Alpha-amino esters of hydroxypropyl thiazolidine carboxamide derivatives and salt forms, crystal polymorphs thereof

Abstract

To provide compositions useful in the treatment of disorders such as preterm labor in early pregnancy.SOLUTION: Pharmaceutical compositions comprising a compound of Formula (I) or (II) and an additional therapeutic agent are provided. Also provided are HCl salts and crystalline forms of the compound of formula (I). The compounds inhibit the prostaglandin F receptor (PGF2 alpha).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to chemical compositions, such as compounds, salts, and crystalline polymorphs, that are capable of binding to and inhibiting the activity of prostaglandin F2α (PGF2α) receptors, and methods for preventing preterm labor during early pregnancy by administering these compositions to a patient in need of such treatment. [Background technology]

[0002] Preterm birth is a common cause of perinatal mortality in the developed world, occurring in approximately 7% to 10% of all births (Berkowitz et al. Epidemiol. Rev. 15:414-443 (1993)). Severe morbidity, particularly respiratory distress syndrome, intraventricular hemorrhage, bronchopulmonary dysplasia, and necrotizing enterocolitis, is much more common in preterm infants than in full-term infants. Long-term disabilities, such as cerebral palsy, visual impairment, and hearing loss, are also more common in preterm infants. Currently, preterm birth remains the leading cause of infant mortality and morbidity in the United States, which, despite significant improvements in obstetric care, has a higher infant mortality rate than many other industrialized countries and costs over $5 billion per year for intensive neonatal care of low-birth-weight infants. The actual costs associated with this treatment are even higher when taking into account medical care for illnesses associated with premature birth, such as respiratory distress syndrome, heart conditions, cerebral palsy, epilepsy, and severe learning disabilities.

[0003] During the past 40 years of clinical investigation, despite the use of multiple therapeutic agents, the rate of preterm birth has not dramatically decreased. Preventing preterm labor is difficult, and although tocolytic therapy remains the cornerstone of management of preterm labor, there is no universal agreement regarding its value in this condition. Available tocolytic agents do not themselves extend labor beyond 48 hours, and most of these agents are poorly uteroselective and therefore may result in potentially serious side effects for both mother and fetus.

[0004] Essentially, term and preterm labor are similar processes in that they share common physiological endpoints characterized by uterine contractions, cervical dilation, and activation of the fetal membranes. The differences lie in the gestational stage at which these processes occur and the mechanisms by which they are activated. While term labor is thought to result from the physiological activation of the terminal pathway, preterm labor is a pathological condition characterized by multiple etiologies in which one or more components of this pathway are abnormally activated.

[0005] Uterine contractions are stimulated or inhibited by various receptors within myometrial cells. It is hypothesized that myometrial activation results from the coordinate expression of receptors for actin, myosin, contraction-associated proteins (CAPs), including connexin-43, as well as oxytocin and prostaglandin receptors. Generally, receptors that trigger calcium influx or calcium release from intracellular stores stimulate contractions. However, receptors coupled to the production of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP), induce uterine relaxation. For example, oxytocin and prostaglandin F (FP) receptors are stimulatory, whereas β2-adrenergic receptors and prostaglandin E2 receptors, which are coupled to cAMP formation, are inhibitory.

[0006] In uterine tissue, prostaglandin E2 (PGE2) and F2α (PGF2α) have been shown to induce cervical changes and uterine contractions, two key events in the physiology of labor and delivery. Activation of FP receptors by PGF2α in human myometrium increases intracellular calcium levels, which in turn leads to contraction of uterine smooth muscle cells (Abramovitz et al. J. Biol. Chem. 269:2632-2636 (1994) and Senior et al. Br. J. Pharmacol. 108:501-506 (1993)). FP receptors are upregulated in uterine tissue toward term (Al-Matubsi et al. Biol. Reprod. 65:1029-1037 (2001)). Although inhibitors of prostaglandin synthesis (e.g., indomethacin and nimesulide) have demonstrated some tocolytic activity, they are not completely free of side effects, and their unauthorized clinical use has raised concerns about fetal safety (Norton et al. New Engl. J. Med. 329:1602-1067 (1993) and Peruzzi et al. New Engl. J. Med. 354:1615 (1999)). There remains a need for the development of therapeutic agents with myometrial selectivity that allow sustained inhibition of uterine contractions leading to labor and prolong pregnancy to a stage where improved fetal maturation increases the likelihood of survival. Summary of the Invention

[0007] The present invention encompasses alpha-aminoesters of hydroxypropylthiazolidinecarboxamide derivatives, as well as salts thereof, that can antagonize the interaction of prostaglandin F2α (PGF2α) with the prostaglandin F receptor. These compounds can be administered to subjects, such as pregnant human female subjects, to treat or prevent preterm labor. The present invention further provides methods for synthesizing these compounds, as well as methods for preparing their crystalline forms.

[0008] In a first aspect, the present invention provides a compound represented by formula (I): [ka] Provided is (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate hydrochloride, represented by formula (III). [ka]

[0009] In some embodiments, the compounds bind to human prostaglandin F2α receptors with an affinity of about 1 nM. The compounds of the present invention exhibit the ability to selectively bind to prostaglandin F receptors, such as prostaglandin F2α, relative to other prostaglandin receptor subtypes. For example, the compounds of the present invention exhibit affinity for the prostaglandin F2α receptor that is about 10-fold higher than that observed for the prostaglandin E2 receptor. Furthermore, the compounds of the present invention exhibit affinity for the prostaglandin F2α receptor that is about 100-fold or higher (e.g., about 100-fold to about 1,000-fold, e.g., about 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 410-fold, 420-fold, 430-fold, 440-fold, 450-fold, 460-fold, 470-fold, 480-fold, 490-fold, 500-fold, 510-fold, 520-fold, 530-fold, 540-fold, 550-fold, 560-fold x, 180x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x , 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 410x, 420x, 430x, 440x, 450x, 460x, 470x, 480x, 490x, 500x, 510x, 520x, 530x, 540x, 550x, 560x, 570x, 580x, 590x, 600x, 610x, 620x, 6 30x, 640x, 650x, 660x, 670x, 680x, 690x, 700x, 710x, 720x, 730x, 740x, 750x, 760x, 770x, 78 In some embodiments, the compound exhibits an affinity for the prostaglandin F2α receptor that is greater than or equal to (0 fold, 790 fold, 800 fold, 810 fold, 820 fold, 830 fold, 840 fold, 850 fold, 860 fold, 870 fold, 880 fold, 890 fold, 900 fold, 910 fold, 920 fold, 930 fold, 940 fold, 950 fold, 960 fold, 970 fold, 980 fold, 990 fold, 1,000 fold, or more). In some embodiments, the compound is soluble in aqueous solution at a concentration of about 300 μg / mL to about 500 μg / mL, e.g., about 380 μg / mL.

[0010] In some embodiments, the compounds inhibit the synthesis of inositol triphosphate in cells, such as mammalian cells. In some embodiments, the mammalian cells are human cells, such as myometrial cells. In some embodiments, the myometrial cells are myometrial cells. In some embodiments, the compounds induce a reduction in the amplitude of uterine contractions in a subject after administration of the compounds to the subject. For example, the compounds may induce a reduction of about 40% to about 50% relative to the measured amplitude of uterine contractions recorded in the subject prior to administration. In some embodiments, the compounds exhibit a half-life of about 1 to about 4 hours in a subject after administration of the compounds to the subject. In some embodiments, the compounds reach a maximum plasma concentration in a subject within about 0.25 to about 2 hours after administration of the compounds to the subject.

[0011] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human, such as a dog or a rat. In some embodiments, the compound is administered orally to the subject. In some embodiments, the compound is administered intravenously to the subject.

[0012] In another aspect, the present invention provides a compound represented by formula (III): [ka] This includes compounds that are in a crystalline state.

[0013] In some embodiments, the compound 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 is characterized by an X-ray powder diffraction spectrum substantially as shown in any one of Figures 19, 22, 29, 45-49, and 54. For example, in some embodiments, the compound is characterized by an X-ray powder diffraction spectrum substantially as shown in Figure 49.

[0014] In some embodiments, the compound is present at concentrations centered at about 1.1 ppm, about 3.3 ppm, about 4.9 ppm, about 5.4 ppm, about 7.1 ppm, about 7.7 ppm, about 7.9 ppm, and about 8.0 ppm. 1 In some embodiments, the compound exhibits a H nuclear magnetic resonance (NMR) peak. 1 Characterized by H NMR spectrum.

[0015] In some embodiments, the compounds exhibit an endotherm of about 145°C to about 147°C as measured by differential scanning calorimetry. In some embodiments, the compounds exhibit an additional endotherm of about 214°C as measured by differential scanning calorimetry. In some embodiments, the compounds are characterized by a differential scanning calorimetry curve substantially as shown in Figure 20. In some embodiments, the compounds exhibit an additional endotherm of about 228°C as measured by differential scanning calorimetry. In some embodiments, the compounds are characterized by a differential scanning calorimetry curve substantially as shown in Figure 23.

[0016] In some embodiments, the compound exhibits a weight loss of about 0.2% to about 0.6% when heated from 25° C. to 100° C., as measured by thermogravimetric analysis. In some embodiments, the compound exhibits a weight loss of about 2.5% to about 3.5% when heated from 100° C. to 160° C., as measured by thermogravimetric analysis. In some embodiments, the compound exhibits a thermogravimetric analysis curve substantially as shown in FIG.

[0017] In a further aspect, the present invention provides a pharmaceutical composition containing a compound of any of the above aspects. The pharmaceutical composition may optionally contain one or more excipients. In some embodiments, the compound has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as determined, for example, by high-pressure liquid chromatography (HPLC) or NMR spectroscopy. In some embodiments, the compound and / or pharmaceutical composition is formulated for oral administration to a subject. In some embodiments, the pharmaceutical composition is a tablet, capsule, gelcap, powder, liquid solution, or liquid suspension. In some embodiments, the compound and / or pharmaceutical composition is formulated for intravenous administration to a subject.

[0018] In some embodiments, the pharmaceutical composition contains two or more therapeutic agents, e.g., a compound of the present invention (e.g., a compound represented by Formula (I), or a pharmaceutically acceptable salt thereof, e.g., a compound represented by Formula (III)), and an additional therapeutic agent. For example, the pharmaceutical composition may contain two or more therapeutic agents admixed with each other for co-administration to a patient, such as for the treatment or prevention of preterm labor. The pharmaceutical compositions of the present invention may be administered to a subject to delay the onset of labor in the subject, for example, by one day or one week or more, for example, from about one 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). In some embodiments, the subject has experienced preterm labor. In some embodiments, the pharmaceutical composition is administered to a subject (e.g., a human subject) before the onset of preterm labor. The pharmaceutical compositions of the present invention may be administered to a subject (e.g., a human subject) to prevent labor prior to cesarean section. The pharmaceutical compositions of the present invention may be administered to a subject (e.g., a human subject) to treat or prevent dysmenorrhea. The pharmaceutical compositions of the present invention may be administered to a subject, such as a pregnant human female subject, to alleviate one or more symptoms associated with labor, such as vaginal bleeding and rupture of the uterine membranes.

[0019] In some embodiments, the additional therapeutic agent is an additional tocolytic agent.

[0020] In some embodiments, the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional tocolytic agent. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (III) and an additional tocolytic agent.

[0021] In some embodiments, the additional tocolytic agent is an oxytocin receptor antagonist, such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, or one or more variants, formulations, crystalline forms, or derivatives thereof.

[0022] In some embodiments, the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and atosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (III) and atosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a variant of atosiban, such as the variants described in U.S. Pat. No. 4,504,469 or U.S. Pat. No. 4,402,942, the disclosures of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (III) and a variant of atosiban, such as the variants described in U.S. Pat. No. 4,504,469 or U.S. Pat. No. 4,402,942.

[0023] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and letosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and letosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and a letosiban variant, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, or US 2016 / 0074413, the disclosures of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and a variant of letosiban, such as a variant described in U.S. Pat. Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, or US2016 / 0074413.

[0024] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and barusiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and barusiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and a variant of barusiban, such as a variant described in U.S. Patent Nos. 6,143,722, 7,091,314, 7,816,489, or US 2016 / 0175283, the disclosures of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and a variant of barusiban, such as a variant described in U.S. Patent Nos. 6,143,722, 7,091,314, 7,816,489, or US 2016 / 0175283.

[0025] In some embodiments, the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and epersiban. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (III) and epersiban. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a variant of epersiban, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, or 8,815,856, the disclosures of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (III) and a variant of epersiban, such as a variant described in U.S. Pat. Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, or 8,815,856.

[0026] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and nolasiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and nolasiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and a mutant, formulation, or crystalline form of nolasiban, such as those described in U.S. Patent No. 7,115,754 or U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, or 2016 / 0002160, the disclosures of each of which are incorporated herein by reference. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and a variant, formulation, or crystalline form of nolasiban, such as a variant, formulation, or crystalline form described in U.S. Pat. No. 7,115,754 or U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, or 2016 / 0002160.

[0027] In some embodiments, the additional tocolytic agent is a betamimetic such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, or orciprenaline.

[0028] In some embodiments, the additional tocolytic agent is a calcium channel blocker such as a dihydropyridine. In some embodiments, the calcium channel blocker is nifedipine. In some embodiments, the calcium channel blocker is nicardipine.

[0029] In some embodiments, the additional tocolytic agent is a magnesium salt, such as magnesium sulfate.

[0030] In some embodiments, the additional tocolytic agent is a nitric oxide donor, such as nitroglycerin.

[0031] In some embodiments, the additional tocolytic agent is an oxytocin receptor antagonist such as atosiban, letosiban, barusiban, epelusiban, nolasiban, or a variant, formulation, crystalline form, or derivative thereof, such as those described herein.

[0032] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the additional tocolytic agent is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the additional tocolytic agent is formulated for intramuscular administration.

[0033] In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and the additional tocolytic agent is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and the additional tocolytic agent is formulated for intramuscular administration.

[0034] In some embodiments, the additional therapeutic agent is progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate.

[0035] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and progesterone or 17-α-hydroxyprogesterone caproate. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the progesterone or 17-α-hydroxyprogesterone caproate is formulated for vaginal administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the progesterone or 17-α-hydroxyprogesterone caproate is formulated for vaginal administration. In some embodiments, both the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof and progesterone or 17-α-hydroxyprogesterone caproate are formulated for oral administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for oral administration.

[0036] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (III) and progesterone or 17-α-hydroxyprogesterone caproate. In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for intravaginal administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for intravaginal administration. In some embodiments, both the compound represented by Formula (III) and progesterone or 17-α-hydroxyprogesterone caproate are formulated for oral administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for oral administration.

[0037] 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 some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration. In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is formulated for intravenous administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration. In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (III) is formulated for oral administration, and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration.In some embodiments, the compound represented by Formula (III) is formulated for intravenous administration and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration.

[0038] In another aspect, the present invention provides a compound represented by formula (I): [ka] or a pharmaceutically acceptable salt thereof, comprising a precursor represented by formula (IV): [ka] with a precursor represented by formula (V) [ka] wherein X is a protecting group to form an amino ester. In some embodiments, the method includes deprotecting the amino ester. In some embodiments, the compound is represented by Formula (III): [ka]

[0039] In some embodiments, the method comprises reacting the amino ester with a reagent capable of deprotecting the amino ester. In some embodiments, the protecting group is tert-butoxycarbonyl, trityl, 4-monomethoxytrityl, 4-methyltrityl, 3,5-dimethoxyphenylisopropoxycarbonyl, 2-(4-biphenyl)isopropoxycarbonyl, 2-nitrophenylsulfenyl, 9-fluorenylmethoxycarbonyl, 2-(4-nitrophoneylsulfonyl)ethoxycarbonyl, (1,1-dioxobenzo[b]thiophen-2-yl)methoxycarbonyl, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl, 2,7-di-tert-butyl-9-fluorenylmethoxycarbonyl, 2-fluoro-9-fluorenylmethoxycarbonyl, 2-monoisooctyl-9-fluorenylmethoxycarbonyl, 2,7-diisooctyl-9-fluorenylmethoxycarbonyl, tetrachlorofluoromethyl, ... and thalloyl, 2-[phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate, ethanesulfonylethoxycarbonyl, 2-(4-sulfophenylsulfonyl)ethoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, o-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, benzothiazole-2-sulfonyl, 2,2,2-trichloroethyloxycarbonyl, dithiasuccinoyl, p-nitrobenzyloxycarbonyl, α-azido acid, propargyloxycarbonyl, 9-(4-bromophenyl)-9-fluorenyl, azidomethoxycarbonyl, hexafluoroacetone, 2-chlorobenzyloxycarbonyl, trifluoroacetyl, 2-(methylsulfonyl)ethoxycarbonyl, phenyldisulfanylethyloxycarbonyl, and 2-pyridyldisulfanylethyloxycarbonyl.

[0040] In some embodiments, the reagent is methanesulfonic acid, hydrochloric acid, trifluoroacetic acid, acetic acid, piperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, morpholine, hexamethyleneimine, ammonia, diethylamine, piperazine, tris(2-aminoethyl)amine, hydrazine, 1-methylpyrrolidine, sodium bicarbonate, sodium hydroxide, barium hydroxide, sodium carbonate, molecular hydrogen, hydrobromic acid, boron tribromide, tetrakis(triphenylphosphine)palladium, thiophene, or the like. The solvent is selected from the group consisting of alcohol, β-mercaptoethanol, 2-mercaptoacetic acid, aluminum amalgam, zinc, hypophosphorous acid, sodium borohydride, N-mercaptoacetamide, tin(II) chloride, trimethylphosphine, tributylphosphine, triphenylphosphine, benzyltriethylammonium tetrathiomolybdate, palladium(II) acetate, hydrofluoric acid, trimethylsilyl chloride, trimethylsilyl trifluoromethanesulfonate, and trifluoromethanesulfonic acid.

[0041] In some embodiments, the protecting group is tert-butoxycarbonyl and the reagent is selected from the group consisting of methanesulfonic acid, hydrochloric acid, and trifluoroacetic acid such as methanesulfonic acid.

[0042] In some embodiments, the method includes exposing the amino ester to electromagnetic radiation. In some embodiments, the protecting group is selected from the group consisting of o-nitrobenzyloxycarbonyl, 4-nitroveratryloxycarbonyl, 2-(2-nitrophenyl)propyloxycarbonyl, and 2-(3,4-methylenedioxy-6-nitrophenyl)propyloxycarbonyl. In some embodiments, the electromagnetic radiation is characterized by a wavelength of about 300 to about 400 nm.

[0043] In some embodiments, the method includes reacting a precursor represented by Formula (IV) with a precursor represented by Formula (V) and a diimide. In some embodiments, the diimide is selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, and N,N'-dicyclohexylcarbodiimide. In some embodiments, the diimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the method includes reacting a precursor represented by Formula (IV) with a precursor represented by Formula (V) and a benzotriazole derivative, such as a benzotriazole derivative selected from the group consisting of 1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole. In some embodiments, the benzotriazole derivative is 1-hydroxybenzotriazole.

[0044] In some embodiments, the method includes reacting a precursor represented by formula (IV) with a precursor represented by formula (V) and a base, such as N,N-dimethylaminopyridine.

[0045] In some embodiments, the method comprises synthesizing a precursor represented by formula (IV), wherein the precursor is represented by formula (VI): [ka] with a precursor represented by (VII) [ka] This includes synthesizing by reacting with

[0046] In some embodiments, the method includes reacting a precursor represented by Formula (VI) with a precursor represented by Formula (VII) and one or more bases. In some embodiments, the one or more bases are selected from the group consisting of diisopropylethylamine, triethylamine, and N,N-dimethylaminopyridine.

[0047] In some embodiments, the method includes reacting a precursor represented by Formula (VI) with a precursor represented by Formula (VII), diisopropylethylamine, and N,N-dimethylaminopyridine.

[0048] In a further aspect, the present invention provides a method of making a compound represented by formula (III): [ka] The method comprises: [ka] with hydrochloric acid.

[0049] In some embodiments, the hydrochloric acid is an aqueous hydrochloric acid solution. The aqueous hydrochloric acid solution can be prepared, for example, by diluting hydrochloric acid in water, such as distilled water or deionized water. In some embodiments, the method includes preparing the compound represented by Formula (III) in a crystalline state.

[0050] In some embodiments, the method includes dissolving a compound represented by Formula (I) in ethanol. In some embodiments, the method includes combining hydrochloric acid with ethanol. In some embodiments, the method includes combining hydrochloric acid with ethyl acetate. In some embodiments, the method includes adding a compound represented by Formula (I) to hydrochloric acid over a period of about 20 to about 30 minutes to form a mixture. In some embodiments, the method includes maintaining the temperature of the mixture at about 15°C to about 25°C during the addition. In some embodiments, the method includes reducing the temperature of the mixture to about 5°C after the addition. In some embodiments, the method includes stirring the mixture at about 0°C to about 5°C for about 50 to about 70 minutes after the reduction.

[0051] In some embodiments, the method comprises combining an equimolar amount of a compound represented by Formula (I) and hydrochloric acid.

[0052] In another aspect, the invention includes a compound produced by any of the above methods.

[0053] In a further aspect, the present invention provides a method of treating preterm labor in a subject by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to any of the above aspects of the invention.

[0054] In a further aspect, the present invention provides a method of preventing preterm labor in a subject by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to any of the above aspects of the invention.

[0055] In another aspect, the invention provides a method of preventing pre-cesarean delivery in a subject by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to any of the above aspects of the invention.

[0056] In another aspect, the present invention provides a method of treating or preventing dysmenorrhea in a subject by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to any of the above aspects of the invention.

[0057] In another aspect, the present invention provides a method of treating or preventing endometriosis in a subject by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to any of the above aspects of the invention.

[0058] In some embodiments, the subject is characterized by a gestational age of about 24 to about 34 weeks. In some embodiments, the subject exhibits, after administration, a reduction in the amplitude of uterine contractions, e.g., a reduction of about 40% to about 50% (e.g., about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%) relative to a measurement of the amplitude of the subject's uterine contractions recorded before administration. In some embodiments, the compound exhibits a half-life in a subject of about 1 to about 4 hours (e.g., about 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3.0 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, or 4.0 hours). In some embodiments, the compound reaches a maximum plasma concentration in the subject within about 0.25 to about 2 hours (e.g., about 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 hours) of administration. In some embodiments, the subject is a mammal, such as a human.

[0059] In some embodiments, the method comprises orally administering the compound or pharmaceutical composition to the subject. In some embodiments, the method comprises intravenously administering the compound or pharmaceutical composition to the subject.

[0060] In some embodiments, the compound is administered to the subject in combination with an additional therapeutic agent. In some embodiments, the compound is administered to the subject in combination with an additional tocolytic agent.

[0061] In some embodiments, the compound is administered to a subject in combination with an oxytocin receptor antagonist. In some embodiments, the method includes orally administering the oxytocin receptor antagonist to the subject. In some embodiments, the method includes intravenously administering the oxytocin receptor antagonist to the subject. The compound may be administered to a subject simultaneously with the administration of an oxytocin receptor antagonist. In some embodiments, the compound is administered to a subject before the administration of an oxytocin receptor antagonist to the subject. In some embodiments, the compound is administered to a subject after the administration of an oxytocin receptor antagonist to the subject. In some embodiments, the compound is mixed with an oxytocin receptor antagonist, and these agents are administered to a subject simultaneously. In some embodiments, the oxytocin receptor antagonist is atosiban, letosiban, barusiban, epersiban, or nolasiban, or a variant, formulation, crystalline form, or derivative thereof.

