Pyrazole compounds as modulators of FSHR and uses thereof

The present invention relates to pyrazole compounds, and pharmaceutically acceptable compositions thereof, useful as positive allosteric modulators of follicle stimulating hormone receptor (FSHR).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to pyrazole compounds useful as agonists of follicle stimulating hormone receptor (FSHR). The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders.

BACKGROUND OF THE INVENTION

Gonadotropins serve important functions in a variety of bodily functions including metabolism, temperature regulation and the reproductive process. Gonadotropins act on specific gonadal cell types to initiate ovarian and testicular differentiation and steroidogenesis. The gonadotropin FSH (follicle stimulating hormone) is released from the anterior pituitary under the influence of gonadotropin-releasing hormone and estrogens, and from the placenta during pregnancy. FSH is a heterodimeric glycoprotein hormone that shares structural similarities with luteinizing hormone (LH) and thyroid stimulating hormone (TSH), both of which are also produced in the pituitary gland, and chorionic gonadotropin (CG), which is produced in the placenta. In the female, FSH plays a pivotal role in the stimulation of follicle development and maturation and in addition, it is the major hormone regulating secretion of estrogens, whereas LH induces ovulation. In the male, FSH is responsible for the integrity of the seminiferous tubules and acts on Sertoli cells to support gametogenesis.

The hormones are relatively large (28-38 kDa) and are composed of a common α-subunit non-covalently bound to a distinct β-subunit that confers receptor binding specificity. The cellular receptor for these hormones is expressed on testicular Sertoli cells and ovarian granulosa cells. The FSH receptor is known to be members of the G protein-coupled class of membrane-bound receptors, which when activated stimulate an increase in the activity of adenylyl cyclase. This results in an increase in the level of the intracellular second messenger adenosine 3′,5′-monophosphate (cAMP), which in turn causes increased steroid synthesis and secretion. Hydropathicity plots of the amino acid sequences of these receptors reveal three general domains: a hydrophilic amino-terminal region, considered to be the amino-terminal extracellular domain; seven hydrophobic segments of membrane-spanning length, considered to be the transmembrane domain; and a carboxy-terminal region that contains potential phosphorylation sites (serine, threonine, and tyrosine residues), considered to be the carboxy-terminal intracellular or cytoplasmic domain. The glycoprotein hormone receptor family is distinguished from other G protein-coupled receptors, such as the β-2-adrenergic, rhodopsin, and substance K receptors, by the large size of the hydrophilic amino-terminal domain, which is involved in hormone binding.

Annually in the U.S. there are 2.4 million couples experiencing infertility that are potential candidates for treatment. FSH, either extracted from urine or produced by recombinant DNA technology, is a parenterally-administered protein product used by specialists for ovulation induction and for controlled ovarial hyperstimulation. Whereas ovulation induction is directed at achieving a single follicle to ovulate, controlled ovarial hyperstimulation is directed at harvesting multiple oocytes for use in various in-vitro assisted reproductive technologies, e.g. in-vitro fertilization (IVF). FSH is also used clinically to treat male hypogonadism and male infertility, e.g. some types of failure of spermatogenesis.

FSHR is a highly specific target in the ovarian follicle growth process and is exclusively expressed in the ovary. However, the use of FSH is limited by its high cost, lack of oral dosing, and need of extensive monitoring by specialist physicians. Hence, identification of a non-peptidic small molecule substitute for FSH that could potentially be developed for oral administration is desirable. Low molecular weight FSH mimetics with agonistic properties are disclosed in the international applications WO 2002/09706 and WO 2010/136438 as well as the U.S. Pat. No. 6,653,338. There is still a need for low molecular weight hormone mimetics that selectively activate FSHR.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, and pharmaceutically acceptable compositions thereof, are effective as modulators of FSHR. Such compounds have general formula I:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A, X, Y, Z, R1, R2, R3, R4, R5, R6, n, and p, is as defined and described in embodiments herein.

Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful for treating a variety of diseases, disorders or conditions, associated with abnormal cellular responses triggered by follicle stimulating hormone events. Such diseases, disorders, or conditions include those described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

1. General Description of Compounds of the Invention

In certain embodiments, the present invention provides modulators of follicle stimulating hormone receptor (FSHR). In certain embodiments, the present invention provides positive allosteric modulators of FSHR. In some embodiments, such compounds include those of the formulae described herein, or a pharmaceutically acceptable salt thereof, wherein each variable is as defined and described herein.

2. Compounds and Definitions

The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Exemplary aliphatic groups are linear or branched, substituted or unsubstituted C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “bivalent C1-8(or C1-6) saturated or unsaturated, straight or branched, hydrocarbon chain”, refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

As described herein, certain compounds of the invention contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g.,

refers to at least

refers to at least

Unless otherwise indicated, an “optionally substituted” group has a suitable substituent at each substitutable position of the group, and when more than one position in any given structure is substituted with more than one substituent selected from a specified group, the substituent is either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

In certain embodiments, the terms “optionally substituted”, “optionally substituted alkyl,” “optionally substituted “optionally substituted alkenyl,” “optionally substituted alkynyl”, “optionally substituted carbocyclic,” “optionally substituted aryl”, “optionally substituted heteroaryl,” “optionally substituted heterocyclic,” and any other optionally substituted group as used herein, refer to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with typical substituents including, but not limited to:

Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.

For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this invention. In some embodiments, the group comprises one or more deuterium atoms.

There is furthermore intended that a compound of the formula I includes isotope-labeled forms thereof. An isotope-labeled form of a compound of the formula I is identical to this compound apart from the fact that one or more atoms of the compound have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs naturally. Examples of isotopes which are readily commercially available and which can be incorporated into a compound of the formula I by well-known methods include isotopes of hydrogen, carbon, nitrogen, oxygen, phos-phorus, fluo-rine and chlorine, for example2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F and36Cl, respectively. A compound of the formula I, a prodrug, thereof or a pharmaceutically acceptable salt of either which contains one or more of the above-mentioned isotopes and/or other isotopes of other atoms is intended to be part of the present invention. An isotope-labeled compound of the formula I can be used in a number of beneficial ways. For example, an isotope-labeled compound of the formula I into which, for example, a radioisotope, such as3H or14C, has been incorporated, is suitable for medicament and/or substrate tissue distribution assays. These radioisotopes, i.e. tritium (3H) and carbon-14 (14C), are particularly preferred owing to simple preparation and excellent detectability. Incorporation of heavier isotopes, for example deuterium (2H), into a compound of the formula I has therapeutic advantages owing to the higher metabolic stability of this isotope-labeled compound. Higher metabolic stability translates directly into an increased in vivo half-life or lower dosages, which under most circumstances would represent a preferred embodiment of the present invention. An isotope-labeled compound of the formula I can usually be prepared by carrying out the procedures disclosed in the synthesis schemes and the related description, in the example part and in the preparation part in the present text, replacing a non-isotope-labeled reactant by a readily available isotope-labeled reactant.

Deuterium (2H) can also be incorporated into a compound of the formula I for the purpose in order to manipulate the oxidative metabolism of the compound by way of the primary kinetic isotope effect. The primary kinetic isotope effect is a change of the rate for a chemical reaction that results from exchange of isotopic nuclei, which in turn is caused by the change in ground state energies necessary for covalent bond formation after this isotopic exchange. Exchange of a heavier isotope usually results in a lowering of the ground state energy for a chemical bond and thus causes a reduction in the rate in rate-limiting bond breakage. If the bond breakage occurs in or in the vicinity of a saddle-point region along the coordinate of a multi-product reaction, the product distribution ratios can be altered substantially. For explanation: if deuterium is bonded to a carbon atom at a non-exchangeable position, rate differences of kM/kD=2-7 are typical. If this rate difference is successfully applied to a com-pound of the formula I that is susceptible to oxidation, the profile of this compound in vivo can be drastically modified and result in improved pharmacokinetic properties.

When discovering and developing therapeutic agents, the person skilled in the art is able to optimize pharmacokinetic parameters while retaining desirable in vitro properties. It is reasonable to assume that many compounds with poor pharmacokinetic profiles are susceptible to oxidative metabolism. In vitro liver microsomal assays currently available provide valuable information on the course of oxidative metabolism of this type, which in turn permits the rational design of deuterated compounds of the formula I with improved stability through resistance to such oxidative metabolism. Significant improvements in the pharmacokinetic profiles of compounds of the formula I are thereby obtained, and can be expressed quantitatively in terms of increases in the in vivo half-life (t/2), concen-tra-tion at maximum therapeutic effect (Cmax), area under the dose response curve (AUC), and F; and in terms of reduced clearance, dose and materials costs.

The following is intended to illustrate the above: a compound of the formula I which has multiple potential sites of attack for oxidative metabolism, for example benzylic hydrogen atoms and hydrogen atoms bonded to a nitrogen atom, is prepared as a series of analogues in which various combinations of hydrogen atoms are replaced by deuterium atoms, so that some, most or all of these hydrogen atoms have been replaced by deuterium atoms. Half-life determinations enable favorable and accurate determination of the extent of the extent to which the improvement in resistance to oxidative metabolism has improved. In this way, it is determined that the half-life of the parent compound can be extended by up to 100% as the result of deuterium-hydrogen exchange of this type.

Deuterium-hydrogen exchange in a compound of the formula I can also be used to achieve a favorable modification of the metabolite spectrum of the starting compound in order to diminish or eliminate undesired toxic metabolites. For example, if a toxic metabolite arises through oxidative carbon-hydrogen (C—H) bond cleavage, it can reasonably be assumed that the deuterated analogue will greatly diminish or eliminate production of the unwanted metabolite, even if the particular oxidation is not a rate-determining step. Further information on the state of the art with respect to deuterium-hydrogen exchange may be found, for example in Hanzlik et al., J. Org. Chem. 55, 3992-3997, 1990, Reider et al., J. Org. Chem. 52, 3326-3334, 1987, Foster, Adv. Drug Res. 14, 1-40, 1985, Gillette et al, Biochemistry 33(10) 2927-2937, 1994, and Jarman et al. Carcinogenesis 16(4), 683-688, 1993.

