EA039545B1 - Methods of preventing preterm labor using a combination of a pgf2 inhibitor and a tocolytic agent - Google Patents

Abstract

The invention provides a method of treating or preventing preterm labor and a method of preventing labor prior to cesarean delivery, comprising administering to a patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an additional therapeutic agent.

Description

The invention relates to methods for the treatment and prevention of preterm labor, as well as methods for preventing labor before delivery by caesarean section by administering to a patient in need of such treatment a compound of formula (I) or a pharmaceutically acceptable salt thereof and an additional tocolytic agent.

Prerequisites for the creation of the invention

Preterm birth is the leading cause of perinatal death in developed countries and occurs in about 7-10% of all births (Berkowitz et al., Epidemiol. Rev. 15:414-443 (1993)). Premature infants are significantly more likely to experience severe illness, especially respiratory distress syndrome, intraventricular hemorrhage, bronchopulmonary dysplasia, and necrotizing enterocolitis, compared to full-term infants. Long-term impairments such as central paralysis, visual impairment and hearing loss are also most common in preterm infants. Currently, preterm birth remains the leading cause of neonatal mortality and morbidity in the United States of America, where, despite significant improvements in obstetric medicine, infant mortality rates are higher than in many other industrialized countries, causing intensive care costs. therapy for extremely low birth weight newborns in excess of $5 billion a year. The actual costs associated with this intensive care are even higher when taking into account the provision of medical services for the treatment of diseases associated with preterm birth, such as respiratory distress syndrome, heart disease, central paralysis, epilepsy, and severe learning disabilities.

Over the past 40 years of clinical research, and despite the use of various therapeutic agents, the incidence of preterm birth has not significantly decreased. Prevention of preterm birth is very difficult and, although tocolytic therapy remains a key element in the containment of preterm birth, there is no universal agreement on its significance for this condition. Available tocolytic agents do not by themselves prolong labor beyond 48 hours, and most of these agents lack uterine selectivity and therefore have potential severe side effects for both mother and fetus.

Fundamentally, term and preterm birth are similar processes because they share a common physiological end point, which is characterized by uterine contractions, cervical dilatation, and membrane activation. The difference lies in the gestational age at which these processes occur and the mechanisms that activate them. It is believed that term delivery is the result of physiological activation of the terminal pathway, while preterm birth is a pathological condition that is characterized by various etiologies, in which one or more components of this pathway are aberrantly activated.

The contractility of the uterus is stimulated or inhibited by various receptors in the cells of the myometrium. It is hypothesized that myometrial activation results from the coordinated expression of contraction-associated proteins (CAPs), including actin, myosin, connexin-43, and receptors for oxytocin and prostaglandins. As a rule, receptors that cause calcium entry or calcium release from the intracellular pool stimulate contractility. However, receptors associated with the production of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP), relax the uterus. For example, oxytocin and prostaglandin F (FP) receptors are stimulatory, while β2 adrenoceptors and prostaglandin E2 receptors associated with cAMP formation are inhibitory.

In uterine tissues, prostaglandins E2 (PGE2) and F2a (PGF2a) have been shown to induce cervical changes and uterine contractility, two key events in the physiology of labor and delivery. Activation of the FP receptor in human myometrium by PGF2a leads to an increase in intracellular calcium concentration, which in turn leads to contraction of uterine smooth muscle cells (Abramovitz et al. J. Biol. Chem. 269:2632-2636 (1994) and Senior and Br J Pharmacol 108:501-506 (1993)). FP receptors are upregulated in uterine tissues at birth (Al-Matubsi et al. Biol. Reprod. 65:1029-1037 (2001)). Prostaglandin synthesis inhibitors (such as indomethacin and nimesulide) show some tocolytic effect, but they have side effects and their unlicensed use in clinical practice raises fetal safety concerns (Norton et al. New Engl. J. Med. 329:1602 -1067 (1993) and Peruzzi et al. NewEngl J. Med 354:1615 (1999)). There remains a need to develop drugs with myometrial selectivity that will allow long-term inhibition of uterine contractions leading to labor and prolong pregnancy to a stage at which increased fetal maturation increases the chances of survival.

The essence of the invention

The invention covers alpha-amino esters of a hydroxypropylthiazolidine derivative

- 1 039545 carboxamide, as well as their salts, which are capable of disrupting the interaction between prostaglandin

F2a (PGF2a) and the prostaglandin F receptor. These compounds may be administered to a subject, such as a pregnant woman, to treat or prevent preterm labor.

In a first aspect, the invention provides a method for treating or preventing preterm labor in a human patient by administering to the patient a therapeutically effective amount of a compound of formula (I)(I) or a pharmaceutically acceptable salt thereof and an additional tocolytic agent.

In another aspect, the invention provides a method for preventing preterm labor before delivery by caesarean section in a human patient by administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an additional tocolytic agent.

In some embodiments, said compound is represented by formula (III)

In some embodiments, said compound is in a crystalline state.

In some embodiments, said compound exhibits characteristic X-ray powder diffraction peaks at 2Θ angles of 7.0°, 8.1°, 10.0°, 12.0°, 13.1°, 14.1°, 16.4°, 18.4°, 20.1°, 21.0°, 23.5° and 29.5°.

In some embodiments, said compound has an X-ray powder diffraction spectrum such as that depicted in FIG. 49.

In some embodiments, said compound exhibits 1H nuclear magnetic resonance (NMR) peaks centered at 1.1 ppm, 3.3 ppm. 4.9 m.h., 5.4 m.h., 7.1 m.h., 7.7 m.h., 7.9 m.h., and 8.0 m.h.

In some embodiments, said compound has a ¹ H NMR spectrum such as that shown in FIG. 21.

In some embodiments, said compound exhibits endotherm at 145 to 147° C., as determined by differential scanning calorimetry.

In some embodiments, said compound is characterized by a differential scanning calorimetry curve such as shown in FIG. 20 or FIG. 23.

In some embodiments, said compound has a weight loss of 0.2 to 0.6% when heated from 25 to 100° C., as measured by thermogravimetric analysis.

In some embodiments, said compound is characterized by a thermogravimetric analysis curve such as shown in FIG. 24.

In some embodiments, the method includes oral administration of the compound.

In some embodiments, said additional tocolytic agent is an oxytocin receptor antagonist. In some embodiments, the method comprises orally administering an oxytocin receptor antagonist to a subject. In some embodiments, the method comprises administering an oxytocin receptor antagonist intravenously to a patient. The compound may be administered to the subject simultaneously with the administration of the oxytocin receptor antagonist. In some embodiments, the compound is administered to the patient prior to administration of the oxytocin receptor antagonist to the patient. In some embodiments, the compound is administered to the patient following administration of the oxytocin receptor antagonist to the subject. In some embodiments, the compound is mixed with an oxytocin receptor antagonist and these agents are administered to the patient simultaneously. In some embodiments, the oxytocin receptor antagonist is atosiban, retosiban, barusiban, epelsiban, or nolasiban.

In some embodiments, the oxytocin receptor antagonist is atosiban.

In some embodiments, said additional tocolytic agent is a calcium channel blocker. In some embodiments, said calcium channel blocker is nifedipine or nicardipine. In some embodiments, said calcium channel blocker is nifedipine. In some embodiments, the method includes orally administering a calcium channel blocker to a patient. The compound may be administered to the subject simultaneously with the administration of the calcium channel blocker. In some embodiments, the compound is administered to the patient prior to administration of the calcium channel blocker to the patient. In some embodiments, the compound is administered to the patient following administration of the calcium channel blocker to the patient. In some embodiments, the compound is mixed with a calcium channel blocker and these agents are administered to the patient simultaneously.

In some embodiments, said additional tocolytic agent is a betamimetic, magnesium salt, nitric oxide donor, progesterone, or a variant thereof. In some embodiments, said additional tocolytic agent is a betamimetic selected from the group consisting of terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine, or orciprenaline. In some embodiments, the method includes oral administration of the betamimetic to a patient. The compound may be administered to the patient simultaneously with the administration of the betamimetic. In some embodiments, the compound is administered to the patient prior to the administration of the betamimetic to the patient. In some embodiments, the compound is administered to the patient following administration of the betamimetic to the patient. In some embodiments, the compound is mixed with a betamimetic and these agents are administered to the patient simultaneously.

In some embodiments, said additional tocolytic agent is magnesium sulfate. In some embodiments, the method includes administering a magnesium salt intravenously to a subject. In some embodiments, the method includes intramuscularly administering a magnesium salt to a patient. In some embodiments, the method includes oral administration of a magnesium salt to a patient. The compound may be administered to the patient simultaneously with the administration of the magnesium salt. In some embodiments, the compound is administered to the subject prior to administration of the magnesium salt to the patient. In some embodiments, the compound is administered to the subject following administration of the magnesium salt to the patient. In some embodiments, the compound is mixed with a magnesium salt and these agents are administered to the patient simultaneously.

In some embodiments, said additional tocolytic agent is nitroglycerin. In some embodiments, the implementation of the method includes oral administration of nitroglycerin to the patient. In some embodiments, the implementation of the method includes intravenous administration of nitroglycerin to the patient. The compound may be administered to the patient simultaneously with the administration of nitroglycerin. In some embodiments, the compound is administered to the patient prior to administration of the nitroglycerin to the patient. In some embodiments, the compound is administered to the patient following administration of nitroglycerin to the patient. In some embodiments, the compound is mixed with nitroglycerin and these agents are administered to the patient simultaneously.

In some embodiments, said additional tocolytic agent is progesterone or 17-a-hydroxyprogesterone caproate. In some embodiments, the method includes oral administration of progesterone, or 17-a-hydroxyprogesterone caproate, to a subject. In some embodiments, the method includes intravaginally administering progesterone, or 17-a-hydroxyprogesterone caproate, to a subject. The compound may be administered to the subject simultaneously with the administration of progesterone or 17-a-hydroxyprogesterone caproate. In some embodiments, the compound is administered to the subject prior to administration of progesterone 17-a-hydroxyprogesterone caproate to the subject. In some embodiments, the compound is administered to the subject following administration of progesterone 17-a-hydroxyprogesterone caproate to the subject. In some embodiments, the compound is admixed with progesterone 17-a-hydroxyprogesterone caproate (eg, in particular in an oral formulation) and these agents are administered to the subject simultaneously.

In some embodiments, the patient is additionally administered a corticosteroid. In some embodiments, the corticosteroid is betamethasone. In some embodiments, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is hydrocortisone. In some embodiments, the method includes oral administration of a corticosteroid to a subject. In some embodiments, the method includes intramuscular administration of a corticosteroid to a subject. The compound may be administered to the subject simultaneously with the administration of the corticosteroid. In some embodiments, the compound is administered to the subject prior to administration of the corticosteroid to the subject. In some embodiments, the compound is administered to the subject following administration of the corticosteroid to the subject. In some embodiments, the compound is mixed with a corticosteroid (eg, in particular in an oral formulation), and these agents are administered to the subject simultaneously.

In some embodiments, the patient has a gestational age of about 24 to about 34 weeks. In some embodiments, the subject exhibits a decrease in the amplitude of uterine contractions after insertion, such as a decrease of from about 40% to about 50% (by

- 3 039545 example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50%) relative to the measurement of the amplitude of uterine contractions in the patient, recorded before the introduction. In some embodiments, the compound exhibits a half-life of about 1 to about 4 hours in a subject (e.g., about 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1 .6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2 .6 h, 2.7 h, 2.8 h, 2.9 h, 3.0 h, 3.1 h, 3.2 h, 3.3 h, 3.4 h, 3.5 h, 3 .6 h, 3.7 h, 3.8 h, 3.9 h or 4.0 h). In some embodiments, the compound reaches its maximum plasma concentration in the subject within about 0.25 to about 2 hours of administration (e.g., about 0.25 hours, 0.3 hours, 0.4 hours, 0.5 hours, 0 .6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1 .6 h, 1.7 h, 1.8 h, 1.9 h or 2.0 h). In some embodiments, the subject is a mammal, such as a human. In some embodiments, said compound binds to the human prostaglandin F2a receptor with an affinity of about 1 nM. The compounds of the invention exhibit the ability to selectively bind to prostaglandin F receptors such as prostaglandin F2a over other prostaglandin receptor subtypes. For example, the compounds of the invention exhibit an affinity for the prostaglandin F2a receptor that is about 10 times greater than that observed for the prostaglandin E2 receptor. Additionally, the compounds of the invention exhibit an affinity for the prostaglandin F2a receptor that is about 100 times or higher (e.g., about 100 times to about 1000 times, for example, about 100 times, 110 times, 120 times, 130 times, 140 times , 150 times, 160 times, 170 times, 180 times, 190 times, 200 times, 210 times, 220 times, 230 times, 240 times, 250 times, 260 times, 270 times, 280 times, 290 times, 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 410 times, 420 times, 430 times, 440 times, 450 times, 460 times, 470 times, 480 times, 490 times, 500 times, 510 times, 520 times, 530 times, 540 times, 550 times, 560 times, 570 times, 580 times, 590 times, 600 times, 610 times, 620 times, 630 times, 640 times , 650 times, 660 times, 670 times, 680 times, 690 times, 700 times, 710 times, 720 times, 730 times, 740 times, 750 times, 760 times, 770 times, 780 times, 790 times, 800 times, 810 times, 820 times, 830 times, 840 times, 850 times, 860 times, 870 times, 880 times, 890 times, 900 times, 910 times, 920 times, 930 times, 940 times, 950 times, 960 times, 970 times, 980 times 990 times, 1000 times, or higher) than for other prostaglandin receptor subtypes, such as the prostaglandin E1, E3, E4, D1, D2, I1, and I2 receptor subtypes. In some embodiments, the compound is soluble in aqueous solution at a concentration of about 300 µg/mL to about 500 µg/mL, for example, at a concentration of about 380 µg/mL.

In some embodiments, said compound inhibits the synthesis of inositol triphosphate in a cell, such as a mammalian cell. In some embodiments, the mammalian cell is a human cell, such as a myometrial cell. In some embodiments, the myometrial cell is a uterine myocyte. In some embodiments, the compound induces a decrease in the amplitude of uterine contractions in a subject following administration of the compound to the subject. For example, a compound can induce a reduction of about 40% to about 50% relative to a subject's uterine contraction amplitude measured prior to administration. In some embodiments, the compound exhibits a half-life in a subject of about 1 to about 4 hours after administration of the compound to the subject. In some embodiments, the compound reaches its maximum plasma concentration in the subject within about 0.25 to about 2 hours after administration of the compound to the subject.

In some embodiments, the patient is a human. In some embodiments, the compound is administered orally to a subject. In some embodiments, the compound is administered to a subject intravenously.

In some embodiments, the compound of formula (III) exhibits characteristic X-ray powder diffraction peaks at about 7.0° 2θ, about 8.1° 2θ, about 10.0° 2θ, about 20.1° 2θ, about 21.0° 2θ, and about 23.5° 2θ. In some embodiments, the compound further exhibits X-ray powder diffraction peaks at about 12.0° 2θ, about 13.1° 2θ, about 14.1° 2θ, about 16.4° 2θ, about 18.4° 2θ, and about 29 .5° 2θ. In some embodiments, the compound has an X-ray powder diffraction spectrum substantially as shown in any of FIGS. 19, 22, 29, 45-49, and 54. For example, in some embodiments, the compound has an X-ray powder diffraction spectrum substantially as depicted in FIG. 49.

In some embodiments, said compound exhibits 1H nuclear magnetic resonance (NMR) peaks centered at about 1.1 ppm, about 3.3 ppm, about 4.9 ppm, about 5.4 about 7.1 ppm, about 7.7 ppm, about 7.9 ppm, and about 8.0 ppm. In some embodiments, the compound is characterized by 1H An NMR spectrum essentially as shown in FIG. 21.

In some embodiments, the compound is endothermic at about 145° C. to about 147° C., as measured by differential scanning calorimetry. In some embodiments, said compound exhibits an additional endotherm at about 214° C. as measured by differential scanning calorimetry. In some embodiments, said compound has a differential scanning calorimetry curve substantially as depicted in FIG. 20. In some embodiments, said compound exhibits an additional endotherm at about 228° C., as measured by differential scanning calorimetry. In some embodiments, said compound has a differential scanning calorimetry curve substantially as depicted in FIG. 23.

In some embodiments, said compound exhibits a weight loss of about 0.2% to about 0.6% when heated from 25 to 100° C., as measured by thermogravimetric analysis. In some embodiments, said compound exhibits a weight loss of about 2.5% to about 3.5% when heated from 100 to 160°C, as measured by thermogravimetric analysis. In some embodiments, said compound exhibits a thermogravimetric analysis curve substantially as depicted in FIG. 24.

Definitions

As used in this application, the term about refers to a value that is within 10% above or below the specified value.

As used in this application, the term affinity refers to the strength of the binding interaction between two molecules, such as a ligand and a receptor. The term Ki, as used herein, refers to the antagonist inhibition constant for a particular molecule of interest and is expressed as a molar concentration (M). Ki values of antagonist-target interactions can be determined, for example, using methods existing in the art. Methods that can be used to determine the K _i of a molecular target antagonist include competitive binding experiments, for example, radioactive ligand competitive binding studies, for example, as described in US 8415480. The term Kd, as used in this application, refers to a dissociation constant, which is can be obtained, for example, from the ratio of the rate constant of dissociation of two molecules (kd) to the rate constant of association of two molecules (k _a ) and is expressed as a molar concentration (M). Kd values for receptor-ligand interactions can be determined, for example, using methods available in the art. Methods that can be used to determine the Kd of the receptor-ligand interaction include surface plasmon resonance, for example by using a biosensor system such as the BIACORE® system.

As used in this application, the term corticosteroid refers to any of the steroid hormones produced by the adrenal cortex, or their synthetic analogues. Exemplary corticosteroids include, in particular, betamethasone, dexamethasone, and hydrocortisone, as well as variants thereof. Corticosteroids for use in combination with the compositions and methods described in this application include those that are capable of inducing fetal lung maturation, for example, to prevent the development of respiratory distress syndrome in preterm infants. Typical corticosteroids for use in combination with the compositions and methods described in this application include those described in Jobe and others. Am. J. Obstet. Gynecol. 190:878-881 (2004) and Miracle et al. J. Pennat. Med. 36:191-196 (2008), the disclosure of each of which is incorporated herein by reference.

As used in this application, the term crystalline or crystalline form means that the compound has a physical state that is a regular spatial lattice of atoms, ions, molecules or molecular aggregates. Crystalline forms have lattice structures of structural elements called asymmetric units, which are arranged in a certain order according to a well-defined symmetry in unit cells that repeat in three dimensions. In contrast, the term amorphous or amorphous form refers to an unorganized (disordered) structure. The physical state of the therapeutic compound can be determined using typical techniques such as X-ray diffraction analysis, polarized light microscopy and/or differential scanning calorimetry.

As used herein, the term endogenous describes a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or at a particular location in an organism (e.g., an organ, tissue, or cell, like a human cell).

As used in this application, the term exogenous describes a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., human) or at a particular location in the body (e.g., organ, tissue, or cell). , such as a human cell). Exogenous materials include those provided from an external source to the organism or cultured material extracted from them.