[0062] In some embodiments, the oxytocin receptor antagonist is atosiban or a variant of atosiban, such as those described in U.S. Pat. No. 4,504,469 or U.S. Pat. No. 4,402,942, the disclosures of each of which are incorporated herein by reference.

[0063] In some embodiments, the oxytocin receptor antagonist is letosiban or a variant of letosiban, such as a variant described in U.S. Pat. Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, or US2016 / 0074413, the disclosures of each of which are incorporated herein by reference.

[0064] In some embodiments, the oxytocin receptor antagonist is barsiban or a variant of barsiban, such as a variant described in U.S. Pat. Nos. 6,143,722, 7,091,314, 7,816,489, or US2016 / 0175283, the disclosures of each of which are incorporated herein by reference.

[0065] In some embodiments, the oxytocin receptor antagonist is epersiban or a variant of epersiban, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, or 8,815,856, the disclosures of each of which are incorporated herein by reference.

[0066] In some embodiments, the oxytocin receptor antagonist is nolasiban, or a variant, formulation, or crystalline form of nolasiban, such as those variants, formulations, or crystalline forms described in U.S. Pat. No. 7,115,754 or U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, or 2016 / 0002160, the disclosures of each of which are incorporated herein by reference.

[0067] In some embodiments, the compound is administered to a subject in combination with a betamimetic, such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, or orciprenaline. In some embodiments, the method comprises orally administering a betamimetic to a subject. In some embodiments, the method comprises intravenously administering a betamimetic to a subject. The compound may be administered to a subject simultaneously with the administration of a betamimetic. In some embodiments, the compound is administered to a subject before the administration of a betamimetic to a subject. In some embodiments, the compound is administered to a subject after the administration of a betamimetic to a subject. In some embodiments, the compound is admixed with a betamimetic, and these agents are administered to a subject simultaneously.

[0068] In some embodiments, the compound is administered to a subject in combination with a calcium channel inhibitor such as a dihydropyridine. In some embodiments, the calcium channel inhibitor is nifedipine. In some embodiments, the calcium channel inhibitor is nicardipine. In some embodiments, the method comprises orally administering a calcium channel inhibitor to a subject. In some embodiments, the method comprises intravenously administering a calcium channel inhibitor to a subject. The compound may be administered to a subject simultaneously with administration of a calcium channel inhibitor. In some embodiments, the compound is administered to a subject before administration of a calcium channel inhibitor to a subject. In some embodiments, the compound is administered to a subject after administration of a calcium channel inhibitor to a subject. In some embodiments, the compound is admixed with a calcium channel inhibitor, and these agents are administered to a subject simultaneously.

[0069] In some embodiments, the compound is administered to the subject in combination with a magnesium salt, such as magnesium sulfate. In some embodiments, the method includes intravenously administering the magnesium salt to the subject. In some embodiments, the method includes intramuscularly administering the magnesium salt to the subject. In some embodiments, the method includes orally administering the magnesium salt to the subject. The compound may be administered to the subject simultaneously with the administration of the magnesium salt. In some embodiments, the compound is administered to the subject before the magnesium salt is administered to the subject. In some embodiments, the compound is administered to the subject after the magnesium salt is administered to the subject. In some embodiments, the compound is admixed with the magnesium salt, and these agents are administered to the subject simultaneously.

[0070] In some embodiments, the compound is administered to the subject in combination with a nitric oxide donor, such as nitroglycerin. In some embodiments, the method includes orally administering the nitric oxide donor to the subject. In some embodiments, the method includes intravenously administering the nitric oxide donor to the subject. The compound may be administered to the subject simultaneously with the administration of the nitric oxide donor. In some embodiments, the compound is administered to the subject before the administration of the nitric oxide donor to the subject. In some embodiments, the compound is administered to the subject after the administration of the nitric oxide donor to the subject. In some embodiments, the compound is admixed with the nitric oxide donor, and these agents are administered to the subject simultaneously.

[0071] In some embodiments, the compound is administered to a subject in combination with progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate. In some embodiments, the method comprises orally administering progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the method comprises intravaginally administering progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. The compound may be administered to a subject simultaneously with the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate. In some embodiments, the compound is administered to a subject before the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the compound is administered to a subject after the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the compound is admixed with progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate (e.g., especially in an oral formulation), and the agents are administered to the subject simultaneously.

[0072] In some embodiments, the compound is administered to the subject in combination with a corticosteroid. In some embodiments, the corticosteroid is betamethasone. In some embodiments, the corticosteroid is dexamethasone. In some embodiments, the method comprises orally administering a corticosteroid to the subject. In some embodiments, the method comprises intramuscularly administering a corticosteroid to the subject. The compound may be administered to the subject simultaneously with administration of a corticosteroid. In some embodiments, the compound is administered to the subject before administration of the corticosteroid to the subject. In some embodiments, the compound is administered to the subject after administration of the corticosteroid to the subject. In some embodiments, the compound is admixed with the corticosteroid (e.g., particularly in an oral formulation) and these agents are administered to the subject simultaneously.

[0073] In some embodiments, the present invention provides a kit comprising a compound or pharmaceutical composition according to any of the above aspects of the present invention and a package insert. In some embodiments, the package insert instructs a user of the kit to administer the compound or pharmaceutical composition to a subject experiencing or at risk of experiencing preterm labor, such as a subject experiencing one or more symptoms of preterm labor described herein. In some embodiments, the subject is characterized by a gestational age of about 24 to about 34 weeks. In some embodiments, the package insert instructs a user of the kit to mix the compound or pharmaceutical composition with an aqueous solution. In some embodiments, the package insert instructs a user of the kit to orally administer the compound to the subject. In some embodiments, the package insert instructs a user of the kit to intravenously administer the compound to the subject.

[0074] In a further aspect, the present invention provides a compound represented by formula (II): [ka] Provided are pharmaceutical compositions containing 3-([1,1'-biphenyl]-4-ylsulfonyl)-N-[1-(4-fluorophenyl)-3-hydroxypropyl]-1,3-thiazolidine-2-carboxamide. In some embodiments, the pharmaceutical compositions contain a compound represented by Formula (II) and an additional therapeutic agent. In some embodiments, the pharmaceutical compositions contain a compound represented by Formula (II) and an additional tocolytic agent. The pharmaceutical compositions may optionally contain one or more excipients. In some embodiments, the compound represented by Formula (II) has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%, as determined by, for example, high pressure liquid chromatography (HPLC) or NMR spectroscopy. In some embodiments, the compounds and / or pharmaceutical compositions are formulated for oral administration to a subject. In some embodiments, the compound and / or pharmaceutical composition is a tablet, capsule, gelcap, powder, liquid solution, or liquid suspension. In some embodiments, the compound and / or pharmaceutical composition is formulated for intravenous administration to a subject.

[0075] In some embodiments, the pharmaceutical composition contains two or more therapeutic agents, for example, a compound represented by Formula (II) and an additional therapeutic agent. For example, the pharmaceutical composition may contain two or more therapeutic agents admixed with each other for co-administration to a patient, such as for treating or preventing preterm labor. The pharmaceutical composition may be administered to a subject to delay the onset of labor in the subject by, for example, one day or one week or more, for example, from about one 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). In some embodiments, the subject has experienced preterm labor. In some embodiments, the pharmaceutical composition is administered to a subject (e.g., a human subject) before the onset of preterm labor. The pharmaceutical composition may be administered to a subject (e.g., a human subject) to prevent labor before a cesarean section. The pharmaceutical composition may be administered to a subject (e.g., a human subject) to treat or prevent dysmenorrhea. The pharmaceutical composition may be administered to a subject, such as a pregnant human female subject, to relieve one or more symptoms associated with labor, such as vaginal bleeding and rupture of the uterine membranes.

[0076] In some embodiments, the additional therapeutic agent is an additional tocolytic agent.

[0077] In some embodiments, the additional tocolytic agent is an oxytocin receptor antagonist such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, or one or more variants, formulations, crystalline forms, or derivatives thereof.

[0078] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and atosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and a variant of atosiban, such as the variants described in U.S. Patent No. 4,504,469 or U.S. Patent No. 4,402,942, the disclosures of each of which are incorporated herein by reference.

[0079] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and letosiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and a variant of letosiban, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, or US2016 / 0074413, the disclosures of each of which are incorporated herein by reference.

[0080] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and barusiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and a variant of barusiban, such as a variant described in U.S. Patent Nos. 6,143,722, 7,091,314, 7,816,489, or US2016 / 0175283, the disclosures of each of which are incorporated herein by reference.

[0081] In some embodiments, the pharmaceutical composition comprises a compound represented by formula (II) and epersiban. In some embodiments, the pharmaceutical composition comprises a compound represented by formula (II) and a variant of epersiban, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, or 8,815,856, the disclosures of each of which are incorporated herein by reference.

[0082] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and nolasiban. In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and a mutant, formulation, or crystalline form of nolasiban, such as those described in U.S. Patent No. 7,115,754 or U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, or 2016 / 0002160, the disclosures of each of which are incorporated herein by reference.

[0083] In some embodiments, the additional tocolytic agent is a betamimetic such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, or orciprenaline.

[0084] In some embodiments, the additional tocolytic agent is a calcium channel blocker such as a dihydropyridine. In some embodiments, the calcium channel blocker is nifedipine. In some embodiments, the calcium channel blocker is nicardipine.

[0085] In some embodiments, the additional tocolytic agent is a magnesium salt, such as magnesium sulfate.

[0086] In some embodiments, the additional tocolytic agent is a nitric oxide donor, such as nitroglycerin.

[0087] In some embodiments, the additional tocolytic agent is an oxytocin receptor antagonist such as atosiban, letosiban, barusiban, epelusiban, nolasiban, or a variant, formulation, crystalline form, or derivative thereof, such as those described herein.

[0088] In some embodiments, the compound represented by Formula (II) is formulated for oral administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (II) is formulated for oral administration, and the additional tocolytic agent is formulated for intravenous administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration, and the additional tocolytic agent is formulated for oral administration. In some embodiments, the compound represented by Formula (II) is formulated for oral administration, and the additional tocolytic agent is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration, and the additional tocolytic agent is formulated for intramuscular administration.

[0089] In some embodiments, the additional therapeutic agent is progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate.

[0090] In some embodiments, the pharmaceutical composition comprises a compound represented by Formula (II) and progesterone or 17-α-hydroxyprogesterone caproate. In some embodiments, the compound represented by Formula (II) is formulated for oral administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for vaginal administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for vaginal administration. In some embodiments, both the compound represented by Formula (II) and progesterone or 17-α-hydroxyprogesterone caproate are formulated for oral administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration, and progesterone or 17-α-hydroxyprogesterone caproate is formulated for oral administration.

[0091] 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 some embodiments, the compound represented by Formula (II) is formulated for oral administration and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for intramuscular administration. In some embodiments, the compound represented by Formula (II) is formulated for oral administration and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration. In some embodiments, the compound represented by Formula (II) is formulated for intravenous administration and the corticosteroid (e.g., betamethasone, dexamethasone, or hydrocortisone) is formulated for oral administration.

[0092] In a further aspect, the present invention provides a compound represented by formula (II) according to any of the above aspects of the invention, [ka] Provided is a method for treating preterm labor in a subject by providing (e.g., administering) to the subject a therapeutically effective amount of 3-([1,1'-biphenyl]-4-ylsulfonyl)-N-[1-(4-fluorophenyl)-3-hydroxypropyl]-1,3-thiazolidine-2-carboxamide, or a pharmaceutical composition containing a compound represented by Formula (II).

[0093] In a further aspect, the present invention provides a method of preventing preterm labor in a subject by providing (e.g., administering) to the subject a therapeutically effective amount of a compound represented by Formula (II), or a pharmaceutical composition containing a compound represented by Formula (II), according to any of the above-mentioned aspects of the present invention.

[0094] In another aspect, the present invention provides a method of preventing pre-cesarean delivery in a subject by providing (e.g., administering) to the subject a therapeutically effective amount of a compound represented by Formula (II), or a pharmaceutical composition containing a compound represented by Formula (II), according to any of the above-mentioned aspects of the present invention.

[0095] In another aspect, the present invention provides a method of treating or preventing dysmenorrhea in a subject by providing (e.g., administering) to the subject a therapeutically effective amount of a compound represented by formula (II), or a pharmaceutical composition containing a compound represented by formula (II), according to any of the above-described aspects of the present invention.

[0096] In another aspect, the present invention provides a method of treating or preventing endometriosis in a subject by providing (e.g., administering) to the subject a therapeutically effective amount of a compound represented by Formula (II), or a pharmaceutical composition containing a compound represented by Formula (II), according to any of the above-described aspects of the present invention.

[0097] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with an additional therapeutic agent. In some embodiments, the compound is provided to a subject in combination with an additional tocolytic agent. In some embodiments, the compound is provided to a subject by administering the compound to the subject. In some embodiments, the compound is provided to a subject by administering to the subject a prodrug that is metabolized in vivo to produce the compound represented by Formula (II).

[0098] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with an oxytocin receptor antagonist. In some embodiments, the method includes orally administering the oxytocin receptor antagonist to the subject. In some embodiments, the method includes intravenously administering the oxytocin receptor antagonist to the subject. The compound represented by Formula (II) may be provided to the subject simultaneously with the administration of the oxytocin receptor antagonist. In some embodiments, the compound represented by Formula (II) is provided to the subject before the administration of the oxytocin receptor antagonist to the subject. In some embodiments, the compound represented by Formula (II) is provided to the subject after the administration of the oxytocin receptor antagonist to the subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is mixed with the oxytocin receptor antagonist, and these agents are administered to the subject simultaneously. In some embodiments, the oxytocin receptor antagonist is atosiban, letosiban, barusiban, epersiban, or nolasiban, or a variant, formulation, crystalline form, or derivative thereof.

[0099] In some embodiments, the oxytocin receptor antagonist is atosiban or a variant of atosiban, such as those described in U.S. Pat. No. 4,504,469 or U.S. Pat. No. 4,402,942, the disclosures of each of which are incorporated herein by reference.

[0100] In some embodiments, the oxytocin receptor antagonist is letosiban or a variant of letosiban, such as a variant described in U.S. Pat. Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, or US2016 / 0074413, the disclosures of each of which are incorporated herein by reference.

[0101] In some embodiments, the oxytocin receptor antagonist is barsiban or a variant of barsiban, such as a variant described in U.S. Pat. Nos. 6,143,722, 7,091,314, 7,816,489, or US2016 / 0175283, the disclosures of each of which are incorporated herein by reference.

[0102] In some embodiments, the oxytocin receptor antagonist is epersiban or a variant of epersiban, such as a variant described in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, or 8,815,856, the disclosures of each of which are incorporated herein by reference.

[0103] In some embodiments, the oxytocin receptor antagonist is nolasiban, or a variant, formulation, or crystalline form of nolasiban, such as those variants, formulations, or crystalline forms described in U.S. Pat. No. 7,115,754 or U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, or 2016 / 0002160, the disclosures of each of which are incorporated herein by reference.

[0104] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with a betamimetic such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, or orciprenaline. In some embodiments, the method includes orally administering a betamimetic to the subject. In some embodiments, the method includes intravenously administering a betamimetic to the subject. The compound represented by Formula (II) may be provided to the subject simultaneously with the administration of the betamimetic. In some embodiments, the compound represented by Formula (II) is provided to the subject before the administration of the betamimetic to the subject. In some embodiments, the compound represented by Formula (II) is provided to the subject after the administration of the betamimetic to the subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is mixed with the betamimetic, and these agents are administered to the subject simultaneously.

[0105] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with a calcium channel inhibitor such as a dihydropyridine. In some embodiments, the calcium channel inhibitor is nifedipine. In some embodiments, the calcium channel inhibitor is nicardipine. In some embodiments, the method comprises orally administering a calcium channel inhibitor to a subject. In some embodiments, the method comprises intravenously administering a calcium channel inhibitor to a subject. The compound represented by Formula (II) may be provided to a subject simultaneously with administration of a calcium channel inhibitor. In some embodiments, the compound represented by Formula (II) is provided to a subject before administration of a calcium channel inhibitor to a subject. In some embodiments, the compound represented by Formula (II) is provided to a subject after administration of a calcium channel inhibitor to a subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is mixed with a calcium channel inhibitor, and these agents are administered to a subject simultaneously.

[0106] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with a magnesium salt, such as magnesium sulfate. In some embodiments, the method includes intravenously administering the magnesium salt to the subject. In some embodiments, the method includes intramuscularly administering the magnesium salt to the subject. In some embodiments, the method includes orally administering the magnesium salt to the subject. The compound represented by Formula (II) may be provided to the subject simultaneously with the administration of the magnesium salt. In some embodiments, the compound represented by Formula (II) is provided to the subject before the administration of the magnesium salt to the subject. In some embodiments, the compound represented by Formula (II) is provided to the subject after the administration of the magnesium salt to the subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is mixed with the magnesium salt, and these agents are administered to the subject simultaneously.

[0107] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with a nitric oxide donor, such as nitroglycerin. In some embodiments, the method includes orally administering the nitric oxide donor to the subject. In some embodiments, the method includes intravenously administering the nitric oxide donor to the subject. The compound represented by Formula (II) may be provided to the subject simultaneously with administration of the nitric oxide donor. In some embodiments, the compound represented by Formula (II) is provided to the subject before administration of the nitric oxide donor to the subject. In some embodiments, the compound represented by Formula (II) is provided to the subject after administration of the nitric oxide donor to the subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is mixed with the nitric oxide donor, and these agents are administered to the subject simultaneously.

[0108] In some embodiments, the compound represented by Formula (II) is provided to a subject in combination with progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate. In some embodiments, the method includes orally administering progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the method includes intravaginally administering progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. The compound represented by Formula (II) may be provided to a subject simultaneously with the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate. In some embodiments, the compound represented by Formula (II) is provided to a subject before the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the compound represented by Formula (II) is provided to a subject after the administration of progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate, to a subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is admixed with progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate (e.g., in an oral formulation, among others), and the agents are administered simultaneously to a subject.

[0109] In some embodiments, the compound represented by Formula (II) is provided to the subject in combination with a corticosteroid. In some embodiments, the corticosteroid is betamethasone. In some embodiments, the corticosteroid is dexamethasone. In some embodiments, the method includes orally administering a corticosteroid to the subject. In some embodiments, the method includes intramuscularly administering a corticosteroid to the subject. The compound represented by Formula (II) may be provided to the subject simultaneously with administration of the corticosteroid. In some embodiments, the compound represented by Formula (II) is provided to the subject before administration of the corticosteroid to the subject. In some embodiments, the compound represented by Formula (II) is provided to the subject after administration of the corticosteroid to the subject. In some embodiments, the compound represented by Formula (II) or a prodrug thereof is admixed with the corticosteroid (e.g., particularly in an oral formulation), and these agents are administered to the subject simultaneously.

[0110] In some embodiments, the subject is characterized by a gestational age of about 24 to about 34 weeks. In some embodiments, the subject exhibits, after administration, a reduction in the amplitude of uterine contractions, e.g., a reduction of about 40% to about 50% (e.g., about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%) relative to a measurement of the subject's uterine contraction amplitude recorded before administration. In some embodiments, the subject is a mammal, such as a human.

[0111] In some embodiments, the method comprises orally administering the compound or pharmaceutical composition to the subject. In some embodiments, the method comprises intravenously administering the compound or pharmaceutical composition to the subject.

[0112] In some embodiments, the present invention provides a kit comprising a compound or pharmaceutical composition according to any of the above aspects of the present invention and a package insert. In some embodiments, the package insert instructs a user of the kit to administer the compound or pharmaceutical composition to a subject experiencing or at risk of experiencing preterm labor, such as a subject experiencing one or more symptoms of preterm labor described herein. In some embodiments, the subject is characterized by a gestational age of about 24 to about 34 weeks. In some embodiments, the package insert instructs a user of the kit to mix the compound or pharmaceutical composition with an aqueous solution. In some embodiments, the package insert instructs a user of the kit to orally administer the compound to the subject. In some embodiments, the package insert instructs a user of the kit to intravenously administer the compound to the subject.

[0113] definition As used herein, the term "about" refers to a value that is within 10% above or below the stated value.

[0114] As used herein, the term "affinity" refers to the strength of the binding interaction between two molecules, such as a ligand and a receptor. i The term " " as used herein is intended to refer to the inhibition constant of an antagonist with respect to a particular molecule of interest, expressed as a molar concentration (M). The K for an antagonist-target interaction i Values ​​can be determined, for example, using methods established in the art. K of an antagonist with respect to a molecular target i Methods that can be used to determine K include competitive binding experiments, such as the competitive radioligand binding assay described in US 8,415,480. d " as used herein refers to, for example, the dissociation rate constant (k d ) is the association rate constant (k a ) and expressed as a molar concentration (M).d The K value can be determined, for example, using methods established in the art. d Methods that can be used to determine β include, for example, surface plasmon resonance by use of a biosensor system such as a BIACORE® system.

[0115] As used herein, the term "corticosteroid" refers to any of the steroid hormones produced by the adrenal cortex or their synthetic equivalents. Exemplary corticosteroids include betamethasone, dexamethasone, and hydrocortisone, among others, and their variants. Corticosteroids used in conjunction with the compositions and methods described herein include those capable of inducing fetal lung maturation, for example, to prevent the development of respiratory distress syndrome in premature infants. Exemplary corticosteroids used in conjunction with the compositions and methods described herein include those described in Jobe et al. Am. J. Obstet. Gynecol. 190:878-881 (2004) and Miracle et al. J. Perinat. Med. 36:191-196 (2008), the disclosures of each of which are incorporated herein by reference.

[0116] As used herein, the term "crystalline" or "crystalline form" means having a physical state that is an ordered three-dimensional arrangement of atoms, ions, molecules, or molecular aggregates. A crystalline form has a lattice arrangement of components called asymmetric units, arranged according to well-defined symmetries and forming a unit cell that repeats in three dimensions. In contrast, the term "amorphous" or "amorphous form" refers to an unorganized (disordered) structure. The physical state of a therapeutic compound can be determined by exemplary techniques such as x-ray diffraction, polarized light microscopy, and / or differential scanning calorimetry.

[0117] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell).

[0118] As used herein, the term "exogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell). Exogenous materials include those provided to an organism or culture extracted from an organism from an external source.

[0119] As used herein, the term "gestational age" describes how far a particular pregnancy has progressed and is measured from the first day of a pregnant female subject's last menstrual cycle to the current day. As used herein, the term "labor" (sometimes referred to as birth) refers to the expulsion of a fetus and placenta from a pregnant female subject's uterus. In a normal pregnancy, labor can occur at approximately 40 weeks of gestation. As used herein, "preterm labor" refers to a situation in which labor begins more than three weeks before full-term gestation, which is usually approximately 40 weeks. That is, preterm labor occurs, for example, at any stage before 38 weeks of gestation. Typically, if untreated, preterm labor leads to the onset of labor or physiological changes associated with labor in a pregnant female subject. Preterm labor may or may not be associated with vaginal bleeding or rupture of the uterine membranes. Preterm labor may also be referred to as premature labor. Preventing preterm labor in a subject can extend the length of pregnancy, thus avoiding preterm labor and thereby reducing the risk of neonatal mortality and morbidity.

[0120] As used herein, "IC 50The term "agonist" refers to the concentration of a substance (antagonist) that reduces the efficacy of a reference agonist or the constitutive activity of a biological target by 50%, as measured, for example, in a competitive ligand binding assay. Exemplary competitive ligand binding assays include competitive radioligand binding assays, competitive enzyme-linked immunosorbent assays (ELISAs), and fluorescence polarization measurement-based assays, among others known in the art.