As used herein, the term “modulator” is defined as a compound that binds to and/or inhibits the target with measurable affinity. In certain embodiments, a modulator has an IC50and/or binding constant of less about 50 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as used herein, means a measurable change in FSHR activity between a sample comprising a compound of the present invention, or composition thereof, and FSHR, and an equivalent sample comprising FSHR, in the absence of said compound, or composition thereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound of formula I,

or a pharmaceutically acceptable salt thereof, wherein:X is O, S, SO, SO2, or NR;Y is O, S, or NR;Z is O, S, SO, SO2, or N; wherein when Z is O, S, SO, or SO2, then p is 0;each R is independently hydrogen, C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted; ortwo R groups on the same atom are taken together with the atom to which they are attached to form a C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;Ring A is a fused C3-10aryl, a fused 3-8 membered saturated or partially unsaturated carbocyclic ring, a fused 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a fused 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;R1is —OR, —SR, —CN, —NO2, —SO2R, —SOR, —C(O)R, —CO2R, —C(O)N(R)2, —NRC(O)R, —NRC(O)N(R)2, —NRSO2R, or —N(R)2;R2is —R, halogen, -haloalkyl, —OR, —SR, —CN, —NO2, —SO2R, —SOR, —C(O)R, —CO2R, —C(O)N(R)2, —NRC(O)R, —NRC(O)N(R)2, —NRSO2R, or —N(R)2;R3is hydrogen, C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;each R4is independently —R, halogen, -haloalkyl, —OR, —SR, —CN, —NO2, —SO2R, —SOR, —C(O)R, —CO2R, —C(O)N(R)2, —NRC(O)R, —NRC(O)N(R)2, —NRSO2R, or —N(R)2;R5is C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;R6is hydrogen, C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;or R5and R6, together with the atom to which each is attached, form a 3-8 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 3-8 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted;n is 0, 1, or 2; andp is 0 or 1.

In certain embodiments, X is O. In certain embodiments, X is S. In certain embodiments, X is SO, or SO2. In certain embodiments, X is NR.

In certain embodiments, Y is O. In certain embodiments, Y is S. In certain embodiments, Y is NR.

In certain embodiments, Z is O. In certain embodiments, Z is S. In certain embodiments, Z is SO or SO2. In certain embodiments, Z is N.

In certain embodiments, Ring A is a fused C3-10aryl. In certain embodiments, Ring A is a fused 3-8 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, Ring A is a fused 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, Ring A is a fused 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, Ring A is phenyl.

In certain embodiments, R1is —OR, and R is hydrogen.

In certain embodiments, R1is —OR, and R is C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R1is —OR, and R is C1-6aliphatic. In certain embodiments, R is methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted.

In certain embodiments, R1is —OR, and R is C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R2is C1-6aliphatic. In certain embodiments, R2is C1-6aliphatic wherein the aliphatic group is a C1-6alkyl. In certain embodiments, R2is methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted. In certain embodiments, R2is C1-6aliphatic wherein the aliphatic group is a C1-6alkenyl.

In certain embodiments, R2is C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R2is

In certain embodiments, R2is

In certain embodiments, R2is

In certain embodiments, R3is C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R3is an optionally substituted C1-6aliphatic. In certain embodiments, R3is an optionally substituted C3-10aryl. In certain embodiments, R3is an optionally substituted 3-8 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R3is an optionally substituted 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R3is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R3is

In certain embodiments, each R4is independently hydrogen.

In certain embodiments, each R4is independently C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, each R4is independently an optionally substituted C1-6aliphatic. In certain embodiments, each R4is independently an optionally substituted C3-10 aryl. In certain embodiments, each R4is independently an optionally substituted 3-8 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, each R4is independently an optionally substituted 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, each R4is independently an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R5is C1-6aliphatic, C3-10aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R5is an optionally substituted C1-6aliphatic. In certain embodiments, R5is an optionally substituted C3-10aryl. In certain embodiments, R5is an optionally substituted 3-8 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R5is an optionally substituted 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R5is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R5is C1-6aliphatic. In certain embodiments, R5is methyl, ethyl, propyl, i-propyl, butyl, s-butyl, t-butyl, straight or branched pentyl, or straight or branched hexyl; each of which is optionally substituted

In certain embodiments, R5and R6, together with the atom to which each is attached, form a 3-8 membered heterocylic 1 ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R5is

In certain embodiments, Z is N and the ring formed by Z, R5and R6is

In certain embodiments, R6is C1-6aliphatic, C3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each of which is optionally substituted.

In certain embodiments, R6is an optionally substituted C1-6aliphatic. In certain embodiments, R6is an optionally substituted C3-10aryl. In certain embodiments, R6is an optionally substituted 3-8 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R6is an optionally substituted 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R6is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.

In certain embodiments, p is 0. In certain embodiments, p is 1.

In certain embodiments, each of R1, R2, R3, R4, R5, R6, X, Y, Z, n, and p is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, R5, R6, X, Y, Z, and p is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the present invention provides a compound of formula I-b,

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, R4, R5, R6, Y, Z, n, and p is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the compound is of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, R5, R6, Z, and p is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the invention provides a compound of formula I-d:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, R5, and R6is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the invention provides a compound of formula I-e:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, and R5is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In other embodiments, the invention provides a compound of formula I-f:

or a pharmaceutically acceptable salt thereof, wherein each of R2, R3, R5, and R6is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In other embodiments, the invention provides a compound of formula I-g:

or a pharmaceutically acceptable salt thereof, wherein each of R2, R3, and R5is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the invention provides a compound of formula I-h:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R5, R6, Z, and p is as defined above and described in embodiments, classes and subclasses above and herein, singly or in combination.

In certain embodiments, the invention provides a compound of formula I-f, wherein R2is 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; which is optionally substituted; R3is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and Z is N.

In certain embodiments, R5is an optionally substituted C1-6aliphatic.

In certain embodiments, R5and R6, together with the atom to which each is attached, form a 3-8 membered heterocylic 1 ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which is optionally substituted.

In certain embodiments, R6is an optionally substituted C1-6aliphatic.

In certain embodiments, the invention provides a compound of formula I-h, wherein R1is —OR and R is C1-6aliphatic; R2is 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; which is optionally substituted; and Z is N.

In certain embodiments, R5is an optionally substituted C1-6aliphatic.

In certain embodiments, R5and R6, together with the atom to which each is attached, form a 3-8 membered heterocylic 1 ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which is optionally substituted.

In certain embodiments, R6is an optionally substituted C1-6aliphatic.

In certain embodiments, the invention provides a compound selected from Table 1:

In some embodiments, the present invention provides a compound selected from those depicted above, or a pharmaceutically acceptable salt thereof.

Various structural depictions may show a heteroatom without an attached group, radical, charge, or counterion. Those of ordinary skill in the art are aware that such depictions are meant to indicate that the heteroatom is attached to hydrogen

is understood to be

In certain embodiments, the compounds of the invention were synthesized in accordance with Schemes A-C below. More specific examples of compounds made utilizing Schemes A-C are provided in the Examples below.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a composition comprising a compound of this invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in compositions of this invention is such that is effective to measurably modulate FSHR, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the amount of compound in compositions of this invention is such that is effective to measurably modulate FSHR, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such composition.

The term “patient” or “subject”, as used herein, means an animal, preferably a mammal, and most preferably a human.

A “pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof.

Compositions of the present invention are administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

Pharmaceutically acceptable compositions of this invention are orally administered in any orally acceptable dosage form. Exemplary oral dosage forms are capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents are optionally also added.

Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches are also used.

For topical applications, provided pharmaceutically acceptable compositions are formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Exemplary carriers for topical administration of compounds of this aremineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Pharmaceutically acceptable compositions of this invention are optionally administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.

In certain embodiments, the invention provides a method for allosterically agonising FSHR, or a mutant thereof, in a positive manner in a patient or in a biological sample comprising the step of administering to said patient or contacting said biological sample with a compound according to the invention.

In certain embodiments, the invention is directed to the use of compounds of the invention and/or physiologically acceptable salts thereof, for modulating a FSH receptor, particularly in the presence of FSH. The term “modulation” denotes any change in FSHR-mediated signal transduction, which is based on the action of the specific inventive compounds capable to interact with the FSHR target in such a manner that makes recognition, binding and activating possible. The compounds are characterized by such a high affinity to FSHR, which ensures a reliable binding and preferably a positive allosteric modulation of FSHR. In certain embodiments, the substances are mono-specific in order to guarantee an exclusive and directed recognition with the single FSHR target. In the context of the present invention, the term “recognition”—without being limited thereto—relates to any type of interaction between the specific compounds and the target, particularly covalent or non-covalent binding or association, such as a covalent bond, hydrophobic/hydrophilic interactions, van der Waals forces, ion pairs, hydrogen bonds, ligand-receptor interactions, and the like. Such association may also encompass the presence of other molecules such as peptides, proteins or nucleotide sequences. The present receptor/ligand-interaction is characterized by high affinity, high selectivity and minimal or even lacking cross-reactivity to other target molecules to exclude unhealthy and harmful impacts to the treated subject.