As used herein, the term gestational age describes how long a particular pregnancy lasts and is measured from the first day of a pregnant female's last menstrual cycle to the present date. As used in this application, the term childbirth (which may also be referred to as birth) refers to the expulsion of the fetus and placenta from the uterus of a pregnant female. For a normal pregnancy, delivery can occur at gestational age around 40 weeks. Prematurity, as used herein, refers to a condition in which delivery occurs earlier than three weeks before the full gestational period, which is usually about 40 weeks. Thus, preterm birth occurs at any stage before, for example, 38 weeks of gestation. Prematurity usually results in labor or physiological changes associated with childbirth in a pregnant female if left untreated. Preterm labor may or may not be associated with vaginal bleeding or uterine membrane rupture. Premature birth can also be referred to as premature termination of pregnancy. Prevention of preterm delivery in the subject will prolong the term of pregnancy and therefore may avoid preterm delivery, thus reducing the risk of neonatal mortality and morbidity.

As used herein, the term IC50 refers to the concentration of a substance (antagonist) that reduces the efficacy of a comparative agonist or the constitutive activity of a biological target by 50%, for example, as measured in a competitive ligand binding assay. Exemplary competitive ligand binding assays include, in particular, competitive radiolabeled ligand binding assays, competitive enzyme-linked immunosorbent assays (ELISAs), and fluorescence anisotropy assays known in the art.

As used herein in the context of providing or administering two or more therapeutic agents to a subject, the phrase in combination with refers to the delivery of two or more therapeutic agents to a subject (e.g., a mammalian subject, e.g., a human), for example, either simultaneously or at different times. . For example, one therapeutic agent may be administered to a subject in combination with another by administering both agents to the subject simultaneously, eg, in the same pharmaceutical composition or in separate compositions that are administered to the subject simultaneously (eg, via different routes of administration). In another example, one therapeutic agent may be administered to a subject in combination with another by first administering one therapeutic agent to the subject and then administering another therapeutic agent, either by the same route of administration or by a different route of administration.

As used herein, the term nolasiban refers to (32.58)-5-(hydroxymethyl)1-[(2'-methyl-1,1'-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O- methyloxime, which is represented by the following structural formula:

Seats OH

Nolasiban

Options, compositions, and crystal form of nolasiban are described, for example, in US patent No. 7115754 and published application for US patent No. 2015/0073032; 2015/0164859; and 2016/0002160, the disclosure of each of which is incorporated herein by reference.

As used herein, the term oral bioavailability refers to the fraction of an administered compound to a subject, e.g., a mammal (e.g., human), that reaches the subject's systemic circulation and is not deposited in a non-target organ or excreted without absorption through the gastrointestinal tract. The term refers to plasma concentration, which is integrated over time and is typically expressed as a percentage of an orally administered dose.

As used herein, the term oxytocin receptor antagonist or oxytocin antagonist refers to a compound capable of inhibiting the interaction between oxytocin and the oxytocin receptor, for example such that the activity of one or more downstream signaling molecules in the oxytocin signaling cascade is inhibited. Oxytocin antagonists for use in the compositions and methods described herein include compounds that bind and inhibit the oxytocin receptor, such as, but not limited to, atosiban, retosiban, barusiban, epelsiban, and nolasiban, as well as variants, formulations, crystalline forms, and derivatives thereof. , including those described in this application.

As used herein, the term pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are suitable for contact with the tissues of a subject, e.g., a mammal (e.g., a human) without undue toxicity, irritation, allergic reaction, and other problematic complications commensurate with an acceptable benefit/risk ratio.

As used herein, the term pharmaceutical composition refers to a mixture containing a therapeutic compound for administration to a subject, e.g., a mammal, e.g., a human, for the prevention, treatment, or control of a specific disease or condition affecting the mammal, e.g., in particular, preterm labor or dysmenorrhea. , for example, as described in this application.

- 6 039545

As used herein, the term protecting group refers to a chemical moiety that, when bound to a functional group, renders the functional group inert to one or more chemical reactions. Such reactions may modify one or more substituents on the compound and, in the absence of a protecting group, will result in undesirable chemical modification (e.g., electron-withdrawing addition, solvolysis, oxidation, reduction, or functional group interconversion) of the component of interest (e.g., amino , hydroxyl, carboxyl or carboxamide component). The protecting groups may, at appropriate times, chemically react in such a way that the original functional group is restored. The identity of the protecting group may be chosen to be compatible with the rest of the molecule, such as such that the protecting group is not removed in subsequent steps in the synthesis or modification of the molecule, and optionally such that the reaction conditions used to effectively remove the protecting group groups do not lead to the removal of other protecting groups located on other substituents in the molecule. Exemplary protecting groups include those groups which may be covalently attached to, for example, an amino substituent, for example the amino group of an a-amino ester. Subsequent deprotection, referred to herein as deprotection of a chemical moiety, can be carried out using reagents and conditions known in the art. Examples of protecting groups include, but are not limited to, benzyl, acetyl, hydroxyacetyl, carboxybenzyl, 9-fluorenyloxycarbonyl, 2-chloro-1-indanylmethoxycarbonyl, benz[G]indene-3methoxycarbonyl, 2-(tert-butylsulfonyl)- 2-propenyloxycarbonyl, benzothiophene sulfone-2-methylcarbonyl, tert-butoxycarbonyl, tert-amyloxycarbonyl, β-trimethylsilylethyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(n-biphenylyl)propyl-2-hydroxycarbonyl, 2-(n-phenylazophenyl)propyl-2 -hydroxycarbonyl, 2-2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, 2-phenylpropyl-2-hydroxycarbonyl, benzyloxycarbonyl, p-toluenesulfonylaminocarbonyl, o-nitrophenylsulfenyl, dithiasuccinoyl, phthaloyl, piperidinooxycarbonyl, formyl, trifluoroacetyl, 2,4,6-trimethoxybenzyl , 2,3,6-trimethyl-4-methoxybenzenesulfonyl, tert-butoxymethyl, pentamethylchromanesulfonyl, adamantly, β-trimethylsilylethyl, β-trimethylylylethyloxycarbonyl, tert-butyl, tert-butylbenzyl, cyclopentyl, triphenylmethyl, benzyloxycarbonyl, formyl, and trifluoro racetyl. Protecting groups may be appropriate for the particular chemical substituent. For example, examples of hydroxyl protecting groups include, but are not limited to, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, dialkylsilyl ethers such as dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl an ether, and tert-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl, benzyl, and p-nitrophenyl. Additional examples of protecting groups can be found, for example, in Greene and Wuts, Protective Groups in Organic Synthesis, 2nd ed., 1991, John Wiley & Sons, and McOmie, Protective Groups in Organic Chemistry, 1975, Plenum Press, disclosure each of which is incorporated into this application by reference. Other examples of protecting groups are described, for example, in US patent No. 3835175; 4508657; 3839396; 4581167; 4460501; and 4,108,846, the disclosure of each of which is incorporated herein by reference.

As used herein in the context of therapeutic treatment, the terms administer and administer refer to the delivery of a therapeutic agent to a subject (e.g., a mammalian subject, e.g., a human) in need of treatment, e.g., a subject at or at risk of developing preterm labor. The therapeutic agent may be administered to a subject in need thereof, for example, by direct administration of the therapeutic agent to the subject, or by administration of a prodrug that is converted in vivo to the therapeutic agent upon administration of the prodrug to the subject. Examples of prodrugs include, but are not limited to, esters, phosphates, and other chemical functionalities susceptible to hydrolysis when administered to a subject. Prodrugs include those known in the art, such as those described in, for example, Vig et al., Adv. drug deliv. Rev. 65:1370-1385 (2013), and Huttunen et al., Pharmacol. Rev. 63:750-771 (2011), the disclosure of each of which is incorporated herein by reference.

As used in this application, the term sample refers to a sample (e.g. blood, blood component (e.g. serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g. placenta or skin), pancreatic juice, villus samples chorion, and cells) isolated from the subject.

As used herein, the phrase specifically binds and binds refers to a binding reaction that determines the presence of a particular protein in a heterogeneous population of proteins and other biological molecules that are recognized, for example, by a ligand with specificity. A ligand (eg, protein, proteoglycan, or glycosaminoglycan) that specifically

- 7 039545 binds to a protein, will bind to a protein, for example, with a K _D of less than 100 nM. For example, a ligand that specifically binds to a protein can bind to a protein with a K _D of up to 100 nM (eg, in the range of 1 pM to 100 nM). A ligand that does not specifically bind to a protein or its domain will exhibit a K _D greater than 100 nM (e.g., greater than 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 µM, 100 µM, 500 µM, or 1 mM) for that particular protein or domain. Various assay formats can be used to determine the affinity of a ligand for a specific protein. For example, solid phase ELISA assays are commonly used to identify ligands that specifically bind to a target protein. See, for example, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description assay formats and conditions that can be used to determine specific protein binding.

As used herein, the terms subject and patient are used interchangeably and refer to an organism that is being treated for a specific disease or condition as described herein (such as preterm labor or dysmenorrhea) or that is diagnosed as having a disease or condition according to in the ways described in this application. Examples of subjects and patients include mammals, such as humans, who have been treated for diseases or conditions, such as premature birth at an early gestational age (eg, 24-34 weeks).

A compound, salt form, crystalline polymorph, therapeutic agent, or other composition as described herein may be referred to as those that are characterized by graphic data essentially as shown in the figure. Such data may include, but are not limited to, for example, X-ray powder diffraction patterns, NMR spectra, differential scanning calorimetry curves, and thermogravimetric analysis curves. As is known in the art, such graphics may provide additional technical information to further define a compound, salt form, crystalline polymorph, therapeutic agent, or other composition. As one of skill in the art would appreciate, such graphical representations of data may be subject to slight variations in, for example, relative peak intensities and peak positions due to factors such as variations in instrument performance and variations in sample concentration and purity. However, one skilled in the art would easily be able to compare the plots in the figures in this application with plots generated for a compound, salt form, crystal polymorph, therapeutic agent, or other composition and confirm whether two sets of plots represent one the same material or two different materials. For example, the crystalline form of (3S)-3-({[(2S)-3-(biphenyl4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}amino)-3-(4-fluorophenyl)propyl L- hydrochloride valate is referred to in this application as that characterized by graphic data essentially as shown in the figure, therefore, will be understood as including any crystal form (3 S)-3-({[(2S)-3-(biphenyl-4 -ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4fluorophenyl)propyl L-valinate hydrochloride, which is characterized by graphical data optionally having one or more minor variations, for example, one or more the number of variations described above or known to a person skilled in the art.

As used herein, the terms treat or treatment refers to therapeutic treatment in which an undesirable physiological change or disorder is prevented or slowed down (reduced) in the subject, e.g., progression of preterm labor at an early gestational age (e.g., 24-34 weeks). Beneficial or desirable clinical results include, but are not limited to, relief of symptoms such as vaginal bleeding or membrane disruption, and delay or delay in labor. Those in need of treatment include, for example, pregnant females already in preterm labor as well as those predisposed to developing the condition.

As used herein, the term tocolytic agent refers to a substance capable of delaying the onset of labor in a subject (eg, a mammalian subject, eg, a human). Tocolytic agents may act by suppressing uterine contractility, for example by increasing cytoplasmic cAMP levels and inhibiting intracellular Ca ²⁺ mobilization. Certain tocolytic agents are described, for example, in Haas et al. Int. J. Women's Health. 6:343-349 (2014), the disclosure of which is incorporated herein by reference. Tocolytic agents for use in combination with the compositions and methods described in this application include, but are not limited to, the substances listed in Table 1 below.

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Table 1 Typical tocolytic agents

Pharmacological Class Typical tocolytic agents Link Betamimetics Terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine, orciprenaline Conde-Agudelo et al. Ash. J. Obstet. Gynecol. 204:ele20 (2011); Creasy et al. Creast and Resnik’s Maternal Fetal Medicine: Principles and Practice. Ed. 6. Philadelphia, PA (2009) Calcium channel blockers Dihydropyridines, eg nifedipine, nicardipine Nassar et al. Am. J. Perinatol. 281:57-66 (2011) Magnesium salts Magnesium sulfate Mercer et al. Obstet. Gynecol. 114:650-668 (2009) Oxytocin receptor antagonists atosiban, retosiban, barusiban, epelsiban, nolasiban Papatsonis et al. Cochrane Database Syst. Rev. 3:CD004452 (2005) Nitric oxide donors Nitroglycerine Duckitt et al. Cochrane Database Syst. Rev. 3:CD002860 (2002)

Brief description of the figures

Fig. 1 is a graph showing the effect of Compound II and Compound III on spontaneous uterine contractility in late pregnant rats following intravenous administration.

Fig. 2 is a graph showing the dose-dependent and reversible effect of Compound I on spontaneous uterine contractions in late pregnant rats.

Fig. 3 is a graph showing the effect of Compound II and Compound III on spontaneous uterine contractility in late pregnant rats following oral administration.

Fig. 4 is a table that summarizes data from the various methods used to prepare the free base of compound I, as well as observations regarding the physical characteristics and NMR spectra of compound I, as obtained using each method.

Fig. 5 is a table that summarizes data from the various methods used to prepare salts of compound I, as well as observations regarding the physical characteristics and NMR spectra of these salts, as obtained using each method.

Fig. 6 is a table summarizing the physical characteristics data as well as powder X-ray diffraction (XRPD) spectra of various salts of Compound I.

Fig. 7 is a table that summarizes data from the methods used to prepare the crystalline forms of the various salts of Compound I, as well as observations regarding the physical characteristics and XRPD spectra of each crystalline form.

Fig. 8 is a table summarizing the solubility data of various salts of Compound I in aqueous solution.

Fig. 9 is a table summarizing the stability data of the crystalline forms of various salts of Compound I at indicated relative humidity (RH).

Fig. 10 is a table summarizing the various characteristics of compound III as determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric (TG) analysis, moisture sorption/desorption (MB), and ¹ H nuclear magnetic resonance (NMR).

Fig. 11 is a table summarizing various characteristics of the hydrosulfate salt of compound I as determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric (TG) analysis, and ¹ H nuclear magnetic resonance (NMR).

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Fig. 12 shows the XRPD spectrum of the mesylate salt of compound I.

Fig. 13 shows the 1H NMR spectrum of the mesylate salt of compound I.

Fig. 14 shows the XRPD spectrum of the free base of compound I.

Fig. 15 shows the 1H NMR spectrum of the free base of compound I.

Fig. 16 shows the Raman infrared spectrum of the free base of compound I.

Fig. 17 shows the 1H NMR spectrum of the mesylate salt of compound I. The mesylate salt was prepared by adding methanesulfonic acid to a solution of the free base of compound I in diethyl ether.

Fig. 18 shows 1H NMR series of the free base spectrum of compound I recorded using homonuclear dissociation experiments.

Fig. 19 shows the XRPD spectrum series of the chloride salt of compound I as obtained from an acetone slurry (top), by evaporation from methylene chloride:ethyl ether (second from top), and by slow evaporation of 1:1 acetone:toluene (second from bottom and bottom) .

Fig. 20 shows the overlap of the differential scanning calorimetry curve (in the range of about -0.5 to about 1.3 W/g) and the thermogravimetric analysis curve (in the range of about 0% to about 100% by weight) recorded for the chloride salt of Compound I as obtained from acetone slurry.

Fig. 21 shows the 1H NMR spectrum of the chloride salt of compound I as prepared from 1:1 acetone:toluene.

Fig. 22 shows a series of XRPD spectra of the chloride salt of compound I as obtained from an acetone slurry (top) and after vacuum drying at about 50° C. for 1 day (bottom).

Fig. 23 shows the overlap of the differential scanning calorimetry curve (in the range of about -1.0 to about 0.2 W/g) and the thermogravimetric analysis curve (in the range of about 30% to about 100% by weight) recorded for the chloride salt of Compound I under vacuum at about 50°C for 1 day.

Fig. 24 shows the overlap of thermogravimetric analysis curves of the chloride salt of compound I as obtained from acetone slurry (top) and after vacuum drying at about 50° C. for 1 day (bottom).

Fig. 25 shows the overlap of differential scanning calorimetry curves recorded for the chloride salt of compound I, as obtained from an acetone slurry (top) and after vacuum drying at about 50° C. for 1 day (bottom).

Fig. 26 shows sorption/desorption curves of moisture recorded for the chloride salt of compound I. The values on the y-axis indicate the percent change in weight of the chloride salt as a function of the relative humidity (RH) in the atmosphere surrounding the salt.

Fig. 27 is a table showing data obtained from moisture sorption/desorption experiments that were carried out with the chloride salt of Compound I.

Fig. 28 shows a moisture sorption/desorption curve recorded for the chloride salt of compound I. The values on the y-axis indicate the percent change in weight of the chloride salt as a function of time during which the relative humidity in the atmosphere surrounding the salt changes.

Fig. 29 shows the XRPD overlaps of the chloride salt of compound I after (top) and before (bottom) moisture sorption/desorption experiments.

Fig. 30 shows the XRPD spectrum overlap of the fumarate salt of compound I obtained by slow evaporation of a 1:1 mixture of methanol:toluene (top) and the XRPD of fumaric acid (bottom).

Fig. 31 shows the XRPD spectrum overlap of the dihydrogen phosphate salt of Compound I (top) and the XRPD of the hydrogen sulfate salt of Compound I (bottom).

Fig. 32 shows the overlap of the differential scanning calorimetry curve (in the range of about -1.9 to about 0 W/g) and the thermogravimetric analysis curve (in the range of about 25% to about 95% by weight) recorded for the hydrosulfate salt of Compound I.

Fig. 33 shows the 1H NMR spectrum of the hydrosulfate salt of compound I.

Fig. 34 shows the 1H NMR spectrum of the sulfate salt of compound I.

Fig. 35 shows the XRPD spectrum of the mesylate salt of compound I.

Fig. 36 shows the XRPD spectrum of the citrate salt of compound I.

Fig. 37 shows the XRPD spectrum of the edisylate salt of compound I.

Fig. 38 shows the XRPD spectrum of the hydrosulfate salt of compound I.

Fig. 39 shows the XRPD spectrum of the citrate salt of Compound I as obtained by slow evaporation of a 1:2 methanol:toluene mixture.

Fig. 40 shows the XRPD spectrum of the hydrosulfate salt of compound I as obtained by slow evaporation of a 6:1 mixture of ethyl acetate-heptane.

Fig. 41 shows the XRPD spectrum of the hydrosulfate salt of compound I, as obtained by slowly evaporating the ethyl acetate mixture.

Fig. 42 shows the XRPD spectrum of the dihydrogen phosphate salt of compound I as obtained by slow evaporation of a 1:2 methanol:acetonitrile mixture.

Fig. 43 shows the XRPD spectrum of the dihydrogen phosphate salt of Compound I as obtained by slow evaporation of a 1:1 mixture of methyl ethyl ketone:n-butyl acetate.