[0121] As used herein in connection with 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, such as a human subject), e.g., at either the same time or 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, such as in a single pharmaceutical composition or in separate compositions that are administered to the subject at the same time (e.g., by 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, followed by the other therapeutic agent, either by the same route of administration or by a different route of administration.

[0122] As used herein, the term "nolasiban" refers to (3Z,5S)-5-(hydroxymethyl)-1-[(2'-methyl-1,1'-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-methyloxime, which is represented by the following structural formula: [ka] Mutants, formulations, and crystalline forms of nolasiban are described, for example, in U.S. Pat. No. 7,115,754 and U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, and 2016 / 0002160, the disclosures of each of which are incorporated herein by reference.

[0123] As used herein, the term "oral bioavailability" refers to the proportion of a compound administered to a subject, such as a mammal (e.g., a human), that reaches the subject's systemic circulation and is neither sequestered in non-target organs nor absorbed in the gastrointestinal tract and excreted. The term refers to the plasma concentration incorporated over time and is usually expressed as a percentage of the orally administered dose.

[0124] As used herein, the term "oxytocin receptor antagonist" or "oxytocin antagonist" refers to a compound that can inhibit the interaction of oxytocin with the oxytocin receptor, such that, for example, the activity of one or more downstream signaling molecules in the oxytocin signaling cascade is inhibited. Oxytocin antagonists for use with the compositions and methods described herein include, among others, compounds that bind to and inhibit the oxytocin receptor, such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, as well as variants, formulations, crystalline forms, and derivatives thereof, including those described herein.

[0125] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0126] As used herein, the term "pharmaceutical composition" means a mixture containing therapeutic compounds that is administered to a mammal, e.g., a human subject, to prevent, treat, or control a particular disease or condition affecting the mammal, such as preterm labor or dysmenorrhea, among others, as described herein.

[0127] As used herein, the term "protecting group" refers to a chemical moiety that, when attached to a functional group, renders the functional group inert to one or more chemical reactions. Such reactions may modify one or more substituents of a compound and, in the absence of the protecting group, may result in undesired chemical modifications (e.g., electrophilic addition, solvolysis, oxidation, reduction, or functional group interconversion) of the moiety of interest (e.g., an amino, hydroxyl, carboxyl, or carboxamide moiety). Protecting groups may, when appropriate, be chemically reacted to regenerate the original functionality. The identity of the protecting group may be selected to be compatible with the remainder of the molecule, e.g., so that the protecting group will not be removed during other synthetic or modification steps of the molecule, and, optionally, so that the reaction conditions used to effect removal of the protecting group do not result in the removal of a different protecting group located on another substituent of the molecule. Exemplary protecting groups include those that can be covalently bonded to an amino substituent, such as, for example, the amino group of an α-amino ester. Subsequent removal of the protecting group is referred to herein as "deprotection" of the chemical moiety and can be accomplished using reagents and conditions known in the art.Examples of protecting groups include, but are not limited to, benzyl, acetyl, oxyacetyl, carboxybenzyl, 9-fluorenyloxycarbonyl, 2-chloro-1-indanylmethoxy-carbonyl, benz[f]indene-3-methoxycarbonyl, 2-(tert-butylsulfonyl)-2-propenyloxycarbonyl, benzothiophenesulfone-2-methylcarbonyl, tert-butoxycarbonyl, tert-amyloxycarbonyl, β-trimethylsilylethyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenylyl)propyl-2-oxycarbonyl, 2-(p-phenylazophenyl)propyl-2-oxycarbonyl, 2-2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, among others. , 2-phenylpropyl-2-oxycarbonyl, benzyloxycarbonyl, p-toluenesulfonylaminocarbonyl, o-nitrophenylsulfenyl, dithiasuccinoyl, phthaloyl, piperidinoxycarbonyl, formyl, trifluoroacetyl, 2,4,6-trimethoxybenzyl, 2,3,6-trimethyl-4 methoxybenzenesulfonyl, tert-butoxymethyl, pentamethylchromansulfonyl, adamantly, β-trimethylsilylethyl, β-trimethylilylethyloxycarbonyl, tert-butyl, tert-butylbenzyl, cyclopentyl, triphenylmethyl, benzyloxycarbonyl, formyl, and trifluoroacetyl. Protecting groups may be suitable for particular chemical substituents.For example, examples of hydroxyl protecting groups include, but are not limited to, dialkylsilyl ethers such as benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether, and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetyl, for example, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, and the like; and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl, benzyl, and p-nitrophenyl. Further 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, the disclosures of which are incorporated herein by reference. Other examples of protecting groups are described, for example, in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167, 4,460,501, and 4,108,846, the disclosures of each of which are incorporated herein by reference.

[0128] As used herein in reference to therapeutic treatment, the terms "provide" and "providing" refer to the delivery of a therapeutic agent to a subject (e.g., a mammalian subject, such as a human) in need of treatment, such as, for example, a subject experiencing or at risk of experiencing preterm labor. The therapeutic agent may be provided 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 to the therapeutic agent in vivo when the prodrug is administered to the subject. Exemplary prodrugs include, but are not limited to, esters, phosphates, and other chemical functionalities that are susceptible to hydrolysis when administered to a subject. Prodrugs include those known in the art, such as, for example, those described in Vig et al., Adv. Drug Deliv. Rev. 65:1370-1385 (2013) and Huttunen et al., Pharmacol. Rev. 63:750-771 (2011), the disclosures of each of which are incorporated herein by reference.

[0129] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental tissue or skin tissue), pancreatic juice, chorionic villus samples, and cells).

[0130] As used herein, the terms "specifically bind" and "binding" refer to a binding reaction that determines, for example, the presence of a particular protein in a heterogeneous population of proteins and other biological molecules that are recognized by a specific ligand. A ligand (e.g., a protein, proteoglycan, or glycosaminoglycan) that specifically binds to a protein has, for example, a K of less than 100 nM. D For example, a ligand that specifically binds to a protein may have a K of up to 100 nM (e.g., 1 pM to 100 nM). DA ligand that does not exhibit specific binding to a protein or domain thereof may have a K of 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 thereof. D A variety of assay formats can be used to determine the affinity of a ligand for a particular protein. For example, solid-phase ELISA assays are routinely used to identify ligands that specifically bind to target proteins. For a description of assay formats and conditions that can be used to determine the binding of a particular 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).

[0131] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism undergoing treatment for a particular disease or condition described herein (e.g., preterm labor or dysmenorrhea), or an organism diagnosed as having a disease or condition by the methods described herein. Examples of subjects and patients include mammals, such as humans, undergoing treatment for a disease or condition, e.g., preterm labor in early pregnancy (e.g., 24-34 weeks).

[0132] The compounds, salt forms, crystalline polymorphs, therapeutic agents, or other compositions described herein may be referred to as characterized by graphical data "substantially as shown" in the figures. Such data may include, but are not limited to, powder X-ray diffractograms, NMR spectra, differential scanning calorimetry curves, and thermogravimetric analysis curves, among others. As is known in the art, such graphical data may provide additional technical information to further define the compounds, salt forms, crystalline polymorphs, therapeutic agents, or other compositions. As will be appreciated by those skilled in the art, graphical representations of such data may be subject to slight variations, for example, in relative peak intensities and peak positions, due to factors such as variations in instrument response and variations in sample concentration and purity. Nevertheless, one skilled in the art would readily be able to compare the graphical data in the figures herein with graphical data generated for a compound, salt form, crystalline polymorph, therapeutic agent, or other composition to determine whether the two sets of graphical data characterize the same or two different substances. Thus, for example, a crystalline form of (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate hydrochloride referred to herein as being characterized by graphical data "substantially as shown" in a Figure is understood to include all crystalline forms of (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate hydrochloride characterized by this graphical data and optionally having one or more minor variations, such as one or more variations described above or known to those of skill in the art.

[0133] As used herein, the terms "treat" or "treatment" refer to therapeutic procedures aimed at preventing or slowing (alleviating) the progression of an undesirable physiological change or disorder, such as preterm labor during early pregnancy (e.g., 24-34 weeks). Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms such as vaginal bleeding or rupture of membranes, and delaying or slowing labor. Those in need of treatment include, for example, pregnant female subjects who have already experienced preterm labor, as well as those prone to developing this condition.

[0134] As used herein, the term "tocolytic agent" refers to a substance that can delay the onset of labor in a subject (e.g., a mammalian subject, such as a human subject). Tocolytic agents can, for example, increase cytosolic cAMP levels and increase intracellular Ca 2+ The tocolytic agent may function to inhibit uterine contractions by inhibiting the recruitment of uterine contractions. Exemplary 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 that may be used in conjunction with the compositions and methods described herein include, but are not limited to, the agents listed in Table 1 below. [Table 1] [Brief explanation of the drawings]