In certain embodiments, the present invention relates to a method for modulating an FSH receptor, and in particular in a positive allosteric manner, wherein a system capable of expressing the FSH receptor is contacted, in the presence of FSH, with at least one compound of formula (I) according to the invention and/or physiologically acceptable salts thereof, under conditions such that said FSH receptor is modulated. In certain embodiments, modulation is in a positive allosteric manner. In certain embodiments, the system is a cellular system. In other embodiments, the system is an in-vitro translation which is based on the protein synthesis without living cells. The cellular system is defined to be any subject provided that the subject comprises cells. Hence, the cellular system can be selected from the group of single cells, cell cultures, tissues, organs and animals. In certain embodiments, the method for modulating an FSH receptor is performed in-vitro. The prior teaching of the present specification concerning the compounds of formula (I), including any embodiments thereof, is valid and applicable without restrictions to the compounds according to formula (I) and their salts when used in the method for modulating FSHR. The prior teaching of the present specification concerning the compounds of formula (I), including any embodiments thereof, is valid and applicable without restrictions to the compounds according to formula (I) and their salts when used in the method for modulating FSHR.

In certain embodiments, the compounds according to the invention exhibit an advantageous biological activity, which is easily demonstrated in cell culture-based assays, for example assays as described herein or in prior art (cf. e.g. WO 2002/09706, which is incorporated herein by reference). In such assays, the compounds according to the invention preferably exhibit and cause an agonistic effect. In certain embodiments, the compounds of the invention have an FSHR agonist activity, as expressed by an EC50standard, of less than 5 μM. In certain embodiments, less than 1 μM. In certain embodiments, less than 0.5 μM. In certain embodiments, less than 0.1 μM. “EC50” is the effective concentration of a compound at which 50% of the maximal response of that obtained with FSH would be obtained.

As discussed herein, these signaling pathways are relevant for various diseases, including fertility disorders. Disorders/diseases treated by the methods of the invention include but are not limited to, hypogonadotropic hypogonadism, Isolated idiopathic hypogonadotropic hypogonadism, Kallmann syndrome, Idiopathic hypogonadotropic hypogonadism, Craniopharyngiomas, Combined pituitary hormone deficiency, Fertile eunuch syndrome, Abnormal beta subunit of LH, Abnormal beta subunit of FSH, mass lesions, pituitary adenomas, cysts, metastatic cancer to the sella (breast in women, lung and prostate in men), Infiltrative lesions, Hemochromatosis, sarcoidosis, histiocytosis, lymphoma, Lymphocytic hypophysitis, Infections, Meningitis, Pituitary apoplexy, Hyperprolactinemia, hypothyroidism, Intentional (iatrogenic) secondary hypogonadism, Empty sella, Pituitary infarction, Sheehan syndrome, Anorexia nervosa, Congenital adrenal hyperplasia, and disorders related to GnRH deficiency. Accordingly, the compounds according to the invention are useful in the prophylaxis and/or treatment of diseases that are dependent on the said signaling pathways by interaction with one or more of the said signaling pathways. The present invention therefore relates to compounds according to the invention as modulators, preferably agonists, more preferably positive allosteric modulators, of the signaling pathways described herein, preferably of the FSHR-mediated signaling pathway. The compounds of the invention are supposed to bind to the intracellular receptor domain without a competitive interaction with FSH, but they act as an allosteric enhancer of FSH on its receptor. The non-competitive interaction refers to the nature of the agonist activity exhibited by the compounds of the invention, wherein the compounds activate FSHR without substantially reducing the magnitude of binding of FSH to FSHR.

In certain embodiments, the invention is directed towards the stimulation of follicular development, ovulation induction, controlled ovarial hyperstimulation, assisted reproductive technology, including in-vitro fertilization, male hypogonadism and male infertility, including some types of failure of spermatogenesis.

It is another object of the invention to provide a method for treating diseases that are caused, mediated and/or propagated by FSHR activity, wherein at least one compound of formula (I) according to the invention and/or physiologically acceptable salts thereof is administered to a mammal in need of such treatment. In certain embodiments, the invention provides a method for treating fertility disorders, wherein at least one compound of formula (I) according to the invention and/or physiologically acceptable salts thereof is administered to a mammal in need of such treatment. In certain embodiments, the compound is administered in an effective amount as defined above. In certain embodiments, the treatment is an oral administration.

In certain embodiments, the method of treatment aims to achieve ovulation induction and/or controlled ovarian hyperstimulation. In still another embodiment, the method of treatment forms the basis for a method for in-vitro fertilization comprising the steps of: (a) treating a mammal according to the method of treatment as described above, (b) collecting ova from said mammal, (c) fertilizing said ova, and (d) implanting said fertilized ova into a host mammal. The host mammal can be either the treated mammal (i.e. the patient) or a surrogate. The prior teaching of the invention and its embodiments is valid and applicable without restrictions to the methods of treatment if expedient.

The method of the invention can be performed either in-vitro or in-vivo. The susceptibility of a particular cell to treatment with the compounds according to the invention can be particularly determined by in-vitro tests, whether in the course of research or clinical application. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period of time which is sufficient to allow the active agents to modulate FSHR activity, usually between about one hour and one week. In-vitro treatment can be carried out using cultivated cells from a biopsy sample or cell line. In a preferred aspect of the invention, a follicle cell is stimulated for maturation. The viable cells remaining after the treatment are counted and further processed.

The host or patient can belong to any mammalian species, for example a primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.

For identification of a signal transduction pathway and for detection of interactions between various signal transduction pathways, various scientists have developed suitable models or model systems, for example cell culture models and models of transgenic animals. For the determination of certain stages in the signal transduction cascade, interacting compounds can be utilized in order to modulate the signal. The compounds according to the invention can also be used as reagents for testing FSHR-dependent signal transduction pathways in animals and/or cell culture models or in the clinical diseases mentioned in this application.

The use according to the previous paragraphs of the specification may be either performed in-vitro or in-vivo models. The modulation can be monitored by the techniques described in the course of the present specification. In certain embodiments, the in-vitro use is preferably applied to samples of humans suffering from fertility disorders. Testing of several specific compounds and/or derivatives thereof makes the selection of that active ingredient possible that is best suited for the treatment of the human subject. The in-vivo dose rate of the chosen derivative is advantageously pre-adjusted to the FSHR susceptibility and/or severity of disease of the respective subject with regard to the in-vitro data. Therefore, the therapeutic efficacy is remarkably enhanced. Moreover, the subsequent teaching of the present specification concerning the use of the compounds according to formula (I) and its derivatives for the production of a medicament for the prophylactic or therapeutic treatment and/or monitoring is considered as valid and applicable without restrictions to the use of the compound for the modulation of FSHR activity if expedient.

The invention also relates to the use of compounds according to formula (I) and/or physiologically acceptable salts thereof for the prophylactic or therapeutic treatment and/or monitoring of diseases that are caused, mediated and/or propagated by FSHR activity. Furthermore, the invention relates to the use of compounds according to formula (I) and/or physiologically acceptable salts thereof for the production of a medicament for the prophylactic or therapeutic treatment and/or monitoring of diseases that are caused, mediated and/or propagated by FSHR activity. In certain embodiments, the invention provides the use of a compound according to formula I or physiologically acceptable salts thereof, for the production of a medicament for the prophylactic or therapeutic treatment of a FSHR-mediated disorder.

Compounds of formula (I) and/or a physiologically acceptable salt thereof can furthermore be employed as intermediate for the preparation of further medicament active ingredients. The medicament is preferably prepared in a non-chemical manner, e.g. by combining the active ingredient with at least one solid, fluid and/or semi-fluid carrier or excipient, and optionally in conjunction with a single or more other active substances in an appropriate dosage form.

Another object of the present invention are compounds of formula (I) according to the invention and/or physiologically acceptable salts thereof for use in the prophylactic or therapeutic treatment and/or monitoring of diseases that are caused, mediated and/or propagated by FSHR activity. Another preferred object of the invention concerns compounds of formula (I) according to the invention and/or physiologically acceptable salts thereof for use in the prophylactic or therapeutic treatment and/or monitoring of fertility disorders. The prior teaching of the present specification concerning the compounds of formula (I), including any preferred embodiment thereof, is valid and applicable without restrictions to the compounds according to formula (I) and their salts for use in the prophylactic or therapeutic treatment and/or monitoring of fertility disorders.

The compounds of formula (I) according to the invention can be administered before or following an onset of disease once or several times acting as therapy. The aforementioned compounds and medical products of the inventive use are particularly used for the therapeutic treatment. A therapeutically relevant effect relieves to some extent one or more symptoms of a disorder, or returns to normality, either partially or completely, one or more physiological or biochemical parameters associated with or causative of a disease or pathological condition. Monitoring is considered as a kind of treatment provided that the compounds are administered in distinct intervals, e.g. in order to booster the response and eradicate the pathogens and/or symptoms of the disease completely. Either the identical compound or different compounds can be applied. The methods of the invention can also be used to reducing the likelihood of developing a disorder or even prevent the initiation of disorders associated with FSHR activity in advance or to treat the arising and continuing symptoms. In certain embodiments, the disorders are fertility disorders.

In the meaning of the invention, prophylactic treatment is advisable if the subject possesses any preconditions for the aforementioned physiological or pathological conditions, such as a familial disposition, a genetic defect, or a previously passed disease.

The invention furthermore relates to a medicament comprising at least one compound according to the invention and/or pharmaceutically usable derivatives, salts, solvates and stereoisomers thereof, including mixtures thereof in all ratios. In certain embodiments, the invention relates to a medicament comprising at least one compound according to the invention and/or physiologically acceptable salts thereof.

A “medicament” in the meaning of the invention is any agent in the field of medicine, which comprises one or more compounds of formula (I) or preparations thereof (e.g. a pharmaceutical composition or pharmaceutical formulation) and can be used in prophylaxis, therapy, follow-up or aftercare of patients who suffer from diseases, which are associated with FSHR activity, in such a way that a pathogenic modification of their overall condition or of the condition of particular regions of the organism could establish at least temporarily.

