WO2008033894A2 - Compounds with activity at estrogen receptors - Google Patents

Compounds with activity at estrogen receptors Download PDF

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Publication number
WO2008033894A2
WO2008033894A2 PCT/US2007/078236 US2007078236W WO2008033894A2 WO 2008033894 A2 WO2008033894 A2 WO 2008033894A2 US 2007078236 W US2007078236 W US 2007078236W WO 2008033894 A2 WO2008033894 A2 WO 2008033894A2
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substituted
unsubstituted
group
erb
cycloalkyl
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PCT/US2007/078236
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French (fr)
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WO2008033894A3 (en
Inventor
Fabrice Piu
Daniel W. Gil
Roger Olsson
Magnus Gustafsson
Lene Hyldtoft
Karyn F. Siemasko
Michael E. Stern
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Acadia Pharmaceuticals Inc.
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Publication of WO2008033894A2 publication Critical patent/WO2008033894A2/en
Publication of WO2008033894A3 publication Critical patent/WO2008033894A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages

Definitions

  • This invention relates to the fields of organic chemistry, pharmaceutical chemistry, biochemistry, molecular biology and medicine. In particular it relates to compounds that modulate the activity of the Estrogen receptors, and to the use of the compounds for the treatment and prevention of diseases and disorders related to the Estrogen beta receptor.
  • Estrogen receptors belong to the family of nuclear hormone receptors. Two estrogen receptor subtypes have been identified: ER alpha (ERa, NR3A1) (Green, 1986, Nature 320: 134; Greene, 1986, Science 231: 1150) and ER beta (ER ⁇ , NR3A2) (Kuiper, 1996, PNAS 93:5925). Both receptors bind to the endogenous natural ligand 17 ⁇ estradiol with comparable high affinity and modulate the transcriptional activity of target genes through classical estrogen response elements (reviewed in Nilsson, 2005, Bas Clin Pharm Tox, 96: 15).
  • estrogens In neuropathic pain is quite controversial, most likely because estrogens lack selectivity towards the two reported receptor subtypes, ERa and ER ⁇ . Indeed, both subtypes are expressed in sensory neurons of the dorsal root ganglia, the effector site of pain sensation (Papka et Storey- Workley, 2002, Neurosci Letters 319:71; Papka et al, 2001, Cell Tissue Res 304: 193). They are also found in interneurons of the spinal cord (Shughrue et al, 1997, J Comp Neeurol, 388:507).
  • Estrogen can induce mechanical hyperalgesia, a hallmark of neuropathic pain through direct action on nociceptive neurons (Hucho et al, 206, Eur J Neurosci, 24:527). Similarly, treatment with estradiol increases pain responsiveness to a thermal stimulus, while acute blockade of estrogen synthesis in the spinal dorsal horn reduces that behavior, consistent with a pro-nociceptive role of non-selective estrogen agonism (Evrard & Balthazart, 2004, J Neurosci 24:7225). However, estrogens have also been described to display beneficial effects in neuropathic pain.
  • ER ⁇ agonist is a compound of formula I
  • FIGURE IA depicts agonist activity of ERB-002 at the estrogen receptor ⁇ as evaluated using the Receptor Selection and Amplification (R-SATTM) technology.
  • FIGURE IB depicts agonist activity of ERB-002 at the estrogen receptor ⁇ as evaluated using the Receptor Selection and Amplification (R-SATTM) technology.
  • FIGURE 2 is a graph depicting rat paw hot plate latency illustrating the reversal of thermal hyperalgesia by ERB-002 in a CFA-induced arthritis model.
  • FIGURE 3 is a graph depicting rat paw thickness illustrating the reversal of edema/inflammation by ERB-002 in a CFA-induced arthritis model.
  • FIGURE 4 is a bar graph depicting uterine weight illustrating that ERB-002 does not display uterotrophic properties in vivo in immature female rats, i.e., lack of ER- ⁇ activity in vivo.
  • FIGURE 5A is a bar graph showing that IFN- ⁇ tear levels are decreased in the presence of ERB-002.
  • FIGURE 5B is a bar graphs showing that IL-4 tear levels are increased in the presence of ERB-002 in a model of T H 1 inflammation.
  • FIGURE 6A is a bar graph showing the clinical scoring for lid edema in the multi-hit antigen challenge mouse model.
  • FIGURE 6B is a bar graph showing the clinical scoring for hyperemia in the multi-hit antigen challenge mouse model.
  • FIGURE 6C is a bar graph showing the clinical scoring for chemosis in the multi-hit antigen challenge mouse model.
  • FIGURE 6D is a bar graph showing the clinical scoring for tearing in the multi-hit antigen challenge mouse model.
  • FIGURE 7A is a bar graphs showing that IL-4 (SRW sensitized mice) tear levels are decreased in the presence of ERB-002 in a T H 2 inflammation model.
  • FIGURE 7B is a bar graphs showing there was a decrease in IL- 12 (SRW sensitized mice) tear levels in the presence of ERB-002.
  • FIGURE 8A is a graph showing the effect of ERB-002 (referred to as ERB- 131) on weight loss of mice with dextran sulphate/indomethacin induced colitis.
  • FIGURE 8B is a graph showing the effect of ERB-002 (referred to as ERB-131) on diarrhea of mice with dextran sulfate/indomethacin induced colitis.
  • FIGURE 9A is a graph showing the effect of ERB-002 (referred to as ERB-131) on response to thermal hyperalgesia as measured by using a hot plate test.
  • FIGURE 9B is a graph showing the effect of ERB-002 (referred to as ERB-131) on inflammation as assessed by the formation of local edema from the treated paw.
  • FIGURE 1OA is a graph showing the effect of gabapentin on response to thermal hyperalgesia as measured by using a hot plate test.
  • FIGURE 1OB is a graph showing the effect of gabapentin on inflammation as assessed by the formation of local edema from the treated paw.
  • FIGURE 11 is a graph showing ERB-002 (referred to as ERB-131) does not alleviate formalin induced inflammatory pain.
  • FIGURE 12 are graphs showing ERB-002 (referred to as ERB-131) does does not affect Phase I or Phase II in the formalin test.
  • FIGURE 12A shows the data from Phase 1 and
  • FIGURE 12B shows the data from Phase II.
  • FIGURE 13 A is a graph showing the effect of ERB-002 (referred to as ERB-131) on hyperalgesia in the CFA model.
  • FIGURE 13B is a graph showing the effect of ERB-002 (referred to as ERB-131) on inflammation in the CFA model.
  • FIGURE 14 shows that ERB-002 (referred to as ERB-131) is a potent selective ER ⁇ agonist.
  • FIGURE 14A is a graph comparing the activity of ERB-002 with that of estrone at estrogen receptors ERa and ER ⁇ .
  • FIGURE 14B is a graph depicting the selectivity of ERb- 131 using R-SAT ® .
  • FIGURE 15 shows the effect of ERB-002 (referred to as ERB-131) on the uterus size of sexually immature female rats at different doses: na ⁇ ve (Fig. 15A), vehicle (Fig. 15B), PPT (Fig. 15C), 10 mg/kg (Fig. 15D), 30 mg/kg (Fig. 15E), 100 mg/kg (Fig. 15F). Quantitative values are shown graphically in Fig. 15G.
  • FIGURE 16 is a graph showing that ERB-002 (referred to as ERB- 131) alleviates capsaicin-induced acute hyperalgesia.
  • FIGURE 17 shows that ERB-002 (referred to as ERB-131) broadly inhibits chemically-induced allodynia, when ERB-002 was injected 15 min prior to injection of the allodynia-inducing agents (Fig. 17A); effects of different doses of ERB- 002 were evaluated (Fig. 17B); and the estrogen receptor pan-antagonist ICI- 182740 was used to confirm that the effects of ERB-002 (Fig. 17C).
  • FIGURE 18A is a graph showing the effects of an acute dose of ERB- 002 (referred to as ERB-131) on inhibition of allodynia 30 min following injection.
  • FIGURE 18B is a graph showing the effects on inhibition of allodynia of different regimens using low doses of ERB-002.
  • FIGURE 19 is a graph showing hot plate latency of rats after injection with ERB-002 (referred to as ERB-131).
  • n is an integer selected from the group consisting of 3, 4, 5 and 6;
  • R 2a is optionally bound to one R3 group to form a substituted or unsubstituted C 4 -C9 heteroalicyclic, C 4 -C9 cycloalkyl, or C 4 -C9 cycloalkenyl;
  • R 4J is optionally bound to one R3 group to form a substituted or unsubstituted C 4 -C9 heteroalicyclic, C 4 -C9 cycloalkyl, or C 4 -C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
  • R 4J , and R 4b are optionally bound together to form an aryl, heteroaryl, or heteroalicyclyl;
  • Z is oxygen or sulfur
  • R 6 , R 6a and R ⁇ are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl.