[0135] [Figure 1] 1 is a graph showing the effects of Compound II and Compound III on spontaneous uterine contractions in late-stage pregnant rats after intravenous administration. [Figure 2] 1 is a graph showing the dose-dependent and reversible effect of Compound I on spontaneous uterine contractions in late-term pregnant rats. [Figure 3] 1 is a graph showing the effects of Compound II and Compound III on spontaneous uterine contractions in late-stage pregnant rats after oral administration. [Figure 4]1 is a table summarizing various methods used to produce the free base of Compound I, as well as the physical characteristics and NMR spectra of Compound I produced by each method. [Figure 5A] 1 is a table summarizing various methods used to produce salts of Compound I, as well as observations regarding the physical characteristics and NMR spectra of those salts produced by each method. [Figure 5B] 1 is a table summarizing various methods used to produce salts of Compound I, as well as observations regarding the physical characteristics and NMR spectra of those salts produced by each method. [Figure 5C] 1 is a table summarizing various methods used to produce salts of Compound I, as well as observations regarding the physical characteristics and NMR spectra of those salts produced by each method. [Figure 6] 1 is a table summarizing the physical characteristics and X-ray powder diffraction (XRPD) spectra of various salts of Compound I. [Figure 7A] 1 is a table summarizing the methods used to generate various crystalline forms of salts of Compound I, as well as observations regarding the physical properties and XRPD spectra of each crystalline form. [Figure 7B] 1 is a table summarizing the methods used to generate various crystalline forms of salts of Compound I, as well as observations regarding the physical properties and XRPD spectra of each crystalline form. [Figure 7C] 1 is a table summarizing the methods used to generate various crystalline forms of salts of Compound I, as well as observations regarding the physical properties and XRPD spectra of each crystalline form. [Figure 8] 1 is a table summarizing the solubility of various salts of Compound I in aqueous solution. [Figure 9] 1 is a table summarizing the stability of various crystalline forms of salts of Compound I at the indicated relative humidity (RH). [Figure 10] 1 is a table summarizing 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 1H nuclear magnetic resonance (NMR). [Figure 11] 1 is a table summarizing various characteristics of the hydrogen sulfate salt of Compound I as determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetry (TG) analysis, and 1H nuclear magnetic resonance (NMR). [Figure 12] 1 shows the XRPD spectrum of the mesylate salt of Compound I. [Figure 13] 1 shows the 1H NMR spectrum of the mesylate salt of Compound I. [Figure 14] 1 shows the XRPD spectrum of the free base of Compound I. [Figure 15] 1 shows the 1H NMR spectrum of the free base of Compound I. [Figure 16] 1 shows the Raman infrared spectrum of the free base of Compound I. [Figure 17] 1 shows the 1H NMR spectrum of the mesylate salt of Compound I, which was prepared by adding methanesulfonic acid to a solution of the free base of Compound I in diethyl ether. [Figure 18] 1 shows a series of 1H NMR spectra of the free base of Compound I recorded during a homonuclear decoupling experiment. [Figure 19] A series of XRPD spectra of the chloride salt of Compound I produced from an acetone slurry (top), from evaporation of a methylene chloride:ethyl ether mixture (second from the top), and from slow evaporation of a 1:1 acetone:toluene mixture (second from the bottom and bottom) are shown. [Figure 20] 1 shows an overlay of differential scanning calorimetry curves (ranging from about −0.5 to about 1.3 W / g) and thermogravimetric curves (ranging from about 0 wt % to about 100 wt %) recorded for the chloride salt of Compound I produced from an acetone slurry. [Figure 21A] 1 shows the 1H NMR spectrum of the chloride salt of Compound I prepared from a 1:1 acetone:toluene mixture. [Figure 21B] 1 shows the 1H NMR spectrum of the chloride salt of Compound I prepared from a 1:1 acetone:toluene mixture. [Figure 21C]1 shows the 1H NMR spectrum of the chloride salt of Compound I prepared from a 1:1 acetone:toluene mixture. [Figure 21D] 1 shows the 1H NMR spectrum of the chloride salt of Compound I prepared from a 1:1 acetone:toluene mixture. [Figure 22] A series of XRPD spectra of the chloride salt of Compound I prepared from an acetone slurry (top) and after drying under vacuum at about 50° C. for 1 day (bottom). [Figure 23] 1 shows an overlay of differential scanning calorimetry curves (ranging from about −1.0 to about 0.2 W / g) and thermogravimetric analysis curves (ranging from about 30 wt % to about 100 wt %) recorded for the chloride salt of Compound I after drying under vacuum at about 50° C. for 1 day. [Figure 24] 1 shows an overlay of thermogravimetric analysis curves for the chloride salt of Compound I prepared from an acetone slurry (top) and after drying under vacuum at about 50° C. for 1 day (bottom). [Figure 25] 1 shows an overlay of differential scanning calorimetry curves recorded for the chloride salt of Compound I produced from an acetone slurry (top) and after drying under vacuum at about 50° C. for 1 day (bottom). [Figure 26] 1 shows the moisture sorption / desorption curve recorded for the chloride salt of Compound I. The values ​​on the y-axis represent the percentage change in weight of the chloride salt as a function of the relative humidity (RH) in the atmosphere surrounding the salt. [Figure 27] 1 is a table reporting data obtained from moisture sorption / desorption experiments conducted with the chloride salt of Compound I. [Figure 28] 1 shows the moisture sorption / desorption curve recorded for the chloride salt of Compound I. The values ​​on the y-axis represent the percentage change in weight of the chloride salt as a function of time as the relative humidity in the atmosphere surrounding the salt is changed. [Figure 29] An overlay of XRPD spectra of the chloride salt of Compound I before (top) and after (bottom) a moisture sorption / desorption experiment is shown. [Figure 30]1 shows an overlay of the XRPD spectrum of the fumarate salt of Compound I (top) and the XRPD of fumaric acid (bottom) produced by slow evaporation of a 1:1 methanol:toluene mixture. [Figure 31] 1 shows an overlay of the XRPD spectrum of the dihydrophosphate salt of Compound I (top) and the XRPD spectrum of the hydrogen sulfate salt of Compound I (bottom). [Figure 32] 1 shows an overlay of differential scanning calorimetry curves (range of about −1.9 to about 0 W / g) and thermogravimetric curves (range of about 25 wt % to about 95 wt %) recorded for the hydrogen sulfate salt of Compound I. [Figure 33] 1 shows the 1H NMR spectrum of the hydrogen sulfate salt of Compound I. [Figure 34] 1 shows the 1H NMR spectrum of the sulfate salt of Compound I. [Figure 35] 1 shows the XRPD spectrum of the mesylate salt of Compound I. [Figure 36] 1 shows the XRPD spectrum of the citrate salt of Compound I. [Figure 37] 1 shows the XRPD spectrum of the edisylate salt of Compound I. [Figure 38] 1 shows the XRPD spectrum of the hydrogen sulfate salt of Compound I. [Figure 39] 1 shows the XRPD spectrum of the citrate salt of Compound I produced by slow evaporation of a 1:2 methanol:toluene mixture. [Figure 40] 1 shows the XRPD spectrum of the hydrogen sulfate salt of Compound I produced by slow evaporation of a 6:1 ethyl acetate:heptane mixture. [Figure 41] 1 shows the XRPD spectrum of the hydrogen sulfate salt of Compound I produced by slow evaporation of the ethyl acetate mixture. [Figure 42] 1 shows the XRPD spectrum of the dihydrogen phosphate salt of Compound I produced by slow evaporation of a 1:2 methanol:acetonitrile mixture. [Figure 43]1 shows the XRPD spectrum of the dihydrogen phosphate salt of Compound I produced by slow evaporation of a 1:1 methyl ethyl ketone:n-butyl acetate mixture. [Figure 44] 1 shows XRPD spectra recorded from duplicate XRPD experiments of the dihydrogen phosphate salt of Compound I produced by slow evaporation of a 1:1 methyl ethyl ketone:n-butyl acetate mixture. [Figure 45] 1 shows the XRPD spectrum of the chloride salt of Compound I produced by slow evaporation of a 1:1 acetone:toluene mixture. [Figure 46] 1 shows XRPD spectra recorded from duplicate XRPD experiments of the chloride salt of Compound I produced by slow evaporation of a 1:1 acetone:toluene mixture. [Figure 47] 1 shows the XRPD spectrum of the chloride salt of Compound I produced by slow evaporation of a diethyl ether:methylene chloride mixture. [Figure 48] 1 shows an XRPD spectrum of the chloride salt of Compound I produced from an acetone slurry. [Figure 49] 1 shows the XRPD spectrum of the chloride salt of Compound I after drying under vacuum. [Figure 50] 1 shows the XRPD spectrum of the fumarate salt of Compound I produced by slow evaporation of a 1:1 methanol:toluene mixture. [Figure 51] 1 shows the XRPD spectrum of the fumarate salt of Compound I produced by slow evaporation of a 1:1 methanol:ethyl acetate mixture. [Figure 52] 1 shows the XRPD spectrum of the fumarate salt of Compound I produced by vacuum drying of a 1:1 methanol:toluene mixture. [Figure 53] 1 shows the XRPD spectrum of the edisylate salt of Compound I produced by slow evaporation of a 1:1:1 methanol:methyl ethyl ketone:toluene mixture. [Figure 54] 1 shows an overlay of XRPD spectra of the chloride salt of Compound I before (bottom) and after (top) storage at 40° C. and 75% relative humidity. [Figure 55]Buffer used in Caco-2 permeation experiments: Table summarizing the stability of Compound I mesylate and Compound II in Hank's Balanced Salt Solution (HBSS) buffer (final concentration of DMSO 2%). [Figure 56A] 1 is a table reporting data obtained from an analysis of the ability of Compound I mesylate to pass from the apical to the basolateral compartment of a transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cells were incubated with the indicated concentrations of Compound I mesylate in the apical compartment of the transwell, and aliquots were sampled from the basolateral compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data are reported as the concentration of Compound II in the basolateral compartment as a percentage of the indicated initial concentration of Compound I mesylate. [Figure 56B] 1 is a table reporting data obtained from an analysis of the ability of Compound I mesylate to pass from the basolateral to the apical compartment of a transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cells were incubated with the indicated concentrations of Compound I mesylate in the basolateral compartment of the transwell, and aliquots were sampled from the apical compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data are reported as the concentration of Compound II in the basolateral compartment as a percentage of the indicated initial concentration of Compound I mesylate. [Figure 56C] 1 is a graph showing the relative concentration of Compound II in the basolateral compartment as a percentage of the initial concentration of Compound I mesylate in the apical compartment. [Figure 56D] 1 is a graph showing the relative concentration of Compound II in the apical compartment as a percentage of the initial concentration of Compound I mesylate in the basolateral compartment. Compound I was not detected in the basolateral compartment after 60 or 120 minutes of incubation in the apical compartment. Furthermore, Compound I was not detected in the apical compartment after 60 or 120 minutes of incubation in the basolateral compartment. Rather, Compound II was detected in each case. [Figure 56E]1 is a table showing the recovery of Compound I in the apical compartment after 120 minutes of incubation. The initial compound was recovered primarily in the form of the deesterified variant, Compound II. [Figure 57A] 1 is a table reporting data obtained from an analysis of the ability of Compound II to pass from the apical to the basolateral compartment of a transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cells were incubated with the indicated concentrations of Compound II in the apical compartment of the transwell, and aliquots were sampled from the basolateral compartment at the indicated sampling times to determine the presence of Compound II. The data are reported as the concentration of Compound II in the basolateral compartment as a percentage of the indicated initial concentration of Compound II. [Figure 57B] 1 is a table reporting data obtained from an analysis of the ability of Compound II to pass from the basolateral to the apical compartment of a transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cells were incubated with the indicated concentrations of Compound II in the basolateral compartment of the transwell, and aliquots were sampled from the apical compartment at the indicated sampling times to determine the presence of Compound II. The data are reported as the concentration of Compound II in the basolateral compartment as a percentage of the indicated initial concentration of Compound II. [Figure 57C] 1 is a table showing the recovery of Compound II in the apical compartment after 60 and 120 minutes of incubation in the basolateral compartment, as well as the permeability of Compound II through Caco-2 cell monolayers. [Figure 57D] 1 is a graph showing the relative concentration of Compound II in the basolateral compartment as a percentage of the initial concentration of Compound II in the apical compartment. [Figure 57E] 1 is a graph showing the relative concentration of Compound II in the apical compartment as a percentage of the initial concentration of Compound II in the basolateral compartment. [Figure 58A]1 is a table reporting data obtained from an analysis of the ability of Compound I mesylate to pass from the apical to the basolateral compartment of a transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cells were incubated with the indicated concentrations of Compound I mesylate in the apical compartment of the transwell, and aliquots were sampled from the basolateral compartment at the indicated sampling times to determine the presence of Compound I or Compound II. The data report the concentration of Compound II in the basolateral compartment as a percentage of the indicated initial concentration of Compound I mesylate. Compound I was not detected in the basolateral compartment after 60 or 120 minutes of incubation in the apical compartment. [Figure 58B] 1 is a graph showing the relative concentration of Compound II in the basolateral compartment as a percentage of the initial concentration of Compound I mesylate in the apical compartment. [Figure 58C] 1 is a table showing the recovery of Compound I in the apical compartment after 120 minutes of incubation. The initial compound was recovered primarily in the form of Compound II, a deesterified variant of the compound. [Figure 59] 1 is a table summarizing the chromatographic and mass spectrometry parameters used for the analysis of Compound I and Compound II concentrations in the Caco-2 cell permeation experiments described herein. [Figure 60A] This graph illustrates the survival rate of offspring of CD-1 mice treated with RU486 or lipopolysaccharide (LPS) at 17 days of gestation to induce birth. Values ​​represent the mean ± standard error of the mean. Asterisks represent p-values ​​of p<0.05. Statistical analysis was performed using the Mann-Whitney test for corresponding groups. [Figure 60B] 1 is a graph illustrating the number of viable and non-viable offspring in CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. [Figure 61A] 1 is a graph illustrating the time from induction to birth of the first pup in CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce birth. Values ​​represent the mean ± standard error of the mean. [Figure 61B] Graphs illustrating the time from induction to complete parturition among CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. Values ​​along the y-axis represent the percentage of CD-1 mice that completed parturition. In each figure, an asterisk represents a p-value of p<0.05. Statistical analysis was performed using the Mann-Whitney test or the log-rank test for corresponding groups. [Figure 61C] Graphs illustrating the time from induction to complete parturition among CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. Values ​​along the y-axis represent the percentage of CD-1 mice that completed parturition. In each figure, an asterisk represents a p-value of p<0.05. Statistical analysis was performed using the Mann-Whitney test or the log-rank test for corresponding groups. [Figure 62A] This graph shows the effect of atosiban (300 mg / kg, subcutaneous administration) and nifedipine (5 mg / kg, oral administration) on the survival rate of offspring in CD-1 mice treated with RU486 or lipopolysaccharide (LPS) at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. Asterisks indicate p<0.05, and "ns" indicates p>0.05. Statistical analysis was performed using a Mann-Whitney test or unpaired t-test against the corresponding vehicle group. [Figure 62B] FIG. 1 is a graph showing the effect of atosiban (300 mg / kg, subcutaneous administration) and nifedipine (5 mg / kg, oral administration) on the number of viable and nonviable offspring in CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. [Figure 63A]This graph shows the effect of Compound III (10 mg / kg, 30 mg / kg, and 100 mg / kg, administered orally) on the survival rate of offspring of CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05. Statistical analysis was performed using the Mann-Whitney test against the corresponding vehicle group. [Figure 63B] 1 is a graph showing the effect of Compound III (10 mg / kg, 30 mg / kg, and 100 mg / kg, orally administered) on the number of viable and nonviable offspring in CD-1 mice treated with RU486 or LPS at 17 days of gestation to induce parturition. [Figure 64A] This graph shows the effects of nifedipine (5 mg / kg, PO), Compound III (100 mg / kg, PO), and their combination on the survival rate of offspring in CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05 relative to the corresponding group, and "NS" indicates a p-value of p>0.05 relative to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test for corresponding groups of interest. [Figure 64B] 1 is a graph showing the effect of nifedipine (5 mg / kg, orally administered), Compound III (100 mg / kg, orally administered), and their combination on the number of viable and non-viable offspring in CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. [Figure 65A]This graph shows the effects of nifedipine (5 mg / kg, orally), Compound III (100 mg / kg, orally), and their combination on the time from induction to delivery of the first pup in CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. Three asterisks represent a p-value of p<0.001 relative to the corresponding group, and two asterisks represent a p-value of p<0.01 relative to the corresponding group. The nifedipine, Compound III, and combination arms exhibited p-values ​​of p=0.0576, p=0.0601, and p<0.001 (indicated by the "$$$" symbol), respectively, relative to the vehicle-treated group. Statistical analysis was performed using a Mann-Whitney test or an unpaired t-test for corresponding groups of interest. [Figure 65B] 1 is a graph showing the effect of nifedipine (5 mg / kg, po), Compound III (100 mg / kg, po), and their combination on the time from induction to completed parturition among CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. The values ​​along the y-axis represent the percentage of CD-1 mice that completed parturition. [Figure 65C] Figure 65B is a graph showing the time from induction to complete offspring birth in the vehicle and combination arms shown in Figure 65B. Three asterisks represent a p-value of p<0.001 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 65D] Figure 65B shows the time from induction to complete birth of offspring in the Compound III and combination arms. Three asterisks represent p-values ​​of p<0.001 for the corresponding groups. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 65E] Figure 65B is a graph showing the time from induction to complete birth of offspring in the nifedipine and combination arms shown in Figure 65B. Two asterisks represent a p-value of p<0.01 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 66A]This graph shows the effects of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the survival rate of offspring in CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05 relative to the corresponding group, and "NS" indicates a p-value of p>0.05 relative to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test for corresponding groups of interest. [Figure 66B] FIG. 1 is a graph showing the effect of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the number of viable and nonviable offspring in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 67A] Graphs showing the effects of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the time from induction to delivery of the first pup in CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05 relative to the corresponding group, and "NS" indicates a p-value of p>0.05 relative to the corresponding vehicle group. The atosiban, Compound III, and combination arms exhibited p-values ​​of p>0.05, p=0.0601, and p>0.05, respectively, relative to the vehicle group. Statistical analysis was performed using an unpaired t-test for corresponding groups of interest. [Figure 67B] 1 is a graph showing the effect of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the time from induction to completed parturition among CD-1 mice treated with RU486 at 17 days of gestation to induce parturition. The values ​​along the Y-axis represent the percentage of CD-1 mice that completed parturition. [Figure 67C]67B is a graph showing the time from induction to complete birth of offspring in the vehicle and combination arms shown in Figure 67B. "ns" represents a p-value of p>0.05 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 67D] Figure 67B shows the graph showing the time from induction to complete birth of offspring in the Compound III and combination arms. The combination arm exhibited a p-value of p=0.0832 compared to the Compound III arm. Statistical analysis was performed using the log-rank test for corresponding groups of interest. [Figure 67E] Figure 67B is a graph showing the time from induction to complete offspring delivery in the atosiban and combination arms. "ns" represents a p-value of p>0.05 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 68A] This graph shows the effects of nifedipine (5 mg / kg, orally administered), Compound III (10 mg / kg, 30 mg / kg, and 100 mg / kg, orally administered), and their combination on the survival rate of offspring in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05 relative to the corresponding group, and "NS" indicates a p-value of p>0.05 relative to the corresponding vehicle group. The nifedipine arm exhibited a p-value of p=0.0859 relative to the group treated with vehicle alone. Statistical analysis was performed using a Mann-Whitney test or an unpaired t-test for corresponding groups of interest. [Figure 68B] 1 is a graph showing the effect of nifedipine (5 mg / kg, orally administered), Compound III (10 mg / kg, 30 mg / kg, and 100 mg / kg, orally administered), and their combination on the number of viable and nonviable offspring in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 69A]This graph shows the effects of nifedipine (5 mg / kg, orally), Compound III (10 mg / kg, 30 mg / kg, and 100 mg / kg, orally), and their combination on the time from induction to delivery of the first pup in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. Two asterisks represent a p-value of p<0.01 relative to the corresponding group as assessed by a Mann-Whitney test for the corresponding group; "ns" represents a p-value of p>0.05 relative to the corresponding group as assessed by a Mann-Whitney test for the corresponding group; "NS" represents a p-value of p>0.05 relative to the corresponding vehicle group as assessed by an unpaired t-test for the corresponding group; and "not tested" indicates that no statistical testing was performed for the indicated pair. [Figure 69B] FIG. 1 is a graph showing the effect of nifedipine (5 mg / kg, orally administered), Compound III (10 mg / kg, orally administered), and their combination on the time from induction to completed parturition among CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 69C] 1 is a graph showing the effect of nifedipine (5 mg / kg, orally administered), Compound III (30 mg / kg, orally administered), and their combination on the time from induction to completed parturition among CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 69D] 1 is a graph showing the effect of nifedipine (5 mg / kg, orally administered), Compound III (100 mg / kg, orally administered), and their combination on the time from induction to completed parturition among CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 69E] 69B is a graph showing the time from induction to complete birth of offspring in the vehicle and combination arms shown in Figure 69B. "ns" represents a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69F]Figure 69B shows the graph showing the time from induction to complete birth of offspring in the Compound III and combination arms. Two asterisks represent p-values ​​of p<0.01 for the corresponding groups. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 69G] 69B is a graph showing the time from induction to complete birth of offspring in the nifedipine and combination arms shown in Figure 69B. "ns" represents a p-value of p>0.05 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding group. [Figure 69H] 69C is a graph showing the time from induction to complete birth of offspring in the vehicle and combination arms. "ns" represents a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69I] Figure 69C shows the time from induction to complete birth of offspring in the Compound III and combination arms. "ns" indicates a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69J] 69C is a graph showing the time from induction to complete delivery of offspring in the nifedipine and combination arms. "ns" represents a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69K] 69D is a graph showing the time from induction to complete birth of offspring in the vehicle and combination arms. "ns" represents a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69L] Figure 69D shows the time from induction to complete birth of offspring in the Compound III and combination arms. "ns" indicates a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 69M]69D is a graph showing the time from induction to complete birth of offspring in the nifedipine and combination arms. "ns" represents a p-value of p>0.05 relative to the corresponding group. Statistical analysis was performed using the log-rank test relative to the corresponding group. [Figure 70A] This graph shows the effects of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the survival rate of offspring in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" indicates a p-value of p>0.05 relative to the corresponding group, and "NS" indicates a p-value of p>0.05 relative to the corresponding vehicle group. Statistical analysis was performed using the Mann-Whitney test or unpaired t-test for corresponding groups of interest. [Figure 70B] FIG. 1 is a graph showing the effect of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the number of viable and nonviable offspring in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 71A] This graph shows the effects of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the time from induction to delivery of the first pup in CD-1 mice treated with LPS at 17 days of gestation to induce parturition. Values ​​represent the mean ± standard error of the mean. "ns" represents a p-value of p>0.05 relative to the corresponding group, "NS" represents a p-value of p>0.05 relative to the corresponding vehicle group, and "$" represents a p-value of p<0.05 relative to the corresponding vehicle group. The combination arm exhibited a p-value of p=0.0909 relative to the atosiban-only arm. Statistical analysis was performed using a Mann-Whitney test or an unpaired t-test for corresponding groups of interest. [Figure 71B]FIG. 1 is a graph showing the effect of atosiban (300 mg / kg, subcutaneous administration), Compound III (100 mg / kg, oral administration), and their combination on the time from induction to completed parturition among CD-1 mice treated with LPS at 17 days of gestation to induce parturition. [Figure 71C] Figure 71B is a graph showing the time from induction to complete offspring birth in the vehicle and combination arms shown in Figure 71B. Two asterisks represent a p-value of p<0.01 for the corresponding group. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 71D] Figure 71B shows the graph showing the time from induction to complete birth of offspring in the Compound III and combination arms. The combination arm exhibited a p-value of p=0.0964 compared to the Compound III arm. Statistical analysis was performed using the log-rank test for corresponding groups of interest. [Figure 71E] Figure 71B is a graph showing the time from induction to complete offspring delivery in the atosiban and combination arms shown in Figure 71B. Asterisks represent p-values ​​of p<0.05 for the corresponding groups. Statistical analysis was performed using the log-rank test for the corresponding groups of interest. [Figure 72A]Graph showing the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on PGF2α-induced smooth muscle contraction frequency in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spontaneous." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on contraction frequency was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on contraction frequency in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent the frequency of contractions as a percentage of the spontaneous baseline contraction frequency. The "#" symbol represents a p-value of p<0.05 versus the DMSO control. [Figure 72B]Graph showing the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the work exerted per contraction (area under the curve, or "AUC") of PGF2α-induced smooth muscle contractions in N=6 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on the work exerted per contraction was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on the work done per contraction in the presence of PGFα was then measured by loading myometrial tissue samples with increasing concentrations of PGFα (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGFα 1 nM," "PGFα 10 nM," and "PGFα 100 nM," respectively. Values ​​along the y-axis represent the work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. A "#" symbol represents a p-value of p<0.05 versus the DMSO control. [Figure 72C]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the peak amplitude of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on peak contraction amplitude was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on peak contraction amplitude in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. The "#" symbol represents a p-value of p<0.05 versus the DMSO control. [Figure 72D]Graph showing the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the duration of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on contraction duration was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on contraction duration in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 72E]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the total work exerted by all contractions (sum of the area under the curve for all contractions) of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution and ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted for all spontaneous contractions were recorded. The measurements of the work exerted for all spontaneous contractions are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on the total work exerted for all subsequent contractions was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on the total work done by contractions in the presence of PGFα was then measured by loading myometrial tissue samples with increasing concentrations of PGFα (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGFα 1 nM," "PGFα 10 nM," and "PGFα 100 nM," respectively. Values ​​along the y-axis represent the total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. A "#" symbol represents a p-value of p<0.05 versus the DMSO control. [Figure 73A]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the frequency of oxytocin (OT)-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions had been established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on contraction frequency was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on contraction frequency in the presence of OT was then measured by loading myometrial tissue samples with 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 spontaneous baseline contraction frequency. Asterisks represent p-values ​​of p<0.05 versus the DMSO control. [Figure 73B]Graph showing the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the work exerted per contraction (area under the curve, or "AUC") of OT-induced smooth muscle contractions in N=6 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on the work exerted per contraction was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on the work done per contraction in the presence of OT was then measured by loading the myometrial tissue samples with 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 work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 73C]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the peak amplitude of OT-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on peak contraction amplitude was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on peak contraction amplitude in the presence of OT was then measured by loading myometrial tissue samples with 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 peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. Asterisks represent p-values ​​of p<0.05 relative to the DMSO control. [Figure 73D]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the duration of OT-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on contraction duration was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on contraction duration in the presence of OT was then measured by loading the myometrial tissue samples with 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 contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 73E]This graph shows the effect of various concentrations of Compound II (6 nM, 60 nM, 600 nM, and 6000 nM) on the total work exerted by all OT-induced smooth muscle contractions (the sum of the areas under the curve for all contractions) in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted on all spontaneous contractions were recorded. The measurements of the work exerted on all spontaneous contractions are represented on the x-axis as "Spon." DMSO control or Compound II was then added to each myometrial sample at the indicated concentrations, and the effect of the control or Compound II on the total work exerted on all subsequent contractions was measured over the following 10 minutes. This time point is represented on the x-axis as "Compound II." The effect of Compound II on the total work done by contractions in the presence of OT was then measured by loading myometrial tissue samples with 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 total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. Asterisks represent p-values ​​of p<0.05 versus the DMSO control. [Figure 74A]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the frequency of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions had been established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spontaneous." DMSO control or atosiban ("Ato") was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on contraction frequency was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on contraction frequency in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at sequential 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent the frequency of contractions as a percentage of the spontaneous baseline contraction frequency. Asterisks represent p-values ​​of p<0.05 versus the DMSO control. [Figure 74B]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the work exerted per contraction (area under the curve, or "AUC") of PGF2α-induced smooth muscle contractions in six full-term prepartum myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on the work exerted per contraction was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on the work done per contraction in the presence of PGFα was then measured by loading the myometrial tissue samples with increasing concentrations of PGFα (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGFα 1 nM," "PGFα 10 nM," and "PGFα 100 nM," respectively. Values ​​along the y-axis represent the work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 74C]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the peak amplitude of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous peak contraction amplitude were recorded. The measured spontaneous peak contraction amplitude is represented on the x-axis as "Spon." 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 the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on peak contraction amplitude in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. [Fig. 74D]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the duration of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spon." 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 duration was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on contraction duration in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 74E]This graph shows the effects of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the total work exerted by all contractions (sum of the area under the curve for all contractions) of PGF2α-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted for all spontaneous contractions were recorded. The measurements of the work exerted for all spontaneous contractions are represented on the x-axis as "Spon." DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on the total work exerted for all subsequent contractions was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on the total work done by contractions in the presence of PGF2α was then measured by loading myometrial tissue samples with increasing concentrations of PGF2α (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals. These time points are represented on the x-axis as "PGF2α 1 nM," "PGF2α 10 nM," and "PGF2α 100 nM," respectively. Values ​​along the y-axis represent the total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. Asterisks represent p-values ​​of p<0.05 versus the DMSO control. [Figure 75A]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the frequency of PGE2-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions had been established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spontaneous." DMSO control or atosiban ("Ato") was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on contraction frequency was measured over the following 10 minutes. This time point is represented on the x-axis as "Atot." The effect of atosiban on contraction frequency in the presence of PGE2 was then measured by loading myometrial tissue samples with 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 frequency of contractions as a percentage of the spontaneous baseline contraction frequency. Three asterisks represent a p-value of p<0.001 relative to the DMSO control. [Figure 75B]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the work exerted per contraction (area under the curve, or "AUC") of PGE2-induced smooth muscle contractions in N=6 full-term prepartum myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on work exerted per contraction was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on the work done per contraction in the presence of PGE2 was then measured by loading the myometrial tissue samples with 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 work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 75C]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the peak amplitude of PGE2-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." 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 the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on peak contraction amplitude in the presence of PGE2 was then measured by loading myometrial tissue samples with 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 peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. Asterisks represent p-values ​​of p<0.05 versus the DMSO control. [Figure 75D]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the duration of PGE2-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spon." 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 duration was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on contraction duration in the presence of PGE2 was then measured by loading myometrial tissue samples with 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 contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 75E]This graph shows the effect of various concentrations of atosiban (6 nM, 60 nM, and 600 nM) on the total work done by all contractions (sum of the area under the curve for all contractions) of PGE2-induced smooth muscle contractions in six full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work done for all spontaneous contractions were recorded. The measurements of the work done for all spontaneous contractions are represented on the x-axis as "Spon." DMSO control or atosiban was then added to each myometrial sample at the indicated concentrations, and the effect of the control or atosiban on the total work done for all subsequent contractions was measured over the following 10 minutes. This time point is represented on the x-axis as "Ato." The effect of atosiban on the total work done by contractions in the presence of PGE2 was then measured by loading myometrial tissue samples with 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 total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. An asterisk represents a p-value of p<0.05 versus the DMSO control. Three asterisks represent a p-value of p<0.001 versus the DMSO control. [Figure 76A]This graph shows the effects of various concentrations of Compound II (60 nM and 600 nM), atosiban (6 nM), and the combination of Compound II and atosiban on the frequency of OT-induced smooth muscle contractions in N=3 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spontaneous." DMSO control, Compound II, and / or atosiban were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or atosiban on contraction frequency were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or atosiban on contraction frequency in the presence of OT were then measured by loading myometrial tissue samples with 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 spontaneous baseline contraction frequency. [Figure 76B]Graph showing the effect of various concentrations of Compound II (60 nM and 600 nM), atosiban (6 nM), and the combination of Compound II and atosiban on the work exerted per contraction (area under the curve, or "AUC") of OT-induced smooth muscle contractions in N=3 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control, Compound II, and / or atosiban were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or atosiban on the work done per contraction were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or atosiban on the work done per contraction in the presence of OT were then measured by loading the myometrial tissue samples with 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 work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 76C]This graph shows the effects of various concentrations of Compound II (60 nM and 600 nM), atosiban (6 nM), and the combination of Compound II and atosiban on the peak amplitude of OT-induced smooth muscle contractions in N=3 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." DMSO control, Compound II, and / or atosiban were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or atosiban on peak contraction amplitude were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or atosiban on peak contraction amplitude in the presence of OT were then measured by loading the myometrial tissue samples with 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 peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. [Figure 76D]Figure 1 shows the effects of various concentrations of Compound II (60 nM and 600 nM), atosiban (6 nM), and the combination of Compound II and atosiban on the duration of OT-induced smooth muscle contractions in N=3 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spontaneous." DMSO control, Compound II, and / or atosiban were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or atosiban on contraction duration were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or atosiban on contraction duration in the presence of OT were then measured by loading myometrial tissue samples with 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 contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 76E]Graph showing the effects of various concentrations of Compound II (60 nM and 600 nM), atosiban (6 nM), and the combination of Compound II and atosiban on the total work exerted by all contractions (sum of the area under the curve for all contractions) of OT-induced smooth muscle contractions in N=3 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution and ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted by all spontaneous contractions were recorded. The measurement of the work exerted by all spontaneous contractions is represented on the x-axis as "Spon." DMSO control, Compound II, and / or atosiban were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or atosiban on the total work done for all subsequent contractions were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The myometrial tissue samples were then loaded with increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals to measure the effects of Compound II and / or atosiban on the total work done by contractions in the presence of OT. 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 total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. Three asterisks represent a p-value of p<0.001 relative to the DMSO control. The two "#" symbols represent a p-value of p<0.01 for treatment with atosiban at a concentration of 6 nM. [Figure 77A]This graph shows the effect of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the frequency of OT-induced smooth muscle contractions in two full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions had been established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spon." 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 frequency was measured over the following 10 minutes. This time point is represented on the x-axis as "Nif." The effect of nifedipine on contraction frequency in the presence of OT was then measured by loading myometrial tissue samples with 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 spontaneous baseline contraction frequency. [Figure 77B]This graph shows the effect of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the work exerted per contraction (area under the curve, or "AUC") of OT-induced smooth muscle contractions in N=2 full-term prepartum myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." 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 work exerted per contraction was measured over the following 10 minutes. This time point is represented on the x-axis as "Nif." The effect of nifedipine on the work done per contraction in the presence of OT was then measured by loading the myometrial tissue samples with 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 work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 77C]This graph shows the effect of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the peak amplitude of OT-induced smooth muscle contractions in two full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." DMSO control or nifedipine was then added to each myometrial sample at the indicated concentrations, and the effect of the control or nifedipine on peak contraction amplitude was measured over the following 10 minutes. This time point is represented on the x-axis as "Nif." The effect of nifedipine on peak contraction amplitude in the presence of OT was then measured by loading myometrial tissue samples with 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 peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. [Figure 77D]This graph shows the effect of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the duration of OT-induced smooth muscle contractions in two full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spon." 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 duration was measured over the following 10 minutes. This time point is represented on the x-axis as "Nif." The effect of nifedipine on contraction duration in the presence of OT was then measured by loading myometrial tissue samples with 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 contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 77E]This graph shows the effects of various concentrations of nifedipine (1 nM, 6 nM, 60 nM, 600 nM, and 10 μM) on the total work exerted by all OT-induced smooth muscle contractions (the sum of the areas under the curve for all contractions) in two full-term prepartum myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution and ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted for all spontaneous contractions were recorded. The measurements of the work exerted for all spontaneous contractions are represented on the x-axis as "Spon." 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 total work exerted for all subsequent contractions was measured over the following 10 minutes. This time point is represented on the x-axis as "Nif." The effect of nifedipine on the total work done by contractions in the presence of OT was then measured by loading myometrial tissue samples with 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 total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. [Figure 78A]

[0023] Figure 1 shows the effects of various concentrations of Compound II (60 nM and 600 nM), nifedipine (6 nM), and the combination of Compound II and nifedipine on the frequency of OT-induced smooth muscle contractions in five full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction frequency were recorded. Spontaneous contraction frequency measurements are represented on the x-axis as "Spontaneous." DMSO control, Compound II, and / or nifedipine were then added to each myometrial sample at the indicated concentrations, and the effects of control, Compound II, and / or nifedipine on contraction frequency were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or nifedipine on contraction frequency in the presence of OT were then measured by loading the myometrial tissue samples with 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 spontaneous baseline contraction frequency. [Figure 78B]Graph showing the effect of various concentrations of Compound II (60 nM and 600 nM), nifedipine (6 nM), and the combination of Compound II and nifedipine on the work exerted per contraction (area under the curve, or "AUC") of OT-induced smooth muscle contractions in N=5 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous work exerted per contraction were recorded. The measurements of spontaneous work exerted per contraction are represented on the x-axis as "Spon." DMSO control, Compound II, and / or nifedipine were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or nifedipine on the work done per contraction were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or nifedipine on the work done per contraction in the presence of OT were then measured by loading the myometrial tissue samples with 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 work done per contraction as a percentage of the work done per contraction in spontaneous baseline contractions. [Figure 78C]

[0023] Figure 1 shows the effects of various concentrations of Compound II (60 nM and 600 nM), nifedipine (6 nM), and the combination of Compound II and nifedipine on the peak amplitude of OT-induced smooth muscle contractions in N=5 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction peak amplitude were recorded. The measured spontaneous contraction peak amplitude is represented on the x-axis as "Spon." DMSO control, Compound II, and / or nifedipine were then added to each myometrial sample at the indicated concentrations, and the effects of control, Compound II, and / or nifedipine on peak contraction amplitude were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or nifedipine on peak contraction amplitude in the presence of OT were then measured by loading the myometrial tissue samples with 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 peak contraction amplitude as a percentage of the peak amplitude of spontaneous baseline contractions. [Figure 78D]