In various embodiments, the active ingredient may be administered alone or in combination with other treatments. A synergistic effect may be achieved by using more than one compound in the pharmaceutical composition, i.e. the compound of formula (I) is combined with at least another agent as active ingredient, which is either another compound of formula (I) or a compound of different structural scaffold. The active ingredients can be used either simultaneously or sequentially. The present compounds are suitable for combination with known fertility-inducing agents. In certain embodiments, the other active pharmaceutical ingredient is selected from the group of FSH, α-FSH (Gonal F), β-FSH, LH, hMG and 2-(4-(2-chloro-1,2-diphenylethenyl)-phenoxy)-N,N-diethyl-ethanamine citrate (Chlomifene citrate). Further ovulation adjuncts are known to those of skill in the art (cf. e.g. WO 2002/09706, which is incorporated herein by reference) and are useful with the compounds of the present invention.

In another aspect, the invention provides for a kit consisting of separate packs of an effective amount of a compound according to the invention and/or pharmaceutically acceptable salts, derivatives, solvates and stereoisomers thereof, including mixtures thereof in all ratios, and optionally, an effective amount of a further active ingredient. The kit comprises suitable containers, such as boxes, individual bottles, bags or ampoules. The kit may, for example, comprise separate ampoules, each containing an effective amount of a compound according to the invention and/or pharmaceutically acceptable salts, derivatives, solvates and stereoisomers thereof, including mixtures thereof in all ratios, and an effective amount of a further active ingredient in dissolved or lyophilized form.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment is administered after one or more symptoms have developed. In other embodiments, treatment is administered in the absence of symptoms. For example, treatment is administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment is also continued after symptoms have resolved, for example to prevent or delay their recurrence.

The compounds and compositions, according to the method of the present invention, are administered using any amount and any route of administration effective for treating or lessening the severity of a disorder provided above. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like.

In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection.

This is accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method of allosterically modulating FSHR activity in a biological sample comprising the step of contacting said biological sample with a compound of this invention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method of allosterically modulating FSHR, or a mutant thereof, activity in a biological sample in a positive manner, comprising the step of contacting said biological sample with a compound of this invention, or a composition comprising said compound.

The compounds of the invention are strong and selective modulators of the FSH receptor. Their selectivity to the FSH receptor is 3 to 10-fold over the LH receptor and even 10 to 100-fold over the TSH receptor while the EC50or IC50amounts to more than 10 μM on unrelated G protein-coupled receptors (GPCR) or non-GPCR targets. The current invention comprises the use of the compounds of the invention in the regulation and/or modulation of the FSHR signal cascade, which can be advantageously applied as research tool, for diagnosis and/or in treatment of any disorder arising from FSHR signaling.

For example, the compounds of the invention are useful in-vitro as unique tools for understanding the biological role of FSH, including the evaluation of the many factors thought to influence, and be influenced by, the production of FSH and the interaction of FSH with the FSHR (e. g. the mechanism of FSH signal transduction/receptor activation). The present compounds are also useful in the development of other compounds that interact with FSHR since the present compounds provide important structure-activity relationship (SAR) information that facilitate that development. Compounds of the present invention that bind to FSHR can be used as reagents for detecting FSHR on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, etc. For example, by labeling such compounds, one can identify cells having FSHR on their surfaces. In addition, based on their ability to bind FSHR, compounds of the present invention can be used in in-situ staining, FACS (fluorescence-activated cell sorting), western blotting, ELISA (enzyme-linked immunoadsorptive assay), etc., receptor purification, or in purifying cells expressing FSHR on the cell surface or inside permeabilized cells.

The compounds of the invention can also be utilized as commercial research reagents for various medical research and diagnostic uses. Such uses can include but are not limited to: use as a calibration standard for quantifying the activities of candidate FSH agonists in a variety of functional assays; use as blocking reagents in random compound screening, i.e. in looking for new families of FSH receptor ligands, the compounds can be used to block recovery of the presently claimed FSH compounds; use in the co-crystallization with FSHR receptor, i.e. the compounds of the present invention will allow formation of crystals of the compound bound to FSHR, enabling the determination of receptor/compound structure by x-ray crystallography; other research and diagnostic applications, wherein FSHR is preferably activated or such activation is conveniently calibrated against a known quantity of an FSH agonist, etc.; use in assays as probes for determining the expression of FSHR on the surface of cells; and developing assays for detecting compounds which bind to the same site as the FSHR binding ligands.

The compounds of the invention can be applied either themselves and/or in combination with physical measurements for diagnostics of treatment effectiveness. Pharmaceutical compositions containing said compounds and the use of said compounds to treat FSHR-mediated conditions is a promising, novel approach for a broad spectrum of therapies causing a direct and immediate improvement in the state of health, whether in human or animal. The impact is of special benefit to efficiently combat infertility, either alone or in combination with other fertility-inducing treatments. In particular, the compounds of the invention potentiate the native FSH effect for both ovulation induction and assisted reproductive technology. The orally bioavailable and active new chemical entities of the invention improve convenience for patients and compliance for physicians.

The compounds of the invention are active in the primary screen (CHO with or without FSH), selective in secondary screen (no or low activity against TSHR and LHR) and potent in the granulosa cell estrodiol assay. Neither hERG nor any toxic effects could be observed in-vitro.

In certain embodiments, the invention provides a method for in-vitro fertilization comprising the steps of:

(a) treating a mammal according to the method as described above,

(b) collecting ova from said mammal,

(c) fertilizing said ova, and

(d) implanting said fertilized ova into a host mammal.

The compounds of formula (I), their salts, isomers, tautomers, enantiomeric forms, diastereomers, racemates, derivatives, prodrugs and/or metabolites are characterized by a high specificity and stability, low manufacturing costs and convenient handling. These features form the basis for a reproducible action, wherein the lack of cross-reactivity is included, and for a reliable and safe interaction with the target structure.

Modulation of FSHR, or a mutant thereof, activity in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, biological specimen storage, and biological assays.

Compound numbers utilized in the Examples below correspond to compound numbers set forth supra.

1H NMR was recorded on a Bruker 400 MHz spectrometer, using residual signal of deuterated solvent as internal reference. Chemical shifts (δ) are reported in ppm relative to the residual solvent signal (δ=2.49 ppm for 1H NMR in DMSO-d6). 1H NMR data are reported as follows: chemical shift (multiplicity, coupling constants, and number of hydrogens). Multiplicity is abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

LCMS-Analysis was performed under the following conditions:

To a stirred suspension of resorcinol (25 g, 0.22 mol) and 3-Bromopropanoic acid (38.3 g, 0.25 mol) was added trifluoromethanesulfonic acid (75 mL, 0.84 mol) drop wise at 0° C. under nitrogen atmosphere. After the addition, the reaction mixture was heated to 80° C. for 30 min. The reaction mixture was cooled to RT and quenched with ice water (200 mL) and extracted with DCM (500 mL). The aqueous layer was re-extracted with DCM (2×100 mL); the combined organic layer was dried over sodium sulphate and concentrated under vacuum to afford the desired compound (40 g, 72%) as an orange solid.

To an ice cold solution of 2M NaOH (92 mL, 2.33 mol), was added 3-Bromo-1-(2,4-dihydroxyphenyl)propan-1-one (38 g, 0.155 mol) in lots over a period of 30 min. The resulting suspension was stirred at RT for 2 h. The reaction mixture was cooled to 0° C.; pH was adjusted to ˜2 using 50% aqueous solution of sulfuric acid. The solid separated out was stirred for additional 10 min at RT, filtered and dried under high vacuum to afford the desired compound (16 g, 63%) as brown solid.

To a stirred solution of 7-Hydroxy-2,3-dihydro-4H-chromen-4-one (27 g, 0.16 mol) in acetone (700 mL) was added dry K2CO3(45.6 g, 0.32 mol) in lots at RT under nitrogen. The reaction mixture was stirred at RT for 10 min and then methyl iodide (65.4 g, 0.46 mol) was added drop wise at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was filtered and filtrate was concentrated under vacuum. The crude product was dissolved in DCM (200 mL), washed with water (100 mL), brine (50 mL), dried over sodium sulphate and concentrated under vacuum to afford the desired compound (15 g, 89%) as light yellow solid.

To a solution of 7-Methoxy-2,3-dihydro-4H-chromen-4-one (30 g, 0.16 mol) in acetonitrile:diethyl ether mixture (100:300 mL) was added silica gel 60-120 mesh (1.5 g) and NBS (33 g, 0.18 mol) in lots at RT under nitrogen. The reaction mixture was stirred at RT for 14 h. The reaction mixture was filtered and concentrated under vacuum. The crude product was purified by column chromatography by using pet ether/ethyl acetate (9:1) as eluent to afford the desired compound (10 g, 72%) as a light brown solid.

Diisopropylamine (7 mL, 0.3958 mol) was taken in dry THF (50 mL) at RT under nitrogen atmosphere. The reaction mixture was cooled to −78° C. and n-Butyl lithium (1.6 M solution in hexane, 29.2 mL, 0.04 mol) was added drop wise over a period of 30 min. After the addition, the reaction mixture was stirred at the same temperature for 15 min and then slowly warmed to −10° C. and stirred further for 30 min. The reaction mixture was again cooled to −78° C., 6-Bromo-7-methoxy-2,3-dihydro-4H-chromen-4-one (10 g, 0.03 mol) in THF (50 mL) was added drop wise over period of 30 min and stirred at −78° C. After 1 h, diethyl oxalate (7.8 mL, 0.05 mol) was added drop wise at −78° C.; the reaction mixture was slowly brought to 0° C. and stirred for 1 h. The reaction mixture was cooled to −5° C., quenched with a solution of 1.5N HCl and extracted with ethyl acetate (100 mL×2). The combined organic layer was washed with water (100 mL), brine (50 mL), dried over sodium sulphate and concentrated to afford the desired compound (10 g, 72%) as pale yellow solid.