Fig. 44 shows an XRPD spectrum recorded from a duplicate XRPD experiment of the dihydrogen phosphate salt of Compound I, as obtained by slow evaporation of a 1:1 mixture of methyl ethyl ketone:nbutyl acetate.

Fig. 45 shows the XRPD spectrum of the chloride salt of Compound I as obtained by slow evaporation of a 1:1 mixture of acetone:toluene.

Fig. 46 shows an XRPD spectrum recorded from a duplicate XRPD experiment of the chloride salt of Compound I, as obtained by slow evaporation of a 1:1 mixture of acetone:toluene.

Fig. 47 shows the XRPD spectrum of the chloride salt of compound I as obtained by slow evaporation of diethyl ether:methylene chloride.

Fig. 48 shows the XRPD spectrum of the chloride salt of Compound I as obtained from an acetone slurry.

Fig. 49 shows the XRPD spectrum of the chloride salt of Compound I after vacuum drying.

Fig. 50 shows the XRPD spectrum of the fumarate salt of compound I as obtained by slow evaporation of a 1:1 methanol:toluene mixture.

Fig. 51 shows the XRPD spectrum of the fumarate salt of compound I as obtained by slow evaporation of a 1:1 mixture of methanol:ethyl acetate.

Fig. 52 shows the XRPD spectrum of the fumarate salt of compound I as obtained by vacuum drying a 1:1 methanol:toluene mixture.

Fig. 53 shows the XRPD spectrum of the edisylate salt of compound I as obtained by slow evaporation of a 1:1:1 mixture of methanol:methyl ethyl ketone:toluene.

Fig. 54 shows the XRPD spectra overlap of the chloride salt of compound I before (bottom) and after (top) storage at 40° C. and 75% relative humidity.

Fig. 55 is a table summarizing the stability data of the mesylate salt of Compound I and Compound II in the buffer used for Caco-2 permeation experiments: Hank's Balanced Buffered Salt Solution (HBSS), 2% DMSO final concentration.

Fig. 56a is a table describing the data obtained from the analysis of the ability of the mesylate salt of compound I to pass from the apical to the basolateral compartment of a transwell coated with a monolayer of Caco-2 cells. Cultured Caco-2 cells were incubated with the indicated concentration of Compound I mesylate salt in the apical transwell compartment, and aliquots were taken from the basolateral compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data describe the concentration of compound II in the basolateral compartment as a percentage of the indicated initial concentration of the mesylate salt of compound I.

Fig. 56b is a table describing the data obtained from the analysis of the ability of the mesylate salt of Compound I to pass from the basolateral to apical compartment of the transwell coated with a monolayer of Caco-2 cells. Cultured Caco-2 cells were incubated with the indicated concentration of Compound I mesylate salt in the transwell basolateral compartment, and aliquots were taken from the apical compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data describe the concentration of compound II in the basolateral compartment as a percentage of the indicated initial concentration of the mesylate salt of compound I.

Fig. 56c is a graph showing the relative concentration of compound II in the basolateral compartment as a percentage of the initial concentration of the mesylate salt of compound I in the apical compartment.

Fig. 56d is a graph showing the relative concentration of compound II in the apical compartment as a percentage of the initial concentration of the mesylate salt of compound I in the basolateral compartment. Compound I was not detected in the basolateral compartment after incubation for 60 or 120 minutes in the apical compartment. Additionally, Compound I was not detected in the apical compartment after incubation for 60 or 120 minutes in the basolateral compartment. In contrast, compound II was found in every case.

Fig. 56e is a table showing the recovery of Compound I in the apical compartment after incubation for 120 minutes. The parent compound was predominantly reduced as the deesterified variant, Compound II.

Fig. 58a is a table describing the data obtained from the analysis of the ability of the mesylate salt of Compound I to pass from the apical to the basolateral compartment of the transwell coated with a monolayer of Caco-2 cells. Cultured Caco-2 cells were incubated with the indicated concentration of Compound I mesylate salt in the apical transwell compartment, and aliquots were taken from the basolateral compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data describe the concentration of compound II in the basolateral compartment as a percentage of the indicated initial concentration of the mesylate salt of the compound

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I. Compound I was not detected in the basolateral compartment after incubation for 60 or 120 minutes in the apical compartment.

Fig. 58b is a graph showing the relative concentration of compound II in the basolateral compartment as a percentage of the initial concentration of the mesylate salt of compound I in the apical compartment.

Fig. 58c is a table showing the recovery of Compound I in the apical compartment after incubation for 120 minutes. The parent compound was predominantly reduced in the form of a deesterified variant of the compound, Compound II.

Fig. 59 is a table summarizing data from the chromatography and mass spectrometry parameters used to analyze the concentration of Compound I and Compound II in the Caco-2 cell penetration experiments described herein.

Fig. 60a is a graph illustrating the fractional viability of progeny CD-1 mice treated with RU486 or lipopolysaccharide (LPS) at a gestational age of 17 days so as to induce labor. Values are the mean plus/minus the standard error of the mean. An asterisk denotes a p value <0.05. Statistical analysis was performed using the Mann-Whitney test for the respective group.

Fig. 60b is a graph illustrating the number of viable and non-viable offspring of CD-1 mice treated with RU486 or LPS at 17 days gestational age so as to induce labor.

Fig. 61a is a graph illustrating the time from induction to first birth for CD-1 mice treated with RU486 or LPS at 17 days gestational age so as to induce labor. Values are the mean plus/minus the standard error of the mean.

Fig. 61b and 61c are graphs illustrating the time from induction to completion of labor among CD-1 mice treated with RU486 or LPS at a gestational age of 17 days so as to induce labor. Values along the y-axis indicate the proportion of CD-1 mice that completed parturition. In each figure, an asterisk denotes a p value of p<0.05. Statistical analysis was performed using the Mann-Whitney test or log-rank test relative to the respective group.

Fig. 62a is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously) and nifedipine (5 mg/kg administered orally) on the fractional viability of progeny CD-1 mice treated with RU486 or lipopolysaccharide (LPS) in gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean. An asterisk denotes a p value <0.05; nd denotes a p value of p>0.05. Statistical analysis was performed using the Mann-Whitney test or an unpaired two-tailed Student's t-test compared to the respective vehicle group.

Fig. 62b is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously) and nifedipine (5 mg/kg administered orally) on viable and non-viable offspring of CD-1 mice treated with RU486 or LPS at gestational age. 17 days of age in such a way as to induce labor.

Fig. 63a is a graph showing the effects of Compound III (10 mg/kg, 30 mg/kg, and 100 mg/kg orally) on the fractional viability of progeny CD-1 mice treated with RU486 or LPS at gestational age 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean, nd denotes a p value of p>0.05. Statistical analysis was performed using the Mann-Whitney test compared to the respective vehicle group.

Fig. 63b is a graph showing the effects of Compound III (10 mg/kg, 30 mg/kg, and 100 mg/kg orally) on viable and non-viable offspring of CD-1 mice treated with RU486 or LPS at gestational age. 17 days of age in such a way as to induce labor.

Fig. 64a is a graph demonstrating the effects of nifedipine (5 mg/kg orally), compound III (100 mg/kg orally), and their combination on the fractional viability of progeny CD-1 mice treated with gestational RU486. 17 days of age in such a way as to induce labor. Values are mean plus/minus standard error of the mean, nd denotes a p value of p>0.05 relative to the corresponding group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test compared to the corresponding group of interest.

Fig. 64b is a graph demonstrating the effects of nifedipine (5 mg/kg orally), Compound III (100 mg/kg orally) and their combination on viable and non-viable offspring of CD-1 mice treated with RU486

- 12 039545 at a gestational age of 17 days in such a way as to induce labor.

Fig. 65a is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (100 mg/kg orally), and their combination on time from induction to first birth in CD-1 mice treated with with RU486 at a gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean. Three asterisks denote a p value <0.001 relative to the respective group; two asterisks denote a p value <0.01 relative to the corresponding group. Nifedipine, Compound III, and the combination thereof exhibited p values p=0.0576, p=0.0601, and p<0.001 (indicated by $$$), respectively, compared to the vehicle treated group alone. Statistical analysis was performed using the Mann-Whitney test or unpaired two-tailed Student's t-test compared with the corresponding group of interest.

Fig. 65b is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (100 mg/kg orally), and their combination on induction to completion of labor in CD-1 mice treated with using RU486 at gestational age 17 days in such a way as to induce labor. Values along the y-axis indicate the proportion of CD-1 mice that completed parturition.

Fig. 65c is a graph showing the time from induction to completion of fruiting for the vehicle and combination shown in FIG. 65b. Three asterisks denote a p value <0.001 relative to the corresponding group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 65d is a graph showing the time from induction to completion of fetuses for Compound III and the combination shown in FIG. 65b. Three asterisks denote a p value <0.001 relative to the corresponding group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 65e is a graph showing the time from induction to completion of fetal delivery for nifedipine and the combination shown in FIG. 65b. Two asterisks denote a p value of p<0.01 relative to the corresponding group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 66a is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), compound III (100 mg/kg administered orally), and their combination on the fractional viability of progeny CD-1 mice treated with RU486 in gestational age of 17 days in such a way as to induce labor. Values are mean plus/minus standard error of the mean, nd denotes a p value of p>0.05 relative to the corresponding group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test compared to the corresponding group of interest.

Fig. 66b is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on viable and non-viable offspring of CD-1 mice treated with LPS at a gestational age of 17 days in such a way as to induce labor.

Fig. 67a is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally) and their combination on time from induction to first birth in CD-1 mice, treated with RU486 at a gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean, nd denotes a p value of p>0.05 relative to the respective group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group. Atosiban, Compound III, and their combination exhibit p values p>0.05, p=0.0601, and p>0.05, respectively, compared to the vehicle group. Statistical analysis was performed using an unpaired two-tailed Student's t-test compared with the corresponding group of interest.

Fig. 67b is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on induction to completion of labor among CD-1 mice treated with with RU486 at a gestational age of 17 days in such a way as to induce labor. Values along the y-axis indicate the proportion of CD-1 mice that completed parturition.

Fig. 67c is a graph showing the time from induction to completion of fetal production for the vehicle and combination shown in FIG. 67b. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a logarithmic rank test compared to the corresponding group of interest.

Fig. 67d is a graph showing the time from induction to completion of gestation for Compound III and the combination shown in FIG. 67b. Their combination exhibits a p value of p=0.0832 relative to the combination with compound III. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 67e is a graph showing the time from induction to completion of fetuses for atosiban and the combination shown in FIG. 67b. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 68a is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (10 mg/kg, 30 mg/kg, and 100 mg/kg orally), and combinations thereof, on offspring fractional viability. CD-1 mice treated with LPS at a gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean, nd denotes a p value of p>0.05 relative to the corresponding group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group. The nifedipine group exhibited a p value of p=0.0859 compared to the vehicle treated group alone. Statistical analysis was performed using the Mann-Whitney test or unpaired two-tailed Student's t-test compared with the corresponding group of interest.

Fig. 68b is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (10 mg/kg, 30 mg/kg, and 100 mg/kg orally), and combinations thereof on viable and nonviable offspring of CD-1 mice treated with LPS at gestational age of 17 days so as to induce labor.

Fig. 69a is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (10 mg/kg, 30 mg/kg and 100 mg/kg orally), and their combinations on time from induction to birth of the first fetus for CD-1 mice treated with LPS at a gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean. Two asterisks denote a p value <0.01 relative to the respective group, as assessed by the Mann-Whitney test relative to the respective group; nd denotes a p value of p>0.05 relative to the corresponding group, as assessed using the Mann-Whitney test relative to the corresponding group; ND denotes p value p>0.05 compared to vehicle matched group as assessed by unpaired two-tailed Student's t-test relative to matched group; no testing means that no statistical testing was performed on the specified pair.

Fig. 69b is a graph showing the effects of nifedipine (5 mg/kg orally), Compound III (10 mg/kg orally), and their combinations on time from induction to completion of labor in CD-1 mice treated with using LPS at 17 days gestational age in such a way as to induce labor.

Fig. 69c is a graph showing the effects of nifedipine (5 mg/kg orally), compound III (30 mg/kg orally), and their combinations on time from induction to completion of labor in CD-1 mice treated with using LPS at 17 days gestational age in such a way as to induce labor.

Fig. 69d is a graph demonstrating the effects of nifedipine (5 mg/kg orally), Compound III (100 mg/kg orally), and their combinations on time from induction to completion of labor in CD-1 mice treated with using LPS at 17 days gestational age in such a way as to induce labor.

Fig. 69e is a graph showing the time from induction to completion of fetal production for the vehicle and combination shown in FIG. 69b. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69f is a graph showing the time from induction to completion of fetuses for Compound III and the combination shown in FIG. 69b. Two asterisks denote a p value of p<0.01 relative to the respective group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 69g is a graph showing the time from induction to completion of fetal delivery for nifedipine and the combination shown in FIG. 69b. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69h is a graph showing the time from induction to completion of fetal delivery for the vehicle and combination shown in FIG. 69s. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69i is a graph showing the time from induction to completion of fetuses for Compound III and the combination shown in FIG. 69s. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69j is a graph showing the time from induction to completion of fetuses for nifedipine and the combination shown in FIG. 69s. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69k is a graph showing the time from induction to completion of birth for the vehicle and combination shown in FIG. 69d. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69l is a graph showing the time from induction to completion of fetuses for Compound III and the combination shown in FIG. 69d. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 69m is a graph showing the time from induction to completion of fetuses for nifedipine and the combination shown in FIG. 69d. nd denotes a p value of p>0.05 relative to the respective group. Statistical analysis was performed using a log-rank test relative to the respective group.

Fig. 70a is a graph demonstrating the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on the fractional viability of progeny CD-1 mice treated with LPS in gestational age of 17 days in such a way as to induce labor. Values are the mean plus/minus the standard error of the mean, nd denotes a p value of p>0.05 relative to the corresponding group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test or unpaired two-tailed Student's t-test compared with the corresponding group of interest.

Fig. 70b is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on viable and non-viable offspring of CD-1 mice treated with LPS at a gestational age of 17 days in such a way as to induce labor.

Fig. 71a is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on time from induction to first birth in CD-1 mice, treated with LPS at a gestational age of 17 days so as to induce labor. Values are the mean plus/minus the standard error of the mean, nd denotes a p value of p>0.05 relative to the respective group; ND denotes a p value of p>0.05 compared to the corresponding vehicle group; $ denotes a p value of p<0.05 compared to the corresponding vehicle group. Their combination exhibits a p value of p=0.0909 compared to the atosiban treated group alone. Statistical analysis was performed using the Mann-Whitney test or unpaired two-tailed Student's t-test compared with the corresponding group of interest.

Fig. 71b is a graph showing the effects of atosiban (300 mg/kg administered subcutaneously), Compound III (100 mg/kg administered orally), and their combination on induction to completion of labor among CD-1 mice treated with with LPS at a gestational age of 17 days in such a way as to induce labor.

Fig. 71c is a graph showing the time from induction to completion of fetal production for the vehicle and combination shown in FIG. 71b. Two asterisks denote a p value of p<0.01 relative to the corresponding group. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 71d is a graph showing the time from induction to completion of fruiting for Compound III and the combination shown in FIG. 71b. Their combination exhibits a p value of p=0.0964 relative to the combination with compound III. Statistical analysis was performed using a log-rank test compared to the corresponding group of interest.

Fig. 71e is a graph showing the time from induction to completion of fetuses for atosiban and the combination shown in FIG. 71b. An asterisk denotes a p value <0.05 relative to the corresponding group. Statistical analysis was carried out using

- 15 039545 logarithmic rank test compared to the corresponding group of interest.

Fig. 74a is a graph demonstrating the effects of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the frequency of PGF2α-induced smooth muscle contractions in N=6 first-term endometrial biopsies at term collected from female subjects, delivered by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, measurements of the baseline frequency of spontaneous contractions were recorded. Frequency measurements of spontaneous contractions are presented on the x-axis as Self. DMSO control or atosiban (Ato) was then added to each myometrial sample at the indicated concentrations and the effect of control or atosiban on heart rate was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on heart rate in the presence of PGF2a were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGF2a (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGF2a 1 nM, PGF2a 10 nM, and PGF2a 100 nM, respectively. Values along the y-axis represent the frequency of contractions as a percentage of the frequency of spontaneous initial contractions. Asterisk denotes p value <0.05 compared to DMSO control.

Fig. 74b is a graph demonstrating the effects of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the work performed on contraction (area under the curve, or AUC) of POP2a-induced smooth muscle contractions in N=6 endometrial biopsies in the first stage of labor at term, selected from female subjects subjected to delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work performed on the contraction were recorded. Measurements of spontaneous work performed for contraction are presented on the x-axis as Self-restraint. A DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of the control or atosiban on contraction work was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on work performed on contraction in the presence of PGF2a were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGF2a (1 nM, 10 nM, and 100 nM) at successive 10 minute intervals. These time points are represented on the x-axis as PGF2a 1 nM, PGF2a 10 nM, and PGF2a 100 nM, respectively. Values along the y-axis represent work done on contraction as a percentage of work done on contraction for spontaneous initial contractions.

Fig. 74c is a graph demonstrating the effects of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the peak amplitude of PCP2a-induced smooth muscle contractions in N=6 first-term endometrial biopsies at term from female subjects who underwent delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After the establishment of regular contractions for at least 20 minutes, measurements of the initial level of the amplitude of the peak of spontaneous contractions were recorded. The spontaneous contraction peak amplitude measurements are presented on the x-axis as Self-adv. DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of the control or atosiban on peak contraction amplitude was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on peak contraction amplitude in the presence of PGF2a were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGF2a (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGF2a 1 nM, PGF2a 10 nM, and PGF2a 100 nM, respectively. The values along the y-axis represent the amplitude of the peak contractions as a percentage of the amplitude of the peak of spontaneous initial contractions.

Fig. 74d is a graph demonstrating the effects of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the duration of PGF2α-induced smooth muscle contractions in N=6 first-term endometrial biopsies at term from female subjects undergoing delivery. by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of the duration of spontaneous contractions were recorded. The measurements of the duration of spontaneous contractions are presented on the x-axis as Self-adv.

- 16 039545

DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of control or atosiban on contraction duration was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on contraction duration in the presence of PGF2a were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGF2a (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGF2a 1 nM, PGF2a 10 nM, and PGF2a 100 nM, respectively. Values along the y-axis represent the duration of contractions as a percentage of the duration of spontaneous initial contractions.

Fig. 74e is a graph demonstrating the effects of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on total work done on all contractions (sum of area under the curve for all contractions) for PGF2α-induced smooth muscle contractions in N= 6 biopsies of the endometrium in the first stage of labor at term, collected from female subjects undergoing delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, baseline measurements of work performed for all spontaneous contractions were recorded. Measurements of the work done for all spontaneous contractions are presented on the x-axis as Self. A DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on total work performed for all subsequent contractions was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on total contraction work in the presence of PGF2a were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGF2a (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as PGF2a 1 nM, PGF2a 10 nM, and PGF2a 100 nM, respectively. The values along the y-axis represent the total work done by contractions as a percentage of the total work done by spontaneous initial contractions. Asterisk denotes p value <0.05 compared to DMSO control.