  • n is an integer selected from the group consisting of 3, 4, and 5;
  • Ri is selected from the group consisting of hydrogen, Ci-C 4 straight chained or branched alkyl, Ci-C 4 straight chained or branched alkenyl, Ci-C 4 straight chained or branched perhaloalkyl, and substituted or unsubstituted aryl;
  • R 4 , R 43 , Rib, R 4 C are separately selected from the group consisting hydrogen, C 1 -C 5 straight chained or branched alkyl, C 1 -C 5 alkenyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -OR 6 , -CN, -C(O)R 6 , -C(O)OR 6 , and -SR 6 ; and
  • R5 is selected from the group consisting of hydrogen, C 1 -C 5 straight chained or branched alkyl, halogen, -CN, -SR 6, sulfonyl, -OCF 3 , and perhaloalkyl.
  • n is 3;
  • Ri is selected from the group consisting of hydrogen, C 1 -C 5 straight chained or branched alkyl, substituted or unsubstituted aryl;
  • R 2 , R2a, R2b, R2c are separately selected from the group consisting of hydrogen, C 1 -C 5 straight chained or branched alkyl, F, Cl, Br, perhaloalkyl, -CN, -OR 6 , -C(O), and -SR 6 ; each R 3 is separately selected from the group consisting of hydrogen, C 1 - C 5 straight chained or branched alkyl, C 1 -C 5 alkenyl, cycloalkyl, halogen, perhaloalkyl, -CN, and -OR 6 , or each R 3 is separately absent to accommodate a double bond; each R 4 , R 43 , Rn,, R 40 is separately selected from the group consisting hydrogen, C 1 -C 5 straight chained or branched alkyl, halogen, sulfonyl, perhaloalkyl, -OR 6 , -CN, , and -SR 6 ; and Rs is selected from the group
  • n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6;
  • R 2a is optionally bound to one Y group to form a substituted or unsubstituted C 4 - C9 heteroalicyclic, C 4 -C9 cycloalkyl, or C 4 -C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond; A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, substituted heteroalicyclyl, unsubstituted heteroalicyclyl, unsubstitued aryl, and substituted aryl;
  • A is optionally bound to one Y group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C 4 -C9 cycloalkyl, or C 4 -C9 cycloalkenyl;
  • Z is oxygen or sulfur; and each R 5 , Rs a and Rsb are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl; provided that when every Y is a substituted or unsubstituted methylene, then A is not a substituted or unsubstituted aryl.
  • A when A is a substituted aryl, it is not substituted at the para position.
  • n is an integer selected from the group consisting of 3, 4, and 5;
  • Ri is selected from the group consisting of hydrogen, C 1 -C 4 straight chained or branched alkyl, C 1 -C 4 straight chained or branched alkenyl, C 1 -C 4 straight chained or branched perhaloalkyl, and substituted or unsubstituted aryl;
  • the compound of formula II is selected from the group consisting of:
  • R group(s) such as, without limitation, R, R a and R b , is(are) independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl (bonded to the indicated group at a ring carbon atom) and heteroalicyclyl (likewise bonded to the indicated group at a ring carbon atom), as these groups are defined herein. If two "R" groups are covalently bonded to the same atom then they may be bound together so as to form a cycloalkyl or heteroalicyclyl group.
  • substituent is a group that may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and
  • C m to C n in which "m” and “n” are integers refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group or the number of carbon atoms in the ring of a cycloalkyl or cycloalkenyl group. That is, the alkyl, alkenyl, alkynyl, ring of the cycloalkyl or ring of the cycloalkenyl can contain from “m” to "n", inclusive, carbon atoms.
  • a "Ci to C 4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, CH 3 CH(CH 3 )-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )-, and (CH 3 ) 3 CH-. If no "m” and "n” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl group, the broadest range described in these definitions is to be assumed.
  • aryl refers to a carbocyclic (all carbon) ring or two or more fused rings (rings that share two adjacent carbon atoms) that have a fully delocalized pi-electron system.
  • aryl groups include, but are not limited to, benzene, naphthalene and azulene.
  • heteroaryl refers to a ring or two or more fused rings that contain(s) one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur in the ring and that have a fully delocalized pi-electron system.
  • heteroaryl rings include, but are not limited to, furan, thiophene, phthalazinone, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, triazole, thiadiazole, pyran, pyridine, pyridazine, pyrimidine, pyrazine and triazine.
  • alkyl refers to a straight or branched chain fully saturated (no double or triple bonds) hydrocarbon group.
  • An alkyl group herein may also be of medium size having 1 to 10 carbon atoms.
  • An alkyl group herein may also be a lower alkyl having 1 to 5 carbon atoms.
  • alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec- butyl, tert-butyl, amyl, tert-amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
  • an alkyl group of this invention may be substituted or unsubstituted.
  • the substituent group(s) is(are) one or more group(s) independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S- sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR a R and protected
  • alkenyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds.
  • An alkenyl group of this invention may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution.
  • alkynyl refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds.
  • An alkynyl group of this invention may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution.
  • cycloalkyl refers to a completely saturated (no double bonds) hydrocarbon ring. Cycloalkyl groups of this invention may range from C 3 to Cs. A cycloalkyl group may be unsubstituted or substituted. If substituted, the substituent(s) may be selected from those indicated above with regard to substitution of an alkyl group.
  • cycloalkenyl refers to a cycloalkyl group that contains one or more double bonds in the ring although, if there is more than one, they cannot form a fully delocalized pi-electron system in the ring (otherwise the group would be "aryl,” as defined herein).
  • a cycloalkenyl group of this invention may unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution.
  • alkylene refers to an alkyl group, as defined here, which is a biradical and is connected to two other moieties.
  • methylene -CH 2 -
  • ethylene - CH 2 CH 2 -
  • proylene -CH 2 CH 2 CH 2 -
  • isopropylene -CH 2 -CH(CH 3 )-
  • isobutylene - CH 2 -CH(CHs)-CH 2 -
  • cycloalkylene refers to an cycloalkyl group, as defined here, which binds in an analogues way to two other moieties. If the alkyl and cycloalkyl groups contains unsaturated carbons, the terms "alkenylene” and "cycloalkenylene” are used.
  • heteroalicyclic or heteroalicyclyl refers to a ring or one or more fused rings having in the ring system one or more heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • the rings may also contain one or more double bonds provided that they do not form a fully delocalized pi-electron system in the rings.
  • Heteroalicyclyl groups of this invention may be unsubstituted or substituted.
  • the substituent(s) may be one or more groups independently selected from the group consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, alkyl, alkoxy, acyl, acyloxy, carboxy, protected carboxy, amino, protected amino, carboxamide, protected carboxamide, alkylsulfonamido and trifluoromethanesulfonamido.
  • a "trihalomethanesulfonyl” group refers to an "X3CSO 2 -" group wherein X is a halogen.
  • a "cyano" group refers to a "-CN” group.
  • a "thiocyanato" group refers to a "-CNS” group.
  • An "isothiocyanato" group refers to an " -NCS” group.
  • a “sulfonyl” group refers to an “SO 2 R” group with R as defined above.
  • S-sulfonamido refers to a "-SO 2 NR a R b " group with R a and R as defined above.
  • N-sulfonamido refers to a "RSO 2 N(R 3 )-" group with R and R a as defined above.
  • a "trihalomethanesulfonamido" group refers to an "X 3 CSO 2 N(R)-" group with X as halogen and R as defined above.
  • perhaloalkyl refers to an alkyl group in which all the hydrogen atoms are replaced by halogen atoms.
  • Any unsubstituted or monosubstituted amine group on a compound herein can be converted to an amide, any hydroxyl group can be converted to an ester and any carboxyl group can be converted to either an amide or ester using techniques well- known to those skilled in the art (see, for example, Greene and Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., John Wiley & Sons, New York, NY, 1999).
  • each center may independently be R or S or a mixture thereof.
  • each double bond may independently be E or Z a mixture thereof.
  • pharmaceutically acceptable salt refers to a salt of a compound that does not cause significant irritation to a patient to which it is administered and does not abrogate the biological activity and properties of the compound.
  • Pharmaceutical salts can be obtained by reaction of a compound disclosed herein with an acid or base.
  • Base-formed salts include, without limitation, ammonium salt (NH 4 + ); alkali metal, such as, without limitation, sodium or potassium, salts; alkaline earth, such as, without limitation, calcium or magnesium, salts; salts of organic bases such as, without limitation, dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine; and salts with the amino group of amino acids such as, without limitation, arginine and lysine.
  • NH 4 + ammonium salt
  • alkali metal such as, without limitation, sodium or potassium
  • alkaline earth such as, without limitation, calcium or magnesium
  • salts of organic bases such as, without limitation, dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine
  • salts with the amino group of amino acids such as, without limitation, arginine and lysine.
  • Useful acid-based salts include, without limitation, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, methanesulfonates, ethanesulfonates, p-toluenesulfonates and salicylates.
  • a "prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may decrease the rate of metabolic degradation for instance by decreasing O-glucuronidation and or O-sulfation.
  • the prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
  • An example, without limitation, of a prodrug would be a compound disclosed herein, which is administered as an ester (the "prodrug") to facilitate absorption over a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial.
  • a further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety.