To a solution of 3-Bromo thiophene (10 g, 0.061 mol) in DMSO (100 mL) was added tert-butyl carbazate (16.3 g, 0.122 mol), cesium carbonate (40 g, 0.122 mol) followed by CuI (1.2 g, 0.006 mol) and 4-Hydroxy-L-Proline (1.6 g, 0.01 mol) at RT under nitrogen. The reaction mixture was stirred at 80° C. for 14 h. The reaction mixture was cooled to RT, quenched with water (100 mL) and extracted with ethyl acetate (3×200 mL). The combined organic layer was washed with water (100 mL×2), brine (100 mL), dried over sodium sulphate and evaporated under vacuum. The crude product was purified by column chromatography by using pet ether and ethyl acetate (7:3) as eluent to afford the title compound (3.5 g, 27%) as pale brown liquid.

To a stirred solution of tert-Butyl 1-(3-thienyl)hydrazinecarboxylate (3.5 g, 0.0163 mol) in diethyl ether (10 mL) was added HCl in dioxane (30 mL) at RT under nitrogen. The reaction mixture was stirred at RT for 8 h. The organic solvent was removed under reduced pressure to afford the desired compound (2.4 g, 97%) as pale brown solid.

To a solution of ethyl-(6-bromo-7-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)(hydroxy)acetate (8.0 g, 0.0224 mol) in a mixture of ethanol (200 mL) and acetic acid (200 mL) was added 3-thienylhydrazine hydrochloride (4.4 g, 0.0291 mol) at RT under nitrogen. The reaction mixture was stirred at 100° C. for 4 h. The reaction mixture was concentrated under high vacuum. The residue was dissolved with ethyl acetate (40 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulphate and concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate as eluent to afford the desired compound (8.5 g, 87%) as a pale yellow solid.

To a solution of Ethyl 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (3 g, 0.0069 mol) in mixture of THF (70 mL), H2O (20 mL), MeOH (10 mL) was added LiOH.H2O (0.857 g, 0.0207 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with a solution of 1.5N HCl. The solid was filtered and dried to afford the desired compound (2.8 g, 99%) as an off-white solid.

To a solution of 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4, 3-c]pyrazole-3-carboxylic acid (2.8 g, 0.0069 mol) in DCM (50 mL) was added N-tert-butyl methyl amine (718 mg, 0.0083 mol), HATU (3.14 g, 0.0083 mol) and diisopropyl ethyl amine (1.8 mL, 0.0103 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched to sodium bicarbonate (10 mL, 10%), extracted with DCM (2×50 mL). The combined organic layer was washed with NaHCO3solution (1×100 mL, 10% solution), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography by using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound (3.2 g, 98%) as a white solid.

To a solution of 8-bromo-N-(tert-butyl)-7-methoxy-N-methyl-1-(thiophen-3-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxamide (1 g, 0.0021 mol) in dioxane (20 mL) was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (611 mg, 0.0031 mol), PdCl2(dppf)CH2Cl2(86 mg, 0.0001 mol) and cesium fluoride (800 mg, 0.0053 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 20 min and water (4 mL) was added at RT. The reaction mixture was stirred at 100° C. for 12 h. The reaction mixture was filtered through celite and washed with DCM (20 mL). The filtrate was concentrated under vacuum; the crude product was dissolved in DCM (200 mL), washed with water (10 ml), brine (10 mL) and dried over sodium sulphate. The organic solvent was removed under vacuum; the crude product was purified by column chromatograph using pet ether: ethyl acetate as eluent to afford the desired compound (0.5 g, 51%) as an off-white solid.

To a solution of 8-bromo-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide (Example 1 step 10) (100 mg, 0.2 mmol) in DME (10 mL) was added pyridine-3-boronic acid (52 mg, 0.4 mmol), tetra kis (triphenylphospine)palladium (13 mg, 0.01 mmol) and potassium carbonate (90 mg, 0.6 mmol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water was added (1 mL). The reaction mixture was stirred at 90° C. for 16 h. The reaction mixture was filtered through celite. Filtrate was concentrated under vacuum; the crude product was dissolved in DCM (200 mL), washed with water (10 ml), brine (10 mL) and dried over sodium sulphate. The solvent was removed under vacuum to provide the crude product. The crude product was slurred with diethyl ether (5 mL), filtered and dried to afford the desired compound (95 mg, 98%) as an off white solid.

To a solution of 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylic acid (example 1 step 9) (0.5 g, 0.001 mol) in DCM (10 mL) was added 1-(cyclobutyl carbonyl)-1,4 diazepane (0.25 g, 0.001 mol), T3P (1 mL, 0.001 mol, 50% soln in EtOAc) and TEA (0.2 mL, 0.003 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched with saturated sodium bicarbonate (10 mL), extracted with DCM (2×25 mL). The combined organic layer was washed with NaHCO3solution (1×100 mL, 100%), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum, the crude product was purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound as (0.4 g, 57%) an off white solid.

To a solution of (4-cyclobutanecarbonyl-[1,4]diazepan-1-yl)-(7-methoxy-8-pyridin-3-yl-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-methanone (250 mg, 0.4 mmol) in DME (10 mL) was added pyridine-3-boronic acid (100 mg, 0.8 mmol), tetra kis (triphenylphospine)palladium (30 mg, 0.02 mmol) and potassium carbonate (150 mg, 0.001 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water (2 mL) was added. The reaction mixture was stirred at 90° C. for 16 h. The reaction mixture was filtered through celite and washed with DCM (20 mL). Filtrate was concentrated under vacuum; crude product was dissolved in DCM (200 mL), washed with water (10 ml), brine (10 mL) and dried over sodium sulphate. The solvent was removed under vacuum; crude product was slurred with diethyl ether (10 mL), filtered and dried to afford the desired compound as (240 mg, 96%) an off white solid.

To a solution of ethyl 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (example 1 step 8) (1 g, 0.002 mol) in THF (20 mL) was added 2,4,6-Tris-(2-methyl-propenyl)-cyclotriboroxanepyridine complex (380 mg, 0.001 mol), bis (triphenylphospine)palladium (II) dichloride (80 mg, 0.1 mmol) and potassium tri phosphate (63 mg, 0.004 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water (2 mL) was added at RT. The reaction mixture was stirred at 70° C. for 4 h. The reaction mixture was filtered through celite and washed with DCM (50 mL). The filtrate was concentrated under vacuum; crude product was dissolved in DCM (200 mL), washed with water (20 ml), brine (20 mL) and dried over sodium sulphate. The organic solvent was removed under vacuum; crude product was purified by column chromatograph with pet ether: ethyl acetate as eluent to afford the desired product as (0.9 g, 96%) a light yellow solid.

To a solution of ethyl 7-methoxy-8-(2-methylprop-1-en-1-yl)-1-(thiophen-3-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (step 1) (1 g, 0.02 mol) in methanol and ethyl acetate mixture (40 mL) was added palladium on carbon (10%, 0.5 g). The reaction mixture was hydrogenated under 3 bar of pressure hydrogen for 4 h at RT. The reaction mixture was filtered through a celite bed and filtrate was concentrated under vacuum. The residue was purified by column chromatography using pet ether: ethyl acetate as eluent to afford the title compound (0.7 g, 70%) as an off white solid.

To a solution of ethyl 8-isobutyl-7-methoxy-1-(thiophen-3-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (250 mg, 0.006 mol) in mixture of THF (21 mL), H2O (6 mL), MeOH (3 mL) was added LiOH (0.08 g, 0.001 mol) at RT. The reaction mixture was stirred at RT for 2 h. The reaction mixture was evaporated and acidified with 1.5N HCl solution. The solid was filtered and dried under high vacuum to afford the desired compound (200 mg, 87%) as a white solid.

To a solution of 8-isobutyl-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylic acid (step 3) (180 mg, 0.4 mmol) in DCM (20 mL) was added N-tert-butyl methyl amine (50 mg, 0.5 mmol), HATU (0.22 g, 5 mmol) and diisopropyl ethyl amine (0.2 mL, 0.7 mmol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched with sodium bicarbonate (10 mL, 10%), extracted with DCM (2×25 mL). The combined organic layer was washed with NaHCO3solution (1×100 mL, 10%), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound (140 mg, 66%) as a white solid.

To a solution of ethyl 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (6 g, 0.0138 mol) in THF (100 mL) was added 2,4,6-Tris-(2-methyl-propenyl)-cyclotriboroxanepyridine complex (5.8 g, 0.018 mol), bis(triphenylphospine)palladium (II) dichloride (1.0 g, 1.38 mmol) and potassium triphosphate (3.8 g, 0.0276 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water (10 mL) was added at RT. The reaction mixture was stirred at 80° C. for 8 h. The reaction mixture was filtered through celite and washed with DCM (50 mL). The filtrate was concentrated under vacuum; crude product was dissolved in DCM (200 mL), washed with water (20 ml), brine (20 mL) and dried over sodium sulphate. The organic solvent was removed under vacuum; crude product was purified by column chromatograph using pet ether: ethyl acetate as eluent to afford the desired compound as (5.5 g, 98%) a pale yellow solid.

To a solution of ethyl 7-methoxy-8-(2-methylprop-1-en-1-yl)-1-(thiophen-3-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (7 g, 0.0171 mol) in mixture of THF (70 mL), H2O (20 mL), MeOH (10 mL) was added LiOH.H2O (2.1 g, 0.0512 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with 1.5N HCl solution. The solid was filtered and dried under high vacuum to afford the desired compound (5.5 g, 85%) as an off-white solid.

The following compounds were prepared using procedures analogous to those disclosed in Example 6.