[0023] Figure 1 shows the effects of Compound II (60 nM and 600 nM), nifedipine (6 nM), and the combination of Compound II and nifedipine on the duration of OT-induced smooth muscle contractions in N=5 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of spontaneous contraction duration were recorded. Spontaneous contraction duration measurements are represented on the x-axis as "Spontaneous." DMSO control, Compound II, and / or nifedipine were then added to each myometrial sample at the indicated concentrations, and the effects of control, Compound II, and / or nifedipine on contraction duration were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The effects of Compound II and / or nifedipine on contraction duration in the presence of OT were then measured by loading the myometrial tissue samples with 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 contraction duration as a percentage of the duration of spontaneous baseline contractions. [Figure 78E]Graph showing the effects of Compound II (60 nM and 600 nM), nifedipine (6 nM), and the combination of Compound II and nifedipine on the total work exerted by all contractions (sum of the area under the curve of all contractions) of OT-induced smooth muscle contractions in N=5 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution and ADI Powerlab software. Once regular contractions were established for at least 20 minutes, baseline measurements of the work exerted by all spontaneous contractions were recorded. The measurement of the work exerted by all spontaneous contractions is represented on the x-axis as "Spon." DMSO control, Compound II, and / or nifedipine were then added to each myometrial sample at the indicated concentrations, and the effects of the control, Compound II, and / or nifedipine on the total work done for all subsequent contractions were measured over the following 10 minutes. This time point is represented on the x-axis as "ANT." The myometrial tissue samples were then loaded with increasing concentrations of OT (1 nM, 10 nM, and 100 nM) at successive 10-minute intervals to measure the effects of Compound II and / or nifedipine on the total work done by contractions in the presence of OT. 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 total work done by contractions as a percentage of the total work done by spontaneous baseline contractions. An asterisk represents a p-value of p<0.05 versus the DMSO control. Two asterisks represent a p-value of p<0.01 relative to the DMSO control. Three asterisks represent a p-value of p<0.001 relative to the DMSO control. Three "+" symbols represent a p-value of p<0.001 relative to treatment with Compound II at a concentration of 60 nM. [Figure 79A]Western blots showing the effects of oxytocin, nolasiban, and their combination on the expression of phosphorylated p65 (p-p65), phosphorylated p38 (p-p38), and phosphorylated extracellular signal-regulated kinase (p-ERK) in N=6 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with nolasiban at a concentration of 1 μM, or treated with both oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. Blots for β-actin were performed as controls. [Figure 79B]

[0023] Figure 1 shows Western blots showing the effect 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 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with Compound II at a concentration of 3 μM, or treated with both oxytocin and Compound II at varying concentrations of Compound II, both in the presence and absence of nolasiban at a concentration of 1 μM, for the indicated time periods. Blots for β-actin were performed as controls. [Figure 79C] Western blots showing the effects of oxytocin, nolasiban, and their combination on the expression of the pro-inflammatory genes cyclooxygenase 2 (COX-2) and calcium-phosphorylated, calcium-dependent phospholipase A2 (p-cPLA2) in N=6 full-term prepartum myometrial biopsies collected from human female subjects undergoing cesarean delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with nolasiban at a concentration of 1 μM, or treated with both oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. Blots for β-actin were performed as controls. [Figure 79D]

[0023] Figure 1 shows Western blots showing the effect 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 full-term pre-labor myometrial biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with Compound II at a concentration of 3 μM, or treated with both oxytocin and Compound II at varying concentrations of Compound II, both in the presence and absence of nolasiban at a concentration of 1 μM, for the indicated time periods. Blots for β-actin were performed as controls. [Figure 79E] 79A and 79B are graphs quantifying the expression of p-p65. [Figure 79F] FIG. 79B is a graph quantifying the expression of p-p38 shown in FIGS. 79A and 79B. [Figure 79G] FIG. 79B is a graph quantifying the expression of p-ERK shown in FIGS. 79A and 79B. [Figure 79H] 79C and 79D are graphs quantifying the expression of COX-2 shown in Figures 79C and 79D. An asterisk represents a p-value of p<0.05 relative to the non-stimulated ("NS") sample. Two asterisks represent a p-value of p<0.01 relative to the non-stimulated sample. Three asterisks represent a p-value of p<0.001 relative to the non-stimulated sample. Three "#" symbols represent a p-value of p<0.001 relative to the oxytocin (OT)-treated sample. [Figure 79I] 79C and 79D are graphs quantifying the expression of p-cPLA2. An asterisk represents a p-value of p<0.05 relative to the unstimulated ("NS") sample. Three asterisks represent a p-value of p<0.001 relative to the unstimulated sample. [Figure 80A]Western blots showing the effects of oxytocin, nolasiban, and their combination on the expression of p-p65, p-p38, and p-ERK in N=3 full-term pre-delivery amniotic membrane biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with nolasiban at a concentration of 1 μM, or treated with both oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. Blots for β-actin were performed as a control. [Figure 80B] 1 is a Western blot showing the effect 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=3 full-term pre-delivery amniotic membrane biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with Compound II at a concentration of 3 μM, or treated with both oxytocin and Compound II at varying concentrations of Compound II, both in the presence and absence of nolasiban at a concentration of 1 μM, for the indicated time periods. Blots for β-actin were performed as a control. [Figure 80C] Western blots showing the effects of oxytocin, nolasiban, and their combination on the expression of the pro-inflammatory genes COX-2 and p-cPLA2 in N=3 full-term pre-delivery amniotic membrane biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with nolasiban at a concentration of 1 μM, or treated with both oxytocin and nolasiban at a concentration of 1 μM for the indicated time periods. Blots for β-actin were performed as controls. [Figure 80D]

[0023] Figure 1 shows Western blots showing the effect 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 full-term pre-delivery amniotic membrane biopsies collected from human female subjects undergoing cesarean section delivery. Samples were either unstimulated ("NS"), stimulated with oxytocin ("OT"), treated with Compound II at a concentration of 3 μM, or treated with both oxytocin and Compound II at varying concentrations of Compound II, both in the presence and absence of nolasiban at a concentration of 1 μM, for the indicated time periods. Blots for β-actin were performed as controls. DETAILED DESCRIPTION OF THE INVENTION

[0136] The present invention provides α-aminoesters of thiazolidinecarboxamides, such as (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate, as well as salt forms and crystalline polymorphs thereof. These compounds can inhibit the activity of proteins of the prostaglandin F receptor (FP-R) family, such as the prostaglandin F2α (PGF2α) receptor. The compounds, salts, and crystalline polymorphs described herein can be used to inhibit prostaglandin F receptor activity in vitro and in vivo, making them effective therapeutic compositions for the treatment of preterm labor. The compounds, salts, and crystalline polymorphs described herein can be administered to a subject (e.g., a mammalian subject, such as a human) experiencing or at risk of experiencing labor early in pregnancy, e.g., before 38 weeks (e.g., from about 20 to about 37 weeks, e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 weeks, preferably from about 24 to about 34 weeks of gestation, e.g., about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 weeks of gestation). The present invention further provides methods of synthesizing (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate, as well as processes for preparing salt forms and crystalline polymorphs thereof. The present invention further encompasses methods of treating preterm labor in a subject, such as a subject experiencing or at risk of experiencing preterm labor, by administering to the subject in need thereof an alpha-amino ester of the present invention, optionally in combination with one or more additional therapeutic agents described herein.

[0137] In addition to the above, the present 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, the compounds, optionally in combination with one or more additional therapeutic agents described herein, may be provided to a subject (e.g., a mammalian subject, such as a human) experiencing or at risk of experiencing labor early in pregnancy, e.g., before 38 weeks (e.g., from about 20 to about 37 weeks, e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 weeks, preferably from about 24 to about 34 weeks of gestation, e.g., about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 weeks of gestation).

[0138] (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate (Compound I) The present invention is based on the discovery that compound I ((3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-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-(4-fluorophenyl)-3-hydroxypropyl]-1,3-thiazolidine-2-carboxamide, represented by formula II below. Compound II, previously described in US Pat. No. 8,415,480, exhibits an inhibition constant (Ki) of 6 nM against human FP-R as determined by a competitive radioligand binding assay, making this compound an antagonist of the prostaglandin F receptor (experimental details of a competitive radioligand binding assay useful for determining Ki values ​​are described, for example, in US Pat. No. 8,415,480, Example 51). After administration to a subject, compound I has been found to be deesterified in vivo to form compound II due to the activity of endogenous esterases, such as those present in the gastrointestinal tract. [ka]

[0139] Compound I was discovered to be an inhibitor of the prostaglandin F receptor, as it inhibited the human FP-R with a K of 1 nM. Compound I exhibits several improved physicochemical characteristics compared to Compound II, including solubility in water, fed-state small intestinal content simulating medium (FeSSIF), and fasted-state small intestinal content simulating medium (FaSSIF). These data are summarized in Table 2 below. [Table 2]

[0140] In addition to exhibiting improved water solubility, Compound I and its salts feature a surprising and beneficial absorption mechanism. As described in the Examples below, Compound I is deesterified by surrounding esterases in the small intestine and then passively permeates the small intestinal epithelium. Surprisingly, Compound I and its salts are not substrates of the Pept1 transporter protein, a protein-linked cotransporter that mediates the absorption of peptide nutrients. This finding represents an unexpected and pharmacologically beneficial property. Pept1 is known to mediate the absorption of various valinate 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 transported across the intestinal epithelium by this protein. Despite the presence of the valinate ester functional group, Compound I and its salts do not rely on this transporter for absorption across the small intestinal epithelium. Thus, Compound I and its salts (e.g., Compound III) are advantageous because they do not compete with natural substrates of Pept1, such as peptide nutrients, for binding to and transport by this protein. Rather, Compound I and its salts are converted in vivo into a form that is readily absorbed in a manner that depends on energy and the local proton gradient. This unexpected property, combined with the high water solubility of Compound I and its salts, collectively results in a beneficial pharmacokinetic profile in which the compounds of the present invention are readily dissolved in an aqueous environment and then converted into a form that is capable of transporter-dependent absorption.

[0141] (3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate hydrochloride (Compound III) The chloride salt of Compound I ((3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propyl L-valinate hydrochloride, hereinafter designated as Formula III), has been discovered to be readily crystallized using several distinct experimental procedures, as described in the Examples below. Compound III adopts a single, reproducible crystalline form when crystallized from various media under different ambient conditions. Furthermore, this crystalline form of Compound III exhibits long-term stability under ambient conditions and in the presence of elevated relative humidity. As described in more detail in the Examples presented below, Compound III exhibits low hygroscopicity and therefore does not exhibit a propensity to absorb moisture from the local atmosphere. Compound III therefore exhibits resistance to chemical transformations such as hydrolysis, as well as resistance to the incorporation of impurities. For example, the crystalline form of Compound III does not readily incorporate impurities associated with atmospheric water. Compound III may be administered to a subject, such as a pregnant female human subject, to delay the onset of labor in the subject, for example, by one day or one week or more, for example, from about one 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 is also administered to a subject, such as a pregnant female human subject, to alleviate one or more symptoms associated with labor, such as vaginal bleeding and rupture of the uterine membranes. [ka]

[0142] Compound I, or a pharmaceutically acceptable salt thereof, such as Compound III, may be administered alone or in combination with one or more additional agents, such as an additional therapeutic agent. Exemplary additional therapeutic agents include additional tocolytic agents, such as the oxytocin receptor antagonists described herein, including, for example, atosiban, letosiban, barusiban, epelusiban, and (3Z,5S)-5-(hydroxymethyl)-1-[(2'-methyl-1,1'-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-methyloxime nolasiban, or variants, formulations, crystalline forms, or derivatives thereof. By suppressing oxytocin signaling, the oxytocin receptor antagonists can cooperate with the prostaglandin F2α receptor antagonists described herein to slow or stop uterine contractions, for example, in patients experiencing or at risk of experiencing preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Exemplary additional tocolytic agents include betamimetics such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, and orciprenaline, which inactivate myosin light chain kinase by upregulating cAMP and / or myometrial Ca 2+ Additionally or alternatively, calcium channel inhibitors such as dihydropyridines (e.g., nifedipine and nicardipine) can act to deplete stores, thereby inhibiting uterine contractions. 2+ ] and regulates Ca in myosin filaments, leading to myometrial contraction 2+ Additionally or alternatively, magnesium salts, such as magnesium sulfate, may be administered in conjunction with the compounds of the present invention to inhibit Ca mediated activation, for example, to hyperpolarize the plasma membrane and / or bind to myosin light chains. 2+ Additionally or alternatively, a nitric oxide donor, such as nitroglycerin, may be administered in conjunction with a compound described herein to, for example, increase cyclic guanosine monophosphate levels in the myometrium, thereby inactivating myosin light chain filaments.

[0143] A compound of the invention, e.g., Compound I, or a pharmaceutically acceptable salt thereof, e.g., Compound III, may also or alternatively be administered in conjunction with progesterone or a variant or derivative thereof, e.g., 17-α-hydroxyprogesterone, to inhibit uterine contractions in a subject experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor).

[0144] Additionally or alternatively, the compounds of the invention may be administered in conjunction with corticosteroids described herein or known in the art, for example, to promote fetal lung maturation and prevent the development of respiratory distress syndrome, among other infantile disorders.

[0145] Additionally, Compound III can be formulated into pharmaceutical compositions, such as those formulated as described below.

[0146] Treatment method Compound I and its salts are potent inhibitors of prostaglandin F receptors and can be used to antagonize the interaction of prostaglandin F family members, such as prostaglandin F2α, with the corresponding prostaglandin F receptor in vivo to attenuate uterine contractions. Compound I and its salts can be administered to subjects, such as pregnant human female subjects, to treat or prevent preterm labor. Endogenous prostaglandin F2α is synthesized in and released by uterine epithelial cells in response to a signaling cascade initiated by oxytocin. Upon binding of PGF2α to PGF2α-R on the extracellular surface of uterine myocytes, phospholipase C cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 then releases intracellular calcium (Ca). 2+) enhances the release of sarcoplasmic reticulum calcium. The sudden increase in calcium stores ultimately leads to contraction of the uterine muscle and necrosis of the endothelial cells of the corpus luteum, a progesterone-secreting structure that supports fetal development. Abnormal initiation of uterine contractions and breakdown of the corpus luteum caused by dysregulation of PGF2α secretion can lead to preterm labor. Compound I and its salts, such as Compound III, can attenuate phospholipase C-mediated IP3 formation and subsequent mobilization of intracellular calcium stores by inhibiting the association of PGF2α with PGF2αR. Thus, Compound I or a salt thereof, e.g., Compound III, may be administered to a subject, such as a pregnant female human subject, to delay the onset of labor in the subject, e.g., by one day or one week or more, e.g., from about one 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, e.g., Compound III, may be administered to a subject to prevent labor before cesarean section. Additionally, Compound I or a salt thereof, e.g., Compound III, may be administered to a subject for the prevention and / or treatment of dysmenorrhea. Compound I or a salt thereof, such as Compound III, may also be administered to a subject, such as a pregnant female human subject, to alleviate one or more symptoms associated with labor, such as vaginal bleeding and rupture of the uterine membranes.

[0147] Furthermore, the compounds of the present invention can be used to treat endometriosis in patients (e.g., human patients). Overexpression of prostaglandin F2α receptors is correlated with abnormal endometrial proliferation. As antagonists of prostaglandin F2α receptor activity, the compounds of the present invention (e.g., compound (I) or its salt, e.g., compound (III)) can be administered to patients suffering from endometriosis to treat this indication. The compounds of the present invention can also be administered to patients to alleviate one or more symptoms of endometriosis, such as menstrual and / or nonmenstrual pain symptoms, including dysmenorrhea, dyspareunia, chronic pelvic pain, urinary disorders, and bowel disorders. The successful treatment of endometriosis by administering the compounds of the present invention to patients can be indicated, for example, by a reduction in endometrial tissue proliferation and / or a reduction in menstrual and / or nonmenstrual pain symptoms.

[0148] In addition to the above, the present invention provides a therapeutic treatment method by providing compound II to a subject who needs to treat the conditions described herein.For example, compound II can be provided to a subject, such as a pregnant human female subject, to treat or prevent premature labor.Compound II is a potent antagonist of PGF2α receptor, and therefore can inhibit the association of this receptor with PGF2α. Thus, compound II may be provided to a subject, such as a pregnant female human subject, to delay the onset of labor in the subject, e.g., by one day or one week or more, e.g., from about one 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 II may be provided to a subject to prevent labor prior to cesarean section. Additionally, compound II may be provided to a subject for the prevention and / or treatment of dysmenorrhea. Compound II may also be provided to a subject, such as a pregnant female human subject, to alleviate one or more symptoms associated with labor and delivery, such as vaginal bleeding and rupture of the uterine membranes.

[0149] In addition, compound II can be provided to a subject (e.g., a human patient) to treat endometriosis in the patient.As a PGF2α receptor antagonist, compound II can be provided to a patient suffering from endometriosis to treat this indication.Compound II can be provided to a patient to alleviate one or more symptoms of endometriosis, such as menstrual and / or non-menstrual pain symptoms, including dysmenorrhea, dyspareunia, chronic pelvic pain, urinary disorders, and defecation disorders.The success of treating endometriosis by providing compound II to a subject can be indicated by, for example, a reduction in endometrial tissue proliferation and / or a reduction in menstrual and / or non-menstrual pain symptoms.

[0150] Combination therapy Although the processes involved in the onset of labor have not yet been fully defined, increasing evidence supports the importance of inflammation in both term and preterm labor. During the onset of labor, several pro-inflammatory factors, including prostaglandins, cytokines, and manganese superoxide dismutase, increase throughout the body. In addition, inflammation has been strongly associated with infection-induced preterm labor.

[0151] Oxytocin is thought to initiate labor by exerting two distinct actions: directly inducing uterine myometrial contractions and enhancing the synthesis and release of contractile prostaglandins from the uterine endometrium / decidua. By inhibiting oxytocin signaling, both the direct (contractile) and indirect (prostaglandin synthesis-enhancing) effects of oxytocin on the uterus can be achieved. Furthermore, treatment of human decidua with oxytocin stimulates prostaglandin F2α production. This suggests that oxytocin signaling in uterine tissue has a complementary role, where oxytocin not only interacts directly with the myometrium in stimulating uterine contractions but can also interact indirectly through the formation of prostaglandins in other tissues.

[0152] Recent evidence correlates the activity of contractile prostaglandin F receptors with the onset and progression of labor. Recent reports have also shown that oxytocin induces the production of prostaglandins in human myometrial cells by enhancing cyclooxygenase 2 (COX-2). Such a mechanism may explain the sustained release of prostaglandins in uterine tissues, which promotes labor. Therefore, a combination therapy comprising a prostaglandin F2α receptor antagonist, such as Compound I or a salt thereof (e.g., Compound III), and an oxytocin receptor antagonist may be useful for treating and / or preventing preterm labor. Furthermore, the combination of an oxytocin receptor antagonist and a prostaglandin F2α receptor antagonist may be more effective than current regimens in treating preterm labor. Because the dose(s) of an oxytocin receptor antagonist administered to a patient when administered in combination with a prostaglandin F receptor antagonist may be lower than the dose that may be administered to a patient receiving an oxytocin receptor antagonist alone, a synergistic effect may be observed and is described herein in preventing both the contractile and inflammatory processes underlying preterm labor.

[0153] Compound I or its salt, such as Compound III, may be administered with one or more additional agents, such as an oxytocin receptor antagonist, to reduce the occurrence of uterine contractions and delay the onset of labor.For example, Compound I or its salt, such as Compound III, may be administered simultaneously with, mixed with, or separately from the oxytocin receptor antagonist.Exemplary oxytocin receptor antagonists used in conjunction with the compositions and methods of the present invention include atosiban, letosiban, barusiban, epelusiban, and nolasiban, or variants, formulations, crystalline forms, or derivatives thereof. For example, Compound I or a salt thereof, e.g., Compound III, may be administered before, after, or simultaneously with nolasiban, or a variant, formulation, crystalline form, or derivative thereof, to delay the onset of labor in a subject, e.g., by one day or one week or more, e.g., from about one 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).

[0154] 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 in conjunction with a betamimetics to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Betamimetics, such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, and orciprenaline, increase intracellular Ca through potentiation of beta-2 adrenergic receptors. 2+ levels (e.g., intracellular myometrial Ca 2+ levels), thereby upregulating cAMP and intracellular Ca that would otherwise be available to stimulate uterine contractions. 2+Exemplary betamimetics for use in conjunction with the compositions and methods described herein, as well as exemplary methods for administering betamimetics in conjunction with the compositions and methods described herein, are described, for example, in Gyetvai et al. Obstet. Gynecol. 94:869-877 (1999), the disclosure of which is incorporated herein by reference.

[0155] 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 in conjunction with a calcium channel blocker, such as an L-type calcium channel blocker, to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Calcium channel blockers, including dihydropyridines such as nifedipine and nicardipine, inhibit the release of calcium from the sarcoplasmic reticulum. 2+ It functions by inhibiting the release of Ca, which stimulates uterine muscle contractions. 2+ Exemplary calcium channel blockers for use in conjunction with the compositions and methods described herein, as well as exemplary methods for administering calcium channel blockers in conjunction with the compositions and methods described 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.

[0156] 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 in conjunction with a magnesium salt, such as magnesium sulfate, to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Magnesium salts, such as magnesium sulfate, are known to induce hyperpolarization of the plasma membrane and / or inhibit Ca ion transport due to binding to myosin light chain. 2+ It can regulate uterine contractions by multiple mechanisms, including competing with ATP, thereby inhibiting the contraction of myosin filaments in uterine muscle cells.

[0157] Additionally or alternatively, a compound of the present invention (e.g., Compound I or a pharmaceutically acceptable salt thereof, e.g., Compound III) may be administered in conjunction with a nitric oxide donor to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Nitric oxide, a vasodilator essential for maintaining normal smooth muscle tone, is produced in a variety of cells. Nitric oxide is synthesized during the oxidation of L-arginine to L-citrulline. This reaction is catalyzed by nitric oxide synthase, which exists in several isoforms. Both inducible (type 2) and brain (type 1) nitric oxide synthases are expressed in myometrial cells and vascular endothelial cells, whereas endothelial (type 3) nitric oxide synthase is expressed exclusively in vascular endothelial cells. The interaction of nitric oxide with soluble guanylyl cyclase present in nearby effector cells is a widespread signaling mechanism that couples diverse extracellular stimuli for nitric oxide formation to the synthesis of cyclic guanosine monophosphate (cGMP) within target cells. Increased cGMP content in smooth muscle cells, such as myometrial cells, inactivates myosin light chain kinase, leading to smooth muscle relaxation. The tocolytic effect of nitric oxide donors, such as nitroglycerin, is described, for example, in Simhan et al., New Engl. J. Med. 357:477-487 (2007), the disclosure of which is incorporated herein by reference.

[0158] 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 in conjunction with progesterone or a variant thereof, such as 17-α-hydroxyprogesterone caproate, to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Progesterone is a steroid hormone secreted by the corpus luteum and placenta after about eight weeks of pregnancy. Progesterone and its variants, such as 17-α-hydroxyprogesterone caproate, have been shown to inhibit myometrial [Ca] levels, 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. Reprod. Biol. 142:3-11 (2009), Bernal. Sem. Cell Dev. Biol. 18:340-347 (2007), and Hubinont et al. J. Pregnancy. 941057 (2011), the disclosures of each of which are incorporated herein by reference. 2+ ] and prostaglandin synthesis, thereby controlling uterine quiescence.

[0159] Additionally or alternatively, a compound of the present invention (e.g., Compound I or a pharmaceutically acceptable salt thereof, e.g., Compound III) may be administered in conjunction with a corticosteroid to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). Antenatal corticosteroids, such as betamethasone, dexamethasone, and hydrocortisone, are a class of therapeutic agents that can be administered to subjects, such as pregnant female subjects in preterm labor, or subjects at risk of preterm labor (e.g., subjects exhibiting one or more symptoms of preterm labor, such as vaginal bleeding and rupture of the uterine membranes), to accelerate fetal lung maturation. Antenatal corticosteroid treatment has been associated with an overall reduction in neonatal death, respiratory distress syndrome, intraventricular hemorrhage, necrotizing enterocolitis, respiratory support, intensive care admission, and systemic infection within the first 48 hours of life. Additionally, antenatal corticosteroid therapy is effective in women with preterm rupture of membranes (PROM) and pregnancy-associated hypertensive syndrome. There is evidence suggesting benefits across a wide range of gestational ages, particularly from about 26 to about 34 weeks (Miracle et al. J. Perinat. Med. 36:191-196 (2008), the disclosure of which is incorporated herein by reference).