  • Another embodiment is a method of identifying a compound that alleviates inflammation in a subject, comprising identifying a subject suffering from inflammation; providing the subject with at least one compound of Formula I or II, as defined herein; and determining if the at least one compound reduces inflammation in the subject.
  • Also disclosed herein is a method of reducing inflammation in a subject comprising identifying a subject in need of the reduction in inflammation; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • the inflammation to be treated is in the eye. In some of these embodiments, the inflammation results in lid edema, hyperemia, chemosis, or tearing.
  • inflammation is due to an ophthalmologic disorder selected from the group consisting of uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration, or is an ocular manifestation of a systemic inflammatory disease such as Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis.
  • an ophthalmologic disorder selected from the group consisting of uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration
  • a systemic inflammatory disease such as Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis.
  • the inflammation is in the gastrointestinal tract. In some of these embodiments, the inflammation is colitis, or is caused by colitis.
  • the inflammation is due to arthritis.
  • the inflammation is acute, whereas in other embodiments, the inflammation is chronic.
  • Also disclosed herein is a method of treating allergic conjunctivitis in a subject comprising identifying a subject in need of such treatment; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • Also disclosed herein is a method of reducing IL-4 levels in a subject, comprising identifying a subject in need of reduction in IL-4 levels; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • Also disclosed herein is a method of reducing IFN- ⁇ levels in a subject, comprising identifying a subject in need of reduction in IFN- ⁇ levels; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • Also disclosed herein is a method of treating neuropathic pain in a subject, comprising identifying a subject in need of the treatment of neuropathic pain; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • Neuropathic pain is caused by abnormalities in the nerves, spinal cord, or brain and includes, without limitation, phantom limb pain, postherpetic neuralgia, reflex sympathetic dystrophy, causalgia, complex regional pain syndrome II, painful HIV-associated neuropathy, diabetic neuropathy.
  • Neuropathic pain is also associated with many medical conditions including, without limitation, traumatic nerve injury, multiple sclerosis, stroke, syringomyelia, epilepsy, spinal cord injury, and cancer. The methods disclosed herein are useful in the treatment of all of the aforementioned manifestations of neuropathic pain.
  • the neuropathic pain is mechanical hyperalgesia. In other embodiments, the neuropathic pain is allydonia.
  • Also disclosed herein is a method of increasing IL- 12 levels in a subject, comprising identifying a subject in need of increase in IL-12 levels; and administering to the subject a pharmaceutically effective amount of an ER ⁇ agonist.
  • the ER ⁇ agonist is a compound of formula I, as described herein.
  • the compound of formula I is selected from the group consisting of
  • the ER ⁇ agonist is 4-(l- phenylcyclohexyl)phenol.
  • the ER ⁇ agonist is a compound of formula II, as described herein.
  • the subject to be treated is a human.
  • the ER ⁇ agonist is administered topically, whereas in other embodiments, the ER ⁇ agonist is administered intraperitoneally. In other embodiments, the ER ⁇ agonist is administered orally.
  • the term "subject” refers to an animal, preferably a mammal, and most preferably a human, who is the object of treatment, observation or experiment.
  • the mammal may be selected from the group consisting of mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, primates, such as monkeys, chimpanzees, and apes, and humans.
  • terapéuticaally effective amount is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. This response may occur in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and includes alleviation of the symptoms of the disease being treated.
  • Another embodiment is a method of identifying a compound which regulates activity of an Estrogen receptor by culturing cells that express the Estrogen receptors; incubating the cells with at least one compound of Formula I or II as defined herein; and determining any change in activity of the Estrogen receptors so as to identify a compound of Formula I or II which regulates activity of a Estrogen receptors.
  • methods are provided for alleviating diseases by administering one or more compounds of Formula I or II. These methods include, but are not limited to methods such as: a method of treating clinical manifestations in which estrogen receptor function is altered; a method of treating or preventing inflammatory bowel syndrome, Crohn's disease, ulcerative proctitis or colitis; a method of treating or preventing prostatic hypertrophy, uterine leiomyomnas, breast carcinoma, endometrial carcinoma, polycystic ovary syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian carcinoma, melanoma, prostate carcinoma, colon carcinoma, brain tumors including but not limited to glioblastoma, astrocytoma, glioma, and meningioma; a method of treating or preventing prostatitis or interstitial cystitis; a method of hormonal replacement therapy; a method of treating or preventing bone density loss including but not limited to osteopo
  • Another embodiment is a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I or II as described above, and a physiologically acceptable carrier, diluent, or excipient, or a combination thereof.
  • composition refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.
  • Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • carrier defines a chemical compound that facilitates the incorporation of a compound into cells or tissues.
  • DMSO dimethyl sulfoxide
  • carrier facilitates the uptake of many organic compounds into the cells or tissues of an organism.
  • diot defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art.
  • One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
  • physiologically acceptable defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.
  • compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., as disclosed in Remington's Pharmaceutical Sciences, cited above.
  • the agents disclosed herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds disclosed herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with pharmaceutical combination disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds disclosed herein is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase.
  • a common co-solvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • VPD co-solvent system which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; and other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone.
  • other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • salts may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acids or base forms.
  • compositions suitable for use in the methods disclosed herein include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the exact formulation, route of administration and dosage for the pharmaceutical compositions disclosed herein can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, which is hereby incorporated by reference in its entirety).
  • the dose range of the composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient's body weight, or 1 to 500 mg/kg, or 10 to 500 mg/kg, or 50 to 100 mg/kg of the patient's body weight.
  • the dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. Where no human dosage is established, a suitable human dosage can be inferred from ED 50 or ID 50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • the daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 mg and 500 mg of each ingredient, preferably between 1 mg and 250 mg, e.g. 5 to 200 mg or an intravenous, subcutaneous, or intramuscular dose of each ingredient between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of each ingredient of the pharmaceutical compositions disclosed herein or a pharmaceutically acceptable salt thereof calculated as the free base, the composition being administered 1 to 4 times per day.
  • compositions disclosed herein may be administered by continuous intravenous infusion, preferably at a dose of each ingredient up to 400 mg per day.
  • the total daily dosage by oral administration of each ingredient will typically be in the range 1 to 2000 mg and the total daily dosage by parenteral administration will typically be in the range 0.1 to 400 mg.
  • the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety, which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compositions should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Compositions comprising a compound disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Procedure 1 The analysis was performed on a combined prep/analytical Waters/Micromass system consisting of a ZMD single quadropole mass spectrometer equipped with electro-spray ionization interface.
  • the HPLC system consisted of a Waters 600 gradient pump with on-line degassing, a 2700 sample manager and a 996 PDA detector.
  • Procedure 2 The analysis was performed on a combined prep/analytical Waters/Micromass system consisting of a ZMD single quadropole mass spectrometer equipped with electro-spray ionization interface.
  • the HPLC system consisted of a Waters 600 gradient pump with on-line degassing, a 2700 sample manager and a 996 PDA detector.
  • GC method 50 was used. Method 50 starts at 50 0 C and has a gradient of 20 °C/min until 250 0 C then holds the temperature for 5 minutes. The analysis was performed on an Aglient 6850 series GC system with capillary S/SL inlet and FID with EPC installation. The column was a 10 m x 0.32 mm x 0.25 ⁇ m HP5 column.
  • Example 3 Synthesis of trifluoromethanesulfonates. general procedure 1 (GPl)
  • Trifluoromethanesulfonates were prepared according to literature procedure by McMurry and Scott (McMurry, J. E.; Scott, W. J., Tetrahedron letters, 1983, 979-982). ⁇ isoPropyl-cyclehexenyl-l-trifluoromethanesulfonate
  • the vinylaromatic compounds were prepared as exemplified below using cycloheptanone and phenylmagnesium chloride.
  • Phenylmagnesium chloride (10.8 mL, 1.2 equivalents in THF) was added over 10 minutes, resulting in a gentle reflux of the solvent. After stirring at 65 0 C for 30 minutes, the mixture was cooled to rt and poured into a mixture of 3 N HCl (30 mL) and pentane (30 mL). The phases were separated, and the aqueous portion was extracted with pentane (30 mL). The combined organic phase was washed sequentially with 3 N HCl (20 mL), 3 M NaOH (2 x 20 mL), and brine (20 mL), and dried over MgSO4. Evaporation of the solvent followed by distillation using a Kugelrohr apparatus (oven temperature 100-140 0 C, 0.065 torr) yielded 1- phenylcycloheptene (1.29 g, 43%).
  • l-(4-Fluorophenyl)-cycloheptene was prepared according to GP3 and GP4 described above and isolated by column chromatography.
  • ERB-030 1 H NMR (400 MHz, CDCl 3 ): ⁇ 7.35-7.08 (m, 7H), 6.82- 6.77 (m, 2H), 4.80 (br. s, IH), 2.80-2.75 (m, 2H), 2.23-2.10 (m, IH), 1.99-1.82 (m, 4H), 1.62-1.55 (m, 2H).
  • LC-MS purity (UV/MS): 100 /100 R t 9.16 min, M-I : 319.19.