The following compounds were prepared using procedures analogous to those disclosed in example 4:

To a stirred suspension of 3, 4-dimethoxy phenol (10 g, 0.06 mol) and chloropropanoyl chloride (12.5 g, 0.12 mol) was added boron trifluorideethylethrate (8.8 mL, 0.06 mol) in drops at 60° C. under nitrogen atmosphere. After the complete addition, the reaction mixture was heated to 70° C. for 1 h. The reaction mixture was cooled to RT and quenched with ice-water (200 mL) and extracted with DCM (500 mL). The aqueous layer was re-extracted (2×100 mL) DCM, the combined organic layer was dried over sodium sulphate and concentrated under vacuum. The crude mass was purified by column chromatography using pet ether/ethyl acetate (8:2) as eluent to afford the desired compound (12 g, 76%) as a light yellow solid.

To a stirred solution of 3-Chloro-1-(2-hydroxy-4,5-dimethoxyphenyl)propan-1-one (13 g, 0.05 mol) in ethanol was added dry K2CO3(16.3 g, 0.10 mol) in lots at RT under nitrogen. The resulting suspension was stirred at RT for 16 h. The reaction mixture was filtered and concentrated under vacuum. The crude mass was dissolved in ethyl acetate (200 mL), washed with 5% sodium bicarbonate (50 mL), brine (50 mL) and dried over sodium sulphate. The organic solvent was concentrated; the residue was purified by column chromatography using pet ether/ethyl acetate (8:2) as eluent to afford the desired compound (9 g, 81%) as a light brown solid.

The mixture of 6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (2.5 g, 0.01 mol) and pyridine hydrochloride (20 g, 0.20 mol) was heated at 170° C. under nitrogen for 12 h. The reaction mixture was slurred with DCM (100 mL), the separated solid was filtered and filtrate was concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate (5:5) as eluent to afford the desired compound (1 g, 50%) as a light white solid.

To a stirred solution of 6,7-dihydroxy-2,3-dihydro-4H-chromen-4-one (5.5 g, 0.0305 mol) in DMF (60 mL) was added dry K2CO3(4.2 g, 0.0305 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 15 min and then added methyl iodide (1.3 mL, 0.0214 mol) in drops at RT. The reaction mixture was stirred at RT for 2 h. The reaction mixture was filtered and filtrate was concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate (9:1) as eluent to afford the desired compound (4 g, 68%) as a light brown solid.

To a stirred solution of 6-hydroxy-7-methoxy-2,3-dihydro-4H-chromen-4-one (4.0 g, 0.0206 mol) in DMF (80 mL) was added dry K2CO3(5.7 g, 0.0412 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 15 min and then added 2-lodo propane (6.2 mL, 0.0618 mol) in drops at RT. The reaction mixture was stirred at 65° C. for 8 h. The reaction mixture was filtered and filtrate was concentrated under vacuum. The crude product was dissolved in ethyl acetate (2×200 mL), washed with water (50 mL), brine (50 mL) and dried over sodium sulphate. The organic solvent was concentrated, crude mass was purified by column chromatography using pet ether/ethyl acetate (8:2) as eluent to afford the desired compound (4.1 g, 87%) as a light brown solid.

Diisopropyl amine (6.9 mL, 0.0495 mol) was taken in dry THF (150 mL) at RT under nitrogen atmosphere. The reaction mixture was cooled to −78° C. and n-Butyl lithium (1.6 M solution in hexane, 28.6 mL, 0.0457 mol) was added in drops over a period of 30 min. After the addition, the reaction mixture stirred at same temperature for 15 min and then slowly warmed to −10° C. and stirred further for 30 min. Reaction mixture was again re-cooled to −78° C., 6-Isopropoxy-7-methoxy-2,3-dihydro-4H-chromen-4-one (9 g, 0.0381 mol) in THF (50 mL) was added in drops over period of 30 min and stirred at −78° C. After 1 h, diethyl oxalate (7.8 mL, 0.0571 mol) was added in drops at −78° C.; the reaction mixture was slowly brought to 0° C. and stirred for 1 h. The reaction mixture was cooled to −5° C., quenched with a solution of 1.5N HCl and extracted with ethyl acetate (100 mL×2). The combined organic layer was washed with water (100 mL), brine (50 mL), dried over sodium sulphate and concentrated to afford the desired compound (10.5 g, 92%) as a pale yellow solid.

To a solution of (ethyl (2Z)-hydroxy(6-isopropoxy-7-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)acetate (6.0 mg, 0.0223 mol) in a mixture of ethanol (150 mL) and acetic acid (150, mL) was added 3-thienylhydrazine hydrochloride (2.7 g, 0.0223 mol) at RT under nitrogen. The reaction mixture was stirred at 100° C. for 4 h. The reaction mixture was concentrated under high vacuum. The residue was dissolved with ethyl acetate (20 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulphate and concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate as eluent to afford desired compound (6.5 g, 88%) as an off white solid.

To a solution of 8-isopropoxy-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (4 g, 0.0108 mol) in mixture of THF (70 mL), H2O (20 mL), MeOH (10 mL) was added LiOH.H2O (1.4 g, 0.0326 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with a solution of 1.5N HCl. The filtrate was dried under high vacuum to afford the desired compound (3.3 g, 79%) as an off white solid.

To a stirred solution of 8-isopropoxy-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylic acid (1.1 g, 0.0028 mol) in DCM (50 mL) was added N-tert-butyl methyl amine (0.3 g, 0.0034 mol), HATU (1.3 g, 0.0034 mol) and DIPEA (0.8 mL, 0.0043 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched to water (20 mL), extracted with dichloromethane (2×100 mL). The combined organic layer was washed with brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography using pet ether/ethyl acetate (8:2) as eluent to afford the desired compound (1.1 g, 85%) as an off white solid.

The following compounds were prepared using procedures analogous to those disclosed in example 7.

The following compounds were prepared using procedures analogous to those disclosed in example 1:

To 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid [2-(2-oxo-oxazolidin-3-yl)-ethyl]-amide (22.00 mg; 0.04 mmol; 1.00 eq.) was added N,N-Dimethyl-formamide (0.50 ml) and sodium hydride (2.67 mg; 0.07 mmol; 1.50 eq.). Reaction was stirred at RT for 10 min then iodomethane (0.00 ml; 0.05 mmol; 1.10 eq.) (1 drop) was added and the reaction was stirred at RT for 15 min. Water was added and product was extracted with EtOAc and washed with water. Organic layer was dried, filtered, concentrated. Residue was dried on high vacuum then lyophilized to afford the desired product (22.5 mg, 99%) as an off white solid.

The following compounds were prepared using procedures analogous to those disclosed in example 8 above:

To 4-[3-(tert-Butyl-methyl-carbamoyl)-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-8-yl]-pyrazole-1-carboxylic acid tert-butyl ester (99.00 mg; 0.18 mmol) in methanol (3.00 ml; 74.06 mmol; 302.68 eq.) was added hydrochloric acid (0.20 ml; 0.80 mmol; 3.27 eq.) (4M in dioxane). After stirring at room temperature for 1 h, the mixture was concentrated and purified by flash chromatography to afford the desired product (33.8 mg, 34%) as a white solid.

To {3-[(8-Isopropoxy-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carbonyl)-amino]-propyl}-carbamic acid tert-butyl ester (172.70 mg; 0.32 mmol) in methanol (4.00 ml; 98.75 mmol; 317.98 eq.) was added hydrochloric acid in dioxane (0.31 ml; 1.24 mmol; 4.00 eq.). After stirring at room temperature for 18 h the reaction mixture was concentrated to dryness and purified by flash chromatography to afford the desired compound (71.3 mg, 52%) as a white solid.

To 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide (30.00 mg; 0.07 mmol; 1.00 eq.) in water (0.10 ml), and acetone (0.40 ml) was added 4-methylmorpholine 4-oxide (23.35 mg; 0.20 mmol; 3.00 eq.) and osmium tetroxide (0.51 mg; 0.00 mmol; 0.03 eq.). After stirring for 1 h at room temperature the mixture was quenched with saturated sodium sulfide and extracted with EtOAc. The organic layer was dried (Na2SO4), filtered, concentrated and purified by flash chromatography to afford the desired compound (22.5 mg, 70%) as a white solid.

To 1-[7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carbonyl]-azepane-4-carboxylic acid (75.00 mg; 0.15 mmol; 1.00 eq.) in DMSO (1.00 ml) was added 1,1′-carbonylbis(1H-imidazole) (119.72 mg; 0.74 mmol; 5.00 eq.). Reaction was stirred at room temperature for 18 h, then ammonium acetate (34.17 mg; 0.44 mmol; 3.00 eq.) was added and the reaction was continued to stir at room temperature for 2 h. After completion, water was added to the mixture and solid formed was filtered, rinsed with water to afford the desired compound (62.5 mg, 84%) as a white solid.

In a similar manner to example 12 step 2, 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (3-amino-propyl)-amide was obtained from (3-{[7-Methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carbonyl]-amino}-propyl)-carbamic acid tert-butyl ester and HCl (4M in dioxane).

In a similar manner to example 12, step 1, 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid [3-(cyclobutanecarbonyl-amino)-propyl]-amide was obtained from 7-Methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (3-amino-propyl)-amide (100 mg, 0.23 mmol) and cyclobutanecarboxylic acid (0.14 ml; 1.50 mmol; 6.58 eq.). The desired compound was obtained in 97% yield (115 mg) as a white solid.

To a mixture of 2-aminothiazole (10.0 g, 100 mmol) and conc. hydrochloric acid (80 mL), was added a solution of sodium nitrite (6.90 g, 100 mmol) in water (50 mL) drop wise at −10° C. The reaction mixture was stirred for 10 min at the same temperature and followed by addition of tin (II)chloride (37.9 g, 200 mmol) in conc. hydrochloric acid (20 mL) drop wise carefully so that the temperature of the solution did not exceed −10.deg.C. After the addition, the reaction mixture stirred for 30 min at the same temperature. The resulting crystals were collected by filtration. The crystal was recrystalized from diethylether to afford the desired compound (11.0 g, 97%) as brown solid.