[0160] Further to the above, according to the methods described herein, Compound II can be provided (e.g., by direct administration or by administration of a prodrug thereof) to a subject in need of treatment (e.g., a human subject experiencing or at risk of experiencing preterm labor, or a human subject suffering from dysmenorrhea or endometriosis) together with one or more additional agents, such as an oxytocin receptor antagonist, for example, to reduce the occurrence of uterine contractions and delay the onset of labor. For example, Compound II can be provided simultaneously with, mixed with, or separately from the oxytocin receptor antagonist. Exemplary oxytocin receptor antagonists for use in conjunction with the compositions and methods of the present invention include atosiban, letosiban, barusiban, epelusiban, and nolasiban, or variants, formulations, crystalline forms, or derivatives thereof. For example, Compound II may be provided before, after, or simultaneously with nolasiban, or a variant, formulation, crystalline form, or derivative thereof, to delay the onset of labor in a subject, e.g., by one day or one week or more, e.g., from about one 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).

[0161] Additionally or alternatively, Compound II may be provided in conjunction with a betamimetics to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). As discussed above, betamimetics such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, and orciprenaline act by enhancing beta-2 adrenergic receptors to increase intracellular Ca. 2+ levels (e.g., intracellular myometrial Ca 2+ levels), thereby upregulating cAMP and intracellular Ca that would otherwise be available to stimulate uterine contractions. 2+Exemplary betamimetics for use in conjunction with the compositions and methods described herein, as well as exemplary methods for administering betamimetics in conjunction with the compositions and methods described herein, are described, for example, in Gyetvai et al. Obstet. Gynecol. 94:869-877 (1999), the disclosure of which is incorporated herein by reference.

[0162] Additionally or alternatively, Compound II may be provided in conjunction with a calcium channel blocker, such as an L-type calcium channel blocker, to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). As discussed above, calcium channel blockers, including dihydropyridines such as nifedipine and nicardipine, block calcium from the sarcoplasmic reticulum. 2+ It functions by inhibiting the release of Ca, which stimulates uterine muscle contractions. 2+ Exemplary calcium channel blockers for use in conjunction with the compositions and methods described herein, as well as exemplary methods for administering calcium channel blockers in conjunction with the compositions and methods described 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.

[0163] Additionally or alternatively, Compound II may be provided to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor) in conjunction with a magnesium salt, such as magnesium sulfate. As discussed above, magnesium salts, such as magnesium sulfate, are known to induce hyperpolarization of the plasma membrane and / or inhibit Ca transport due to binding to myosin light chains. 2+ It can regulate uterine contractions by multiple mechanisms, including competing with ATP, thereby inhibiting the contraction of myosin filaments in uterine muscle cells.

[0164] Additionally or alternatively, Compound II may be administered in conjunction with a nitric oxide donor to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor).As described above, nitric oxide, a vasodilator essential for maintaining normal smooth muscle tone, is produced in various cells, and nitric oxide-induced increases in cGMP content in smooth muscle cells, such as uterine myocytes, result in smooth muscle relaxation.The tocolytic effect of nitric oxide donors, such as nitroglycerin, is described, for example, in Simhan et al., New Engl. J. Med. 357:477-487 (2007), the disclosure of which is incorporated herein by reference.

[0165] Additionally or alternatively, Compound II may be provided in conjunction with progesterone or a variant thereof, such as 17-α-hydroxyprogesterone caproate, to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). As mentioned above, progesterone and variants thereof, such as 17-α-hydroxyprogesterone caproate, can increase [Ca] levels in the myometrium, 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. Reprod. Biol. 142:3-11 (2009), Bernal. Sem. Cell Dev. Biol. 18:340-347 (2007), and Hubinont et al. J. Pregnancy. 941057 (2011), the disclosures of each of which are incorporated herein by reference. 2+ ] and prostaglandin synthesis, thereby controlling uterine quiescence.

[0166] Additionally or alternatively, Compound II may be provided in conjunction with a corticosteroid to a patient experiencing or at risk of preterm labor (e.g., exhibiting one or more symptoms of preterm labor). As discussed above, antenatal corticosteroids, such as betamethasone, dexamethasone, and hydrocortisone, are a class of therapeutic agents that can be administered to subjects, such as pregnant female subjects in preterm labor, or subjects at risk of preterm labor (e.g., subjects exhibiting one or more symptoms of preterm labor, such as vaginal bleeding and rupture of the uterine membranes), to accelerate fetal lung maturation; treatment with antenatal corticosteroids has been associated with an overall reduction in neonatal deaths, respiratory distress syndrome, intraventricular hemorrhage, necrotizing enterocolitis, respiratory support, intensive care admission, and systemic infections within the first 48 hours of life.

[0167] Pharmaceutical Compositions Compound I or a salt thereof, such as Compound III, can be formulated into a pharmaceutical composition for administration to a subject, such as a pregnant female human subject, in a biocompatible form suitable for in vivo administration. Thus, in one aspect, the present invention provides a pharmaceutical composition containing Compound I or a salt thereof, such as Compound III, mixed with a suitable diluent, carrier, or excipient. Compound I or a salt thereof, such as Compound III, can be administered, for example, orally or by intravenous infusion.

[0168] The present invention further provides pharmaceutical compositions containing Compound II. Such compositions may include Compound II in admixture with a suitable diluent, carrier, or excipient.

[0169] Under ordinary conditions of storage and use, pharmaceutical compositions may contain a preservative, for example, to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations can be found, for example, in Remington: The Science and Practice of Pharmacy (2012, 22 nded.) and The United States Pharmacopeia: The National Formulary (2015, USP 38 NF 33).

[0170] The pharmaceutical compositions may include sterile aqueous solutions, dispersions, or powders, such as those for the extemporaneous preparation of sterile solutions or dispersions. In all cases, the form can be sterilized using techniques known in the art and can be fluidized to the extent that it can be easily administered to the subject in need of treatment.

[0171] As described herein, pharmaceutical compositions may be administered to a subject, e.g., a human subject, alone or in combination with a pharmaceutically acceptable carrier, the proportions of which can be determined by the solubility and / or chemical properties of the compound, the selected route of administration, and standard pharmaceutical practice.

[0172] 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 for inhibiting uterine contractions and / or luteal involution, such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, or variants, formulations, crystalline forms, or derivatives thereof, among other therapeutic agents (e.g., tocolytics) described herein. Compound I or a salt thereof, e.g., Compound III, may be combined with an additional active agent, such as an oxytocin receptor antagonist, betamimetic, calcium channel blocker, magnesium salt, nitric oxide donor, progesterone or a variant thereof, or corticosteroid, described herein, and administered to a patient in a single composition. Alternatively, Compound I or a salt thereof, e.g., Compound III, may be administered to a patient separately from the additional active agent. For example, Compound I or a salt thereof, e.g., Compound III, and the additional active agent may be administered to a patient sequentially.

[0173] In addition to the above, Compound II may be provided to a subject alone or in combination with one or more additional agents useful for inhibiting uterine contractions and / or luteal regression, such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, or variants, formulations, crystalline forms, or derivatives thereof, among other therapeutic agents (e.g., tocolytics) described herein. Compound II may be mixed with an additional active agent, such as an oxytocin receptor antagonist, betamimetics, calcium channel blocker, magnesium salt, nitric oxide donor, progesterone or a variant thereof, or corticosteroid, described herein, and administered to the patient in a single composition. Alternatively, Compound II may be provided to the patient separately from the additional active agent. For example, Compound II and the additional active agent may be provided to the patient sequentially, for example, by providing Compound II to the patient and then administering the additional active agent to the patient.

[0174] Compositions for combination therapy described herein, e.g., pharmaceutical compositions described herein, may be administered to a subject to delay the onset of labor in the subject by, e.g., one day or one week or more, e.g., from about one 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). In some embodiments, the subject has experienced preterm labor. In some embodiments, the pharmaceutical composition is administered to a subject (e.g., a human subject) before the onset of preterm labor. The pharmaceutical compositions of the present invention may also be administered to a subject (e.g., a human subject) to prevent labor prior to cesarean section. The pharmaceutical compositions of the present invention may be administered to a subject (e.g., a human subject) to treat or prevent dysmenorrhea. The pharmaceutical compositions of the present invention may be administered to a subject, such as a pregnant human female subject, to alleviate one or more symptoms associated with labor, such as vaginal bleeding and rupture of the uterine membranes.

[0175] The additional therapeutic agent present in the composition for combination therapy can be, for example, another tocolytic agent. The additional tocolytic agent can be, for example, an oxytocin receptor antagonist, such as atosiban, letosiban, barusiban, epelusiban, and nolasiban, and one or more variants, formulations, crystalline forms, or derivatives thereof. For example, atosiban and its variants are described in, for example, U.S. Patent Nos. 4,504,469 and 4,402,942, the disclosures of which are incorporated herein by reference. Letosiban and its variants are described in, for example, U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 8,071,594, 8,357,685, 8,937,179, and US2016 / 0074413, the disclosures of which are incorporated herein by reference. Barusiban and its variants are described, for example, in U.S. Patent Nos. 6,143,722, 7,091,314, 7,816,489, and US 2016 / 0175283, the disclosures of which are incorporated herein by reference. Epersiban and its variants are described, for example, in U.S. Patent Nos. 7,514,437, 8,367,673, 8,541,579, 7,550,462, 7,919,492, 8,202,864, 8,742,099, 9,408,851, 8,716,286, and 8,815,856, the disclosures of which are incorporated herein by reference. Nolasiban and its variants, formulations, and crystalline forms are described, for example, in U.S. Pat. No. 7,115,754 and U.S. Patent Application Publication Nos. 2015 / 0073032, 2015 / 0164859, and 2016 / 0002160, the disclosures of each of which are incorporated herein by reference.

[0176] In some embodiments, the additional tocolytic agent is a betamimetic such as terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, or orciprenaline. In some embodiments, the additional tocolytic agent is a calcium channel inhibitor such as a dihydropyridine, e.g., nifedipine and 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.

[0177] In some embodiments, the additional therapeutic agent is progesterone or a variant or derivative thereof, such as 17-α-hydroxyprogesterone caproate.

[0178] 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.

[0179] In combination treatment, the dosage of one or more of the therapeutic compounds may be reduced from its standard dosage when administered alone, e.g., dosage may be empirically determined from drug combinations and permutations or estimated by a physician of ordinary skill in the art. [Example]

[0180] The following examples are put forth to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0181] Example 1. Preparation of Compounds I and III Compound I and its chloride salt (Compound III) were prepared according to Scheme 1, shown below. This example describes each of the steps, designated Steps 1-6, performed to synthesize Compound I. Scheme 1. Preparation of Compound I and its Chloride Salt [ka]

[0182] Step 1: Preparation of 2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid tert-butyl ester [ka] To a suitably sized flask (vessel A) was added 3-(butoxycarbonyl)-1,3-thiazolidine-(2S)-carboxylic acid (1 wt), followed by tetrahydrofuran and the flask contents were then cooled to -35°C to about 45°C. N-methylmorpholine (1.18 vol) was then added to the flask while maintaining the temperature at -30°C to -40°C. Isobutyl chloroformate (0.58 vol) was then added to the flask while maintaining the temperature at -30°C to -40°C.

[0183] To a separate vessel (vessel B) was added (3S)-amino-3-(4-fluorophenyl)propan-1-ol (0.76 wt) and THF and the vessel was mixed thoroughly until the bulk solids were dissolved.

[0184] The (3S)-amino-3-(4-fluorophenyl)propan-1-ol solution from vessel B was then added to reaction vessel A while maintaining the temperature at -30°C to -40°C. The contents of the flask were then warmed to 15°C to 25°C over 1 hour to 24 hours. The reaction mixture was stirred at 15°C to 25°C until completion of the reaction was observed. The reaction mixture was concentrated to dryness, after which 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. The organic phase was then separated and washed with saturated aqueous sodium bicarbonate. The organic phase was then dried over sodium sulfate, filtered, and the filtrate was concentrated at 35°C to 40°C until the ethyl acetate content was 10% by weight or less to give 2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid tert-butyl ester.

[0185] Step 2: Preparation of 3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylic acid [1-(4-fluorophenyl)-3-hydroxypropyl]-amide [ka] To an appropriately sized flask (vessel A) was added 2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylic acid tert-butyl ester (1 wt), followed by dichloromethane. The flask contents were then cooled to -15°C to -20°C. Hydrochloric acid (3.3 vol) was then added to the flask while maintaining the temperature at -15°C to -20°C until completion of the reaction was observed. The reaction mixture was then cooled to -35°C to -40°C, and tetrahydrofuran was added to the mixture while maintaining the temperature at -30°C to -40°C. N,N-diisopropylethylamine was then added to this mixture (8.16 vol) while maintaining the temperature at -15°C to -45°C. 4-Dimethylaminopyridine (0.032 wt) was then added to the vessel while maintaining the temperature at -15°C to -45°C.

[0186] To a separate vessel (vessel B) was added 4-biphenylsulfonyl chloride (0.85 wt) followed by THF.

[0187] The 4-biphenylsulfonyl chloride solution from vessel B was added to reaction vessel A while maintaining the temperature between -15°C and -45°C. The contents of the reaction mixture were then warmed to 15°C to 25°C over 1 hour to 24 hours. Ethyl acetate, followed by saturated aqueous ammonium chloride, was then added to the flask. The organic phase was separated and washed with saturated aqueous ammonium chloride, followed by saturated aqueous bicarbonate. The organic phase was then dried over sodium sulfate and filtered. The filtrate was concentrated at 35°C to 40°C until a solid residue was obtained. Dichloromethane was then added to the residue and mixed at 30°C to 35°C. After evaporation, ethyl acetate was then added to the residue, and the slurry was transferred to a suitable container. The stirred slurry was then warmed to reflux and then cooled to 0°C to 5°C. The precipitated solid was collected by filtration. The filter cake was washed with ethyl acetate followed by tert-butyl methyl ether and the filter cake was pulled dry under nitrogen for 1 to 24 hours to give 3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylic acid [1-(4-fluorophenyl)-3-hydroxypropyl]-amide.

[0188] Step 3A: Preparation of 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester [ka] To a suitably sized flask (vessel A) was added Boc-L-valine (0.48 wt), dichloromethane, and N,N-dimethylformamide, and the mixture was then stirred under nitrogen at 15° C. to 25° C. 1-Hydroxybenzotriazole (HOBt, 0.3 wt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 0.42 wt) were then added to the vessel while maintaining the temperature at 15° C. to 25° C. The mixture was then stirred at 15° C. to 25° C. until the bulk solids were dissolved to obtain solution A.

[0189] Into a separate vessel (vessel B) was added 3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylic acid [1-(4-fluorophenyl)-3-hydroxypropyl]amide (1.0 wt), dichloromethane, and N,N-dimethylformamide, and the mixture was then stirred under nitrogen at 15° C. to 25° C. 4-Dimethylaminopyridine (0.27 wt) was then added to the vessel while maintaining the temperature at 15° C. to 25° C. The mixture was stirred at this temperature until the bulk solids dissolved (usually 5 to 15 minutes) to provide solution B.

[0190] Solution A was then added to solution B while maintaining the temperature between 15°C and 30°C. The mixture was stirred at this temperature until completion of the reaction was observed. The reaction mixture was concentrated to remove volatile solvents. Ethyl acetate, followed by 10% w / w aqueous citric acid, was then added to the flask. The aqueous phase was separated and extracted with ethyl acetate. The combined organic phase was washed with a mixture of 10% w / w aqueous citric acid, followed by saturated aqueous sodium chloride, saturated aqueous ammonium chloride, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride. The organic phase was then dried over magnesium sulfate, filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to a solid residue to give crude 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester.

[0191] Step 3B: Purification of 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester [ka] To purify 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester, the crude product (1 wt) and dichloromethane were mixed in a vessel until the bulk solid dissolved. This solution was then loaded onto silica, followed by the addition of dichloromethane. The product was eluted with ethyl acetate:heptane. The product-containing fractions were combined and concentrated to dryness under vacuum at a water bath temperature of 35°C to 40°C to afford purified 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester.

[0192] Step 4: Preparation of 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate [ka] To a suitably sized flask was added 2-tert-butoxycarbonylamino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propyl ester (1 wt), followed by 1,4-dioxane, and the mixture was stirred under nitrogen. Methanesulfonic acid (0.18 wt) was then added, and the flask contents were heated to 68°C-73°C. The reaction was complete. 1The reaction was stirred at this temperature until the solids were observed by H NMR analysis. The reaction mixture was then cooled to 35-40°C and concentrated to dryness at this temperature. The residue was then dissolved in THF and concentrated to dryness at 35-40°C. This azeotropic drying cycle was repeated until the 1,4-dioxane content was less than 1.0% w / w to give 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate.

[0193] Step 5: Preparation of 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (Compound I) [ka] To an appropriately sized flask was added 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate (1 wt), followed by dichloromethane. The contents of the flask were then cooled to 5°C to 15°C. Aqueous sodium bicarbonate solution was added to the mixture while maintaining the temperature between 5°C and 25°C. The phases were then separated, and the organic phase was re-added to the vessel, followed by saturated aqueous sodium bicarbonate solution, while maintaining the temperature between 5°C and 25°C. The aqueous and organic layers were then 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 at 40-45°C until the dichloromethane content was 2% w / w or less to give 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (Compound I).

[0194] Step 6: Preparation of 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester hydrochloride (Compound III) [ka] To a suitable sized flask was added water (1.66 vol), followed by hydrochloric acid (0.18 vol), and the temperature of the mixture was adjusted to 15°C-25°C. The solution was then filtered, and to a suitable sized flask (Container A) was added the filtered solution, followed by ethanol and ethyl acetate. The resulting mixture was stirred at 15°C-25°C under nitrogen for at least 5 minutes.

[0195] To a suitably sized vessel (vessel B) was added 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (1 wt), followed by ethanol. The contents of the flask were then mixed to dissolve the bulk solids and clarify the solution.

[0196] The solution in vessel B was then added to vessel A while maintaining the temperature at 15°C to 25°C. The stirred mixture was cooled to 0°C to 5°C and stirred at this temperature for 50 to 70 minutes. The solid was collected by filtration, and the filter cake was sucked dry under nitrogen for at least 12 hours to give crude 2-amino-3-methylbutyric acid 3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester hydrochloride.

[0197] Example 2. Pharmacological properties of Compound I and its salts Nonclinical Pharmacology Compound I and its salts are rapidly converted to Compound II after gastrointestinal administration. Compound II is a competitive and reversible prostaglandin F2α receptor antagonist (human F2α receptor K) that is being developed for the management of preterm labor through the inhibition of premature uterine contractions. i= 6 nM). Efficacy pharmacology (tocolytic activity) has been demonstrated in a model of spontaneous uterine activity in late-term pregnant rats.

[0198] In vitro pharmacology The inhibitory potency of Compound I and Compound II against the prostaglandin F2α receptor was evaluated by analyzing the affinity of these compounds for the recombinant FP receptor expressed in HEK293-EBNA cells. The results show high binding affinity of Compound I and Compound II for the human receptor (see Table 2).

[0199] The selectivity of Compound II was tested against all eight prostaglandin receptor subtypes. Selectivity was approximately 10-fold against the prostaglandin E receptor 2 (EP2) and greater than 100-fold against other receptors. Testing the effect of 1 μM Compound II against a panel of 50 receptors, channels, and enzyme binding sites showed high selectivity for FP.

[0200] Functional characterization of Compound II against human FP was performed in transfected HEK293-EBNA cells. Compound II inhibited IP3 synthesis with an IC of 60 nM. 50 When added alone to FP / HEK293-EBNA cells, compound II tested at concentrations up to 10 μM did not induce any IP3 synthesis, indicating that this compound had no agonist activity.

[0201] In vivo pharmacology The tocolytic effects of Compound I and Compound II were investigated in a model of spontaneous uterine activity in anesthetized pregnant rats at late stage (19-21 days of gestation) (Kawarabayashi et al. Am. J. Obstet. Gynecol. 175:1348-1355 (1996) and Shinkai et al. J. Pharm. Pharmacol. 52:1417-1423 (2000)). Briefly, female rats in late stage pregnancy were anesthetized with urethane. One pregnant uterine horn was exposed, and a polyethylene catheter tipped with a saline-filled latex balloon was inserted into the lumen. The catheter was connected to an amplification / recording system via a pressure transducer. Increasing doses of Compound I (as the mesylate salt) or Compound II were administered orally or infused by 10-minute intravenous infusion. For intravenous administration, uterine contractile activity was quantified by calculating the area under the curve (AUC) during the 10-minute infusion period.

[0202] The percentage change in AUC values ​​for spontaneous uterine contractions observed after each compound administration was calculated relative to the value recorded before the first dose (basal value). The effects of Compound I or Compound II were evaluated by comparing intrauterine lumen pressure values ​​before and after treatment. For oral administration, the same calculation procedure was applied at different time points after 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, significantly reduced spontaneous uterine contractions by approximately 40-50% (maximum effect was achieved at 30 mg / kg intravenously and 60 mg / kg orally). Intravenous activity was comparable to or slightly higher than that of atosiban, a tocolytic drug approved in the European Union.

[0203] The inhibitory effect after oral administration appeared with rapid onset (5–15 min after administration) and remained at a sustained level until the end of the 3-h observation period (Figure 3).

[0204] A single oral dose of 30 mg / kg achieves significant inhibition of uterine contractions.

[0205] Thus, in vitro pharmacological studies demonstrated the high affinity of Compound I and Compound II for the human FP receptor. When administered by intravenous or oral routes, these compounds were able to significantly reduce spontaneous uterine contractions by approximately 40-50% when investigated in a model of spontaneous uterine activity in anesthetized pregnant rats at late stage (19-21 days of pregnancy).

[0206] Example 3. Crystallization screening of salts of Compound I This example describes experiments performed to generate and characterize crystalline salt forms of Compound I.

[0207] Overview The mesylate salt of Compound I was determined to be amorphous by XRPD. Attempts to crystallize this material were unsuccessful. The free base was synthesized from the mesylate salt and used to prepare various salts. The crystalline hydrogen sulfate salt was obtained directly from this salt synthesis. Using different solvent mixtures and crystallization techniques, three salts were crystallized: the hydrochloride, fumarate, and dihydrogen phosphate. The hydrochloride salt appears to exhibit low hygroscopicity, extended stability at high relative humidity (RH), and assumes a single crystalline form when crystallized from a variety of distinct experimental conditions.

[0208] The crystalline HCl salt was obtained in two evaporation experiments and one slurry experiment. The same XRPD pattern was observed in each case. Based on thermal data, this material had some residual solvent, and the estimated melting point was approximately 146-147 °C. Partial decomposition likely occurred during melting. The hydrochloride salt was non-hygroscopic based on water equilibrium data.

[0209] The crystalline hydrogen sulfate salt presumably solvated and decomposed above approximately 100° C. The material was stable up to approximately 65% ​​relative humidity.

[0210] The crystalline dihydrogen phosphate and fumarate salts were hygroscopic at approximately 65% ​​RH. Attempts to scale up the salts were unsuccessful due to high laboratory humidity. Therefore, only partial characterization was available for these salts.

[0211] The hydrochloride, hydrogen sulfate, and fumarate salts showed comparable water solubility (less than 1 mg / mL, see Figure 8).