  • ERB-031 1 H NMR (400 MHz, CDCl 3 ): ⁇ 7.18-7.11 (m, 2H), 7.10- 7.07 (m, 2H), 6.92-6.87 (m, 2H), 6.83-6.80 (m, 2H), 4.80 (br. s, IH), 2.72-2.64 (m, 2H), 2.22-2.08 (m, IH), 1.95-1.85 (m, 4H), 1.59-1.47 (m, 2H).
  • LC-MS purity UV/MS: 100/100%, Rt 9.28 min, M-I : 337.17.
  • ERB-032 1 H NMR (400 MHz, CDCl 3 ) ⁇ 7.48-7.44 (m, IH), 7.24-7.13 (m, 2H), 7.08-7.03 (m, 2H), 6.96-6.90 (m, IH), 6.72-6.67 (m, 2H), 4.73 (br. s, IH), 2.94- 2.86 (m, 2H), 2.20-2.08 (m, IH), 1.98-1.90 (m, 2H), 1.88-1.78 (m, 2H), 1.60-1.48 (m, 2H).
  • LC-MS purity UV/MS: 100/100%, R t 9.21 min, M-I: 337.17.
  • ERB-039 1 H NMR (400 MHz, CDCl 3 ): ⁇ 7.68-7.52 (m, IH), 7.24- 7.10 (m, 2H), 7.10-7.04 (m, 2H), 6.95-6.86 (m, IH), 6.71-6.64 (m, 2H), 4.66 (br. s, IH), 2.84-2.75 (m, 3H), 1.90-1.73 (m, 4H), 1.40-1.25 (m, 3H), 0.83 (d, 6H, 7 Hz).
  • ERB-038 1 H NMR (400 MHz, CDCl 3 ): ⁇ 7.28-7.20 (m, 2H), 7.08- 7.02 (m, 2H), 6.96-6.92 (m, IH), 6.73-6.62 (m, 3H), 4.58 (br. s, IH), 3.80 (s, 3H), 2.68- 2.60 (m, 2H), 1.94-1.82 (m, 2H), 1.38-1.08 (m, 4H), 0.82 (d, 6H, 7 Hz).
  • LC-MS purity UV/MS: 100/ 100 , R t 6.87 min, M-I: 323.
  • Example 16 - l-Methoxy-4-(l-phenyl-cvclohexyl)-benzene, procedure A [00235] A mixture Of AuCl 3 (7.6 mg, 0.025 mmol) and AgOTf (19.3 mg, 0.075 mmol) was stirred in dichloromethane (2 mL) for 30 min. Anisole (54 mg, 0.5 mmol) and 1 -Phenyl- 1-cyclohexene (158 mg, 1 mmol) were then added sequentially. The resulting mixture was stirred at room temperature overnight. Evaporation of the solvent under reduced pressure gave 130 mg of crude material.
  • R-SATTM Receptor Selection and Amplification Technology
  • R-SAT assays were typically performed by transfecting 30 ug/bottle of receptor and 50 ug/bottle of ⁇ -galactosidase plasmid DNA. All receptor and helper constructs used were in mammalian expression vectors. Helpers are defined as signaling molecules that modulate both ligand-dependent and/or ligand-independent function of the ER receptors, typically co-activators and kinases.
  • NIH3T3 cells were transfected for 12-16 h, then trypsinized and frozen in DMSO. Frozen cells were later thawed, plated at 10,000-40,000 cells per well of a 96 well plate containing 4-(l-Phenyl- cyclohexyl)-phenol.
  • Efficacy is relative to the reference ligand Estrone.
  • FIG. 1 illustrates the dose dependent reversal of thermal hyperalgesia in this model.
  • Figure 3 illustrates the dose dependent reversal of edema in this model.
  • %TBW [(uterus weight ( in mg) / 1000) / (body weight (in g))] * 100.
  • Figure 4 illustrates that ERB-002 does not display uterotrophic properties in vivo in immature female rats.
  • the blower model treatment used for this experiment includes running fans directly at mouse cages with wire screens, along with scopolamine injections subcutaneously TID for the total of four days.
  • the timeline for desiccating treatment was for four days before tissues were collected.
  • Mice used for this experiment were BALB/c Wild type female at the approximate age range of 6-8 weeks old. Tear production was measured for all animals before and after blower treatment. All animals' tear were collected before and after blower treatment for the evaluation of inflammation and signaling markers using the Luminex IS 100 system.
  • the tear collection process was as follows:
  • ER ⁇ agonist (ERB-002) given IP at 1 mg/kg or 5 mg/kg BID to mice for the 4 days of blower treatment had a decrease in tear production (81% and 73%, respectively) from baseline as compared to a 43% decrease of blower only treatment or a 60% decrease in tear production of blower with PEG300 (vehicle) treatment.
  • Topical ERB-002 (1 mg/mL, BID, 5 ⁇ L/eye) had no effect over topical PEG300.
  • Topical ERB-002 had no effect on IFN- ⁇ levels as compared to the blower only group.
  • IL-4 levels were increased in the tears of blower mice treated with 1 mg/kg of ERB-002 administered IP.
  • Topical ERB-002 resulted in increased IL-4 levels (2 pg/mL).
  • ER ⁇ agonists in some models have been reported to increase TH2 cytokine production.
  • Example 42 Effect of ER ⁇ Agonist (ERB-002) in the Mouse Multi-Hit Allergic Conjunctivitis Model (TH2)
  • ERB-002 was applied to the mouse model of multi-hit allergic conjunctivitis using BALB/c background female mice with age range at approximately 6- 8 weeks old. Animals in all groups, with the exception of the control group, were sensitized with Short Ragweed (SRW) in Alum, via left hind paw injection of 50 ⁇ L per mouse, ten days prior to the secondary challenge. One day before the animals were subjected to secondary challenge, mice were pre-dosed with ERB-002 and vehicle at different concentrations and administration routes. For intra-peritoneal, the tested concentrations were 100 ⁇ g/kg/dose/mouse and 500 ⁇ g/kg/dose/mouse at BID.
  • SRW Short Ragweed
  • the concentration was 5 ⁇ g/5 ⁇ L/dose/eye at BID for both eyes.
  • the animals were dosed with drug and vehicle for a total of 8 days while they were topically challenged with SRW/PBS for 7 days. Eye pictures were obtained at various time points, which occurred before sensitization, after 1st SRW/PBS challenge, and after 7th challenge. Clinical scoring of the subjects' eyes was recorded by two operators after the 1 st and 7th challenge.
  • S+C refers to the group of mice who received SRW sensitization and challenge.
  • ERB-002 (100 ⁇ g/kg IP) treatment of SRW sensitized and multi-hit challenged mice reduced IL-4 levels in mouse tears to basal levels (10 pg/mL). IL- 12 tear levels were reduced by 75% in the presence of topical ERB-002.
  • ERB-002 reduced IL- 12 levels in the tears of SRW multi-hit mice.
  • IL- 12 induces IFN- ⁇ production.
  • IFN- ⁇ is required for upregulation of VCAM-I on endothelial cells.
  • VCAM-I binds to T cells and eosinophils and permits their access to the site of inflammation.
  • ERB-002 was tested in models in which sensory nerve pathways are sensitized by chemical or surgical insults, resulting in either spontaneous pain (capsaicin model) or pain sensation to normally non-noxious tactile stimuli (chemical allodynia and nerve ligation models).
  • spontaneous pain capsaicin model
  • pain sensation to normally non-noxious tactile stimuli
  • chemical allodynia and nerve ligation models The pain sensitivity takes anywhere from 15 min to 1 week to fully develop.
  • These models are distinct from models of acute nociceptive pain in which there is an immediate pain response to a noxious stimulus such as heat or inflammatory mediators.
  • ERB-002 was effective over a wide dose range (0.5 mg/kg - 10 mg/kg) while the non-selective estrogen receptor agonist, ⁇ -estradiol, was not active even at a high dose of 10 mg/kg.
  • the estrogen receptor antagonist ICI 182,780 could block the actions of ERB-002 (see mouse allodynia model data), demonstrating that its actions are estrogen receptor mediated.
  • mice Male Black6-C57 mice (20-30 grams) were intrathecally injected according to the method devised by Hylden and Wilcox (Eur J Pharmacol, 67: 313-316, 1980).
  • a sterile 30-gauge 1 A inch needle attached to a microsyringe was inserted between the L5 and L6 vertebrae.
  • the mouse was held firmly by the pelvic girdle in one hand, while the syringe was held in the other hand at an angle of approximately 200 above the vertebral column.
  • the needle was inserted into the tissue to one side of the L6 spinous process so that it slipped into the groove between the spinous and transverse processes. The needle angle was then decreased to about 100 and slowly advanced forward into the intervertebral space.
  • Allodynia were carried out essentially according to the method of Yaksh and Harty (1998). All compounds were injected intrathecally in 5 ⁇ L volume or IP in a 1 mL/kg volume, as described above. The test compounds were injected IP 15 min prior to injection of the allodynia- inducing agents (300 ng/kg IP sulprostone in DMSO; 100 ng/kg IP phenylephrine (PE) in H 2 O; 100 ng IT N-methyl D- aspartate (NMDA) in DMSO). The mice were then assessed for allodynia once every 5 minutes over a 15-50 min period post injection of allodynic agent by light stroking of the flank with a paintbrush.