To a solution of ethyl-(6-bromo-7-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)(hydroxy)acetate (3.0 g, 0.0084 mol) in a mixture of Ethanol (100 mL) and Acetic acid (100 mL) was added 2-Hydrazino-1,3-thiazole hydrochloride (1.9 g, 0.0126 mol) at RT under nitrogen. The reaction mixture was stirred at 100° C. for 4 h. The reaction mixture was concentrated under high vacuum. The residue was dissolved with ethyl acetate (40 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulphate and concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate as eluent to afford the desired compound (1.5 g, 41%) as pale yellow solid.

To a solution of ethyl 8-bromo-7-methoxy-1-(1,3-thiazol-2yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (2.6 g, 0.0059 mol) in THF (100 mL) was added 2,4,6-Tris-(2-methyl-propenyl)-cyclotriboroxanepyridine complex (2.9 g, 0.0089 mol), bis(triphenylphospine)palladium (II) dichloride (417 mg, 0.0006 mol) and potassium tri phosphate (2.0 g, 0.0149 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water (10 mL) was added at RT. The reaction mixture was stirred at 90° C. for 12 h. The reaction mixture was filtered through celite and washed with DCM (50 mL). The filtrate was concentrated under vacuum; crude product was dissolved in DCM (200 mL), washed with water (20 ml), brine (20 mL) and dried over sodium sulphate. The organic solvent was removed under vacuum; crude product was purified by column chromatography over (60-120) mesh silica gel and pet ether: ethyl acetate as eluent to afford the desired compound (2.2 g, 90%) as a pale yellow solid.

To a solution of ethyl 7-methoxy-8-(2-methylprop-1-en-1-yl)-1-(1,3-thiazol-2-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (1 g, 0.0024 mol) in mixture of THF (35 mL), H2O (10 mL), MeOH (5 mL) was added LiOH.H2O (302 mg, 0.0073 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with 1.5N HCl solution. The separated solid was filtered and dried under high vacuum to afford the desired compound (900 mg, 97%) as an off-white solid.

To a solution of 7-Methoxy-8-(2-methylprop-1-en-1-yl)-1-(1,3-thiazol-2-yl)-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (900 mg, 0.0023 mol) in DCM (50 mL) was added N-tert-butyl methyl amine (225 mg, 0.0028 mol), HATU (1.1 g, 0.0028 mol) and diisopropyl ethyl amine (0.6 mL, 0.0035 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched to sodium bicarbonate (10 mL, 10%), extracted with DCM (2×50 mL). The combined organic layer was washed with NaHCO3solution (1×100 mL, 10% solution), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound (850 mg, 80%) as an off-white solid.

To a solution of 2-Bromo thiophene (10 g, 0.0613 mol) in DMSO (200 mL) was added tert-butyl carbazate (16.3 g, 0.1227 mol), cesium carbonate (40 g, 0.1227 mol) followed by CuI (1.2 g, 0.0061 mol) and 4-Hydroxy-L-Proline (1.6 g, 0.0123 mol) at RT under nitrogen. The reaction mixture was stirred at 80° C. for 14 h. The reaction mixture was cooled to RT, quenched with water (100 mL) and extracted with ethyl acetate (3×200 mL). The combined organic layer was washed with water (100 mL×2), brine (100 mL), dried over sodium sulphate and evaporated under vacuum. The crude product was purified by column chromatography by using pet ether and ethyl acetate (7:3) as eluent to afford the desired compound (3.0 g, 40%) as a brown liquid.

To a stirred solution of tert-butyl 1-(2-thienyl)hydrazinecarboxylate (4.3 g, 0.0201 mol) in dichloromethane (20 mL) was added HCl in dioxane (30 mL) at RT under nitrogen. The reaction mixture was stirred at RT for 8 h. The organic solvent was removed under reduced pressure to afford the desired compound (2.9 g, 96%) as pale brown solid.

To a solution of ethyl-(6-bromo-7-methoxy-4-oxo-2H-chromen-3(4H)-ylidene)(hydroxy)acetate (6.0 g, 0.0168 mol) in a mixture of Ethanol (100 mL) and acetic acid (100 mL) was added 2-thienylhydrazine hydrochloride (2.9 g, 0.0252 mol) at RT under nitrogen. The reaction mixture was stirred at 100° C. for 4 h. The reaction mixture was concentrated under high vacuum. The residue was dissolved with ethyl acetate (40 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulphate and concentrated under vacuum. The crude product was purified by column chromatography using pet ether/ethyl acetate as eluent to afford desired compound (4.0 g, 55%) as a pale yellow solid.

To a solution of Ethyl 8-bromo-7-methoxy-1-(2-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (3.8 g, 0.0087 mol) in THF (100 mL) was added 2,4,6-Tris-(2-methyl-propenyl)-cyclotriboroxanepyridine complex (4.3 g, 0.0131 mol), bis(triphenylphospine)palladium (II) dichloride (306 mg, 0.0004 mol) and potassium tri phosphate (2.4 g, 0.0174 mol) at RT under nitrogen. The reaction mixture was degassed with nitrogen for 10 min and water (10 mL) was added at RT. The reaction mixture was stirred at 90° C. for 12 h. The reaction mixture was filtered through celite and washed with DCM (50 mL). The filtrate was concentrated under vacuum; the crude product was dissolved in DCM (200 mL), washed with water (20 ml), brine (20 mL) and dried over sodium sulphate. The organic solvent was removed under vacuum; crude product was purified by column chromatography using pet ether: ethyl acetate as eluent to afford the desired compound (2.5 g, 70%) as pale yellow solid.

To a solution of 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-2-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid ethyl ester (1 g, 0.0024 mol) in mixture of THF (35 mL), H2O (10 mL), MeOH (5 mL) was added LiOH.H2O (303 mg, 0.0073 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with 1.5N HCl solution. The separated solid was filtered and dried under high vacuum to afford the desired compound (800 mg, 75%) as an off-white solid.

To a solution of 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-2-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (800 mg, 0.0021 mol) in DCM (50 mL) was added N-tert-butyl methylamine (220 mg, 0.0025 mol), HATU (950 mg, 0.0025 mol) and diisopropylethylamine (0.6 mL, 0.0032 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched to sodium bicarbonate (10 mL, 10%), extracted with DCM (2×50 mL). The combined organic layer was washed with NaHCO3solution (1×100 mL, 10% solution), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound (850 mg, 90%) as a white solid.

To a solution of 7-methoxy-8-(2-methyl-propenyl)-1-thiophen-2-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid ethyl ester (1.2 g, 0.0027 mol) in methanol and ethyl acetate mixture (100 mL) was added palladium on carbon (20%, 0.24 g). The reaction mixture was hydrogenated under 3 bar of pressure for 8 h at RT. The reaction mixture was filtered through celite to remove the catalyst and the filtrate was concentrated under vacuum. The residue was purified by column chromatography using pet ether/ethyl acetate as eluent to afford the desired compound (1.1 g, 90%) as an off white solid.

To a solution of 8-isobutyl-7-methoxy-1-thiophen-2-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid ethyl ester (1.1 g, 0.0027 mol) in mixture of THF (35 mL), H2O (10 mL), MeOH (5 mL) was added LiOH.H2O (332 mg, 0.0080 mol) at RT. The reaction mixture was stirred at RT for 4 h. The reaction mixture was evaporated and acidified with 1.5N HCl solution. The separated solid out was filtered to afford the desired compound (900 mg, 88%) as an off-white solid.

To a solution of 8-isobutyl-7-methoxy-1-thiophen-2-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (900 mg, 0.0023 mol) in DCM (50 mL) was added N-tert-butyl methyl amine (245 mg, 0.0028 mol), HATU (1.0 g, 0.0028 mol) and diisopropyl ethyl amine (0.6 mL, 0.0038 mol) at RT under nitrogen. The reaction mixture was stirred at RT for 16 h. The reaction mixture was quenched to sodium bicarbonate (10 mL, 10%), extracted with DCM (2×50 mL). The combined organic layer was washed with NaHCO3solution (100 mL, 10% solution), brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed under vacuum; the crude product was purified by column chromatography using pet ether and ethyl acetate (9:1) as eluent to afford the desired compound (800 mg, 75%) as a white solid.

To 8-isopropylsulfanyl-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide (24 mg, 0.05 mmol) in DCM (2 mL) was added 3-chloroperbenzoic acid (11.40 mg; 0.05 mmol; 1.00 eq.). The reaction was stirred at RT for 2 h. The mixture was purified by flash chromatography to afford the desired product (8 mg, 32%) as a white solid. m/z=504 [M+H]+, HPLC retention time=4.40 min.

In a similar manner to example 23 above step 2, (8-cyclopropanesulfonyl-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-(3,3-dimethyl-morpholin-4-yl)-methanone was obtained from 8-(cyclopropylsulfonyl)-7-methoxy-1-(thiophen-3-yl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylic acid (30.00 mg; 0.07 mmol; 1.00 eq.) and 3,3-dimethyl-morpholine (0.05 ml; 0.10 mmol; 1.50 eq.). The desired compound was obtained in a yield of 12 mg (33%) as a blue solid.

To (3,3-dimethyl-morpholin-4-yl)-[7-methoxy-8-(2-methyl-propenyl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl]-methanone (70.00 mg; 0.15 mmol; 1.00 eq.) suspended in THF (3.00 ml), was added methylsulfanylmethane; with borane (16.63 mg; 0.22 mmol; 1.50 eq.). The reaction was stirred at RT for 2 h. Then NaOH (2N solution) was added very slowly. The reaction mixture was purified using reverse phase prep-HPLC (45-60% CH3CN in 0.1% NH4OH in H2O) to afford the desired product (19 mg, 26%) as a white solid.