[0212] experiment X-ray powder diffraction analyses described herein were performed on a Shimadzu XRD-6000 X-ray powder diffractometer using CuKα radiation. The instrument was equipped with a long fine focus X-ray tube. The tube voltage and amperage were set at 40 kV and 40 mA, respectively. The divergence and scattering slits were set at 1°, and the receiving slit was set at 0.15 mm. Diffracted radiation was detected by a NaI scintillation detector. A θ-2θ continuous scan was used from 2.5 to 40° 2θ at 3° / min (0.4 s / 0.02° step). A silicon standard was analyzed daily to check instrument alignment. Samples were analyzed using a silicon sample holder.

[0213] The X-ray powder diffraction analyses described herein were also performed on an Inel XRG-3000 diffractometer equipped with a curved position-sensitive detector with a 120° 2θ range. Real-time data collection was performed using CuKα radiation starting at approximately 4° 2θ with a resolution of 0.03° 2θ. The tube voltage and amperage were set to 40 kV and 30 mA, respectively. The monochromator slit was set to 5 mm x 160 μm. Patterns were displayed from 2.5 to 40° 2θ. Samples were prepared for analysis by filling thin-walled glass capillaries. Each capillary was mounted on a motorized goniometer head, which allowed for capillary rotation during data acquisition. Samples were analyzed for 5 or 10 min. Instrument calibration was performed daily using silicon standards.

[0214] The DSC analyses described herein were performed on a TA Instruments 2920 Differential Scanning Calorimeter. The instrument was calibrated using indium as the reference material. Samples were placed in standard aluminum DSC pans, the pans were crimped, and the weights were accurately recorded. Samples were equilibrated at 25°C and heated under a nitrogen purge at a rate of 10°C / min up to 350°C. Indium metal was used as the calibration standard.

[0215] The TG analyses described herein were performed on a TA Instruments 2950 Thermogravimetric Analyzer. Calibration standards were nickel and ALUMEL™. Samples were placed in aluminum sample pans and inserted into the TG furnace. The samples were first equilibrated at 25°C and then heated under nitrogen flow at a heating rate of 10°C / min up to 350°C.

[0216] 399.8MHz 1 The solutions described herein were analyzed using a Varian UNITYINOVA-400 spectrometer at the H Larmor frequency. 1 H nuclear magnetic resonance (NMR) spectra were acquired at ambient temperature. Samples were dissolved in methanol-d4, methylene chloride-d2, or chloroform-d3. The spectra were 1 Spectra were acquired using a 1H pulse width of 7.8 or 8.6 μs, an acquisition time of 2.50 s, a 5 s interscan delay, a spectral width of 4095 or 6400 Hz with 20,474 or 32,000 data points, and 16 or 40 co-added scans. Free induction decays (FIDs) were processed using Varian VNMR 6.1C software with 65,536 points and an exponential line broadening factor of 0.2 Hz to improve signal-to-noise. Spectra were referenced to internal tetramethylsilane (TMS) at 0.0 ppm or the residual solvent peak.

[0217] The FT-Raman spectra described herein were acquired on an FT-Raman 960 or 860 spectrometer (Thermo Nicolet). This spectrometer uses an excitation wavelength of 1064 nm. Approximately 0.5–0.7 W of Nd:YVO4 laser power was used to irradiate the sample. Raman spectra were measured using an indium gallium arsenide (InGaAs) detector. Samples were prepared for analysis by placing the material in a glass capillary, which was then placed in an attached gold-coated capillary holder. A total of 256 sample scans were collected from 3600 to 100 cm-1 using Happ-Genzel apodization at a spectral resolution of 4 cm-1. Wavelength calibration was performed using sulfur and cyclohexane.

[0218] Moisture sorption / desorption (MB) data were collected on a VTI SGA-100 Vapor Sorption Analyzer. Sorption and desorption data were collected under a nitrogen purge over a range of 5% to 95% relative humidity (RH) at 10% RH intervals. Samples were not dried prior to analysis. The equilibration criterion used for analysis was a weight change of less than 0.0100% in 5 minutes; if the weight criterion was not met, a maximum equilibration time of 3 hours was used. Data were not corrected for the initial moisture content of the samples. NaCl and PVP were used as calibration standards.

[0219] Preparation of Compound I Several attempts were made to produce the free base of Compound I from the mesylate salt. The results are shown in Figure 4. Initially, one equivalent of sodium hydroxide was used per equivalent of salt. Proton NMR showed the presence of a methanesulfonic acid peak. Complete reaction was achieved when a solution of the mesylate salt in methylene chloride was mixed with aqueous NaOH at a salt:base ratio of 1:2. The organic layer was separated after several washes and evaporated. The resulting paste-like or viscous oil was dried in vacuo to yield an amorphous solid. This free base was then purified by the following procedure: 1 It was analyzed by H NMR and Raman spectroscopy (Figures 15 and 16, respectively). Subsequent salt screening studies used this free base as the starting material (summarized in Figures 5A-7C).

[0220] Salt screening of compound I Twelve salts of Compound I were prepared. The crystalline hydrogen sulfate salt was precipitated by adding approximately a 25 molar excess of sulfuric acid to an acetone solution of the free base. The other salts in the synthesis steps appeared non-birefringent by microscopy or amorphous by XRPD (Figures 5A-7C). The benzenesulfonate, citrate, ethanesulfonate, hydrochloride, hydrogen sulfate, and sulfate salts were analyzed by proton NMR.

[0221] The crystallization experiments for Compound I are summarized in Figures 5A-7C. The following salts were crystallized: hydrochloride, fumarate, and dihydrogen phosphate.

[0222] The chloride salt was crystallized from a 1:1 mixture of acetone:toluene, a mixture of methylene chloride:ethyl ether, and an acetone slurry. The same XRPD pattern was observed in all experiments and designated Form A (Figures 7A to 7C). The crystalline fumarate salt was obtained from slow evaporation of a 1:1 methanol:toluene solution. This X-ray pattern was designated Pattern B. The hydrogen sulfate and dihydrogen phosphate salts exhibited very similar XRPD patterns (designated Pattern X). HSO4 - and H2PO4 - The counterions of the mesylate salts are similar in size and small compared to the organic base molecules, so the crystal structures of the hydrogen sulfate and dihydrogen phosphate salts are likely to be similar. Attempts to crystallize the mesylate salt have resulted in viscous or glassy solid materials.

[0223] Characterization of the free base and mesylate salt of compound I The proton NMR spectra of the organic bases showed two doublets at approximately 1 ppm, corresponding to the methyl groups of the valine fragment. The methyl groups are at chiral carbon centers and therefore are not equivalent in proton NMR. The two doublets of the methyl groups were observed in the following salts of Compound I: besylate, citrate, esylate, hydrogen sulfate (more overlapping), and sulfate (more overlapping). The mesylate and chloride salts 1In the 1 H NMR spectrum, the doublet at approximately 1 ppm corresponding to six hydrogen atoms arises from the perfect overlap of two doublets of methyl groups (FIGS. 13 and 21A-21D).

[0224] Homonuclear decoupling for the free base 1 H NMR experiments confirmed the methine (CH) hydrogen multiplet at approximately 2 ppm (Figure 18). 1 The H NMR spectrum is shown at the bottom of Figure 18. Irradiation of each methyl group (top, center) resulted in a simplified methine multiplet with the same line number (5). If the two doublets corresponded to different diastereoisomers, two multiplets would be observed: the original and the simplified one.

[0225] Characterization of the chloride salt of compound I (compound III) thermal technology, 1 The crystalline chloride salt was analyzed by H NMR and automated moisture sorption / desorption analysis. The endotherm at approximately 147 °C in DSC appeared broader than that typically observed for melting endotherms. Approximately 4% weight loss was observed between 25 and 160 °C (analyzed acetone slurry sample, Figure 20). 1 H NMR was consistent with the structure (Figures 21A-21D). However, because a different sample was analyzed (slow evaporation of the 1:1 acetone:toluene mixture), this data cannot be correlated with the weight loss in the thermal analysis. The chloride salt from the acetone slurry was dried under vacuum at approximately 50 °C for 1 day. The resulting sample was similar to the original salt by XRPD (Figure 22). The thermal data are presented in Figure 23. Based on a comparison of the thermal data, the dried material had lower weight losses between 25 and 100 °C (0.2% vs. 0.6% for the original chloride salt) and between 100 and 160 °C (2.5% vs. 3.5%) (Figure 24). This indicated that some solvent was removed during vacuum drying. However, the endotherm at approximately 146-147 °C in the DSC was still broad (Figure 25). Partial decomposition likely occurred during melting (note the decomposition baseline and corresponding weight loss in the TG).

[0226] The chloride salt of Compound I did not deliquesce after 2 days at approximately 95% RH. Moisture sorption / desorption data are summarized in Figure 27 and shown in Figures 26 and 28. Minimal weight loss was observed upon equilibration at 5% RH. A weight gain of approximately 0.9% occurred upon sorption from 5 to 95% relative humidity. This sample showed a weight gain of approximately 0.7% upon desorption. XRPD analysis of the post-MB sample exhibited an X-ray pattern similar to that of the starting material (Figure 29).

[0227] Characterization of the hydrogen sulfate and sulfate salts of compound I. Both the hydrogen sulfate and sulfate salts of Compound I were prepared. The hydrogen sulfate salt was precipitated from an acetone solution of the free base by adding approximately a 25 molar excess of sulfuric acid. This precipitate was found to be crystalline by XRPD (Figure 38). Thermal data for the hydrogen sulfate salt is shown in Figure 32. A broad endotherm at approximately 68 °C corresponds to approximately 1% weight loss and is likely due to desolvation (dehydration). Decomposition occurred at higher temperatures. It did not deliquesce after 3 days at approximately 65% ​​RH (Figure 32). The sulfate salt was prepared using 2 equivalents of free base per equivalent of acid. Attempts to crystallize the sulfate salt of Compound I were unsuccessful (Figures 5A-7C). The hydrogen sulfate and sulfate salts were analyzed by proton NMR (Figures 33 and 34). Differences were observed in the NMR spectra. For example, the methyl groups of the valine fragment appeared to have different couplings.

[0228] Characterization of the dihydrogen phosphate salt of compound I The dihydrogen phosphate salt was crystallized from a 1:1 methyl ethyl ketone:n-butyl acetate mixture (Figures 5A-7C). It exhibited an X-ray pattern similar to that of the hydrogen sulfate salt (Figure 4C). Due to sample loss during analysis, characterization of the dihydrogen phosphate salt was limited to XRPD. Attempts to prepare additional quantities of the crystalline salt were unsuccessful. A poorly crystalline material was produced during the first attempt (Figures 5A-7C). Recrystallization of the poorly crystalline salt resulted in a viscous solid. This material remained viscous after drying in vacuo. Laboratory humidity was approximately 62% RH during the scale-up crystallization, which likely affected the material due to its hygroscopic nature. No further attempts to crystallize the dihydrogen phosphate salt were made.

[0229] Characterization of the fumarate salt of compound I A small amount of the fumarate salt was crystallized from a 1:1 mixture of methanol and toluene (Figures 5A-7C). Attempts to scale up the crystalline salt at approximately 62% RH laboratory humidity were unsuccessful. Microscopy revealed some crystalline solid, but a primarily oily material resulted. Drying this viscous solid in vacuo yielded a primarily amorphous material. The originally prepared crystalline salt was used in seeding experiments; however, no crystalline material was produced. The hygroscopic nature of the fumarate salt was confirmed in relative humidity studies.

[0230] The fumarate salt appeared to be moisture sensitive. The crystalline salt was stable at approximately 43 and 53% relative humidity and began to deliquesce within the first day at approximately 65% ​​RH. A yellow oil formed after 3 days at 65% RH (approximately 4% moisture gain).

[0231] conclusion The mesylate salt of Compound I was found to be amorphous by XRPD, and attempts to crystallize this material were unsuccessful.

[0232] The free base of Compound I was synthesized from the mesylate salt and used to prepare 12 salts. Crystalline hydrogen sulfate salt was obtained directly from this salt synthesis. Using different solvent mixtures and crystallization techniques, three salts were crystallized: hydrochloride, fumarate, and dihydrogen phosphate. The chloride salt appeared to be the best candidate for further development. The crystalline hydrogen sulfate salt likely solvated and decomposed above approximately 100°C. This material was stable up to approximately 65% ​​relative humidity. The crystalline HCl salt was obtained in two evaporation experiments and one slurry experiment. Identical XRPD patterns were observed. Based on thermal data, this material contained some residual solvent, and the estimated melting point was approximately 146-147°C. Partial decomposition likely occurred during melting. The chloride salt was non-hygroscopic based on water equilibrium data. The crystalline dihydrogen phosphate and fumarate salts were hygroscopic at approximately 65% ​​RH. Attempts to scale up the salts were unsuccessful due to high laboratory humidity. Therefore, only partial characterization was available for these salts.

[0233] Example 4. Monitoring Caco-2 cell permeability of Compound I mesylate salt The bioavailability of orally administered drugs depends largely on their ability to be transported across the intestinal barrier. Caco-2 cells, derived from a human colon adenocarcinoma established by J. Fogh due to their ability to achieve a higher degree of enterocyte differentiation, can be used as an in vitro model to investigate drug transport across the intestinal epithelium. These cells form a polarized epithelial cell monolayer when grown on collagen-coated polycarbonate membranes. This differentiated cell monolayer serves as an appropriate model for the small intestinal epithelium. The differentiation process, beginning with cell confluence, leads to 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.

[0234] The objectives of this study were, in a first step, to evaluate the nonspecific binding of compound I in a Caco-2 cell test system (cell-free) and, in a second step, to evaluate the conversion of compound I to compound II and to determine whether the transport of compound I across Caco-2 cell monolayers is mediated by the PepT1 transporter protein.

[0235] material Caco-2 cell line (human colon adenocarcinoma cells) was obtained from a control cell bank (Biosearch SpA, Gerenzano, Italy). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, non-essential amino acid solution, 200 mM L-glutamine, penicillin / streptomycin solution, and calcium- and magnesium-free trypsin-EDTA solution were purchased from Celbio (Milan, Italy). HEPES, Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and glycine-sarcosine (Gly-Sar) were purchased from Sigma (Milan, Italy).

[0236] experiment 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.

[0237] These cells were frozen and stored in cryotubes under liquid nitrogen as a cell suspension in 1 mL of 10% DMSO-containing fetal bovine serum. Cells used in experiments were not kept in culture for longer than one month.

[0238] If necessary, frozen vials of Caco-2 cells were rapidly thawed at 37°C in a water bath by gently swirling until semi-complete thawing. The cell suspension was then added dropwise to 10 mL of culture medium. The cell suspension was then centrifuged at 900-1000 rpm for 7 minutes, the supernatant removed, and the cell pellet reconstituted in medium and placed in a 75 cm 2The cells were distributed into 1000 cells / ml flasks. The flasks were incubated at 37°C in a 5% CO2 atmosphere. When a nearly confluent monolayer was obtained, the cells were serially subcultured. The medium from each flask was removed, and the monolayer was washed with 10–15 mL of Dulbecco's phosphate-buffered saline (PBS).

[0239] Trypsin-EDTA solution was added to the cell monolayer and incubated at 37°C, gently tapping at intervals to dislodge the cells. Complete dissociation and disaggregation of the cell monolayer was confirmed by microscopic examination. The cells were then resuspended in 10 mL of complete medium and centrifuged at 900-1000 rpm for 7 minutes. The supernatant was discarded, and the cells were resuspended in culture medium and centrifuged at 175 cm. 2 2.5 x 10 cells in a flask 5 cells / mL.

[0240] Cells from a flask of near-confluent culture were dissociated and disaggregated by trypsinization as described above. Cells were resuspended in medium and counted. Approximately 1 x 10 6 The cell suspension was diluted with medium to 100 cells / mL, and 300 μL of the cell suspension was placed on the apical compartment of each transwell (6.5 mm diameter, 0.4 μm pore size). 600 μL of culture medium was placed in the basolateral compartment. The plates were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air for 15–21 days, with medium changes every 48–72 h.

[0241] The integrity of each Caco-2 cell monolayer was assessed by transepithelial electrical resistance (TEER) both before the experiment and at the end of the incubation period. Ω×cm 2 TEER, expressed as ρ, was measured in transwells using Millicell-ERS (Millipore). Monolayers had a TEER value of 800 Ω × cm. 2 When it is higher, it is considered well-differentiated.

[0242] The integrity of each Caco-2 cell monolayer was assessed by Lucifer Yellow at the end of the incubation period. After the experiment, the 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, and 400 μL of HBSS was added to the basolateral compartment. The transwells were incubated at 37°C for 1 hour. The amount of Lucifer Yellow in the basolateral compartment was quantified at 535 nm wavelength using a microplate spectrofluorometer (EG & G WALLAC) against a standard Lucifer Yellow curve in the same saline solution. The monolayer was considered intact if less than 1% Lucifer Yellow was detected in the basolateral compartment.

[0243] Assessment of nonspecific binding to cell-free transwells Nonspecific binding and recovery were assessed in cell-free transwells. Compound I was tested at 1.5, 3, and 6 μM in duplicate cell-free transwells. The test was performed in a pH gradient between the apical and basolateral compartments. The apical compartment (donor) had a buffer pH of 6.5, while the basolateral compartment (receiver) had a buffer pH of 7.4. Sampling was performed at the following times: 60 and 120 minutes for the basolateral compartment (receiver), and 120 minutes for the apical compartment (donor). The resulting samples were analyzed by LC-MS to monitor both compound I and compound II for recovery assessment.

[0244] Evaluation of the stability of Compound I and Compound II The stability of both Compound I and Compound II was evaluated during the study. These compounds were dissolved in HBSS buffer (final concentration of 1% DMSO) at concentrations of 1.5, 3, and 6 μM. An aliquot of each solution was sampled at time zero (t=0) to assess the starting concentration of the compound. The solutions were incubated at 37° C. for the duration of the transport experiment. An aliquot of each solution was sampled at the end of the experiment (t=120) to assess the final concentrations of Compound I and Compound II. The samples were analyzed by LC-MS.

[0245] Evaluation of bidirectional permeability of Compound I Compound I was dissolved in HBSS buffer (final concentration of 1% DMSO) at concentrations of 1.5, 3, and 6 μM. Each concentration / sampling time was used in duplicate wells. The study was performed at a gradient pH: the apical compartment (mucosal) was at pH 6.5 and the basolateral compartment (serosal) was at pH 7.4.

[0246] Apical to basolateral (A→B, mucosa to serosal) transport: 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. Plates were incubated at 37°C. Aliquots of the basolateral compartment were sampled after 60 and 120 minutes (t=60 and t=120). Aliquots of the apical compartment were sampled at the start (t=0) and after 120 minutes (t=120).

[0247] Basolateral to apical (B→A, serosal to mucosal) transport: 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. Plates were incubated at 37°C. Aliquots of the apical compartment were sampled after 60 and 120 minutes (t = 60 and t = 120). Aliquots of the basolateral compartment were sampled at the start (t = 0) and after 120 minutes (t = 120). All samples were analyzed by LC / MS, monitoring the appearance of both compound I and compound II.

[0248] Evaluation of bidirectional permeability of Compound II Compound II was dissolved in HBSS buffer (final concentration of 1% DMSO) at concentrations of 1.5, 3, and 6 μM. Each concentration / sampling time was used in duplicate wells. The study was performed at a gradient pH: the apical compartment (mucosal) was at pH 6.5, and the basolateral compartment (serosal) was at pH 7.4.

[0249] Apical to basolateral (A→B, mucosa to serosal) transport: 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. Plates were incubated at 37°C. Aliquots from the basolateral compartment were sampled after 60 and 120 minutes (t=60 and t=120). Aliquots from the apical compartment were sampled at the start (t=0) and after 120 minutes (t=120).

[0250] Basolateral to apical (B→A, serosal to mucosal) transport: 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. Plates were incubated at 37°C. Aliquots from the apical compartment were sampled after 60 and 120 minutes (t = 60 and t = 120). Aliquots from the basolateral compartment were sampled at the start (t = 0) and after 120 minutes (t = 120). All samples were analyzed by LC / MS while monitoring Compound II.

[0251] Inhibition of Compound I transport from mucosa to serosal membrane by PepT1 substrate (Gly-Sar) To block the active transporter PepT1, differentiated cells were pretreated with 10 mM Gly-Sar for 30 min.

[0252] Compound I was dissolved in HBSS buffer (final concentration of 1% DMSO) at concentrations of 1.5, 3, and 6 μM. Each concentration / sampling time was used in duplicate wells. The study was performed at a gradient pH: the apical compartment (mucosal) was at pH 6.5 and the basolateral compartment (serosal) was at pH 7.4.

[0253] Apical to basolateral (A→B, mucosa to serosal) transport: 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. Plates were incubated at 37°C. Aliquots of the basolateral compartment were sampled after 60 and 120 minutes (t=60 and t=120). Aliquots of the apical compartment were sampled at the start (t=0) and after 120 minutes (t=120). All samples were analyzed by LC / MS, monitoring the appearance of both Compound I and Compound II.

[0254] analytical decision The concentrations of Compound II and Compound I in the post-incubation samples were determined without any further dilution by the high performance liquid chromatography / mass spectrometry (LC / MS) method reported in the Appendix (Section 7.1).

[0255] result The pre-experiment TEER values ​​of the Caco-2 cell monolayers used were 850-1160 Ω×cm 2 , indicating a tightly junctioned, confluent monolayer. TEER values ​​at the end of the experiment averaged 170 Ω×cm, with no effect on the integrity of the cell monolayer. 2 (680 to 990 Ω×cm 2The Lucifer Yellow assay confirmed the integrity of all monolayers after the experiment; indeed, the amount of Lucifer Yellow detected in the basolateral compartment after the experiment was always less than 1% in all wells. Figure 55 reports data obtained in a nonspecific binding assay for Compound I. Under these test conditions, Compound I was demonstrated to be recovered in the apical compartment at all doses tested. Compound I was not detected in the basolateral compartment at any dose tested. Nonspecific binding of Compound I was excluded. Compound II was not detected in either compartment. Figure 55 reports data obtained in a stability assay for Compound I and Compound II. Both compounds were demonstrated to be stable at 37°C in HBSS buffer (final concentration of 2% DMSO) for 60 and 120 minutes under the test conditions. Figures 56A-56E report data obtained in a bidirectional permeability assay for Compound I. This compound did not cross the cell monolayer. In the apical-to-basolateral study, compound I was not detected in the receptor compartment after either 60 or 120 minutes, whereas increasing concentrations of compound II were detected in the basolateral compartment at the end of the experiment. The permeation rates of compound II are reported in the table. At the end of the apical-to-basolateral study, low recovery of compound I was observed in the apical compartment, whereas increasing concentrations of compound II were detected (high recovery). The increased concentration of compound II in the apical compartment after 120 minutes may be explained by the presence of extracellular and intracellular esterases in Caco-2 cells that can deesterify the compound (Kern et al. J. Agric. Food Chem. 51:7884-7891 (2003)). In the basolateral-to-apical study, compound I was not detected in the receptor compartment, whereas low concentrations of compound II were detected. Therefore, compound I, like compound II, is likely transported and transported across Caco-2 monolayers. Figures 57A-57E report data obtained in bidirectional permeability studies for Compound II. This compound exhibited good apical-to-basolateral permeability and low basolateral-to-apical permeability. Because the concentration in the donor compartment was known, Papp was calculated. Compound II has good passive permeability across Caco-2 monolayers. No efflux was detected.Figures 58A-58C report data obtained in an inhibition study in which Caco-2 cell monolayers were pretreated with 10 mM Gly-Sar (to saturate the PepT1 transporter). Compound I was not detected in the receptor compartment, but passage of compound II was observed. The rate of passage was not linear in this study.