Fig. 75a is a graph demonstrating the effects of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the frequency of PGE2-induced smooth muscle contractions in N=6 first-term endometrial biopsies collected from female subjects, delivered by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, measurements of the baseline frequency of spontaneous contractions were recorded. Frequency measurements of spontaneous contractions are presented on the x-axis as Self. DMSO control or atosiban (Ato) was then added to each myometrial sample at the indicated concentrations and the effect of control or atosiban on heart rate was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on heart rate in the presence of PGE2 were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGE2 (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGE2 1 nM, PGE2 10 nM, and PGE2 100 nM, respectively. Values along the y-axis represent the frequency of contractions as a percentage of the frequency of spontaneous initial contractions. Three asterisks denote p value <0.001 compared to DMSO control.

Fig. 75b is a graph demonstrating the effect of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on work done on contraction (area under the curve, or AUC) of PGE2-induced smooth muscle contractions in N=6 endometrial biopsies in the first stage of labor at term, selected from female subjects subjected to delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work performed on the contraction were recorded. Measurements of spontaneous work performed for contraction are presented on the x-axis as Self-restraint. A DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of the control or atosiban on contraction work was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on work performed on contraction in the presence of PGE2 were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGE2 (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGE2 1 nM, PGE2 10 nM, and PGE2 100 nM, respectively. Values along the y-axis represent work done on contraction as a percentage of work done on contraction for spontaneous initial contractions.

Fig. 75c is a graph showing the effects of various

- 17,039,545 doses of atosiban (6 nM, 60 nM, and 600 nM) on PGE2-induced smooth muscle contraction peak amplitude in N=6 first-term endometrial biopsies collected from female subjects delivered by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After the establishment of regular contractions for at least 20 minutes, measurements of the initial level of the amplitude of the peak of spontaneous contractions were recorded. The spontaneous contraction peak amplitude measurements are presented on the x-axis as Self-adv. DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of the control or atosiban on peak contraction amplitude was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on peak contraction amplitude in the presence of PGE2 were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGE2 (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGE2 1 nM, PGE2 10 nM, and PGE2 100 nM, respectively. The values along the y-axis represent the amplitude of the peak contractions as a percentage of the amplitude of the peak of spontaneous initial contractions. Asterisk denotes p value <0.05 compared to DMSO control.

Fig. 75d is a graph demonstrating the effects of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the duration of PGE2-induced smooth muscle contractions in N=6 first-term endometrial biopsies at term from female subjects undergoing delivery. by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of the duration of spontaneous contractions were recorded. The measurements of the duration of spontaneous contractions are presented on the x-axis as Self-adv. DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations and the effect of control or atosiban on contraction duration was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on contraction duration in the presence of PGE2 were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGE2 (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as PGE2 1 nM, PGE2 10 nM, and PGE2 100 nM, respectively. Values along the y-axis represent the duration of contractions as a percentage of the duration of spontaneous initial contractions.

Fig. 75e is a graph demonstrating the effects of different concentrations of atosiban (6 nM, 60 nM, and 600 nM) on total work done on all contractions (sum of area under the curve for all contractions) for PGE2-induced smooth muscle contractions in N= 6 biopsies of the endometrium in the first stage of labor at term, collected from female subjects undergoing delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, baseline measurements of work performed for all spontaneous contractions were recorded. Measurements of the work done for all spontaneous contractions are presented on the x-axis as Self. A DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on total work performed for all subsequent contractions was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Ato. The effects of atosiban on total contraction work in the presence of PGE2 were subsequently measured by exposing myometrial tissue samples to increasing concentrations of PGE2 (1 nM, 10 nM, and 100 nM) at subsequent 10 minute intervals. These time points are represented on the x-axis as PGE2 1 nM, PGE2 10 nM, and PGE2 100 nM, respectively. The values along the y-axis represent the total work done by contractions as a percentage of the total work done by spontaneous initial contractions. Asterisk denotes p value <0.05 compared to DMSO control. Three asterisks denote p value <0.001 compared to DMSO control.

Fig. 77a is a graph showing the effects of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the frequency of RT-induced smooth muscle contractions in N=2 first-term endometrial biopsies at term, selected from subjects of women subjected to delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, measurements of the baseline frequency of spontaneous contractions were recorded. Frequency measurements of spontaneous contractions are presented on the x-axis as Self. DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations and effects of the control.

- 18 039545 or nifedipine on the frequency of contractions was measured during the subsequent 10-minute period. This time point is represented on the x-axis as Nif. The effects of nifedipine on heart rate in the presence of OT were subsequently measured by exposing myometrial tissue samples to increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as OT 1 nM, OT 10 nM, and OT 100 nM, respectively. Values along the y-axis represent the frequency of contractions as a percentage of the frequency of spontaneous initial contractions.

Fig. 77b is a graph demonstrating the effects of different concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on work done on contraction (area under the curve, or AUC) of RT-induced smooth muscle contractions in N=2 first-term endometrial biopsies taken from female subjects undergoing caesarean delivery. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work performed on the contraction were recorded. Measurements of spontaneous work performed for contraction are presented on the x-axis as Self-restraint. A DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations and the effect of the control or nifedipine on contraction work was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Nif. The effects of nifedipine on contraction work performed in the presence of OT were subsequently measured by exposing myometrial tissue samples to increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10 minute intervals. These time points are represented on the x-axis as OT 1 nM, OT 10 nM, and OT 100 nM, respectively. Values along the y-axis represent work done on contraction as a percentage of work done on contraction for spontaneous initial contractions.

Fig. 77c is a graph showing the effects of different concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 µM) on the peak amplitude of RT-induced smooth muscle contractions in N=2 endometrial biopsies in the first stage of labor at term. taken from female subjects undergoing delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After the establishment of regular contractions for at least 20 minutes, measurements of the initial level of the amplitude of the peak of spontaneous contractions were recorded. The spontaneous contraction peak amplitude measurements are presented on the x-axis as Self-adv. DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations and the effect of control or nifedipine on peak contraction amplitude was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Nif. The effects of nifedipine on peak contraction amplitude in the presence of OT were subsequently measured by exposing myometrial tissue samples to increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as OT 1 nM, OT 10 nM, and OT 100 nM, respectively. The values along the y-axis represent the amplitude of the peak contractions as a percentage of the amplitude of the peak of spontaneous initial contractions.

Fig. 77d is a graph showing the effects of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the duration of RT-induced smooth muscle contractions in N=2 first-term endometrial biopsies at term, selected from female subjects undergoing delivery by caesarean section. Experiments were performed using DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After regular contractions were established for at least 20 minutes, baseline measurements of the duration of spontaneous contractions were recorded. The measurements of the duration of spontaneous contractions are presented on the x-axis as Self-adv. DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations and the effect of control or nifedipine on contraction duration was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Nif. The effects of nifedipine on contraction duration in the presence of OT were subsequently measured by exposing myometrial tissue samples to increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as OT 1 nM, OT 10 nM, and OT 100 nM, respectively. Values along the y-axis represent the duration of contractions as a percentage of the duration of spontaneous initial contractions.

Fig. 77e is a graph showing the effects of different concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 µM) on total work done on all contractions (sum of area under the curve for all contractions) for RT-induced smooth muscle contractions in N=2 first-term endometrial biopsies collected from female subjects delivered by caesarean section. The experiments were carried out using

- 19 039545 DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. After establishing regular contractions for at least 20 minutes, baseline measurements of work performed for all spontaneous contractions were recorded. Measurements of the work done for all spontaneous contractions are presented on the x-axis as Self. A DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations and the effect of the control or nifedipine on the overall work performed for all subsequent contractions was measured over a subsequent 10 minute period. This time point is represented on the x-axis as Nif. The effects of nifedipine on total contraction work in the presence of OT were subsequently measured by exposing myometrial tissue samples to increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as OT 1 nM, OT 10 nM, and OT 100 nM, respectively. The values along the y-axis represent the total work done by contractions as a percentage of the total work done by spontaneous initial contractions.

Fig. 79a is a Western blot showing the effects of oxytocin, nolasiban, and combinations thereof on the expression of phosphorylated p65 (p-p65), phosphorylated p38 (p-p38), and phosphorylated extracellular signal-regulated kinase (p-ERK) in N=6 biopsies. endometrium in the first stage of labor at term, selected from female subjects subjected to delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with nolasiban at a concentration of 1 μM, or treated with both drugs, oxytocin and nolasiban, at a concentration of 1 μM for the indicated time periods. A β-actin blot was performed as a control. Fig. 79b is a Western blot showing the effects of oxytocin and/or various concentrations of compound II, optionally in combination with nolasiban, on the expression of p-p65, p-p38, and p-ERK in N=6 endometrial biopsies in the first stage of labor at term. taken from female subjects undergoing delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with compound II at a concentration of 3 μM, or treated with both drugs, oxytocin and compound II at various concentrations of compound II, both in the presence and absence of nolasiban at a concentration of 1 µM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 79c is a Western blot showing the effects of oxytocin, nolasiban, and their combination on the expression of the pro-inflammatory genes cyclooxygenase 2 (COX-2) and phosphorylated calcium-dependent phospholipase A2 (p-cPLA2) in N=6 endometrial biopsies in the first stage of labor at term. taken from female subjects undergoing delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with nolasiban at a concentration of 1 μM, or treated with both drugs, oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 79d is a Western blot showing the effects of oxytocin and/or various concentrations of compound II, optionally in combination with nolasiban, on the expression of the pro-inflammatory genes COX-2 and p-cPLA2 in N=6 first-term endometrial biopsies collected from female subjects undergoing delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with compound II at a concentration of 3 μM, or treated with both drugs, oxytocin and compound II at various concentrations of compound II, both in the presence and absence of nolasiban at a concentration of 1 µM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 79e is a plot of the quantification of the expression of p-p65 shown in FIG. 79a and 79b.

Fig. 79f is a plot of the quantification of the expression of p-p38 shown in FIG. 79a and 79b.

Fig. 79g is a plot of the p-ERK expression quantification shown in FIG. 79a and 79b.

Fig. 79h is a plot of the quantification of COX-2 expression shown in FIG. 79c and 79d. An asterisk denotes p value <0.05 compared to unstimulated (NA) samples. Two asterisks denote p value <0.01 compared to unstimulated samples. Three asterisks denote p value <0.001 compared to unstimulated samples. Three # symbols denote a p value of p<0.001 compared to oxytocin (OT) treated samples.

Fig. 79i is a plot of the p-cPLA2 expression quantification shown in FIG. 79c and 79d. An asterisk denotes p value <0.05 compared to unstimulated (NA) samples. Three asterisks denote p value <0.001 compared to unstimulated samples.

- 20 039545

Fig. 80a is a Western blot showing the effects of oxytocin, nolasiban, and combinations thereof on the expression of p-p65, p-p38, and p-ERK in N=3 first-term amnion biopsies at term collected from female subjects undergoing delivery. by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with nolasiban at a concentration of 1 μM, or treated with both drugs, oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 80b is a Western blot showing the effects of oxytocin and/or various concentrations of compound II, optionally in combination with nolasiban, on the expression of pp65, pp38, and p-ERK in N=3 first-term amnion biopsies at term selected in female subjects undergoing delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with compound II at a concentration of 3 μM, or treated with both drugs, oxytocin and compound II at various concentrations of compound II, both in the presence and absence of nolasiban at a concentration of 1 µM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 80c is a Western blot showing the effects of oxytocin, nolasiban, and their combination on the expression of the pro-inflammatory COX-2 and p-cPLA2 genes in N=3 first-term amnion biopsies taken at term from female subjects delivered by caesarean section. . Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with nolasiban at a concentration of 1 μM, or treated with both drugs, oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. A β-actin blot was performed as a control.

Fig. 80d is a Western blot showing the effects of oxytocin and/or various concentrations of Compound II, optionally in combination with nolasiban, on the expression of the pro-inflammatory genes COX-2 and p-cPLA2 in N=3 first-term amnion biopsies taken from female subjects undergoing delivery by caesarean section. Samples were either unstimulated (NS), stimulated with oxytocin (OT), treated with compound II at a concentration of 3 μM, or treated with both drugs, oxytocin and compound II at various concentrations of compound II, both in the presence and absence of nolasiban at a concentration of 1 µM for the indicated time periods. A β-actin blot was performed as a control.

Detailed description

The invention provides α-amino esters of thiazolidine carboxamide, for example, (3S)-3({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3 -(4-fluorophenyl)propyl Lvalinate, as well as its salt forms and crystalline polymorphs. These compounds are capable of inhibiting the activity of prostaglandin F receptor (FP-R) family proteins, for example the prostaglandin F2a receptor (PGF2a). Compounds, salts and crystalline polymorphs described in this application can be used to inhibit the activity of the prostaglandin F receptor in vitro and in vivo, and are effective therapeutic compositions for the treatment of preterm labor. The compounds, salts, and crystalline polymorphs described herein may be administered to a subject (e.g., a mammalian subject, e.g., a human) who is having or is at risk of giving birth at an early gestational age, e.g., before 38 weeks (e.g., from about 20 to about 37 weeks, e.g., gestational age about 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks , 33 weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks, preferably from about 24 to about 34 weeks, for example, a gestational age of about 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, or 34 weeks). The invention further provides methods for the synthesis of (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}amino)-3-(4fluorophenyl)propyl L -valinate, as well as methods for obtaining its salt forms and crystalline polymorphs. The invention further encompasses methods of treating preterm labor in a subject by administering an alpha-aminoester of the invention to a subject in need of treatment, such as a subject in preterm labor or a subject at risk of developing preterm labor, optionally in combination with one or several additional therapeutic agents, as described in this application.

In addition to the above, the invention encompasses compositions and methods relating to 3-([1,1'-biphenyl]-4-ylsulfonyl)-N-[1-(4-fluorophenyl)-3-hydroxypropyl]-1,3-thiazolidine -2-carboxamide. As described herein, this compound may be administered to a subject (e.g., a mammalian subject, e.g., a human) who is having or is at risk of giving birth at an early gestational age, e.g., before 38 weeks (e.g., about 20 to about 37 weeks, for example, gestational age is about 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks, preferably about 24 to about 34 weeks, e.g. gestational age

- 21 039545 at about 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, weeks, or 34 weeks), optionally in combination with one or more additional therapeutic agents as described in this application.

(3S)-3-({[(2S)-3-(Biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl) propyl L-valinate (Compound I).

The invention is based on the discovery that compound I ((3S)-3-({[(2S)-3-(biphenyl-4ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-( 4-fluorophenyl)propyl L-valinate represented by formula I, below) and its salts are converted in vivo to 3-([1,1'-biphenyl]-4-ylsulfonyl)-N-[1-(4fluorophenyl)-3- hydroxypropyl]-1,3-thiazolidine-2-carboxamide (represented by Formula II, below). Compound II, previously described in US 8415480, is a prostaglandin F receptor antagonist as this compound exhibits an inhibition constant (Ki) of 6 nM for human FP-R as determined by competitive radiolabeled ligand binding assays (experimental details of competitive assays radiolabeled ligand bindings useful for determining Ki values are described, for example, in US 8,415,480, example 51). It has been found that after administration to a subject, Compound I is deesterified in vivo such that Compound II is formed due to the activity of endogenous esterases, such as those present in the gastrointestinal tract.

Compound I was discovered to be a prostaglandin F receptor inhibitor since Compound I inhibits human FP-R with a Ki of 1 nM. Compound I shows improvements in several physicochemical characteristics relative to compound II, including solubility in water, as well as in media that simulate the content of the small intestine in fed (FeSSIF) and hungry (FaSSIF) states. These data are summarized in Table 2 below.

Table 2. Comparison of physicochemical properties of compound I and compound II

Parameter Compound I Compound II Solubility in water (µg/ml) 380 0.4 Solubility in FaSSIF (µg/mL) pH 6.5 70 0.4 Solubility in FeSSIF (µg/mL) pH 5.0 90 ten

Human Ki FP-R (nM) 1 6

In addition to exhibiting an increased solubility wave, Compound I and its salts exhibit an unexpected and favorable absorption mechanism. As described in the examples below, Compound I is deesterified by environmental esterases in the small intestine and then passively passes through the epithelium of the small intestine. Surprisingly, Compound I and its salts are not substrates for the PepT 1 carrier protein, a proton-coupled co-transporter that mediates the absorption of peptide nutrients. This discovery represents an unexpected and pharmacologically beneficial property. PepT1 is known to mediate the absorption of various valate esters, as described, for example, in Vig et al., Adv. drug deliv. Rev. 65:1370-1385 (2013), the disclosure of which is incorporated herein by reference. PepT1 exhibits a wide range of substrate specificity, as evidenced by the structural diversity of compounds that are carried by this protein across the intestinal epithelium. Despite the presence of a valinate ester functional group, Compound I and its salts are not dependent on this transporter for absorption through the intestinal epithelium. This is a favorable property because Compound I and its salts (eg Compound III) thus do not compete with natural PepT1 substrates, eg peptide nutrients, for binding and transport by this protein. In contrast, Compound I and its salts are converted in vivo to a form that is readily absorbed in a manner independent of energy and local proton gradient. This unexpected property, associated with the high aqueous solubility of compound I and its salts, collectively provides a favorable pharmacokinetic profile whereby the compounds of the invention readily dissolve in an aqueous medium and in turn are converted to a carrier-independent absorbable form.

(3S)-3-({[(2S)-3-(Biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl) propyl L-valinate hydrochloride (Compound III).

- 22 039545

It was discovered that the chloride salt of compound I ((3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3thiazolidin-2-yl]carbonyl}amino)-3-(4- fluorophenyl)propyl L-valinate hydrochloride, designated as III below) crystallizes easily using a variety of different experimental techniques, as described in the examples below. Compound III assumes a single, reproducible crystalline form upon crystallization from various media and under various environmental conditions. In addition, this crystalline form of compound III exhibits increased stability under ambient conditions and in the presence of elevated relative humidity. As described in more detail in the Examples below, Compound III exhibits low hygroscopicity and therefore does not tend to absorb moisture from the local atmosphere. Thus, compound III exhibits resistance to chemical changes, such as hydrolysis, as well as resistance to the incorporation of impurities. For example, impurities associated with atmospheric water are not easily integrated into the crystalline form of compound III. Compound III may be administered to a subject, e.g., a pregnant woman, to delay the onset of labor in the subject, e.g., one or more days or weeks, e.g., from about 1 day to about 16 weeks (e.g., 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks). Compound III may be administered to a subject, such as a pregnant woman, to relieve one or more of the symptoms associated with childbirth, such as vaginal bleeding and rupture of the uterine membranes.