  • the allodynia- inducing agents 300 ng/kg IP sulprostone in DMSO; 100 ng/kg IP phenylephrine (PE) in H 2 O; 100 ng IT N-methyl D- aspartate (NMDA) in DMSO.
  • the allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape. Data was expressed as the average total score for each group. (Each animal can have a maximum score of 16 over the 50- min period.) Each group of mice comprised 5-6 animals per group. The allodynic agents typically caused a pain score of 14 and the vehicle controls typically caused a pain score of 4-5.
  • Allodynia was assessed by applying a light tactile stimulus (Von Frey hairs) to the affected surgical paw. Von Frey hairs were applied in an up-down manner depending on the response (Dixon et al 1980) until the 50% threshold was established. The Von Frey hairs were applied to the plantar surface of the surgical paw with just enough force to bend them. A positive response was recorded if the paw was sharply withdrawn. Eight VonFrey hairs used 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18.
  • ERB-002 and ⁇ -estradiol were dosed IP (in 50% DMSO) using 3 different regimens at indicated concentrations:
  • ⁇ -Estradiol was not active using either regimen 1 or regimen 3.
  • ERB- 002 was active with a single 10 mg/kg dose assessed 30 min later (regimen 1; 91 ⁇ 6% allodynia reversal) and with a lower 0.5 mg/kg dose given 3 times over 6 hrs and assessed 30 min after the last dose (regimen 3; 72 ⁇ 10% allodynia reversal).
  • a solution of 0.3% caspacin was prepared as follows. Caspaicin (1 g) was discsolved in 2 mL of ethanol. To this solution, 10 mL of a cocktail of 0.7 mL TWEEN 80 and 9.3 mL of saline was slowly added while stirring on a hot plate until the volume is reduced to 10 mL. The solution is then dissoved in enough ethanol to result in a 0.3% solution.
  • Capsaicin (0.3%) was injected into the plantar surface of the left hind paw in a 10 ⁇ L volume and the left paw was then tested in the manner described above at 15, 30 and 60 minutes post capsaicin. [00303] A single 10 mg/kg IP dose of ERB-002 (in 50% DMSO vehicle) completely prevented the tactile hyperalgesia occurring 30 min after capsaicin injection.
  • One group of mice received ERB-002, injected concomittently (10 mg/kg), whereas another group of mice received vehicle only. Mice were then monitored daily for body weight, diarrhea, food/water intake, and alertness. Table 3 shows the results of the comparison between the two groups.
  • the results comparing the control group (vehicle) with the treatment group (ERB-002) are shown in Figures 9A (weight loss) and 9B (diarrhea). In the figures, ERB-002 is referred to as ERB-131.
  • estrogens have their ability to modulate inflammatory as well as nociceptive processes.
  • non selective estrogens such as 17 ⁇ -estradiol display anti-inflammatory properties in certain animal models (Josefsson et al. 1992; Jansson and Holmdahl 1998; Miyamoto et al. 1999; Cuzzocrea et al. 2001).
  • animal studies have emphasized that the sole activation of estrogen receptor beta is sufficient to alleviate inflammation in a number of in vivo paradigms (Harris 2006).
  • estrogens have been ascribed both pro- and anti-nociceptive properties.
  • female rats display increased flinching responses, when compared to males (Gaumond et al. 2002).
  • estradiol administration in female rats attenuates both inflammatory and pain behaviors in different models (Kuba et al. 2005; Mannino et al. 2006).
  • Estrogens classically mediate their actions through two distinct estrogen receptors (ERs): ER alpha (NR3A1, ESRl, ERa) and ER beta (NR3A2, ESR2, ER ⁇ ), which act as inducible transcription factors.
  • ER alpha NR3A1, ESRl, ERa
  • ER beta NR3A2, ESR2, ER ⁇
  • Expression studies indicate that estrogen receptors are expressed in a number of tissues and cells that are implicated in inflammation and pain sensation. For instance, both estrogen receptors are expressed in classical immunocompetent cells such as peripheral macrophages, leukocytes and microglial cells (Baker et al. 2004; Ghisletti et al. 2005; Stygar et al. 2006).
  • Estrogen receptors are also present in immune-like dorsal root ganglia cells including endothelial and dendritic cells (Evans et al. 2002; Nalbandian and Kovats 2005). Expression of ERs is also evident in DRG sensory neurons, with neurons expressing either or both subtypes (Papka and Storey- Workley 2002).
  • Acute inflammatory pain was produced by injecting 0.1 ml of 2% ⁇ - carrageenan (Sigma, St. Louis, MO) into the left hind paw of rats. Three hours after carrageenan injection, rats were tested for their responsiveness to noxious thermal stimuli. Test compounds were administered 3 hr following carrageenan injection. Rats were tested at various time-points following compounds administration for up to 2 hours. Response latencies to a noxious thermal stimulus were measured using the 52 0 C hot plate test. Additionally, paw thickness was measured, using a micrometer, immediately following testing as a measure of potential anti-inflammatory activity.
  • the hot plate latency was determined by placing rats in a plexiglass enclosure on a thermostatically controlled metal plate maintained at 52°C (Columbus Instruments, Columbus, OH). The time elapsed until the rat demonstrated an obvious nociceptive response (i.e., licking/elevating the hind paw) was measured. Animals were tested before and at various time-points following drug administration. A significant (p ⁇ 0.05) reduction in the hot plate latency was interpreted as the presence of thermal hypersensitivity. A cut-off time of 25 sec was employed in order to prevent tissue damage.
  • ERB-131 A class of non-steroidal ER ⁇ selective agonists, of which ERB-131 (also referred to herein as ERB-002) represents a prototype lead have been previously reported (Olsson et al. 2005; Piu et al. 2007).
  • ERB- 131 is a selective ERb agonist
  • the natural estrogen ligands genistein and daidzein exhibit potencies at ER ⁇ of 29 ⁇ 12 nM and 250 ⁇ 180 nM, respectively.
  • ERB-131 presents properties of a classical nuclear receptor ligand as evidenced by its ability to modulate ER ⁇ -dependent transcriptional activity and to physically interact with the ERb receptor.
  • K; 50 ⁇ 30 nM
  • EC 50 21 ⁇ 15 nM
  • no significant activities at up to 10 ⁇ M were found at other nuclear receptors, including the steroid hormone receptors (Piu et al. 2007). Therefore, ERB- 131 constitutes a potent ER ⁇ agonist with a selectivity over ERa and other nuclear receptors of over 100 fold.
  • ERB-131 was found to lack any significant ERa activity in vivo at doses as high as 100 mg/kg after several days of exposure (Piu et al. 2007).
  • Carrageenan model of acute inflammatory pain ERB-131 was administered at the doses of 1, 3 and 10 mg/kg based on previous studies showing that ERB-131 is efficacious in animal models of neuropathic pain at doses ranging from 1-10 mg/kg (Piu et al. 2007).
  • Thermal hyperalgesia was assessed using the hot plate latency test. Rats were injected once with carrageenan (2%) to induce a stable state of inflammation and thermal hyperalgesia. Three hours later, ERB-131 was injected (1, 3, 10 mg/kg) and its effects followed for a period of 2 hrs.
  • sham + vehicle filled square, dotted line
  • sham + 10 mg/kg ERB-131 filled diamond, dotted line
  • carrageenan + vehicle filled circle, dotted line
  • carrageenan + 1 mg/kg ERB-131 filled square, plain line
  • carrageenan + 3 mg/kg ERB-131 filled circle, plain line
  • carrageenan + 10 mg/kg ERB- 131 filled diamond, plain line
  • Base na ⁇ ve response latency
  • PIB Post inflammatory latency, determined 3 hours after injection of carrageenan or sham.
  • Carrageenan treated animals had a significantly decreased latency compared to vehicle treated animals (p value ⁇ 0.0001), with values averaging 6.9 ⁇ 0.2 sec vs 11.0 ⁇ 0.3 sec, respectively.
  • the effect of carrageenan was stable over the 2hr course of the experiment.
  • ERB-131 capable of alleviating hyperalgesia.
  • rats injected with carrageenan presented a significant edema compared to vehicle treated animals: 148.8 ⁇ 3.1 % vs 105.7 ⁇ 1.8 % (p value ⁇ 0.0001), respectively.
  • Inflammation was assessed by the formation of local edema from the treated paw.
  • the edema was quantified at the end of the experiment, i.e. at 120 min.
  • the paw width was normalized to the change seen in the contralateral (untreated) paw.
  • the data are shown in Fig. 9B and Table 5.
  • ERB-131 did not improve the inflammatory response, at either doses tested.
  • the pain suppressor gabapentin (10, 30, 100 mg/kg i.p.) was used as a positive control.