To diazomethyl-trimethyl-silane (0.17 ml; 0.33 mmol; 3.00 eq.) suspended in Ethoxy-ethane (3.00 ml) at 0° C. was added n-butyl lithium (0.09 ml; 0.22 mmol; 2.00 eq.). The reaction mixture was stirred at 0° C. for 30 min then 3-(3,3-Dimethyl-morpholine-4-carbonyl)-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-8-carbonitrile (50.00 mg; 0.11 mmol; 1.00 eq.) was added and the reaction was stirred at 0° C. for another 30 min. The reaction mixture was warmed to RT and stirred at RT for 1 h. The mixture was concentrated and purified by column chromatography to afford the desired product (18 mg, 29%) as a white solid.

To (3,3-dimethyl-morpholin-4-yl)-[7-methoxy-1-thiophen-3-yl-8-(5-trimethylsilanyl-5H-[1,2,4]triazol-3-yl)-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl]-methanone (34.00 mg; 0.06 mmol; 1.00 eq.) dissolved in THF (3.00 ml) was added tetrabutyl-ammonium fluoride (1M in THF) (0.30 ml; 0.30 mmol; 5.00 eq.) and the reaction was stirred at RT for 18 h. Reaction mixture was concentrated and purified by reverse phase prep-HPLC (35-40% CH3CN in 0.1% NH4OH in H2O) to afford the desired product (36 mg, 82%) as a white solid.

To a solution of ethyl 8-bromo-7-methoxy-1-(3-thienyl)-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxylate (2 g, 0.0459 mol) in NMP (50 mL), was added CuI (90 mg, 0.5 mmol) and followed by CuCN (825 mg, 9.1 mmol) in a sealed tube. The reaction mixture was heated to 160° C. for 16 h. The reaction mixture was filtered through celite and the filtrate was concentrated. The crude product was purified by column chromatography by using dichloromethane/methanol (9:1) as eluent. The product was triturated with acetonitrile and filtered, to afford of the desired compound (0.7 g, 83%) as a white solid.

To 8-cyano-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid ethyl ester (300.00 mg; 0.79 mmol; 1.00 eq.) dissolved in Methanol (6.00 ml; 177.52 mmol; 225.69 eq.) was added potassium hydroxide (66.20 mg; 1.18 mmol; 1.50 eq.) and water (0.60 ml). The reaction mixture was heated to 50° C. for 2 h. The reaction was concentrated and lyophilized to afford the crude desired product as a gray solid.

3-(3,3-dimethyl-morpholine-4-carbonyl)-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-8-carbonitrile (50.00 mg; 0.11 mmol; 1.00 eq.) was dissolved in amonia in methanol (10.00 ml; 20.00 mmol; 180.20 eq.) and the reaction mixture was run through an H-cube under full H2pressure using Raney nickel cartridge at 70° C. for 2 h. The mixture was concentrated and purified by reverse phase prep-HPLC (35-45% CH3CN in 0.1% NH4OH in H2O) to afford the desired product as white solid (7 mg, 14%). LCMS: m/z=455 [M+H]+, HPLC retention time=2.71 min.

To 8-(2,5-dihydro-furan-3-yl)-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide (30.00 mg; 0.06 mmol; 1.00 eq.) suspended in acetic acid (3.00 ml) was added palladium on carbon (0.02 ml; 0.32 mmol; 5.00 eq.). The flask was capped with a rubber septum and topped with a hydrogen balloon. The reaction mixture was stirred at RT for 18 h. Triethylamine was added, the mixture was filtered through celite and the mixture was purified by reverse phase prep-HPLC (55-63% CH3CN in 0.1% NH4OH in H2O) to afford the desired product (11 mg, 37%) as a white solid.

In a similar manner to example 39, 7-methoxy-8-(tetrahydro-furan-2-yl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide was obtained from 8-(4,5-Dihydro-furan-2-yl)-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid tert-butyl-methyl-amide (65.00 mg; 0.14 mmol; 1.00 eq.) as a white solid in 9% yield (6 mg).

In a similar manner to example 41, 7-methoxy-8-(1-methyl-1H-pyrrol-3-yl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (1S,3S)-3-amino-cyclopentyl ester was obtained from 8-bromo-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (1S,3S)-3-amino-cyclopentyl ester, in 3% yield (3 mg) as a white solid.

In a similar manner to example 43, (2-methoxymethyl-2-methyl-pyrrolidin-1-yl)-(7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-methanone was obtained from (2-Hydroxymethyl-2-methyl-pyrrolidin-1-yl)-(7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-methanone as a white solid (13 mg, 50%).

In a similar manner to example 43, (3,3-Dimethyl-morpholin-4-yl)-(7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-methanone was obtained from (8-Bromo-7-methoxy-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazol-3-yl)-(3,3-dimethyl-morpholin-4-yl)-methanone as a white solid (13 mg, 46%).

The following compounds were prepared using procedures analogous to those disclosed in Example 1:

Compound 116 (MSC2501240) (30.00 mg; 0.06 mmol; 1.00 eq.) was dissolved in hydrochloric acid in water (2.00 ml). The reaction mixture was stirred at room temperature overnight. The mixture was applied to a prep-HPLC (32-38% CH3CN in 0.1% NH4OH in H2O) to afford the desired product 7-Methoxy-8-(1-methyl-1H-pyrazol-3-yl)-1-thiophen-3-yl-1,4-dihydro-chromeno[4,3-c]pyrazole-3-carboxylic acid (2-hydroxy-1-hydroxymethyl-1-methyl-ethyl)-methyl-amide (19.00 mg; 0.04 mmol) as a white solid (61%). LCMS: m/z=513 [M+H]+, HPLC retention time: 2.67 min.

2500 Cho-FSHR-LUC-1-1-43 cells were plated per well in 5 μl of phenol red free DMEM/F12+1% FBS. Cells were plated in 384 well, solid white low volume plates (Greiner 784075) by Multidrop. Cells were assayed by adding 100 μl of 2× EC20FSH/IBMX in DMEM/F12+0.1% BSA) by Multidrop to 2 μl of test compound stamped in 384 well plates (compounds are diluted 1:50). The final FSH concentration was 0.265 μM, and the final IBMX concentration was 200 μM. The compound plate map was as follows: Column 1: 2 μl of DMSO; Column 2: 2 μl of DMSO; Columns 3-12 and 13-24: 2 μl of test compound, diluted 1:4 in 100% DMSO, or 2 μl of FSH, diluted 1:4 in DMEM/F12+0.1% BSA. The starting concentration for FSH was 50 nM (final concentration was 0.5 nM). Furthermore, Column 23 contained 2 μl of EC100FSH reference (100×) (diluted in DMEM/F12+0.1% BSA) at a final concentration of 0.5 nM, and Column 24 contained 2 μl of 1 mM AS707664/2 reference compound 2. 5 μl of compound+EC20FSH mixture were transferred to cell plates (1:2 dilution into 5 μl of cell media) The plates were incubated at 37° C. for 1 h. 10 μl of mixed HTRF (CisBio #62AM4PEC) reagents were added per well and incubated at room temperature for 1 h. The plates were read on Envision using the cAMP HTRF—low volume 384 well protocol. The readout was the calculated fluorescence ratio (665 nm/620 nm). Values given in percent (%) indicate the percental effect (response) at a certain concentration of agonist relative to the maximum response of the FSH standard. The results are provided below.

The assay was performed pursuant to the teaching of Yanofsky et al. (2006) Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists (JBC 281(19): 13226-13233, which is incorporated by reference in the disclosure of the invention). The results are provided below.

The data is interpreted according to the following:

Pharmaceutical Preparations

(A) Injection vials: A solution of 100 g of an active ingredient according to the invention and 5 g of disodium hydrogen phosphate in 3 l of bidistilled water is adjusted to pH 6.5 using 2 N hydrochloric acid, sterile filtered, transferred into injection vials, is lyophilized under sterile conditions and is sealed under sterile conditions. Each injection vial contains 5 mg of active ingredient.

(B) Suppositories: A mixture of 20 g of an active ingredient according to the invention is melted with 100 g of soy lecithin and 1400 g of cocoa butter, is poured into moulds and is allowed to cool. Each suppository contains 20 mg of active ingredient.

(D) Ointment: 500 mg of an active ingredient according to the invention is mixed with 99.5 g of Vaseline under aseptic conditions.

(E) Tablets: A mixture of 1 kg of an active ingredient according to the invention, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is pressed to give tablets in a conventional manner in such a way that each tablet contains 10 mg of active ingredient.

(F) Coated tablets: Tablets are pressed analogously to Example E and subsequently are coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and dye.

(G) Capsules: 2 kg of an active ingredient according to the invention are introduced into hard gelatin capsules in a conventional manner in such a way that each capsule contains 20 mg of the active ingredient.

(H) Ampoules: A solution of 1 kg of an active ingredient according to the invention in 60 l of bidistilled water is sterile filtered, transferred into ampoules, is lyophilized under sterile conditions and is sealed under sterile conditions. Each ampoule contains 10 mg of active ingredient.

(I) Inhalation spray: 14 g of an active ingredient according to the invention are dissolved in 10 l of isotonic NaCl solution, and the solution is transferred into commercially available spray containers with a pump mechanism. The solution could be sprayed into the mouth or nose. One spray shot (about 0.1 ml) corresponds to a dose of about 0.14 mg.

While a number of embodiments of this invention are described herein, it is apparent that the basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by t appended claims rather than by the specific embodiments that have been represented by way of example.