Compound I, or a pharmaceutically acceptable salt thereof, eg Compound III, may be administered alone or in combination with one or more additional agents, eg an additional therapeutic agent. Typical additional therapeutic agents include additional tocolytic agents, for example, the oxytocin receptor antagonist described in this application, including, for example, atosiban, retosiban, barusiban, epelsiban and nolasiban, which is (3Z, 5S)-5-(hydroxymethyl)-1 -[(2'-methyl-1,1'-biphenyl-4-yl)carbonyl]pyrrolidin-3one O-methyl oxime, or a variant, drug, crystalline form, or derivative thereof. By inhibiting signal transduction with oxytocin, oxytocin receptor antagonists can act synergistically with the prostaglandin F2a receptor antagonists described herein to slow or stop uterine contractions, for example, in a patient who is experiencing or at risk of developing (e.g., one or more of the symptoms present) ) preterm birth. Typical additional tocolytic agents include betamimetics, eg terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine, and orciprenaline, which may act by inactivating myosin light chain kinase and/or by depleting myometrial Ca ²⁺ reserves by upregulating cAMP, downregulating, thus, the contractility of the uterus. Calcium channel blockers, such as dihydropyridines (eg, nifedipine and nicardipine), may additionally or alternatively be administered in combination with a compound of the invention, for example, to modulate [Ca ²⁺ ] in the myometrium and suppress Ca ²⁺ -mediated activation of myosin filaments, which leads to contraction of the myometrium. Magnesium salts, eg magnesium sulfate, may additionally or alternatively be administered in combination with a compound of the invention, eg to hyperpolarize the plasma membrane and/or compete with Ca ²⁺ for binding to the myosin light chain. Additionally or alternatively, nitric oxide donors, such as nitroglycerin, may be administered in combination with a compound described herein, for example, to increase myometrial cyclic guanosine monophosphate levels, thereby inactivating myosin filament light chains.

A compound of the invention, e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III, may additionally or alternatively be administered in combination with progesterone or a variant or derivative thereof, e.g. 17-a-hydroxyprogesterone, to suppress uterine contractility in a subject, who develops or is at risk of developing (for example, one or more symptoms are present) preterm birth.

Additionally or alternatively, a compound of the invention may be administered in combination with a corticosteroid as described herein or known in the art, for example, to promote fetal lung maturation such that development of respiratory distress syndrome, among other neonatal disorders, is prevented. .

Additionally, Compound III may be formulated into a pharmaceutical composition,

- 23 039545 for example, a pharmaceutical composition prepared as described below.

Methods of treatment

Compound I, as well as its salts, are potent inhibitors of the prostaglandin F receptor and can be used to antagonize the interaction between members of the prostaglandin F family, eg prostaglandin F2a, with the corresponding prostaglandin F receptor in vivo to attenuate uterine contractions. Compound I and salts thereof may be administered to a subject, such as a pregnant woman, to treat or prevent preterm labor. Endogenous prostaglandin F2a is synthesized and released by uterine epithelial cells in response to signaling cascades initiated by oxytocin. Upon binding of PGF2a to PGF2a-R on the extracellular surface of the uterine myocyte, phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP ₃ ). IP ₃ in turn potentiates the release of intracellular calcium (Ca ²⁺ ) by the sarcoplasmic reticulum. The sudden increase in calcium stores eventually leads to contractions of the uterine muscles and necrosis of the endothelial cells of the corpus luteum, the progesterone-producing structure that supports fetal development. Aberrant initiation of uterine contractions and degradation of the corpus luteum caused by dysregulation of PGF2a secretion can lead to preterm labor. Compound I and its salts, eg compound III, can attenuate phospholipase C-mediated IP ₃ formation, and subsequent mobilization of intracellular calcium stores, by inhibiting the association of PGF2a with PGF2aR. Compound I, or a salt thereof, e.g., compound III, can therefore be administered to subjects, e.g., pregnant women, to delay the onset of labor in the subject, e.g., by one or more days or weeks, e.g., from about 1 day to about 16 weeks ( e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks) . For example, Compound I or a salt thereof, eg Compound III, may be administered to a subject to prevent delivery prior to delivery by caesarean section. Additionally, Compound I or a salt thereof, eg Compound III, may be administered to a subject for the prevention and/or treatment of dysmenorrhea. Compound I or a salt thereof, eg Compound III, may also be administered to a subject, eg a pregnant woman, to relieve one or more symptoms associated with childbirth such as vaginal bleeding and uterine membrane rupture.

Additionally, the compounds of the invention may be administered to treat endometriosis in a patient (eg, a human patient). Overexpression of the prostaglandin F2a receptor correlates with aberrant endometrial growth. As antagonists of prostaglandin F2a receptor activity, the compounds of the invention (eg compound (I) or a salt thereof, eg compound (III)) can be administered to a patient suffering from endometriosis for the treatment of this indication. The compounds of the invention may also be administered to a patient to relieve one or more symptoms of endometriosis, such pain symptoms including dysmenorrhea, dyspareunia, chronic pelvic pain, dysuria, and dyschesia during and/or after menstruation. Successful treatment of endometriosis by administering a compound of the invention to a patient can be judged, for example, by a reduction in endometrial tissue growth and/or a reduction in pain symptoms during and/or independent of menstruation. Combination Therapy

Although the processes involved in the onset of labor are still not fully defined, there is a growing body of evidence supporting the importance of inflammation for both term and preterm birth. At the onset of labor, there is a systemic increase in pro-inflammatory factors, including prostaglandins, cytokines, and manganese peroxide dismutases. Additionally, inflammation is significantly implicated in preterm labor triggered by infection.

It is believed that oxytocin initiates labor by causing two distinct effects: directly inducing contraction of the uterine myometrium, and increasing the synthesis and release of contractile prostaglandins from the uterine endometrium/decidua. By inhibiting oxytocin signaling, direct (contractile) and indirect (increased prostaglandin synthesis) effects of oxytocin on the uterus can be achieved. Additionally, treatment of human decidua with oxytocin results in stimulation of prostaglandin F2a production. This confirms the existence of a complementary role for oxytocin signaling in uterine tissues, by which oxytocin can interact not only directly on the myometrium, stimulating uterine contractions, but also indirectly, through the formation of prostaglandins in other tissues.

Recently, evidence has been obtained for the correlation of the activity of the contractile prostaglandin F receptor with the onset of labor and during the progression of labor. Recent research reports also indicate that oxytocin induces the production of prostaglandins in human myometrial cells by potentiating cyclooxygenase 2 (COX-2). This mechanism may explain the delayed release of prostaglandins in the uterine tissue, which stimulates labor. Thus, combination therapy including a prostaglandin receptor antagonist

- 24 039545

F2a, eg Compound I or a salt thereof (eg Compound III) and an oxytocin receptor antagonist can be used to treat and/or prevent or preterm labor. Additionally, the combination of an oxytocin receptor antagonist and a prostaglandin F2a receptor antagonist may be more effective for the treatment of preterm labor than current therapeutic regimens. Synergistic effects can be observed and are described herein to prevent both the contractile and inflammatory processes that underlie preterm labor as the dose(s) of an oxytocin receptor antagonist administered to a patient may be lower when administered in combination with an antagonist. prostaglandin F receptor in relation to the dose that may be administered to a patient receiving an oxytocin receptor antagonist alone.

Compound I or a salt thereof, eg Compound III, may be administered with one or more additional agents, eg an oxytocin receptor antagonist, to reduce the frequency of uterine contractions and to delay the onset of labor. For example, Compound I or a salt thereof, eg Compound III, may be administered simultaneously with, in admixture with, or administered separately from the oxytocin receptor antagonist. Exemplary oxytocin receptor antagonists for use in combination with the compositions and methods of the invention include atosiban, retosiban, barusiban, epelsiban, and nolasiban, or a variant, formulation, crystalline form, or derivative thereof. For example, Compound I, or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with nolasiban, or a variant, drug, crystalline form, or derivative thereof, to delay the onset of labor in a subject, for example, by one or more days. or weeks, for example, from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks).

Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm labor in combination with a betamimetic. . Betamimetics, eg, terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine, and orciprenaline, may act by depleting intracellular Ca ²⁺ levels (eg, intracellular myometrial Ca ²⁺ levels) by potentiating β-2 adrenergic receptors, thus upregulating cAMP and depleting intracellular reserves of Ca ²⁺ , which under other circumstances can stimulate uterine contractility. Typical betamimetics for use in combination with the compositions and methods described in this application, as well as examples of methods for administering betamimetics in combination with the compositions and methods disclosed in this application, are described, for example, in Gyetvai et al. Obstet. Gynecol. 94:869-877 (1999), the disclosure of which is incorporated herein by reference.

Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm labor in combination with a blocker calcium channels, such as an L-type calcium channel blocker. Calcium channel blockers, including dihydropyridines, such as nifedipine and nicardipine, may act by inhibiting Ca ²⁺ release from the sarcoplasmic reticulum, thus preventing Ca ²⁺ mobilization, which stimulates uterine muscle contraction. Exemplary calcium channel blockers for use in combination with the compositions and methods described herein, as well as exemplary methods for administering calcium channel blockers in combination with the compositions and methods disclosed herein, are described, for example, in Wojcieszek et al. Cochrane Database Syst. Rev. 6:CD002255 (2014), the disclosure of which is incorporated herein by reference.

Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm labor in combination with magnesium salt, such as magnesium sulfate. Magnesium salts, such as magnesium sulfate, can modulate uterine contractility through various mechanisms, for example, by inducing hyperpolarization of the plasma membrane and/or by competing with Ca ²⁺ for binding to myosin light chains, thus inhibiting contraction of myosin filaments in uterine myocytes. Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm birth in association with a donor. nitric oxide. Nitric oxide, a vasodilator that is essential for maintaining normal smooth muscle tone, is produced by various cells. Nitric oxide is synthesized by the oxidation of L-arginine to L-citrulline. This reaction is catalyzed by nitric oxide synthase, which exists in several forms. Both forms of nitric oxide synthase, both inducible (type 2) and brain (type 1), are expressed in myometrial cells and endothelial cells of blood vessels.

- 25 039545 vessels, while endothelial nitric oxide synthase (type 3) is expressed exclusively in endothelial cells of blood vessels. The interaction between nitric oxide and soluble guanylyl cyclase, which is present in nearby effector cells, is a widespread signaling mechanism that links various extracellular nitric oxide production stimuli to cyclic guanosine monophosphate (cGMP) synthesis in target cells. An increase in cGMP in smooth muscle cells, such as uterine myocytes, inactivates myosin light chain kinases, which leads to smooth muscle relaxation. The tocolytic effects of nitric oxide donors, such as nitroglycerin, are described, for example, in Simhan et al. New Engl. J. Med. 357:477-487 (2007), the disclosure of which is incorporated herein by reference.

Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm labor in combination with progesterone. or a variant thereof, for example 17-α-hydroxyprogesterone caproate. Progesterone is a steroid hormone secreted by the corpus luteum and placenta around 8 weeks of gestation. Progesterone and its variants, eg 17-αhydroxyprogesterone caproate, can regulate uterine dormancy by directly modulating myometrial [Ca ²⁺ ] and prostaglandin synthesis, as described, for example, in Muglia et al. New Engl. J. Med. 362:529-535 (2010); Simhan et al. New Engl. J. Med. 357:477-487 (2007); Smith et al. Eur. J. Obstet. Gynecol. reproduction. Biol. 142:3-11 (2009); Bernal. Sem. celldev. Biol. 18:340-347 (2007); and Hubinont et al. J. Pregnancy. 941057 (2011), the disclosure of each of which is incorporated herein by reference.

Additionally or alternatively, a compound of the invention (e.g. compound I or a pharmaceutically acceptable salt thereof, e.g. compound III) may be administered to a patient who is experiencing or at risk of developing (e.g. exhibiting one or more symptoms) preterm labor in combination with a corticosteroid. . Antenatal corticosteroids, e.g., betamethasone, dexamethasone, and hydrocortisone, are a class of therapeutic agents that can be administered to a subject, e.g., a pregnant female in preterm labor or a subject at risk of developing preterm labor (e.g., a subject who has one or more symptoms of preterm labor, such as vaginal bleeding and uterine membrane rupture) to accelerate fetal lung maturation. Treatment with antenatal corticosteroids is associated with an overall reduction in neonatal mortality, respiratory distress syndrome, intraventricular hemorrhage, necrotizing enterocolitis, assisted ventilation, the need for resuscitation and intensive care, and systemic infections during the first 48 hours of life. Additionally, treatment with antenatal corticosteroids is effective in women with premature rupture of membranes (PROM) and pregnancy-associated hypertensive syndromes. There is evidence supporting benefits for a wide range of gestational ages, for example, in particular from about 26 to about 34 weeks (Miracle et al. J. Perinat. Med. 36:191-196 (2008), the disclosure of which is incorporated into this application by links).

Compositions for Combination Therapy

Compound I or a salt thereof, e.g. compound III, may be used alone or in combination with one or more additional agents useful in inhibiting uterine contractions and/or luteolysis, such as, for example, atosiban, retosiban, barusiban, epelsiban and nolasiban, or variant, drug, crystalline form, or derivative, along with other therapeutic agents (eg, tocolytic agents) described in this application. Compound I or a salt thereof, e.g. compound III, may be mixed with an additional active agent, e.g. an oxytocin receptor antagonist, betamimetic, calcium channel blocker, magnesium salt, nitric oxide donor, progesterone or a variant thereof, or a corticosteroid described herein , and be administered to the patient in a single composition, or Compound I or a salt thereof, eg Compound III, may be administered to the patient separately from the additional active agent. For example, Compound I or a salt thereof, eg Compound III, and the additional active agent may be separately administered to a patient.

A combination therapy composition described herein, e.g., a pharmaceutical composition described herein, may be administered to a subject to delay the onset of labor in the subject, e.g., by one or more days or weeks, e.g., from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks ). In some embodiments, the subject goes into preterm labor. In some embodiments, the pharmaceutical composition is administered to a subject (eg, a human subject) prior to the onset of preterm labor. The pharmaceutical composition according to the invention may be administered to a subject (on

- 26 039545 example, human subject) to prevent childbirth before delivery by caesarean section. The pharmaceutical composition of the invention may be administered to a subject (eg, a human subject) to treat or prevent dysmenorrhea. The pharmaceutical composition according to the invention may be administered to a subject, for example, a pregnant woman, to relieve one or more symptoms associated with childbirth, such as vaginal bleeding and rupture of the uterine membranes.

The additional therapeutic agent present in the combination therapy composition may be, for example, another tocolytic agent. The additional tocolytic agent may be, for example, an oxytocin receptor antagonist such as atosiban, retosiban, barusiban, epelsiban, and nolasiban, as well as one or more variants, formulations, crystalline forms, or derivatives thereof. For example, atosiban and its variants are described, for example, in the patent

US No. 4504469 and 4402942, the disclosure of each of which is included in this application by reference. Retosiban and its variants are described, for example, in US patent No. 7514437; 8367673; 8541579; 8071594; 8357685; 8937179; and US 2016/0074413, the disclosure of which is incorporated herein by reference. Barusiban and its variants are described, for example, in US patent No. 6143722; 7091314; 7816489; and US 2016/0175283, the disclosure of each of which is incorporated herein by reference. Epelsiban and its variants are described, for example, in US patent No. 7514437; 8367673; 8541579; 7550462; 7919492; 8202864; 8742099; 9408851; 8716286; and 8,815,856, the disclosure of each of which is incorporated herein by reference. Nolasiban and variants, formulations, and crystalline forms thereof are described in, for example, US Pat. No. 7,115,754 and US Published Application No. 2015/0073032; 2015/0164859; and 2016/0002160, the disclosure of each of which is incorporated herein by reference.

In some embodiments, the additional tocolytic agent is a betamimetic, such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine, or orciprenaline. In some embodiments, the additional tocolytic agent is a calcium channel blocker, such as a dihydropyridine, such as nifedipine or nicardipine. In some embodiments, the additional tocolytic agent is a magnesium salt, such as magnesium sulfate. In some embodiments, the additional tocolytic agent is a nitric oxide donor, such as nitroglycerin.

In some embodiments, the additional therapeutic agent is a progesterone or a variant or derivative thereof, such as 17-a-hydroxyprogesterone caproate.

In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the corticosteroid is betamethasone. In some embodiments, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is hydrocortisone.

In combination treatments, dosages of one or more therapeutic compounds may be reduced from standard dosages when administered alone. For example, dosages may be empirically determined from drug combinations and permutations, or may be deduced by one of skill in the art.

Examples

The following examples are provided to provide those skilled in the art with a description of how the compositions and methods disclosed in this application may be used, prepared and evaluated, and are only exemplary embodiments of the invention and are not intended to limit the scope of the invention as claimed by the inventors.

Example 1 Preparation of compounds I and III.

Compound I, and its chloride salt (compound III), were prepared according to Scheme 1 below. This example will be described for each of the steps carried out for the synthesis of compound I, designated as steps 1-6.

- 27 039545

Scheme 1. Preparation of compound I and its chloride salt

Step 1. Preparation of 2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid tert-butyl ester

MW:

^he MW: 384.46

To an appropriately sized flask (vessel A), 3-(butoxycarbonyl)-1,3-thiazolidine-(2S)carboxylic acid (1 wt.) was added, then tetrahydrofuran was added and thereafter the components of the flask were cooled to -35° C. to about - 45°C. After that, N-methylmorpholine (1.18 vol.) was added to the flask, while the temperature was maintained in the range from -30 to -40°C. After that, isobutyl chloroformate (0.58 vol.) was added to the flask, while the temperature was maintained in the range from -30 to -40°C.

In a separate vessel (Vessel B), (3S)-amino-3-(4-fluorophenyl)propan-1-ol (0.76 wt.) and THF were added and the vessel stirred until large solids dissolved.

A solution of (38)-amino-3-(4-fluorophenyl)propan-1-ol from vessel B was then added to reaction vessel A while the temperature was maintained in the range of -30 to -40°C. The contents of the flask were then allowed to warm to 15-25° C. over a period of 1-24 hours. The reaction mixture was stirred at 15-25° C. until the reaction was complete. The reaction mixture was concentrated to dryness, and then ethyl acetate was added to the residue, followed by saturated aqueous ammonium chloride. The organic phase was separated and washed with saturated aqueous ammonium chloride. Thereafter, the organic phase was separated and washed with saturated aqueous sodium hydrogen carbonate solution. The organic phase was then dried over sodium sulfate, filtered and the filtrate was concentrated at 35-40° C. until the ethyl acetate content was <10% w/w to give 2-[ tert-butyl ester 1-(4-fluorophenyl)-3hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid.

Step 2: Preparation of 3-(biphenyl-4-sulfonyl)thia [1-(4-fluorophenyl)-3-hydroxypropyl]amide

- 28 039545 zolidine-2-carboxylic acid

MW: 304.46

To an appropriately sized flask (vessel A), 2-[1-(4-fluorophenyl)-3hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid tert-butyl ester (1 wt) was added, followed by dichloromethane. After that, the components of the flask were cooled to -15 - -20°C. Then hydrochloric acid (3.3 vol.) was added to the flask, while the temperature was maintained in the range of -15 and -20°C until the reaction was completed. After that, the reaction mixture was cooled to -35 - -40°C and tetrahydrofuran was added to the mixture, while the temperature was maintained in the range of -30 and -40°C. Then N,N-diisopropylethylαmin (8.16 vol) was added to the mixture while the temperature was maintained between -15 and -45°C. Then 4-dimethylaminopyridine (0.032 wt.) was added to the vessel while the temperature was maintained between -15 and -45°C.