  • Gabapentin dose-dependently alleviated hyperalgesia with an ED50 of approximately 43 mg/kg (p value ⁇ 0.0001).
  • Hyperalgesia was assessed using the 52°C hot plate test, measured at 60, 120, 180, 240 and 300 min. The data are shown in Fig.
  • Formalin model of persistent inflammatory pain measures pain sensations to a continuous noxious stimulus and results in a characteristic biphasic response, with a transient Phase I (10 min) relating to behaviors associated with acute pain and nociception and a longer-lasting Phase II (> 60 min) corresponding to inflammatory pain.
  • Phase I 10 min
  • Phase II Phase II
  • formalin induced significant pain with a biphasic modality, the phases I and II averaging 222 ⁇ 21 and 1293 ⁇ 110 flinches, respectively.
  • ERB-131 was unable to reduce the number of flinches at all doses tested.
  • Formalin treated animals were injected with vehicle or ERB-131 at 1, 3 and 10 mg/kg as described in the methods. Data was pooled into Phase I and Phase II. Phase I represents data from 5 and 10 min time points.
  • Phase II incorporates remaining timepoints (from 15 to 60 min). P values were calculated using a one-way ANOVA test. Figs. 11-12 and Table 6 show the data. Legends are: formalin + vehicle (filled circle, dotted line), formalin + 1 mg/kg ERb-131 (filled square, plain line), formalin + 3 mg/kg ERB-131 (filled circle, plain line), formalin + 10 mg/kg ERb-131 (filled diamond, plain line).
  • Fig. 12A For the data shown on Fig. 12A, data from 5 and 10 min timepoints was pooled and reported as Phase I. The number of flinches per 5 min interval represents cumulative data.
  • Fig. 12B For the data shown on Fig. 12B, data from 15-60 min timepoints was pooled and reported as Phase II. Data is thus a cumulative representation of 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min timepoints.
  • CFA model of chronic inflammatory pain The Complete Freund's Adjuvant (CFA) rat model is a well characterized model, producing inflammation as well as chronic pain primarily in the form of hyperalgesia. Rats were injected with Complete Freund's Adjuvant (CFA) to induce a stable state of inflammation and thermal hyperalgesia. Subsequently, control (iCFA: inactivated CFA) or CFA animals were treated with ERB-131 once daily, injected s.c. Response to thermal hyperalgesia was measured daily from day 4 to day 8. Latencies for paw withdrawal are expressed in seconds (sec). The assessment of therrmal hyperalgesia is presented in Fig. 13A and Table 7. Rats were treated as described in the methods. Hyperalgesia and inflammation measures are presented . Data is summarized as AVG ⁇ STD. P values were calculated compared to vehicle treated animals, using a two-tailed unpaired t-test.
  • the ED50 for ERB-131 for the thermal hyperalgesia endpoint was approximately 3 mg/kg. Inflammation was assessed by the formation of local edema from the treated paw. The edema was quantified daily from day 4 on using a micrometer. The paw width was normalized to the change seen in the contralateral (untreated) paw. Administration of CFA into the hind paws of rats produced significant edema formation (Fig. 13B and Table 7).
  • iCFA + vehicle filled square, dotted line
  • iCFA + 10 mg/kg ERB-131 filled diamond, dotted line
  • CFA + vehicle filled circle, dotted line
  • CFA + 1 mg/kg ERB-131 filled square, plain line
  • CFA + 3 mg/kg ERB-131 filled circle, plain line
  • CFA + 10 mg/kg ERB-131 filled diamond, plain line
  • iCFA resulted in a 19.4 ⁇ 1.4 % increase in paw thickness relative to the contralateral paw (non-injected paw), whereas CFA triggered an increase of 53 ⁇ 1.3 % (p value ⁇ 0.0001).
  • ERB-131 Systemic administration of ERB-131 produced a dose-dependent reversal of CFA-induced edema.
  • the ED 50 for ERB-131 in the inflammation assay was approximately 1 mg/kg.
  • ERB-131 had no effect on measures of inflammation and pain sensation.
  • the lack of efficacy was not a consequence of irreversible effects of carrageenan as the pain suppressor gabapentin dose-dependently inhibited thermal hyperalgesia but not edema, consistent with published literature.
  • the selective ERb agonist ERB-041 did not inhibit edema in a reverse paradigm of the carrageenan model (Leventhal et al. 2006), and only partially resolved the thermal hyperalgesia (reversal ⁇ 50% at 30 mg/kg).
  • NF-kB NF-kB
  • NF-kB a master switch of inflammation.
  • NF-kB can be activated by a large variety of stimuli and, in response, regulates a multitude of inflammatory genes including cytokines, chemokines, adhesion molecules and acute phase proteins.
  • cytokines cytokines
  • chemokines chemokines
  • adhesion molecules a multitude of inflammatory genes
  • proinflammatory cytokines act at many levels to increase pain sensitivity, directly on sensory neurons (Fukuoka et al.
  • estrogens through activation of ERb, can modulate directly inflammation as well as nociception.
  • inflammatory processes can also directly influence the initiation and maintenance of pain states (reviewed in Watkins and Maier 2005)
  • ER ⁇ activation presents a unique advantage in resolving noxious pain states.
  • inflammation is a critical event in the establishment of neuropathic pain, it would follow that ER ⁇ activation could be a novel and effective way of addressing neuropathies, consistent with our recent findings that selective activation of ER ⁇ using ERB-131 is extremely effective in alleviating allodynia and hyperlagesia in various
  • Example 46 Broad Modulation of Neuropathic Pain States by a Selective Estrogen Receptor ⁇ Agonist
  • the effects of estrogens in various animal models of pain, including neuropathic pain are often conflicting.
  • estrogens can display pro- or anti-nociceptive effects, depending on the animal model considered (Tsao et al. 1999; Liu and Gintzler 2000; Shir et al. 2002; Evrard and Balthazart 2004; Hucho et al. 2006).
  • soy diet rich in isoflavones and phytoestrogens
  • ER ⁇ has been reported to decrease ERa transcriptional potential (Lindberg et al. 2003). This finding has led to the description of a yin/yang balance of estrogen function, by which ER ⁇ , through the formation of heterodimers ER ⁇ /ER ⁇ , can antagonize ERa function (Weihua et al. 2003). Second, both ERs differ in their spatial and temporal tissue distribution. Both ER subtypes are synthesized by autonomic and sensory neurons (Papka et al. 2001).
  • ER ⁇ mRNAs are widely expressed in sensory neurons, while ERa expression is mostly restricted to small size sensory neurons (Taleghany et al. 1999). Immunoreactivity studies also revealed the presence of DRG neurons that express both ERa and ER ⁇ , while others express either subtype (Papka and Storey- Workley 2002). Temporal regulation was also evident in long term studies of ovariectomized rats. Estrogen treatment downregulated ERa mRNA levels while upregulating ER ⁇ (Taleghany et al. 1999). Similarly, 17 ⁇ -estradiol enhanced regeneration of the sciatic nerve after crush injury, in part through accumulation of both ER proteins in motor neurons and regenerating neurites of the lumbar spinal cord (Islamov et al. 2003).
  • R-SAT ® Receptor Selection and Amplification Technology
  • GPCRs Brain-Osborne and Brann 1996)
  • RTKs Burstein et al. 1998)
  • cytokine receptors Piu et al. 2002
  • nuclear receptors Piu et al. 2005; Piu et al. 2006. This process is achieved by partial cellular transformation via loss of contact inhibition and growth factor dependency.
  • NIH-3T3 fibroblasts were plated overnight in 96-wells plates in DMEM 10 % calf serum (Hyclone) and grown to 60-70 % confluency prior to transfection.
  • Transient transfections were performed using Polyfect (Qiagen) according to manufacturer's instructions. Typically a transfection mix would consist of the receptor and the ⁇ -galactosidase expression vectors.
  • HEK293 cells were grown to 70% confluency in DMEM containing 10 % calf serum (Hyclone) prior to transfection.
  • day 1 expression vectors for ERa or ER ⁇ were cotransfected along a construct containing a synthetic 3*ERE upstream of the luciferase gene (Panomics), using Polyfect (Qiagen) per manufacturer's recommendations.
  • Phenomics 3*ERE upstream of the luciferase gene
  • Qiagen Polyfect
  • cells were incubated in serum free DMEM.
  • cells were incubated with the test compounds for 48 hours in serum free DMEM.
  • Cells extracts were then obtained by lyzing and the Luciferase activity measured, all of these steps performed using a commercially available kit (Promega).
  • HEK293T cells were transiently transfected for 48 hours with expression vectors encoding ERa or ER ⁇ , before being serum starved for 4-6 hours. Cells were then harvested by scraping in ice-cold PBS and subsequently lysed using a cold buffer containing 1OmM Tris pH 7.4, 1 mM EDTA, 1 mM DTT before being subjected to polytron twice for 10 seconds. Cytosolic extracts were isolated by centrifugation at 15000 g for 30 minutes at 4 0 C.
