1 -yl)-2,5-dimethyl-

41 0.049 0.139

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(3-phenylpropyl)piperidin-1 -yl)-

42 0.009 0.213

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(2-fluorophenethyl)piperidin-1 -yl)-2,5-dimethyl-

43 0.013 0.067

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(3-fluorophenethyl)piperidin-1 -yl)-2,5-dimethyl-

44 0.013 0.038

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(benzyloxy)-3,3-dimethylpiperidin-1 -yl)-2,5-

45 0.016 0.052 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(4-

46 (trifluoromethoxy)phenyl)piperidin-1 -yl)- 0.002393 0.02797

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one Mouse

Buckner Macrophage infection

Example infectious

with L.d. No. T. cruzi

Ethiopian ECso (μΜ) HU3

ECso (μΜ)

6-(4-(4-chlorophenyl)piperazin-1 -yl)-2,5-dimethyl-

47 0.0371 0.434

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-fluorophenyl)piperidin-1 -yl)-2,5-dimethyl-

48 0.01272 0.0841

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chlorophenyl)piperidin-1 -yl)-2,5-dimethyl-

49 0.01407 0.1 182

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-methoxyphenyl)piperidin-1 -yl)-2,5-dimethyl-

50 0.32 ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-fluorophenoxy)piperidin-1 -yl)-2,5-dimethyl-

51 0.1 1 19 ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chlorophenyl)piperazin-1 -yl)-2,5-dimethyl-7-

52 oxo-4, 7-dihydropyrazolo[1 ,5-a]pyrimidine-3- 0.0877 0.1 169 carbonitrile

6-(4-(4-chlorophenyl)piperazin-1 -yl)-2,3,5-

53 0.001791 0.02319 trimethylpyrazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(2-methyl-4-

54 (trifluoromethoxy)phenyl)piperazin-1 -yl)- 0.01951 0.2584

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(2-cyclopropyl-4-fluorophenyl)piperazin-1 -yl)-

55 0.677 17.14 2,5-dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chloro-2-methylphenyl)piperazin-1 -yl)-2,5-

56 0.0495 0.2799 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-fluorophenyl)piperazin-1 -yl)-2,5-dimethyl-

57 0.1656 2.166

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(3-

58 (trifluoromethoxy)phenyl)piperazin-1 -yl)- 0.0567 1 .201

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-fluoro-2-methylphenyl)piperazin-1 -yl)-2,5-

59 0.1314 1 .581 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(2,4-difluorophenyl)piperazin-1 -yl)-2,5-dimethyl-

60 0.1299 ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chloro-3-fluorophenyl)piperazin-1 -yl)-2,5-

61 0.0518 0.495 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-methoxyphenyl)piperazin-1 -yl)-2,5-dimethyl-

62 1 .325 5.03

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-(difluoromethoxy)phenyl)piperazin-1 -yl)-2,5-

63 0.442 2.84 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chloro-2-methoxyphenyl)piperazin-1 -yl)-2,5-

64 4.5 19.98 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(2,4-dichlorophenyl)piperazin-1 -yl)-2,5-dimethyl-

65 0.203 2.764

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one Mouse

Buckner Macrophage infection

Example infectious

with L.d. No. T. cruzi

Ethiopian ECso (μΜ) HU3

ECso (μΜ)

2,5-dimethyl-6-(4-(4-

66 (trifluoromethyl)phenyl)piperazin-1 -yl)- 0.1335 0.3026

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(3-chlorophenyl)piperazin-1 -yl)-2,5-dimethyl-

67 1 .292 9.34

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(4-

68 (trifluoromethoxy)benzyl)piperazin-1 -yl)- 1 .48 14.83

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-(4-

69 (trifluoromethoxy)phenyl)piperazin-1 -yl)- 0.0208 0.1383

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5 dimethyl-6-(4-(4-

70 (trifluoromethoxy)phenoxy)piperidin-1 -yl)- 0.0341 0.21 19

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(3-fluorophenyl)piperidin-1 -yl)-2,5-dimethyl-

71 0.01571 0.1307

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-chlorophenoxy)piperidin-1 -yl)-2,5-dimethyl-

72 0.0359 0.1779

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(4-fluorobenzyl)piperidin-1 -yl)-2,5-dimethyl-

73 0.1044 1 .081

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-((4-methylbenzyl)oxy)piperidin-1 -

74 0.02816 0.0351 yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(benzyloxy)-3,3-difluoropiperidin-1 -yl)-2,5-

75 0.1357 ND dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(1 -(3-fluorophenyl)ethoxy)piperidin-1 -yl)-2,5-

76 0.1491 0.513 dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(3-fluorophenethoxy)piperidin-1 -yl)-2,5-dimethyl-

77 ND ND

[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

2,5-dimethyl-6-(4-((phenyl-d5)methoxy-d2)piperidin-

78 ND ND

1 -yl)-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-((3-fluorobenzyl)oxy)-3,3-dimethylpiperidin-1 -yl)-

79 0.00504 ND 2,5-dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

6-(4-(benzyloxy)-3,5-dimethylpiperidin-1 -yl)-2,5-

80 ND ND dimethyl-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-7(4H)-one

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.