In a separate vessel (vessel B), 4-biphenylsulfonyl chloride (0.85 wt.) was added, followed by THF.

The 4-biphenylsulfonyl chloride solution from vessel B was added to reaction vessel A while the temperature was maintained between -15 and -45°C. The components of the reaction mixture were then allowed to warm to 15-25°C over a period of 1 to 24 hours. Ethyl acetate was then added to the flask followed by a saturated aqueous ammonium chloride solution. The organic phase was separated and washed with a saturated aqueous ammonium chloride solution, then with a saturated aqueous hydrogen carbonate solution. The organic phase was then dried over sodium sulfate and filtered. The filtrate was concentrated at 35-40°C to obtain a solid residue. Then dichloromethane was added to the residue and mixed at 30-35°C. After evaporation, ethyl acetate was then added to the residue and the slurry was transferred to a suitable vessel. Thereafter, the stirred slurry was heated at reflux and then cooled to 0-5°C. The precipitated solid was collected by filtration. The filter cake was washed with ethyl acetate, then with tert-butyl methyl ether and the filter cake was pumped dry for 1-24 hours under nitrogen to give [1-(4-fluorophenyl)-3-hydroxypropyl]amide 3(biphenyl-4-sulfonyl)thiazolidine- 2-carboxylic acid.

Stage 3A. Preparation of 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4fluorophenyl)-3-propyl ester of 2-tert-butoxycarbonylamino-3-methylbutyric acid

mw: 50(3.60 WW: 6Y.M

In an appropriately sized flask (vessel A), Boc-L-valine (0.48 wt.), dichloromethane, and N,N-dimethylformamide were added and the mixture was then stirred under nitrogen at 15-25°C. After that, 1-hydroxybenzotriazole (HOBt, 0.3 wt.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 0.42 wt.) were added to the vessel, while the temperature was maintained at 15-25°C . The mixture was then stirred at 15-25°C until all the solids were dissolved to give solution A.

In a separate vessel (vessel B), 3-(biphenyl-4sulfonyl)thiazolidine-2-carboxylic acid [1-(4-fluorophenyl)-3-hydroxypropyl]amide (1.0 wt.), dichloromethane, and N,N -dimethylformamide, and then the mixture was stirred at 15-25° C. under a nitrogen atmosphere. Then 4-dimethylaminopyridine (0.27 wt.) was added to the vessel, while the temperature was maintained in the range of 15-25°C. The mixture was stirred at this temperature until all solids had dissolved (typically 5-15 minutes) to give solution B.

Solution A was then added to solution B, while the temperature was maintained in the range of 15-30°C. The mixture was stirred at this temperature until completion of the reaction. The reaction mixture was concentrated to remove volatile solvents. Then ethyl acetate was added to the flask, after which 10% wt./wt. aqueous solution of citric acid. The aqueous phase was separated and extracted with ethyl acetate. The combined organic phases were washed with a mixture of 10% wt./wt. an aqueous citric acid solution and a saturated aqueous sodium chloride solution, then a saturated aqueous ammonium chloride solution, a saturated aqueous sodium hydrogencarbonate solution and a saturated aqueous sodium chloride solution were added. The organic phase was then dried over magnesium sulfate, filtered and the filter cake washed with ethyl acetate. The filtrates were concentrated until a solid residue was obtained, giving the crude 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-29 039545 fluorophenyl)-3-propyl ether 2-tert-butoxycarbonylamino-3-methylbutyric acid.

Stage 3B. Purification of 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)3-propyl ester of 2-tert-butoxycarbonylamino-3-methylbutyric acid

c ₃₅ h ₄₂ fn ₃ o ₇ s ₂ c ₃₅ h ₄₂ fn ₃ o ₇ s ₂

MB: 599.85 MB: 699.85

For purification of 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3propyl ester of 2-tert-butoxycarbonylamino-3-methylbutyric acid, crude product (1 wt .) and dichloromethane were mixed in a vessel until all solids were dissolved. The solution was then loaded onto silica, after which dichloromethane was added. The product was eluted with ethyl acetate:heptanes. Fractions containing the product were combined and concentrated to dryness in vacuo at a water bath temperature of 35-40°C to give purified 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4- fluorophenyl)-3-propyl ester of 2-tert-butoxycarbonylamino-3-methylbutyric acid.

Step 4: Preparation of 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate

G ₃₅ H ₄₂ FN ₃ O ₇ S ₂

MW 699.85

MW: 695.83

To an appropriately sized flask, 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester was added (1 wt.), then added 1,4-dioxane and the mixture was stirred under nitrogen atmosphere. After that was added methanesulfonic acid (0.18 wt.), and the contents of the flask was heated to 68-73°C. The reaction was stirred at this temperature until completion of the reaction using 1H NMR analysis. After that, the reaction mixture was cooled to 35-40°C and concentrated to dryness at this temperature. Then the residue was dissolved in THF and concentrated to dryness at 35-40°C. This cycle of aze drying was repeated until the content of 1,4-dioceane was less than 1.0% w/w to give methanesulfonate 3{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino }-3-(4-fluorophenyl) propyl ester of 2-amino-3-methylbutyric acid.

Step 5. Preparation of 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4fluorophenyl)propyl ester of 2-amino-3-methylbutyric acid (Compound I)

CagHjjFNjOjSj .CH ₃ SO ₃ H Ο ₃₀ Η ₃₄ ΡΝ ₃ Ο ₅ 8 ₂

MB: 695.83 MB: 599.75

To an appropriately sized flask, 2-amino-3-methylbutyric acid propyl ester methanesulfonate 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl) (1 wt.) then dichloromethane was added. After that, the components of the flask were cooled to 5-15°C. To the mixture was added an aqueous solution of sodium bicarbonate, while the temperature was maintained in the range of 5°C and 25°C. Thereafter, the phases were separated and the organic phase was re-added to the vessel, then a saturated aqueous sodium hydrogen carbonate solution was added while the temperature was maintained at 5-25°C. Thereafter, the aqueous and organic layers were separated and the organic phase was dried over magnesium sulfate, filtered and the filter cake was washed with dichloromethane. The combined organic layers were then concentrated to dryness at 40-45° C. until the dichloromethane content was < 2% w/w to give 2-amino-3-methylbutyric acid propyl ester 3-{[3-(biphenyl- 4sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl) (compound I).

- 30 039545

Step 6. Preparation of 2-amino-3-methylbutyric acid propyl ester hydrochloride 3{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl) (Compound III)

In a suitably sized flask, water (1.66 vol) was added followed by hydrochloric acid (0.18 vol) and the temperature of the mixture was brought to 15-25°C. The solution was then filtered and the filter solution was added to an appropriately sized flask (vessel A), followed by the addition of ethanol and ethyl acetate. The resulting mixture was stirred under nitrogen at 15-25° C. for at least 5 minutes.

To an appropriately sized vessel (Vessel B), 2-amino-3-methylbutyric acid propyl ester 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl) was added ( 1 wt.), then ethanol was added. The contents of the flask were then stirred to dissolve all solids and obtain a clear solution.

The solution from vessel B was then added to vessel A while the temperature was maintained at 15-25°C. The stirred mixture was cooled to 0-5°C and stirred at this temperature for 50-70 minutes. The solid was collected by filtration and the filter cake was pumped dry under nitrogen for at least 12 h to give the crude hydrochloride 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4 -fluorophenyl) propyl ester of 2-amino-3-methylbutyric acid.

Example 2 Pharmacodynamic properties of compound I and its salts.

non-clinical pharmacology.

Compound I and its salts are rapidly converted to compound II after administration to the gastrointestinal tract. Compound II is a competitive and reversible prostaglandin F2a receptor antagonist (FP2a human receptor Ki=6 nM) that is under development to control preterm labor by inhibiting premature uterine contractions. An effective pharmacology (tocolytic effect) has been shown in a model of spontaneous uterine activity in pregnant rats at a late stage of pregnancy.

Pharmacology in vitro.

The inhibition efficacy of Compound I and Compound II on the prostaglandin F2a receptor was assessed by analyzing the affinity of these compounds for the recombinant FP receptor expressed in HEK293-EBNA cells. The results showed a high binding affinity of Compound I and Compound II to the human receptor (see Table 2).

The selectivity of compound II was tested for all eight prostaglandin receptor subtypes. The selectivity was about 10 times relative to the prostaglandin E receptor 2 (EP2) and more than 100 times relative to other receptors. Testing the effect of 1 μM compound II on a panel of binding sites for 50 receptors, channels and enzymes showed high selectivity for FP.

Functional characterization of compound II on human FP was performed in transfected HEK293-EBNA cells. Compound II is also able to dose-dependently inhibit the synthesis of IP3 with an IC ₅₀ value of 60 nM. When added alone to FP/HEK293-EBNA cells, compound II tested up to 10 uM did not induce any IP3 synthesis, indicating that the compound lacked agonist activity.

Pharmacology in vivo.

The tocolytic effects of Compound I and Compound II were studied in a late term (19-21 days of gestation) spontaneous uterine activity model of anesthetized pregnant rats (Kawarabayashi et al. Am. J. Obstet. Gynecol. 175:1348-1355 (1996) and Shinkai et al. J Pharm Pharmacol 52:1417-1423 (2000)). Briefly, late-stage pregnant rats were anesthetized with urethane. One horn of the pregnant uterus was released and a polyethylene catheter carrying a latex chamber filled with saline at the end was inserted into the uterine cavity. The catheter was attached to the amplifying/recording system using a pneumatic transducer. Increasing doses of Compound I (as the mesylate salt) or Compound II were administered orally or injected by 10 minute IV infusion. For intravenous administration, uterine contractility was quantified by calculating the AUC over a 10 minute injection period.

Percentage variation in AUC relative to spontaneous uterine response observed after administration of each compound was calculated by comparison with the value recorded before the first dose (baseline). The effect of Compound I or Compound II was assessed by comparing the pre- and post-treatment pressure values in the uterine cavity. For oral administration, a similar computational method was used to process data at different time points after

- 31 039545 treatment. Statistical differences between treatment groups at each time point were determined using one-way ANOVA followed by Tukey's test. Both compounds, administered intravenously or orally, are able to markedly reduce spontaneous uterine contractions by about 40-50% (maximum effect obtained with a dose of 30 mg/kg intravenously and 60 mg/kg orally). Intravenous activity was comparable to or slightly higher than that of the tocolytic atosiban licensed in the European Union.

The inhibitory effect after oral administration had a rapid onset (5-15 min after administration) and remained at a sustained level until the end of the 3 hour observation period (FIG. 3).

With a single oral dose, significant inhibition of uterine contractions is achieved at a dose of 30 mg/kg.

Thus, the high affinity of Compound I and Compound II for the human FP receptor was shown by in vitro pharmacological studies. When administered intravenously or orally, these compounds are able to significantly reduce spontaneous uterine contractions by about 40-50% when tested in anesthetized pregnant rat model of spontaneous uterine activity at the late stage of pregnancy (19-21 days of gestation).

Example 3. Crystal lattices of salts of compound I.

This example describes experiments to create and characterize crystalline salt forms of compound I.

Essence.

The mesylate salt of compound I was determined to be amorphous according to XRPD. Attempts to crystallize the material were unsuccessful. The free base was synthesized from the mesylate salt and used to prepare various salts. The crystalline hydrosulfate salt was obtained directly from the synthesized salt. The three salts were crystallized using mixtures of different solvents and crystallization techniques: hydrochloride, fumarate, and dihydrogen phosphate. The hydrochloride salt is believed to exhibit low hygroscopicity, increased stability at elevated relative humidity (OB), and is a single crystalline form when crystallized from various different experimental conditions.

The crystalline HCl salt was obtained in two evaporation and suspension experiments. In each case, the same pattern of XRPD was observed. Based on thermal data, the material had some residual solvent and a possible melting point of about 146-147°C. It is likely that partial decomposition occurs during melting. The hydrochloride salt was non-hygroscopic based on moisture balance data.

The crystalline hydrosulfate salt is likely to solvate and decompose above about 100°C. The material was stable at relative humidity up to about 65%.

The crystalline dihydrogen phosphate and fumarate salt were hygroscopic at about 65% RH. Attempts to industrially obtain salts were unsuccessful due to high laboratory humidity. Thus, only a partial characterization is available for these salts.

The hydrochloride, hydrosulfate and fumarate salts exhibit comparable solubilities in water (below 1 mg/mL, see FIG. 8).

Experimental part.

The X-ray powder diffraction analyzes described herein were performed on a Shimadzu XRD-6000 X-ray powder diffractometer using Cu Ka radiation. The instrument is equipped with a long, fine-focus X-ray tube. The voltage and current on the tube were set to 40 kV and 40 mA, respectively. The divergence and scattering slits were set to 1° and the receiving slit was set to 0.15 mm. The diffracted beams were determined using a Nal scintillation detector. Theta-two theta continuous scan at 3°/min (0.4 s/0.02° step) up to 2.5-40°2θ was used. The silicon standard was analyzed every day to control instrument settings. Samples were analyzed using a silicon sample holder.

The powder x-ray diffraction analyzes described herein were also performed on an Inel XRG-3000 diffractometer equipped with a curved position sensitive detector with a 2θ 120° range. Real time data was collected using Cu Ka radiation starting at about 4°2θ with a resolution of 0.03°2θ. The voltage and current on the tube were set to 40 kV and 30 mA, respectively. The monochromator slit was set to 5 mm by 160 µm. The charts represented from 2.5 to 40°2θ. Samples were prepared for analysis by packing them into thin-walled glass capillaries. Each capillary was mounted on a goniometric head, which was switched to the motor mode to enable the capillary to rotate during the measurement and recording of parameters. Samples were analyzed for 5 or 10 min. The instrument was calibrated daily with a silicone reference standard.

The DSC analyzes described herein were performed on a TA Instruments 2920 Differential Scanning Calorimeter. The instrument was calibrated using indium as the reference material. The samples were placed in a standard aluminum DSC crucible, the crucible was clamped, and the weight was accurately recorded. The samples were equilibrated at 25°C and heated with a nitrogen purge at a rate of 10°C/min up to 350°C. Indium metal was used as a calibration standard.

The TG analyzes described herein were performed on a TA Instruments 2950 thermogravimetric analyzer. The calibration standards were nickel and ALUMEL™. Samples were placed in an aluminum sample pan and inserted into a TG oven. The samples were first equilibrated at 25°C, then heated under nitrogen flow at a heating rate of 10°C/min up to 350°C.

Spectra of the 1 H nuclear magnetic resonance (NMR) solutions described in the present invention were recorded at ambient temperature using a Varian UNITYINOVA400 spectrometer at 1 H Larmor frequency of 399.8 MHz. Samples were dissolved in methanol-d4, methylene chloride-d2, or chloroform-d3. Spectra were acquired with 1H pulse widths of 7.8 or 8.6 µs, exposure time of 2.50 s, 5 second delay between scans, spectral width of 4095 or 6400 Hz with 20474 or 32000 observations, and 16 or 40 co-merged scans. . Free induction signal (FID) was performed using Varian VNMR 6.1C software with 65536 points and exponential line broadening factor of 0.2 Hz to improve signal to noise ratio. The spectra were compared with internal standard tetramethylsilane (TMS) at 0.0 ppm. or residual solvent peak.

The FT-Raman spectrum described in this application was recorded on an FT-Raman 960 or 860 spectrometer (Thermo Nicolet). This spectrometer uses an excitation wavelength of 1064 nm. About 0.5-0.7 W of Nd:YVO ₄ laser powder was used to irradiate the samples. Raman spectra were measured with an indium gallium arsenide (InGaAs) detector. Samples were prepared for analysis by placing the material in a glass capillary and then in a gold plated capillary holder in the accessory. A total of 256 sample scans were collected from 3600 to 100 cm ^-1 at a spectral resolution of 4 cm ^-1 using Happ-Gensel apodization. Wavelength calibration was performed using sulfur and cyclohexane.

Moisture uptake/desorption (MB) data were collected on a VTI SGA-100 Vapor Sorption Analyzer. Sorption and desorption data were collected over a range of 5 to 95% relative humidity (RH) at 10% RH intervals under a nitrogen purge. Samples were not dried prior to analysis. The equilibrium criteria used for analysis was less than 0.0100% weight change over 5 minutes, with a maximum equilibration time of 3 hours if the weight criterion was not met. The data were not corrected for the initial moisture content of the samples. NaCl and PVP were used as calibration standards.

Preparation of compound I.

Numerous attempts have been made to prepare the free base of Compound I from the mesylate salt, the results of which are described in FIG. 4. Initially, one equivalent of sodium hydroxide was used per equivalent of salt. Proton NMR indicated the presence of methanesulfonic acid peaks. A complete reaction was obtained when the mesylate salt in methylene chloride and a solution of NaOH in water were mixed at a ratio of 1:2 salt:base. The organic layer was separated after several washes and evaporated. The resulting pasty or viscous oily material was dried in vacuo to give an amorphous solid. The free base was analyzed by 1 H NMR and Raman spectroscopy (FIG. 15 and FIG. 16, respectively). Subsequent salt screening studies used the free base as starting material (summary results are shown in FIGS. 5-7).

Selection of salts of compound I.

Twelve salts of compound I were prepared. The crystalline hydrogen sulfate salt was precipitated by adding about 25 molar excess of sulfuric acid to a solution of the free base in acetone. Other salts from the synthesis step are believed to be incapable of birefringence by microscopy or amorphous according to XRPD (FIGS. 5-7). Benzenesulfonate, citrate, ethanesulfonate, hydrochloride, hydrosulfate and sulfate salts were analyzed by proton NMR.

Crystallization experiments on salts of compound I are summarized in FIG. 5-7. The following salts crystallized: hydrochloride, fumarate and dihydrogen phosphate.

The chloride salt was crystallized from 1:1 acetone:toluene, methylene chloride:ethyl ether, and an acetone slurry. A similar XRPD pattern was observed in all experiments and was designated form A (FIG. 7). The crystalline fumarate salt was obtained by slow evaporation from a 1:1 methanol:toluene solution. The X-ray diffraction pattern is designated as pattern B. The hydrogen sulfate and dihydrogen phosphate salt exhibit very similar XRPD patterns (marked as pattern X). The counterions HSO4 ^- and H ₂ PO ₄ ^- are similar in size and small compared to the free base molecule, thus, similar crystal structures are quite likely for the hydrosulfate and dihydrophosphate salts. Attempts to crystallize the mesylate salt produced viscous or glassy solids.

Characteristics of the free base and mesylate salt of compound I.

- 33 039545

The proton NMR spectrum of the free base showed two doublets at about 1 m.h., which corresponds to the methyl groups of the valine fragment. The methyl groups are on the chiral carbon center and therefore are not equivalent in proton NMR. Two doublets for methyl groups are observed for the following salts of compound I: besylate, citrate, esylate, hydrosulfate (more overlapping) and sulfate (more overlapping). In the 1H NMR spectrum of the mesylate salt and the chloride salt, a doublet at ~ 1 m.h. corresponds to six hydrogen atoms, which occurs when two doublets of methyl groups completely overlap (Figs. 13 and 21).