  • Rats received daily subcutaneous injections of vehicle (100% DMSO), (l,3,5-tris(4-hydroxyphenyl)-4-propyl-lH-pyrazole (PPT, 1 mg/kg) or various doses of ERB-131 (10, 30 or 100 mg/kg) for a total of 3 days. Approximately 24 hours after the final injection, the rats were sacrificed, the uteri removed, trimmed of adhesions, fluid expelled and then weighed.
  • vehicle 100% DMSO
  • PPT l,3,5-tris(4-hydroxyphenyl)-4-propyl-lH-pyrazole
  • ERB-131 10, 30 or 100 mg/kg
  • i.p. intraperitoneal
  • Allodynia was assessed at least one week after surgery by applying a light tactile stimulus (Von Frey hairs) to the affected surgical paw.
  • a light tactile stimulus Von Frey hairs
  • Eight calibrated Von Frey filaments (Stoelting, Wood Dale, IL) were used in logarithmically spaced increments ranging from 0.41 to 15 gm (4-150 mN). Each filament was applied perpendicularly to the plantar surface of the ligated paw of rats kept in suspended wire- mesh cages. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength ("up and down" method). A positive response was recorded if the paw was sharply withdrawn. Von Frey hairs were applied in an up-down manner depending on the response until the 50% threshold was established (Dixon 1980).
  • % Allodynia reversal ((PoDT - PrDT) / (15-PrDT))*100, where PoDT is Post Drug Threshold and PrDT is Pre Drug Threshold.
  • N N-methyl D-aspartate
  • the mice were then assessed for allodynia once every 5 minutes over a 15-50 min period post injection of the allodynic agent by light stroking of the flank with a paintbrush.
  • the allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape.
  • Data is expressed as the average total score for each group (Each animal can have a maximum score of 16 over the 50-min period).
  • the sensitizing agents typically elicit pain scores of 14 while the vehicle controls typically show pain score of 4-5.
  • Test compounds were slowly injected in the subarachnoid space of conscious mice in a volume of 5 ⁇ L.
  • ERB-131 is an ER ⁇ agonist identified using the functional cell-based assay R-SAT ® (Receptor Selection and Amplification Technology) and is representative of a novel class of non steroidal ER ⁇ ligands (Olsson et al. 2005). ERB-131 displays a strong affinity for the ER ⁇ receptor (pEC 50 7.5 ⁇ 0.4) versus ERa (pECso 5.5 ⁇ 0.3) as shown in R-SAT ® . The activity of ERB-131 was compared to that of several estrogen ligands.
  • Estrone is a non-selective agonist, while genistein, daidzein and 5a-androstan 3b, 17b diol are to various degrees selective towards ER ⁇ .
  • ERB-131 For the data shown in Figure 14A, The activity of ERB-131 was compared to estrone, a reference non-selective estrogen ligand. Compounds were evaluated for activity at estrogen receptors ERa and ER ⁇ . Dose response curves were generated using R-SAT ® . Data is reported as efficacy (%eff) normalized to estrone. Results are a representative experiment performed in triplicates. Estrone (filled square), ERB-131 (filled triangle). ERB-131 behaves as a full agonist at ER ⁇ and a partial agonist at ERa.
  • ERB-131 displays properties of a classical nuclear receptor ligand and to expand upon the R-SAT ® data
  • a transcriptional reporter gene assay was performed (Table 8).
  • ERB-131 stimulated the transcriptional activity of ER ⁇ and to a lesser extent that of ERa (pEC50 7.7 ⁇ 0.5 vs 4.8 ⁇ 0.0, respectively).
  • ERB-131 demonstrated partial agonism at both ERa and ER ⁇ subtypes (% efficacy 58 ⁇ 0.0 vs 80 ⁇ 21, respectively).
  • a direct physical interaction between ERB-131 and the ER receptors was evident from competitive binding assays (Table 8).
  • ERB-131 displayed an affinity of 50 nM towards ER ⁇ (pKi 7.3 ⁇ 0.5) and only of 7900 nM towards ERa (PK 1 5.1 ⁇ 0.0). Thus, ERB-131 is more selective towards ER ⁇ (100-800 X) than the purportedly ER ⁇ selective phytoestrogens genistein and daidzein.
  • ER ⁇ and hyperalgesia and allodynia were investigated in a variety of animal models of hyperalgesia and allodynia, symptoms that are characteristic of neuropathic pain.
  • the allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape.
  • the allodynic agents typically cause a pain score of 14 (out of a maximum of 16) and the vehicle controls typically cause a pain score of 4- 5.
  • the results are shown in Fig. 17A, where ERB-131 (10 mg/kg i.p.) was injected 15 min prior to injection of the allodynia- inducing agents and the pain score of treated animals quantified.
  • S Sulprostone
  • PE Phenylephrine
  • N NMDA.
  • ERB-131 (10 mg/kg) was administered 15 min prior to the injection of the pain sensitizers.
  • ERB-131 reversed the tactile allodynia induced by sulprostone (4.2 ⁇ 0.6; p value ⁇ 0.0001), phenylephrine (3.5 ⁇ 0.2; p value ⁇ 0.0001 ) and NMDA (4.5 ⁇ 0.8; p value ⁇ 0.0001). ERB-131 treatment restored the pain thresholds of these animals to levels comparable to vehicle treated animals.
  • a dose response of ERB-131 (Fig.
  • ERB-131 almost completely reversed the tactile allodynia experienced by the surgically ligated animals (91.0 ⁇ 6.0 %).
  • the magnitude of the anti-allodynic effects mediated by ERB-131 was significantly stronger than the ones exhibited by the reference pain suppressors tested, with p values ranging from 0.003 to ⁇ 0.0001.
  • a low dose of ERB-131 (0.5 mg/kg) did not reverse the allodynia experienced by the surgically ligated animals.
  • the same low ERB-131 dosing markedly inhibited allodynia in a regimen of continuous drug effect. In that regimen (Fig.
  • ERB-131 produced a significant reversal of 76.0 ⁇ 6.0 % (p value ⁇ 0.0001).
  • Normal pain threshold As ERB-131 broadly modulates neuropathic pain behavior in various models of hyperalgesia and allodynia, we investigated whether ERB- 131 would affect the pain threshold of normal rats. Naive rats were injected with vehicle or ERB-131 (10 mg/kg s.c.) and their responses to a noxious stimulus (hot plate 52°C) followed over time. Their hot plate latency, expressed in seconds (sec), were defined as the time needed for the animal to physically remove the treated paw from the hot surface, was followed overtime. The results are shown in Figure 19.
  • the 10 mg/kg dose was chosen as it consistently alleviated neuropathic pain in the various animal models.
  • Vehicle treated animals exhibited a hot plate latency of 8.8 ⁇ 0.9 sec, which did not vary over the 2 hour course of the testing (Fig. 19).
  • administration of ERB-131 did not modify the pain threshold of normal animals.
  • ERB-131 A novel class of non-steroidal ER ⁇ agonists has been identified, of which ERB-131 represents a prototype lead.
  • ERB-131 constitutes a potent ER ⁇ agonist with a potency of 20-50 nM, and a high degree of selectivity versus other nuclear receptors, especially the related ERa (>100X).
  • Biochemical characterization indicated that ERB-131 displays classical properties of a nuclear receptor ligand, including the ability to directly modulate transcription through direct binding and activation of ER ⁇ . In vivo, ERB-131 lacks any significant ability to stimulate ERa activity, even at doses as high at 100 mg/kg.
  • ERB-131 at doses ranging from 0.5 to 10 mg/kg, efficiently reverses allodynia and hyperalgesia in several models of altered pain sensation consequent to chemical and surgical insults.
  • this work is the first demonstration of a role for selective ER ⁇ activation in the alleviation of neuropathic pain.
  • Non selective estrogens have been reported to exhibit pro- and antinociceptive properties.
  • Normal male rats treated acutely with estrogen exhibit dose-dependent mechanical hyperalgesia (Hucho et al. 2006).
  • estradiol administration to quails increases pain sensitivity to a noxious thermal stimulus (Evrard and Balthazart 2004).
  • ERB- 131 does not affect the pain threshold of normal animals.
  • treatment with ERB- 131 inhibits allodynia induced by several chemicals, namely sulprostone, phenylephrine and NMDA.
  • estrogen attenuates substance P induced-antinociception in the NMDA model (Nag and Mokha 2004; Claiborne et al. 2006).
  • treatment of ovariectomized rats with estradiol following sciatic nerve resection results in decreased autotomy (Tsao et al. 1999).
  • ERB- 131 reverses tactile allodynia resulting from spinal nerve ligation.
  • the lack of selectivity of estrogen ligands most likely explain these discrepancies. Indeed, the studies described herein highlight the fact that administration of ERB-131 significantly and consistently alleviated altered pain sensation in a variety of animal models associated with nerve injury and hyperexcitability.
  • ER ⁇ agonism protects from inflammatory pain in vivo, as we and others demonstrated (Gardell et al. 2007; Leventhal et al. 2006).