[0256] Consideration In this study, nonspecific binding of Compound I in a Caco-2 cell test system (cell-free) was evaluated and excluded. Compound I was stable under these test conditions. The conversion of Compound I to Compound II was evaluated and confirmed in a bidirectional permeability test. Compound I did not cross the cell monolayer under the tested conditions. Therefore, Compound I is likely transported and transported across Caco-2 cell monolayers as deesterified Compound II.

[0257] In bidirectional permeability studies, Compound II exhibited good passive permeability through Caco-2 cell monolayers, and no evidence was found that Compound II could be a substrate for efflux transporters.

[0258] Studies using Gly-Sar pretreatment (to saturate the PepT1 transporter) showed no penetration rate of Compound I or Compound II. Transport of Compound I across Caco-2 cell monolayers is likely not mediated by PepT1.

[0259] These experiments demonstrate that intestinal absorption of Compound I and its salts is not mediated by the Pept1 transporter protein. Rather, the above results demonstrate that Compound I is deesterified by surrounding esterases in the small intestine and then passively permeates the small intestinal epithelium. The fact that Compound I and its salts are not substrates for Pept1 is an unexpected and pharmacologically beneficial property. Pept1 is a pH-dependent cotransporter known to mediate the absorption of various valinate 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 transported across the intestinal epithelium by this protein. Unexpectedly, despite the presence of the valinate ester functional group, Compound I and its salts do not rely on this transporter for absorption across the small intestinal epithelium. Therefore, Compound I and its salts are advantageous because they do not compete with natural substrates of Pept1, such as peptide nutrients, for binding to and transport by this protein. Rather, Compound I and its salts are converted in vivo into a form that is readily absorbed in a manner that depends on energy and the local proton gradient. This unexpected property, combined with the high water solubility of Compound I and its salts, collectively results in a beneficial pharmacokinetic profile in which these therapeutic agents are readily dissolved in an aqueous environment and then converted into a form that is capable of transporter-dependent absorption.

[0260] Example 5. Combination therapy including an additional tocolytic agent Compound I or a salt thereof, e.g., Compound III, may be administered in combination with one or more additional agents, such as, for example, an oxytocin receptor antagonist, a betamimetic, a calcium channel blocker, a magnesium salt, or a nitric oxide donor, to a subject, such as a human subject, to reduce the occurrence of uterine contractions and delay the onset of labor.

[0261] A skilled practitioner may administer Compound I or a salt thereof, such as Compound III, simultaneously with, in admixture with, or separately from an oxytocin receptor antagonist. Exemplary oxytocin receptor antagonists for use in conjunction with the compositions and methods of the present invention include atosiban, letosiban, barusiban, epelusiban, and nolasiban, or variants, formulations, crystalline forms, or derivatives thereof. For example, Compound I or a salt thereof, e.g., Compound III, may be administered before, after, or simultaneously with nolasiban, or a variant, formulation, crystalline form, or derivative thereof, to delay the onset of labor in a subject, e.g., by one day or one week or more, e.g., from about one 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).

[0262] Additionally or alternatively, a skilled practitioner may administer Compound I or a salt thereof, such as Compound III, simultaneously with, in admixture with, or separately from a betamimetic, such as a betamimetic 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 by, for example, one day or more than one week, for example, from about one 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).

[0263] Additionally or alternatively, a skilled practitioner may administer Compound I or a salt thereof, e.g., Compound III, simultaneously with, in admixture with, or separately from a calcium channel inhibitor, such as a calcium channel inhibitor described herein. For example, Compound I or a salt thereof, e.g., Compound III, may be administered before, after, or simultaneously with a calcium channel inhibitor described herein or known in the art to delay the onset of labor in a subject by, for example, one day or more than one week, e.g., from about one 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).

[0264] Additionally or alternatively, a skilled physician may administer Compound I or a salt thereof, such as Compound III, simultaneously with, in admixture with, or separately from a magnesium salt, such as magnesium sulfate. For example, Compound I or a salt thereof, such as Compound III, may be administered before, after, or simultaneously with magnesium sulfate to delay the onset of labor in a subject by, for example, one day or more than one week, for example, from about one 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).

[0265] Additionally or alternatively, a skilled physician may administer Compound I or a salt thereof, e.g., Compound III, simultaneously with, in admixture with, or separately from a nitric oxide donor such as nitroglycerin. For example, Compound I or a salt thereof, e.g., Compound III, may be administered before, after, or simultaneously with nitroglycerin to delay the onset of labor in a subject by, for example, one day or more than one week, e.g., from about one 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).

[0266] Additionally or alternatively, a skilled practitioner may administer Compound I or a salt thereof, such as Compound III, simultaneously with, in admixture with, or separately from progesterone or a derivative or variant thereof, such as a derivative or variant described herein or known in the art. For example, Compound I or a salt thereof, e.g., Compound III, may be administered before, after, or simultaneously with progesterone or a variant or derivative thereof described herein or known in the art to delay the onset of labor in a subject, e.g., by one day or one week or more, e.g., from about one 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).

[0267] Example 6. Tocolytic activity of Compound I and its pharmaceutically acceptable salts in combination with nifedipine and atosiban in a mouse model of preterm labor To investigate the therapeutic effects of Compound I in combination with calcium channel blockers or oxytocin receptor antagonists in an animal model of preterm labor, primary pregnant CD-1 mice were treated with an established inducer of labor on day 17 of gestation and then administered various doses of the chloride salt of Compound I (Compound III; 10 mg / kg, 30 mg / kg, or 100 mg / kg, orally) alone or in combination with nifedipine (5 mg / kg, orally) or atosiban (300 mg / kg, subcutaneously). Tocolytic activity was assessed by measuring the time from induction to delivery of the first pup in each mouse in the treatment and control cohorts, the time from induction to delivery across all mice in each cohort, and the survival rate of offspring across mice in each cohort. The inducers of preterm labor used in this study were RU486 (also known as mifepristone), a steroidal antiprogestin that promotes cervical dilation and causes uterine contractions and increased sensitivity to prostaglandins, and lipopolysaccharide (LPS), a mediator of inflammation.

[0268] To induce labor during early pregnancy, a single dose of RU486 was administered subcutaneously to each mouse 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 300 mg / kg at two distinct sites. These injections were performed at 5 hours (t = 5) and 29 hours (t = 29), followed by treatment with the inducer RU486 or LPS. Nifedipine was administered orally to CD-1 mice at 5 hours (t = 5), 19 hours (t = 19), 29 hours (t = 29), and 43 hours (t = 43) at 5 mg / kg, followed by treatment with the inducer RU486 or LPS. Compound III was administered orally to CD-1 mice at 10 mg / kg, 30 mg / kg, or 100 mg / kg at 5 hours (t = 5), 19 hours (t = 19), 29 hours (t = 29), and 43 hours (t = 43), followed by treatment with the inducers RU486 or LPS. After induction with RU486 or LPS and subsequent administration of atosiban, nifedipine, and / or Compound III, mouse cohorts were subjected to continuous visual monitoring to assess the time elapsed between induction and delivery of the first pup for each mouse, as well as the proportion of mice in each cohort that gave birth as a function of time. The survival rate of pups delivered in each cohort was assessed by Galenic hydrostatic neonatal pulmonary docimas.

[0269] Treatment of CD-1 mice with RU486 at 17 days of gestation resulted in a mean time to delivery of approximately 21 hours after induction (t = 21; calculated mean = 21 ± 1.00 hours), whereas CD-1 mice treated with LPS at 17 days of gestation exhibited a mean time to delivery of approximately 26 hours after induction (t = 26; calculated mean = 26 ± 2.34 hours), confirming the ability of RU486 and LPS to induce preterm delivery. In contrast, full-term delivery in CD-1 mice occurs at approximately 19 to 21 days of gestation, more than 50 hours past day 17 of gestation. Of the pups delivered to RU486-treated mice, 96% were delivered alive, compared with 48% of pups delivered to LPS-treated mice (Figures 60A to 61C). Three percent of the RU486-treated mice were excluded from the study due to death or sacrifice during the study, and 34% of the LPS-treated mice were excluded from the study due to death or sacrifice during the study.

[0270] During the study, it was observed that treatment with nifedipine alone induced a significant increase in the mean time to birth compared to vehicle in RU486-treated mice (23.53±0.99 hours vs. 21.19±1.00 hours; Figures 65A to 65E). Treatment with nifedipine alone further promoted the increase in time to birth and significantly increased the survival rate of offspring in LPS-treated mice compared to vehicle (90.39%±5.34% vs. 48.20%±16.45%; Figures 68A to 69M). Administration of atosiban similarly increased the time to birth in LPS-treated mice (Figures 70A to 70B).

[0271] Compound III was found to promote an increase in time to birth in RU486-treated mice compared to vehicle (Figures 65A to 65E and 67A to 67E). In particular, RU486-treated mice orally administered Compound III at 30 mg / kg and 100 mg / kg exhibited an increase in time to birth compared to vehicle (p=0.0871 and p=0.0601, respectively). Furthermore, administration of Compound III to LPS-treated mice resulted in a dose-dependent increase in offspring survival (48.20% ± 16.45% survival observed in response to vehicle, compared to 69.41% ± 15.76% survival observed in response to 100 mg / kg Compound III; Figures 68A to 68B).

[0272] The combination of nifedipine and Compound III produced particularly pronounced tocolytic activity (Figures 65A to 65E and 69A to 69M). This combination produced a clear synergistic effect, as oral administration of nifedipine (5 mg / kg) and Compound III (100 mg / kg) to RU486-treated mice induced a significant increase in the time to delivery compared with vehicle (27.91 ± 0.35 h vs. 21.19 ± 1.00 h), the same dose of nifedipine alone (27.91 ± 0.35 h vs. 23.53 ± 0.99 h), and the same dose of Compound III alone (27.91 ± 0.35 h vs. 23.70 ± 0.60 h). Furthermore, oral administration of nifedipine (5 mg / kg) and Compound III (10 mg / kg) to LPS-treated mice significantly increased the time to birth compared with the cohort treated with 10 mg / kg Compound III alone (31.01 ± 1.89 h vs. 23.98 ± 0.66 h). Oral administration of 10 mg / kg Compound III in combination with 5 mg / kg nifedipine also promoted increased survival of pups delivered by LPS-treated mice compared with mice receiving the same dose of Compound III alone (94.23% ± 3.68% vs. 57.90% ± 14.89%) and compared with mice receiving vehicle alone (94.23% ± 3.68% vs. 48.20% ± 16.45%; Figures 68A-68B).

[0273] The combination of atosiban and Compound III further enhanced the tocolytic activity 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 time to delivery compared with mice treated with vehicle alone (33.23 ± 2.95 h vs. 26.17 ± 1.98 h) and with the same dose of atosiban alone (33.23 ± 2.95 h vs. 28.41 ± 2.99 h; Figures 71A-71E). This combination also increased offspring survival compared with mice treated with vehicle alone, the same dose of atosiban alone, or the same dose of Compound III alone (Figures 70A-70B).

[0274] This study further illustrates the tocolytic activity of salts of the FP antagonist Compound I in two distinct animal models of preterm labor, supporting the use of Compound I and its salts to treat and prevent preterm labor, regardless of the underlying biochemical etiology. This investigation further supports the use of FP antagonists, such as Compound I and its salts (e.g., Compound III), in combination with calcium channel antagonists and oxytocin receptor antagonists to prevent preterm labor. The use of Compound III in combination with nifedipine and atosiban significantly exceeds the therapeutic effects of the individual components, demonstrating that Compound I and its salts, such as Compound III, can synergize with additional tocolysis.

[0275] Example 7. Tocolytic activity of Compound II in combination with nifedipine, atosiban, and nolasiban in human tissue samples To investigate the therapeutic effects of Compound II, the active metabolite of Compound I and its salts (e.g., Compound III), in combination with an oxytocin receptor antagonist and a calcium channel blocker, myometrial biopsies were obtained from full-term, prepartum human female subjects undergoing cesarean section delivery. Among the objectives of this study were to characterize the effects of Compound II alone or in combination with additional tocolysis on the frequency, peak amplitude, and duration of myometrial contractions, as well as the work exerted per contraction and the total work exerted by all contractions. To achieve this goal, experiments were performed using a DMT Myograph 800 MS (ADINSTRUMENTS™) in oxygenated Krebs solution with ADI Powerlab software, which facilitates simultaneous measurements of multiple muscle specimens in parallel.

[0276] The myometrial biopsy experiment was initiated by establishing a baseline for smooth muscle contraction for at least 20 minutes.After this period, baseline measurements were recorded for spontaneous contraction frequency, peak amplitude, duration, work done per contraction, and total work done by all contractions.The myometrial biopsy samples were then treated with DMSO control, Compound II, atosiban, nifedipine, a combination of Compound II and atosiban, or a combination of Compound II and nifedipine.The effects of these agents on the frequency, amplitude, and duration of myometrial contractions and the work done by myometrial contractions were then measured over the next 10 minutes.Next, the myometrial samples were stressed by adding increasing concentrations of contractile stimulants, such as oxytocin, PGF2α, or PGE2, over successive 10-minute intervals, and the contraction frequency, peak amplitude, duration, work done per contraction, and total work done by all contractions were measured accordingly. Oxytocin, PGF2α, and PGE2 are distinct regulators of uterine contraction and preterm labor. Oxytocin directly induces uterine myometrial contractions and enhances the synthesis and release of contractile prostaglandins from the uterine endometrium and decidua. Oxytocin has also been associated with promoting prostaglandin production in human myometrial cells by enhancing cyclooxygenase-2 (COX-2). The prostaglandins PGF2α and PGE2 have been shown to induce cervical changes and induce uterine contractions, two key events in the physiology of labor and delivery. Activation of FP receptors by PGF2α in the human myometrium increases intracellular calcium levels, which in turn leads to contraction of uterine smooth muscle cells. Therefore, another objective of this study was to evaluate the ability of Compound II to attenuate uterine contractile activity induced by three distinct biochemical modalities.

[0277] The results of these experiments demonstrate that Compound II alone is capable of inhibiting both PGF2α- and OT-induced myometrial contractions in a dose-dependent manner (Figures 72A-72E and 73A-73E). Furthermore, it has now been discovered that Compound II exhibits a surprising synergistic effect on reducing myometrial contractions when used in combination with the oxytocin receptor antagonist atosiban (Figures 76A-76E) and the calcium channel blocker nifedipine (Figures 78A-78E). Surprisingly, doses of Compound II (e.g., 60 nM, Figures 72A-72E and 73A-732E) that exhibited lower efficacy in reducing myometrial contractions when used in the absence of additional tocolysis exhibited significantly increased inhibitory activity when combined with atosiban (Figures 76A-76E) and nifedipine (Figures 78A-78E). Similarly, doses of atosiban (6 nM, Figures 74A-74E and 75A-75E) and nifedipine (6 nM, Figures 77A-77E), which were found to be suboptimal for reducing myometrial contractions when used in the absence of Compound II, exhibited an unexpected increase in anticontractile potency when combined with Compound II (Figures 76A-76E and 78A-78E). These data demonstrate that Compound II has the ability to synergize with additional tocolytics, such as oxytocin receptor antagonists and calcium channel blockers, to suppress uterine contractile activity that can lead to preterm labor.

[0278] In addition to inhibiting myometrial contractions, the tocolytic activity of Compound II is also evident in its ability to attenuate downstream pro-inflammatory gene expression in human myometrial and amniotic biopsies (Figures 79A-79E and 80A-80E). Western blots were performed to characterize the ability of Compound II, alone or in combination with additional tocolysis, to modulate the expression of various proteins in myometrial and amniotic samples isolated from full-term prepartum human female subjects undergoing cesarean delivery. The results of these studies demonstrate that Compound II can reduce the expression of various pro-inflammatory proteins and exhibits a surprising synergistic effect on reducing COX-2 expression when used in combination with nolasiban.

[0279] Collectively, the data generated from these experiments demonstrate that Compound II has the ability to suppress smooth muscle activity, which can lead to preterm labor induced by distinct regulators of uterine contraction.Furthermore, when Compound II is used in combination with an oxytocin receptor antagonist and a calcium channel blocker, it exhibits an unexpected synergistic effect on attenuating uterine contraction.This synergistic effect is evident both in the level of smooth muscle activity and in the reduction of pro-inflammatory gene expression in myometrial and amniotic membrane biopsies, demonstrating the various benefits of providing Compound II in combination with one or more additional tocolysis agents to subjects in need of treatment, such as those experiencing or at risk of experiencing preterm labor.

[0280] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0281] While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow the principles of the invention and which are within the scope of known or customary practice in the art to which the invention pertains, including departures therefrom which may be applied to the essential features described above, and which fall within the scope of the appended claims.

[0282] Other embodiments are within the scope of the claims.

[0283] An example of an embodiment is described below as an item. [Item 1] Compounds represented by formula (I) [C1] A pharmaceutical composition comprising TIFF2026000959000028.tif81102 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition further comprises an additional therapeutic agent. [Item 2] Item 1, wherein the compound is represented by formula (III). [C2] TIFF2026000959000029.tif79120[Item 3] 3. The pharmaceutical composition of item 1 or 2, wherein the additional therapeutic agent is an additional tocolytic agent. [Item 4] 4. The pharmaceutical composition according to any of items 1 to 3, wherein the pharmaceutical composition comprises one or more excipients. [Item 5] 5. The pharmaceutical composition according to any of items 1 to 4, wherein the compound has a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%. [Item 6] 6. The pharmaceutical composition of item 5, wherein the purity is confirmed by high pressure liquid chromatography (HPLC). [Item 7] 6. The pharmaceutical composition of item 5, wherein the purity is confirmed by NMR spectroscopy. [Item 8] 8. The pharmaceutical composition according to any of items 1 to 7, wherein the compound or pharmaceutical composition is formulated for oral administration to a subject. [Item 9] 9. The pharmaceutical composition according to any of items 1 to 8, wherein the compound or pharmaceutical composition is a tablet, capsule, gelcap, powder, liquid solution, or liquid suspension. [Item 10] 8. The pharmaceutical composition according to any of items 1 to 7, wherein the compound or pharmaceutical composition is formulated for intravenous administration to a subject. [Item 11] 11. The pharmaceutical composition according to any of items 1 to 10, wherein the pharmaceutical composition comprises an oxytocin receptor antagonist. [Item 12] 12. The pharmaceutical composition of item 11, wherein the oxytocin receptor antagonist is selected from the group consisting of atosiban, letosiban, barusiban, epelusiban, and nolasiban. [Item 13] 13. The pharmaceutical composition according to any of items 1 to 12, wherein the pharmaceutical composition comprises a betamimetic. [Item 14] 14. The pharmaceutical composition of claim 13, wherein the betamimetic is selected from the group consisting of terbutaline, ritodrine, hexoprenaline, albuterol, fenoterol, nylidrin, and orciprenaline. [Item 15] 15. The pharmaceutical composition according to any of items 1 to 14, wherein the pharmaceutical composition comprises a calcium channel inhibitor. [Item 16] 16. The pharmaceutical composition of item 15, wherein the calcium channel inhibitor is a dihydropyridine. [Item 17] 17. The pharmaceutical composition of claim 16, wherein the dihydropyridine is selected from the group consisting of nifedipine and nicardipine. [Item 18] 18. The pharmaceutical composition according to any of items 1 to 17, wherein the pharmaceutical composition comprises a magnesium salt. [Item 19] 19. The pharmaceutical composition of item 18, wherein the magnesium salt is magnesium sulfate. [Item 20] 20. The pharmaceutical composition according to any of items 1 to 19, wherein the pharmaceutical composition comprises a nitric oxide donor. [Item 21] 21. The pharmaceutical composition of item 20, wherein the nitric oxide donor is nitroglycerin. [Item 22] 22. The pharmaceutical composition according to any of items 1 to 21, wherein the pharmaceutical composition comprises progesterone or 17-α-hydroxyprogesterone caproate. [Item 23] 23. The pharmaceutical composition according to any of items 1 to 22, wherein the pharmaceutical composition comprises a corticosteroid. [Item 24] 24. The pharmaceutical composition of claim 23, wherein the corticosteroid is selected from the group consisting of betamethasone, dexamethasone, and hydrocortisone. [Item 25] 25. The pharmaceutical composition according to any of items 1 to 24, wherein the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and nifedipine. [Item 26] 26. The pharmaceutical composition according to item 25, wherein the compound is represented by formula (III): [Item 27] 27. The pharmaceutical composition according to any of items 1 to 26, wherein the pharmaceutical composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and atosiban. [Item 28] 28. The pharmaceutical composition according to item 27, wherein the compound is represented by formula (III): [Item 29] 29. The pharmaceutical composition according to any of items 1 to 28, wherein the compound binds to the human prostaglandin F2α receptor with an affinity of about 1 nM. [Item 30] 30. The pharmaceutical composition according to any of items 1 to 29, wherein the compound is soluble in aqueous solution at a concentration of about 300 μg / mL to about 500 μg / mL. [Item 31] 31. The pharmaceutical composition of item 30, wherein the compound is soluble in aqueous solution at a concentration of about 380 μg / mL. [Item 32] 32. The pharmaceutical composition according to any of items 1 to 31, wherein the compound inhibits the synthesis of inositol triphosphate in cells. [Item 33] 33. The pharmaceutical composition of item 32, wherein the cells are mammalian cells. [Item 34] 34. The pharmaceutical composition of item 33, wherein the mammalian cells are human cells. [Item 35] 35. The pharmaceutical composition of item 34, wherein the human cells are myometrial cells. [Item 36] 36. The pharmaceutical composition of item 35, wherein the myometrial cells are myometrial cells. [Item 37] 37. The pharmaceutical composition according to any of items 1 to 36, wherein the compound induces a reduction in the amplitude of uterine contractions in a subject after administration of the compound to the subject. [Item 38] 38. The pharmaceutical composition of item 37, wherein the reduction is about 40% to about 50% relative to the measured amplitude of uterine contractions of the subject recorded before the administration. [Item 39] 39. The pharmaceutical composition according to any of items 1 to 38, wherein the compound exhibits a half-life of about 1 hour to about 4 hours in a subject following administration of the compound to the subject. [Item 40] 40. The pharmaceutical composition according to any one of items 1 to 39, wherein the compound reaches a maximum plasma concentration in the subject within about 0.25 hours to about 2 hours after administration of the compound to the subject. [Item 41] 41. The pharmaceutical composition according to any one of items 37 to 40, wherein the subject is a mammal. [Item 42] 42. The pharmaceutical composition of claim 41, wherein the mammal is a human. [Item 43] 42. The pharmaceutical composition of claim 41, wherein the mammal is a dog. [Item 44] 42. The pharmaceutical composition of claim 41, wherein the mammal is a rat. [Item 45] 45. The pharmaceutical composition according to any of items 37 to 44, wherein the administration is oral. [Item 46] 45. The pharmaceutical composition according to any of items 37 to 44, wherein the administration is intravenous. [Item 47] The compound is represented by formula (III): [C3] TIFF2026000959000030.tif81123 The pharmaceutical composition according to any one of items 1 to 46, wherein the compound is in a crystalline state. [Item 48] 48. The pharmaceutical composition of item 47, wherein the compound 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. [Item 49] 49. The pharmaceutical composition of item 48, wherein 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. [Item 50] 50. The pharmaceutical composition according to any of items 47 to 49, wherein the compound is characterized by an X-ray powder diffraction spectrum substantially as shown in any one of Figures 19, 22, 29, 45-49, and 54. [Item 51] 51. The pharmaceutical composition of item 50, wherein the compound is characterized by an X-ray powder diffraction spectrum substantially as shown in Figure 49. [Item 52] The compounds are centered at about 1.1 ppm, about 3.3 ppm, about 4.9 ppm, about 5.4 ppm, about 7.1 ppm, about 7.7 ppm, about 7.9 ppm, a...

Claims

[Claim 1] The invention described in the specification.

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