A 1H NMR homonuclear dissociation experiment on the free base confirmed the methine (CH) hydrogen multiplet at about 2 m.h. (Fig. 18). The 1H NMR spectrum of the free base, recorded in the absence of prior irradiation of any methyl group, is shown at the bottom of FIG. 18. Irradiation of each methyl group (top, middle) leads to a simplified methine multiplet with the same number of lines (5). If two doublets correspond to different diastereoisomers, then two types of multiplets will be observed, original and simplified.

Characterization of the chloride salt of compound I (compound III).

The crystalline chloride salt was analyzed by thermal techniques, 1H NMR and automated moisture sorption/desorption analysis. The endotherm at about 147° C. in DSC appears more widely than it is typically observed for the melting endotherm. A weight loss of about 4% was observed from 25 to 160° C. (analysis of an acetone slurry sample, Fig. 20). 1 H NMR of the chloride salt was consistent with the structure (FIG. 21). However, the data cannot be correlated with weight loss in thermal analyses, since different samples were analyzed (slow evaporation of 1:1 acetone:toluene mixture). The chloride salt from the acetone slurry was dried under vacuum at about 50° C. for 1 day. The resulting sample was similar to the original salt according to XRPD (Fig. 22). Thermal data are shown in Fig. 23. Based on a comparison of thermal data, the dried material had lower losses in the range of 25 to 100°C (0.2% rel. 0.6% for the original chloride salt) and 100 and 160°C (2.5% rel. .3.5%) (Fig. 24). This indicated that some of the solvent had been removed by vacuum drying. However, the endotherm around 146-147° C. in DSC was still wide (FIG. 25). Partial decomposition possibly occurs on melting (note: initial decomposition and corresponding weight loss in TG).

The chloride salt of compound I does not liquefy after 2 days at about 95% RH. Moisture sorption/desorption data are summarized in FIG. 27 and shown in FIG. 26 and 28. Minimal weight loss was observed when equilibrated at 5% RH. About 0.9% weight gain occurred with sorption from 5 to 95% relative humidity. The sample showed about 0.7% weight loss on desorption. XRPD analysis on the post-MW sample showed an X-ray diffraction pattern similar to that of the starting material (FIG. 29).

Characteristics of the hydrosulfate and sulfate salts of compound I.

Both salts were prepared: the hydrosulfate and sulfate salts of compound I. The hydrogensulfate salt was precipitated from the free base acetone solution by adding about 25 molar excess of sulfuric acid. The precipitate was found to be crystalline according to XRPD (FIG. 38). Thermal data for the hydrosulfate salt are shown in FIG. 32. A wide endotherm at about 68°C corresponds to a weight loss of about 1% and is probably due to desolvation (dehydration). Decomposition occurs at higher temperatures. It liquefies after 3 days at about 65% RH (FIG. 32). The sulfate salt was prepared using two equivalents of free base to one equivalent of acid. Attempts to crystallize the sulfate salt of Compound I were unsuccessful (FIGS. 5-7). Hydrosulfate and sulfate salt were analyzed by proton NMR (Fig. 33 and Fig. 34). No differences were observed in NMR spectra. For example, the methyl groups of the valine moiety are believed to have different binding.

Characteristics of the dihydrophosphate salt of compound I.

The dihydrogen phosphate salt was crystallized from a 1:1 mixture of methyl ethyl ketone: n-butyl acetate (FIGS. 5-7). It exhibits an X-ray diffraction pattern similar to that of the hydrosulfate salt (FIG. 43). Characterization of the dihydrogen phosphate salt is limited by XRPD due to sample loss during analysis. Attempts to prepare additional amounts of crystalline salt have not been successful. Low crystalline material was obtained at the first attempt (Fig. 5-7). Recrystallization of the low crystalline salt gave a viscous solid. The material remained viscous after drying under vacuum. Laboratory humidity was about 62% RH during commercial scale crystallization and is likely to damage the material due to its hygroscopicity. No further attempts were made to crystallize the dihydrogen phosphate salt.

Characterization of the fumarate salt of compound I.

A small amount of the fumarate salt was crystallized from methanol:toluene 1:1 (FIGS. 5-7). Attempts to prepare a commercial scale crystalline salt were carried out at laboratory humidity of about 62% RH and were unsuccessful. Most oily materials were obtained although some crystalline solid was present by microscopy. At

- 34 039545 drying a viscous solid in vacuo gave a substantially amorphous material. The initially prepared crystalline salt was used for seed experiments. However, no crystalline materials were obtained. The hygroscopic nature of the fumarate salt was confirmed in relative humidity studies.

The fumarate salt is believed to be more sensitive to moisture. The crystalline salt was stable at about 43 and 53% RH, and began to liquefy within the first day at about 65% RH. A yellow oil formed after 3 days at 65% RH (about 4% increase in humidity).

Findings.

The mesylate salt of compound I was found to be amorphous according to XRPD. Attempts to crystallize the material were unsuccessful.

The free base of compound I was synthesized from the mesylate salt and used to prepare 12 salts. The crystalline hydrosulfate salt was obtained directly from the synthesized salt. The three salts were crystallized using mixtures of different solvents and crystallization techniques: hydrochloride, fumarate, and dihydrogen phosphate. The chloride salt is believed to be the best candidate for further development. The crystalline hydrosulfate salt is likely to solvate and decompose above about 100°C. The material was stable at relative humidity up to about 65%. The crystalline HCl salt was prepared in two evaporation and slurry experiments. A similar picture of XRPD was observed. Based on thermal data, the material had some residual solvent; and the possible melting point was about 146147°C. It is likely that partial decomposition occurs during melting. The chloride salt was non-hygroscopic based on moisture balance data. The crystalline dihydrogen phosphate and fumarate salt were hygroscopic at about 65% RH. Attempts to industrially obtain salts were unsuccessful due to high laboratory humidity. Thus, only a partial characterization is available for these salts.

Example 4 Monitoring Caco-2 cell permeability of the mesylate salt of Compound I.

The bioavailability of orally administered drugs mainly depends on the ability to penetrate the intestinal barriers. Caco-2 cells derived from human colon adenocarcinoma, adapted by J. Fogh for their ability to achieve a high degree of enterocyte differentiation, can be used as an in vitro model to study drug transport through the intestinal epithelium. These cells form a monolayer of polarized epithelial cells when grown on a collagen-coated polycarbonate membrane. The monolayer of differentiated cells is a relevant model for the epithelium of the small intestine. The process of differentiation, initiated by cell fusion, results in the formation of a brush border with well-developed microvilli, tight apical junctions, and a polarized distribution of membrane components, including enzymes, receptors, transport systems, ion channels, and lipid molecules.

The aim of the study was, firstly, to assess the non-specific binding of compounds I in the test system based on Caco-2 cells (without cells) and, at the second stage, to evaluate the conversion of compound I to compound II and determine whether the transport of compound I through Caco monolayers is mediated. -2 cells PepT1 transport protein.

Materials.

The Caco-2 cell line (human colon adenocarcinoma cells) was obtained from controlled cell banks (Biosearch S.p.A, Gerenzano-Italy). Dulbecco's Modified Eagle's Medium (DMEM), fetal calf serum, essential amino acid solution, L-Glutamine 200 mM, Penicillin/Streptomycin solution, Trypsin-EDTA solution without calcium and magnesium were obtained from Celbio (Milan, Italy). HEPES, Hank's Balanced Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (PBS), Dimethyl Sulfoxide (DMSO), Glycine Sarcosine (Gly-Sar) were obtained from Sigma (Milan, Italy).

Experimental part.

Caco-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2% L-Glutamine 200 mM, and 1% non-essential amino acid solution.

Cells were stored frozen in cryovials in liquid nitrogen as a 1 ml cell suspension in fetal bovine serum containing 10% DMSO. The cells used for the experiments were kept in culture for no more than one month.

If necessary, frozen vials of Caco-2 cells were rapidly thawed at 37° C. in a water bath by gentle swirling until half-completed thawing. Thereafter, the cell suspension was added dropwise to 10 ml of cell culture medium. After that, the cell suspension was centrifuged for 7 min at 900-1000 rpm, the supernatant was removed and the cell pellet after centrifugation was reconstituted in the medium and distributed into flasks containing the medium with a volume of 75 cm ² . The flasks were incubated at 37°C in an atmosphere of 5% CO ₂ . Cells were serially subcultured to

- 35 039545 obtaining almost continuous monolayers. The medium from each flask was removed and the monolayer was washed with 10-15 ml of Dulbecco's phosphate buffered medium (PBS).

The trypsin-EDTA solution was added to the cell monolayer, incubated at 37° C. and cut neatly at intervals to shear the cells. Completely detached and disaggregated cell monolayer was confirmed by examination under a microscope. The cells were then resuspended in 10 ml of complete medium and centrifuged for 7 min at 900-1000 rpm. The supernatant was discarded; cells were resuspended in culture medium and plated at 2.5x105 cells/ml in 175 cm ² flasks.

Cells from bulk culture mail flasks were detached and disaggregated by trypsin treatment as described above. Cells were resuspended in culture medium and the number of cells was counted. The cell suspension was diluted with medium to obtain about 1x10 ⁶ cells/ml and 300 μl of the cell suspension was placed on the Transwell apical compartment (diameter 6.5 mm, pore size 0.4 μm). 600 μl of cell medium was placed in the basolateral compartment. The plates were incubated at 37°C in a humidified atmosphere of 5% CO ₂ in air for 15-21 days, changing the medium every 48-72 hours.

The integrity of each monolayer of Caco-2 cells was assessed using transepithelial electrical resistance (TEER), both before the start of the experiment and after the end of the incubation time. TEER, expressed as OMxcm ² , was measured in Transwells using Millicell-ERS (Millipore). A monolayer was considered differentiated if the TEER value was greater than 800 Ωxcm ² .

The integrity of each monolayer of Caco-2 cells was assessed after the end of the incubation time using Lucifer yellow. After the experiment, Transwells were washed twice with transport buffer. 200 µl of Lucifer yellow at a concentration of 100 µM in HBSS was dispensed into the apical compartment, while 400 µl of HBSS was added to the basolateral compartment. Transwells were incubated at 37°C for 1 hour. Lucifer yellow was quantified in the basolateral compartment at 535 nm against a standard Lucifer yellow curve in the same saline using a microplate assay spectrophotometer (EG & G WALLAC). A monolayer was considered intact if <1% Lucifer yellow was found in the basolateral compartment.

Assessment of non-specific binding in a cell-free transwell.

Non-specific binding and recovery was assessed in cell-free. Compound I was tested at 1.5, 3 and 6 μM in duplicate in a cell-free transwell. Testing was carried out in a pH gradient between the apical and basolateral compartment. The apical compartment (donor) was buffered at 6.5, while the basolateral compartment (receiver) was buffered at pH 7.4. Samples were taken at the following times: 60 and 120 min for the basolateral compartment (recipient) and 120 min for the apical compartment (donor). The resulting samples were analyzed by LC-MS, both components, compound I and compound II were monitored to assess the percent recovery.

Evaluation of the stability of compound I and compound II.

The stability of both components, compound I and compound II was evaluated during the test. These compounds were dissolved in HBSS buffer (final concentration 1% DMSO) at concentrations of 1.5, 3 and 6 μm. Aliquots of each solution were taken at time zero (t=0) to assess the initial concentrations of the compounds. Solutions were incubated at 37°C for a long transport experiment. An aliquot of each solution was taken at the end of the experiment (t=120) to evaluate the final concentrations of Compound I and Compound II. Samples were analyzed by LC-MS.

Evaluation of Bidirectional Permeability of Compound I.

Compound I was dissolved in HBSS buffer (1% DMSO final concentration) at concentrations of 1.5, 3 and 6 μM. Each concentration/sampling time was run in the wells in duplicate. Testing was performed in a pH gradient: the apical compartment (mucous) was performed at pH 6.5, the basolateral compartment (serous) was performed at pH 7.4.

Apical to basolateral transport (A^B, mucosal to serous): 200 μl of each concentration of Compound I was added to the apical compartment and 400 μl of HBSS was added to the basolateral compartment. The plates were incubated at 37°C. An aliquot of the basolateral compartment was taken at 60 and 120 minutes (t=60 and t=120). An aliquot of the apical compartment was taken at the initial time (t=0) and after 120 min (t=120).

Basolateral to apical transport (BiA, serous to mucosal): 400 μl of each concentration of Compound I was added to the basolateral compartment and 200 μl of HBSS was added to the apical compartment. The plates were incubated at 37°C. An aliquot of the apical compartment was taken after 60 and 120 minutes (t=60 and t=120). An aliquot of the basolateral compartment was taken at the initial time (t=0) and after 120 min (t=120). All samples were analyzed by LC-MS, monitoring both components, compound I and the appearance of compound II.

Evaluation of Bidirectional Permeability of Compound II.

Compound II was dissolved in HBSS buffer (1%DMSO final concentration) at concentrations of 1.5, 3 and 6 μM. Each concentration/sampling time was run in the wells in duplicate. Testing was carried out in a pH gradient: the apical compartment (mucous) was carried out at pH

6.5, the basolateral compartment (serous) was performed at pH 7.4.

Apical to basolateral transport (A^B, mucosal to serous): 200 μl of each concentration of Compound II was added to the apical compartment and 400 μl of HBSS was added to the basolateral compartment. The plates were incubated at 37°C. An aliquot of the basolateral compartment was taken at 60 and 120 minutes (t=60 and t=120). An aliquot of the apical compartment was taken at the initial time (t=0) and after 120 min (t=120).

Transport from basolateral to apical direction (BJA, serous to mucosal): 400 μl of each concentration of Compound II was added to the basolateral compartment and 200 μl of HBSS was added to the apical compartment. The plates were incubated at 37°C. An aliquot of the apical compartment was taken after 60 and 120 minutes (t=60 and t=120). An aliquot of the basolateral compartment was taken at the initial time (t=0) and after 120 min (t=120). All samples were analyzed by LC-MS monitoring compound II.

Inhibition of mucosal-serosa transport of compound I by PepT1 substrate (GlySar)

Differentiated cells were pretreated for 30 min with 10 mM Gly-Sar to block the active PepT1 transporter.

Compound I was dissolved in HBSS buffer (1% DMSO final concentration) at concentrations of 1.5, 3 and 6 μM. Each concentration/sampling time was run in the wells in duplicate. Testing was performed in a pH gradient: the apical compartment (mucous) was performed at pH 6.5, the basolateral compartment (serous) was performed at pH 7.4.

Apical to basolateral transport (A^B, mucosal to serous): 200 μl of each concentration of Compound I was added to the apical compartment and 400 μl of HBSS was added to the basolateral compartment. The plates were incubated at 37°C. An aliquot of the basolateral compartment was taken at 60 and 120 minutes (t=60 and t=120). An aliquot of the apical compartment was taken at the initial time (t=0) and after 120 min (t=120). All samples were analyzed by LC-MS, monitoring both components, compound I and the appearance of compound II.

Analytical definitions.

The concentrations of compound II and compound I in the samples after incubation were determined using the method of high performance liquid chromatography/mass spectrometry (LC/MS) described in the appendices (section 7.1) without any additional dilution.

Results.

The values of preliminary experiments TEER monolayers of Caco-2 cells were used in the range from 850 to 1160 Ωχ cm ² , indicating a confluent monolayer with tight junctions. After the end of the experiments, the TEER values decreased on average by 170 Ω χ cm ² (from 680 to 990 Ω χ cm ² ) without affecting the integrity of the cell monolayer. The Lucifer yellow test confirmed the integrity of all monolayers after the experiments, in fact, the detectable amount of lucifer yellow in the basolateral compartments after the experiments was always <1% in all wells. in fig. 55 describes the data obtained in the non-specific binding test for Compound I. Under the conditions tested, Compound I was proven to be reduced in the apical compartment at all tested doses. Compound I was not detected in the basolateral compartment at any tested dose. Non-specific binding of compound I is excluded. Compound II is not found in any compartment. in fig. 55 describes the data obtained in the stability study for Compound I and Compound II. Both compounds are proven to be stable under the conditions tested: HBSS buffer (2% DMSO final concentration) at 37°C for 60 and 120 min in FIG. 56a-56e are data obtained from a bidirectional permeability study for Compound I. This compound does not pass through a cell monolayer. In an apical to basolateral transport study, Compound I was not detected in the receiving compartment at both 60 min and 120 min, while increasing concentrations of Compound II were detected after the end of the experiment in the basolateral compartment. The passage percentages of compound II are described in the table. After the end of the apical to basolateral transport experiment, low transport of Compound I was observed in the apical compartment, while increased concentrations of Compound II were detected (high transport). Increasing concentrations of compound II after 120 min in the apical compartment can be explained by the presence of extra- and intracellular esterases in Caco-2 cells capable of deesterifying compounds (Kern et al. J. Agric. Food Chem. 51: 78847891 (2003)). When examining transport from the basolateral to the apical direction, Compound I was not detected in the receiving compartment, while low Compounds II were found. Thus, Compound I is likely to be transported and transported as Compound II across the Caco-2 monolayer. in fig. 57a-57e show data from a bi-directional permeation study for Compound II. This compound exhibited a high percentage of passage from the apical to the basolateral direction and a low rate of passage from the basolateral to the apical compartment. Papp was calculated because the concentration in the donor compartments was known. Compound II has good passive passage through the Caco-2 monolayer. No leaks were found. in fig. 58a-58c show data from an inhibition assay in which a monolayer of Caco-2 cells was pre-treated with 10 mM Gly-Sar (to saturate the PepT1 transporter). Compound I was not detected in the receiving compartment while the passage of compound II was observed. The passage percentages are not linear in this experiment.

Discussion.

In this study, non-specific binding of Compound I in the tested Caco-2 cell system (without cells) was evaluated and ruled out. Compound I was stable under the conditions tested.

The conversion of compound I to compound II was evaluated and confirmed in a bidirectional permeability study. Compound I does not pass through the cell monolayer under the conditions tested. Thus, it is likely that compound I is transported and transported as deesterified compound II through the monolayer of Caco-2 cells.

In a bidirectional permeability study, Compound II exhibited good passive passage through a monolayer of Caco-2 cells. No evidence was found that Compound II could be a substrate for the efflux transporter.

Gly-Sar pretreatment testing (to saturate the PepT1 transporter) showed no passage of Compound I and normal passage of Compound II. Probably, the transport of compound I through monolayers of Caco-2 cells is not mediated by PepT1.

These experiments indicate that intestinal absorption of Compound I and its salts is not mediated by the protein PepT1 transporter. Instead, the above results indicate that Compound I is deesterified by surrounding esterases in the small intestine and subsequently passively permeates through the small intestine epithelium. This compound I and its salts are not substrates for PepT1, which is an unexpected and pharmacologically favorable property. PepT1 is a pH-independent co-transporter known to mediate the absorption of various valate esters, as described, for example, in Vig et al., Adv. drug deliv. Rev. 65:1370-1385 (2013), the disclosure of which is incorporated herein by reference. PepT1 exhibits broad substrate specificity, as evidenced by the structural diversity of compounds that are transported across the intestinal epithelium by this protein. Surprisingly, despite the presence of valinate ester functionality, Compound I and its salts are not dependent on this transporter for absorption through the intestinal epithelium. This is a favorable property since Compound I and its salts thus do not compete with natural PepT1 substrates, eg peptide nutrients, for binding and transport by this protein. Preferably, compound I and its salts are converted in vivo to a form that is readily absorbed in a manner independent of energy and local proton gradient. This is an unexpected property associated with the high aqueous solubility of Compound I and its salts, which together provide a favorable pharmacokinetic profile whereby these therapeutic agents readily dissolve in an aqueous environment and in turn are converted to a form capable of carrier-independent absorption. .

Example 5 Combination Therapy Including an Additional Tocolytic Agent.

Compound I or a salt thereof, such as Compound III, may be administered to a subject, such as a human, in combination with one or more additional agents, such as an oxytocin receptor antagonist, a betamimetic, a calcium channel blocker, a magnesium salt, or a nitric oxide donor, such as to reduce the frequency of uterine contractions and to delay the onset of labor.

A person skilled in the art can administer Compound I or a salt thereof, eg Compound III, simultaneously with, as a mixture with, or separately from the oxytocin receptor antagonist. Exemplary oxytocin receptor antagonists for use in combination with the compositions and methods of the invention include atosiban, retosiban, barusiban, epelsiban, and nolasiban, or a variant, formulation, crystalline form, or derivative thereof. For example, Compound I, or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with nolasiban, or a variant, drug, crystalline form, or derivative thereof, to delay the onset of labor in a subject, for example, by one or more days. or weeks, for example, from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks).

Additionally or alternatively, a person skilled in the art may administer Compound I or a salt thereof, eg Compound III, simultaneously with, as a mixture with, or separately from a betamimetic, eg betamimetic, as described herein. For example, Compound I or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with a betamimetic described herein or known in the art to delay the onset of labor in a subject, for example, by one or more days or weeks, for example, from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

- 38 039545 or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks).

Additionally or alternatively, one of skill in the art may administer Compound I or a salt thereof, such as Compound III, simultaneously with, as a mixture with, or separately from a calcium channel blocker, such as the calcium channel blocker described herein. For example, Compound I or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with a calcium channel blocker described herein or known in the art to delay the onset of labor in a subject, for example, by one or more days or weeks, such as from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 weeks).

Additionally or alternatively, a person skilled in the art may administer Compound I or a salt thereof, eg Compound III, simultaneously with, as a mixture with, or separately from a magnesium salt, eg magnesium sulfate. For example, Compound I, or a salt thereof, such as Compound III, may be administered before, after, or concurrently with magnesium sulfate to delay the onset of labor in a subject, e.g., one or more days or weeks, e.g., from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks).

Additionally or alternatively, a person skilled in the art may administer Compound I or a salt thereof, eg Compound III, simultaneously with, as a mixture with, or separately from a nitric oxide donor, eg nitroglycerin. For example, Compound I or a salt thereof, such as Compound III, may be administered before, after, or concurrently with nitroglycerin to delay the onset of labor in a subject, e.g., one or more days or weeks, e.g., from about 1 day to about 16 weeks. (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks ).

Additionally or alternatively, one skilled in the art may administer Compound I or a salt thereof, such as Compound III, simultaneously with, as a mixture with, or separately from progesterone or a derivative or variant thereof, such as a derivative or variant described herein. application or known in the art. For example, Compound I or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with progesterone, or a variant or derivative thereof, as described herein or known in the art to delay the onset of labor in a subject, for example, by one or more days or weeks, for example, from about 1 day to about 16 weeks (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks).

Example 6 Tocolytic Effects of Compound I and Their Pharmaceutically Acceptable Salts in Combination with Nifedipine and Atosiban in Mouse Preterm Labor Models.

To investigate the therapeutic effects of Compound I in combination with a calcium channel blocker or an oxytocin receptor antagonist in animal models of preterm labor, CD-1 primigravida mice were treated with established labor inducers at an early gestational age of 17 days and then given various doses of the chloride salt of Compound I ( compound III, 10 mg/kg, 30 mg/kg, or 100 mg/kg, each administered orally) alone or in combination with nifedipine (5 mg/kg orally) or atosiban (300 mg/kg, subcutaneously). Tocolytic effects were assessed by measuring time from induction to first birth for each mouse in the treated and control groups, time from induction to completion of parturition for all mice in each group, and offspring viability among mice in each group. The preterm inducers used in this study were RU486 (also referred to as mifepristone), a steroidal antiprogestin that promotes cervical dilatation and stimulates increased uterine contractility and prostaglandin sensitivity, and lipopolysaccharide (LPS), an inflammatory mediator.

To induce labor at early gestational age, a single dose of RU486 was administered to each mouse subcutaneously at 2.5 mg/kg (t=0). Mice treated with LPS received a single intraperitoneal injection of LPS at 2 mg/kg (t=0). Atosiban was administered to CD-1 mice by subcutaneous injection at a dose of 300 mg/kg at two different sites. These injections were made at 5 hours (t=5) and 29 hours (t=29) after treatment with the inducing agent RU486 or LPS. Nifedipine was administered orally to CD-1 mice at a dose of 5 mg/kg at 5 h (t=5), 19 h (t=19), 29 h (t=29), and 43 h (t=43) after treatment with inducing agent RU486 or LPS. Compound III was administered orally to CD-1 mice at either 10 mg/kg, 30 mg/kg, or 100 mg/kg at 5 h (t=5), 19 h (t=19), 29 h (t=29) , and 43 hours (t=43) after treatment with inducing agent RU486 or LPS. After induction with RU486 or LPS and subsequent administration of atosiban, nifedipine and/or Compound III, groups of mice were subjected to continuous visual control to assess the time elapsed between induction and birth of the first baby for each mouse, as well as the proportion of mice in each group in which childbirth occurred depending on the time. The viability of born fetuses in each group was assessed

- 39 039545 using a hydrostatic lung vital test.

The ability of RU486 and LPS to induce preterm labor was confirmed as treatment of CD-1 mice with RU486 at a gestational age of 17 days resulted in a mean time of delivery of about 21 h after induction (t=21; calculated mean=21±1.00 h ), while LPS-treated CD-1 mice at a gestational age of 17 days showed a mean delivery time of about 26 hours after induction (t=26; calculated mean=26±2.34 hours). In contrast, term deliveries in CD-1 mice occur at gestational age from about 19 days to about 21 days, more than 50 hours after day 17 of gestation. Among mice born from mice treated with RU486, 96% were born alive and 48% of mice born from mice treated with LPS were born alive (FIGS. 60 and 61). 3% of mice treated with RU486 were excluded from this study due to death or sacrifice during the study; 34% of mice treated with LPS were excluded from this study due to death or sacrifice during the study.

During the study, treatment with nifedipine alone was shown to induce a significant increase in mean time to delivery compared with vehicle (23.53±0.99 h vs. 21.19±1.00 h; FIG. 65) in mice treated with using RU486. Treatment with nifedipine alone further improved mean time to delivery and significantly improved fractional offspring viability in LPS-treated mice compared to vehicle (90.39±5.34% vs. 48.20±16.45%; FIG. .68 and 69). Atosiban administration similarly results in an increase in mean time to delivery in LPS-treated mice (FIG. 70).

Compound III was found to increase mean time to delivery compared to vehicle in mice treated with RU486 (FIGS. 65 and 67). Specifically, RU486-treated mice given Compound III orally at 30 mg/kg and 100 mg/kg showed increased time to delivery compared to vehicle (p=0.0871 and p=0.0601, respectively). ). Additionally, administration of Compound III to LPS-treated mice resulted in a dose-dependent increase in fractionated offspring viability (69.41 ± 5.76% viability observed in response to 100 mg/kg Compound III versus 48.20 ± 16.45% observed in response to the introduction of the filler, Fig. 68).

The combination of nifedipine and compound III resulted in a particularly pronounced tocolytic effect (FIGS. 65 and 69). Oral administration of nifedipine (5 mg/kg) and Compound III (100 mg/kg) to RU486-treated mice resulted in an apparent synergistic effect, as the combination induces a significant increase in mean time to delivery compared to vehicle (27.91 ±0.35 h vs. 21.19 ± 1.00 h), the same dosage of nifedipine alone (27.91 ± 0.35 h vs. 23.53 ± 0.99 h), and the same dosage of compound III alone (27 .91±0.35 h versus 23.70±0.60 h). Additionally, oral administration of nifedipine (5 mg/kg) and compound III (10 mg/kg) to LPS-treated mice resulted in a significant increase in mean time to delivery relative to the group treated with 10 mg/kg of compound III alone (31 .01±1.89 h relative to 23.98±0.66 h). Oral administration of 10 mg/kg of Compound III in combination with 5 mg/kg of nifedipine also improved fetal viability in LPS-treated mice compared to mice administered the same dose of Compound III alone (94.23±3. 68% vs. 57.90±14.89%) and vs. mice treated with vehicle alone (94.23±3.68% vs. 48.20±16.45%; FIG. 68).

The combination of atosiban and Compound III further potentiates the tocolytic effect of each compound used alone. Subcutaneous administration of atosiban (300 mg/kg) and oral administration of Compound III (100 mg/kg) to LPS-treated mice induced a significant increase in mean time to delivery relative to mice treated with vehicle alone (33.23±2.95 h). relative to 26.17 ± 1.98 hours) and relative to mice administered the same dosage of atosiban alone (33.23 ± 2.95 hours relative to 28.41 ± 2.99 hours; Fig. 71). This combination also showed the property to increase fractional offspring viability compared to mice treated with vehicle alone, the same dose of atosiban alone, or the same dose of Compound III alone (FIG. 70).

This study further illustrates the tocolytic effect of the Compound I antagonist FP salt in two different animal models of preterm labor and validates the utility of Compound I and its salt in the treatment and prevention of preterm labor regardless of the underlying biochemical etiology. This study further supports the use of FP antagonists, eg Compound I and its salts (eg Compound III) in combination alone with a calcium channel antagonist and an oxytocin receptor antagonist to prevent preterm labor. The use of compound III in combination alone with nifedipine and atosiban significantly exceeds the therapeutic effects of the individual components, and demonstrates that compound I and its salts, for example compound III, can synergize with additional tocolytic agents.

Example 7 Tocolytic Effects of Compound II in Combination with Nifedipine, Atosiban and No

- 40 039545 lasiban on human tissue samples.

To investigate the therapeutic effects of Compounds II, the active metabolite of Compound I and its salts (such as Compound III), in combination with oxytocin receptor antagonists and calcium channel blockers, endometrial biopsies were obtained from term, preterm women who were delivered by caesarean section. One of the objectives of this study was to characterize the effects of Compound II, alone and in combination with additional tocolytic agents, on the frequency, peak amplitude, and duration of myometrial contractions, as well as on work performed per contraction and total work performed on all contractions. For this, experiments were performed using the DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with the ADI Powerlab software, which facilitates the simultaneous measurement of many muscle preparations in parallel.

Endometrial biopsy experiments were initiated by allowing smooth muscle contractions to establish baseline values for at least 20 minutes. After this period of time, baseline measurements of the frequency of spontaneous contractions, peak amplitude, duration, work done per contraction, and total work done on all contractions were recorded. Thereafter, endometrial biopsy specimens were treated with DMSO control, compound II, atosiban, nifedipine, a combination of compound II and atosiban, or a combination of compound II and nifedipine. Thereafter, the effects of these agents on frequency, amplitude, and duration, as well as on myometrial contraction performance, were measured over a subsequent 10 minute period. Myometrial samples were then loaded by adding increasing concentrations of a contraction-stimulating agent, such as oxytocin, PGF2a, or PGE2, over consecutive 10-minute intervals, and the contraction rate, peak amplitude, duration, work done per contraction, and total work were measured. performed for all contractions, respectively. Oxytocin, PGF2a, and PGE are each different modulators of uterine contractility and preterm labor. Oxytocin directly induces contraction of the uterine myometrium and increases the synthesis and release of contractile prostaglandins from the uterine endometrium and decidua. Oxytocin is also involved in promoting the production of prostaglandins in myometrial cells through the potentiation of cyclooxygenase 2 (COX-2). The prostaglandins PGF2a and PGE2 have been shown to induce cervical measurements and excite uterine contractility, two key events in the physiology of labor and delivery. Activation of the FP receptor in human myometrium by PGF2a leads to an increase in intracellular calcium concentration, which in turn leads to contraction of uterine smooth muscle cells. Thus, another aim of the study was to evaluate the ability of compound II to attenuate uterine contractions induced by three different biochemical agents.

The results of these experiments indicate that Compound II alone is able to suppress both PCP2a-induced and OT-induced myometrial contractility in a dose dependent manner (FIGS. 72 and 73). In addition, in this case, it was discovered that compound II exhibits an unexpected synergistic effect in reducing myometrial contractility when used in combination with the oxytocin receptor antagonist atosiban (Fig. 76) and the calcium channel blocker nifedipine (Fig. 78). Surprisingly, doses of Compound II that show lower efficacy in reducing myometrial contractility when used in the absence of an additional tocolytic agent (such as 60 nM, Figs. 72 and 73) show a strong increase in inhibitory activity when combined with atosiban (Fig. 76) and nifedipine (Fig. 78). Similarly, doses of atosiban (6 nM, Figs. 74 and 75) and nifedipine (6 nM, Fig. 77) were found to be suboptimal for reducing myometrial contractility when used in the absence of Compound II, exhibiting an unexpected increase in protease efficacy when combined with compound II (FIGS. 76 and 78). These data suggest that Compound II is able to synergize with additional tocolytic agents, eg, oxytocin receptor antagonists and calcium channel blockers, to suppress uterine contractions, which may lead to preterm labor.

In addition to suppressing myometrial contractility, the tocolytic effects of Compound II also confirm the ability of this agent to attenuate the expression of downstream pro-inflammatory genes in human endometrial and amion biopsies (FIGS. 79 and 80). Western blots were performed to characterize the ability of Compound II, alone and in combination with additional tocolytic agents, to modulate the expression of various proteins in myometrial and amnion samples isolated from term, preterm women delivered by caesarean section. The results of these studies indicate that Compound II is able to reduce the expression of various pro-inflammatory proteins and exhibits unexpected synergy when used in combination with nolasiban in reducing COX-2 expression.

Taken together, the data obtained from these experiments suggest that the compound

- 41 039545 ion II is able to suppress the activity of smooth muscles, which can lead to preterm labor, which is induced by various modulators of uterine contractility. In addition, Compound II exhibits an unexpected synergistic effect with respect to attenuation of uterine contractions when used in combination with oxytocin receptor antagonists and calcium channel blockers. This synergy manifests itself both at the level of smooth muscle activity, and at the level of reduction of pro-inflammatory gene expression in myometrial and amnion biopsies, and shows various advantages, providing compound II in combination with one or more additional tocolytic agents to a subject in need of treatment, for example, a subject who are starting or at risk of developing preterm labor.

Other embodiments

All publications, patents, and patent applications referred to in this application are incorporated by reference herein as if each independent publication or patent application were specifically and individually incorporated by reference.

Although the invention has been described using its specific embodiments, it is understood that further modifications are possible and the present description is intended to cover any variations, applications or adaptations of the invention, following, in general, the principles of the invention and including such deviations from inventions that fall within known or customary practice in the field to which the invention relates and can be used in the essential characteristics set forth earlier in this application and hereinafter, are covered by the scope of the claims.

Other embodiments are covered by the claims.

CLAIM

Claims (28)

CLAIM 1. A method of treating or preventing preterm labor in a human patient, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an additional tocolytic agent. 2. A method for preventing pre-delivery delivery by caesarean section in a human patient, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an additional tocolytic agent. 3. The method according to claim 1 or 2, characterized in that said compound is represented by formula (III) 4. The method according to claim 3, characterized in that said compound is in a crystalline state. 5. Method according to claim 4, characterized in that said compound exhibits characteristic X-ray powder diffraction peaks at 2Θ angles of 7.0°, 8.1°, 10.0°, 12.0°, 13.1°, 14, 1°, 16.4°, 18.4°, 20.1°, 21.0°, 23.5° and 29.5°. 6. The method according to claim 5, characterized in that the specified compound is characterized by a spectrum of powder - 42 039545 Codex X-ray diffraction, such as shown in FIG. 49. 7. The method according to claim 4, characterized in that said compound exhibits 1H nuclear magnetic resonance (NMR) peaks centered at 1.1 ppm, 3.3 ppm, 4.9 ppm, 5.4 m.h., 7.1 m.h., 7.7 m.h., 7.9 m.h., and 8.0 m.h. 8. The method according to claim 7, characterized in that said compound is characterized by a 1 H NMR spectrum such as shown in FIG. 21. 9. The method of claim 4 wherein said compound exhibits an endotherm at 145 to 147°C as determined by differential scanning calorimetry. 10. The method of claim 9, wherein said compound is characterized by a differential scanning calorimetry curve such as shown in FIG. 20 or 23. 11. The method according to claim 4, characterized in that the specified connection is characterized by a weight loss of 0.2 to 0.6% when heated from 25 to 100°C, as measured by thermogravimetric analysis. 12. The method according to claim 11, characterized in that said compound is characterized by a thermogravimetric analysis curve such as shown in FIG. 24. 13. The method of claim 1 or 2, wherein said additional tocolytic agent is an oxytocin receptor antagonist. 14. The method of claim 13 wherein said oxytocin receptor antagonist is atosiban, retosiban, barusiban, epelsiban or nolasiban. 15. The method of claim 14 wherein said oxytocin receptor antagonist is atosiban. 16. The method of claim 1 or 2, wherein said additional tocolytic agent is a calcium channel blocker. 17. The method of claim 16 wherein said calcium channel blocker is nifedipine or nicardipine. 18. The method of claim 17 wherein said calcium channel blocker is nifedipine. 19. The method according to claim 1 or 2, characterized in that said additional tocolytic agent is a betamimetic, magnesium salt, nitric oxide donor, progesterone, or a variant thereof. 20. The method of claim 19 wherein said additional tocolytic agent is a betamimetic selected from the group consisting of terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nilidrine and orciprenaline. 21. The method of claim 19 wherein said additional tocolytic agent is magnesium sulfate. 22. The method of claim 19 wherein said additional tocolytic agent is nitroglycerin. 23. The method of claim 19 wherein said additional tocolytic agent is progesterone or 17-a-hydroxyprogesterone caproate. 24. The method according to claim 1 or 2, characterized in that the corticosteroid is additionally administered to the patient. 25. The method of claim 24, wherein said corticosteroid is selected from the group consisting of betamethasone, dexamethasone, and hydrocortisone. 26. The method according to claim 1 or 2, characterized in that said compound is administered orally to said patient. 27. The method of claim 1 or 2, wherein said patient has a gestational age of about 24 weeks to about 34 weeks. 28. The method according to claim 1 or 2, characterized in that the patient shows a decrease in the amplitude of uterine contractions after said administration.