  • estrogens trigger neurochemical changes that modulate pain responses. For instance, estradiol influences opioid neurotransmission through the mu opioid receptor (Smith et al. 2006). By activating ER ⁇ -bearing inhibitory neurons, estrogen also modulates GABAergic neurons that innervate BDNF expressing neurons (Blurton- Jones and Tuszynski 2006). Whether activation of both peripheral and central sensory neurons is required for the antinociceptive properties of ER ⁇ still remains an open question.
  • ERB- 131 displays significant CNS penetration (FP unpublished).
  • ERB- 131 alleviated pain sensation triggered by the various chemicals that act either centrally or peripherally.
  • Both sulprostone and phenylephrine act through peripheral activation of sensory neurons of the dorsal root ganglia (Minami et al. 1994; Lee et al. 2000), while activation of the NMDA glutamate receptors in the spinal dorsal horn is essential for central sensitization (Riedel and Neeck 2001).
  • ERB-131 The selective activation of ER ⁇ by ERB-131 appears sufficient to provide beneficial effects in animal models of symptoms associated with neuropathic pain.
  • the robustness of ERB-131 effects across a variety of neuropathic pain models suggests a strong potential and clinical utility of ER ⁇ selective agonists in treating neuropathic pain disorders in humans. Additionally, the lack of ERa and thus of "classical" estrogenic actions could provide for optimal efficacy and enhanced safety profile.
  • Gardell LR Hyldtoft L, Del Tredici AL, Andersen CB, Fairbain LC, Ohrmund SR, Lund BW, Gustaffson M, Brann MR, Olsson R, Piu F. Protection against inflammatory pain by a selective estrogen receptor beta agonist. 2007 submitted.
  • Harkonen PL Vaananen HK. Monocyte-macrophage system as a target for estrogen and selective estrogen receptor modulators. Ann N Y Acad Sci 2006;1089:218-227.
  • Harris HA The unexpected science of estrogen receptor-beta selective agonists: a new class of anti-inflammatory agents? Nucl Recept Signal 2006;4:e012.
  • Hucho TB Dina OA, Kuhn J, Levine JD. Estrogen controls PKCepsilon-dependent mechanical hyperalgesia through direct action on nociceptive neurons. Eur J Neurosci 2006;24(2):527-534.
  • Kim SH Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50(3):355-363.
  • Piu F Magnani M, Ader ME. Dissection of the cytoplasmic domains of cytokine receptors involved in STAT and Ras dependent proliferation. Oncogene 2002;21(22):3579-3591.
  • ER-X a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 2002;22(19):8391-8401.
  • Tremblay GB Tremblay A
  • Copeland NG Gilbert DJ
  • Jenkins NA Labrie F
  • Giguere V Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. MoI Endocrinol 1997;11(3):353-365.
  • Watkins LR Maier SF. Immune regulation of central nervous system functions: from sickness responses to pathological pain. J Intern Med 2005;257(2): 139- 155.

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Abstract

Disclosed herein are methods of treating neuropathic pain, reducing inflammation, reducing IL-4 levels, and reducing IFN-γ levels, using various di-aromatic compounds for use as estrogen receptors β agonists.

Description

COMPOUNDS WITH ACTIVITY AT ESTROGEN RECEPTORS
RELATED APPLICATIONS
[001] This application claims priority to to U.S. Provisional Application Serial No. 60/825,682 filed September 14, 2006, entitled "COMPOUNDS WITH ACTIVITY AT ESTROGEN RECEPTORS", and U.S. Provisional Application Serial No. 60/948,181 filed July 5, 2007, entitled "COMPOUNDS WITH ACTIVITY AT ESTROGEN RECEPTORS", both of which are incorporated by reference herein in their entirety, including any drawings.
FIELD OF THE INVENTION
[002] This invention relates to the fields of organic chemistry, pharmaceutical chemistry, biochemistry, molecular biology and medicine. In particular it relates to compounds that modulate the activity of the Estrogen receptors, and to the use of the compounds for the treatment and prevention of diseases and disorders related to the Estrogen beta receptor.
BACKGROUND
[003] Estrogen receptors (ER) belong to the family of nuclear hormone receptors. Two estrogen receptor subtypes have been identified: ER alpha (ERa, NR3A1) (Green, 1986, Nature 320: 134; Greene, 1986, Science 231: 1150) and ER beta (ERβ, NR3A2) (Kuiper, 1996, PNAS 93:5925). Both receptors bind to the endogenous natural ligand 17β estradiol with comparable high affinity and modulate the transcriptional activity of target genes through classical estrogen response elements (reviewed in Nilsson, 2005, Bas Clin Pharm Tox, 96: 15).
[004] The role of estrogens in neuropathic pain is quite controversial, most likely because estrogens lack selectivity towards the two reported receptor subtypes, ERa and ERβ. Indeed, both subtypes are expressed in sensory neurons of the dorsal root ganglia, the effector site of pain sensation (Papka et Storey- Workley, 2002, Neurosci Letters 319:71; Papka et al, 2001, Cell Tissue Res 304: 193). They are also found in interneurons of the spinal cord (Shughrue et al, 1997, J Comp Neeurol, 388:507).
[005] Estrogen can induce mechanical hyperalgesia, a hallmark of neuropathic pain through direct action on nociceptive neurons (Hucho et al, 206, Eur J Neurosci, 24:527). Similarly, treatment with estradiol increases pain responsiveness to a thermal stimulus, while acute blockade of estrogen synthesis in the spinal dorsal horn reduces that behavior, consistent with a pro-nociceptive role of non-selective estrogen agonism (Evrard & Balthazart, 2004, J Neurosci 24:7225). However, estrogens have also been described to display beneficial effects in neuropathic pain. Treatment of rats with estradiol starting prior to sciatic nerve resection results in decreased autotomy (self- mutilation), which could reflect a reduction in the magnitude of the injury state (Tsao et al, 1999, Pharmacology, 59: 142). In addition, phytoestrogens reduce neuropathic pain behavior (both allodynic and hyperalgesic) in rats undergoing partial nerve ligation injury (Shir et al, 2002, Anesth Analg, 94:421). The effect was variable and associated with medium, but not low or high plasma levels of phytoestrogens.
[006] The clinical use of estrogen replacement therapy unexpectedly failed to provide clear answers as well. For example, clinical trials examining the impact of soy diet (rich in isoflavones and phytoestrogens) on mastalgia reported opposing results (Ingram et al, 2002, Breast, 11: 170; McFaydyen et al, 2000, Breast, 9:271). A Phase I study of methoxyestradiol, a catabolite of the estrogen pathway, showed significant improvement in bone pain (Lakhani et al, 2003, Pharmacotherapy, 23: 165). In addition, a recent study of women inversely correlated their estrogen levels to pain sensation (Smith et al, 2006, J Neurosci, 26:5777), even though there is a much higher incidence ofchronic pain in women than in men. Finally, a cross-sectional strudy of over 10,000 women strongly associated factors increasing estrogen levels with a higher incidence of chronic lower back pain (Winjhoven et al, 2006, Spine, 31 : 1496).
SUMMARY OF THE DISCLOSURE
[007] Disclosed herein are methods of treating neuropathic pain, reducing inflammation, reducing IL-4 levels, and reducing IFN-γ levels, comprising administering an ERβ agonist to a subject in need thereof. In some embodiments, the ERβ agonist is a compound of formula I
Figure imgf000005_0001
(I) or a pharmaceutically acceptable salt or prodrug thereof, as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[008] FIGURE IA depicts agonist activity of ERB-002 at the estrogen receptor β as evaluated using the Receptor Selection and Amplification (R-SAT™) technology.
[009] FIGURE IB depicts agonist activity of ERB-002 at the estrogen receptor α as evaluated using the Receptor Selection and Amplification (R-SAT™) technology.
[0010] FIGURE 2 is a graph depicting rat paw hot plate latency illustrating the reversal of thermal hyperalgesia by ERB-002 in a CFA-induced arthritis model.
[0011] FIGURE 3 is a graph depicting rat paw thickness illustrating the reversal of edema/inflammation by ERB-002 in a CFA-induced arthritis model.
[0012] FIGURE 4 is a bar graph depicting uterine weight illustrating that ERB-002 does not display uterotrophic properties in vivo in immature female rats, i.e., lack of ER-α activity in vivo.
[0013] FIGURE 5A is a bar graph showing that IFN-γ tear levels are decreased in the presence of ERB-002.
[0014] FIGURE 5B is a bar graphs showing that IL-4 tear levels are increased in the presence of ERB-002 in a model of TH1 inflammation.
[0015] FIGURE 6A is a bar graph showing the clinical scoring for lid edema in the multi-hit antigen challenge mouse model.
[0016] FIGURE 6B is a bar graph showing the clinical scoring for hyperemia in the multi-hit antigen challenge mouse model. [0017] FIGURE 6C is a bar graph showing the clinical scoring for chemosis in the multi-hit antigen challenge mouse model.
[0018] FIGURE 6D is a bar graph showing the clinical scoring for tearing in the multi-hit antigen challenge mouse model.
[0019] FIGURE 7A is a bar graphs showing that IL-4 (SRW sensitized mice) tear levels are decreased in the presence of ERB-002 in a TH2 inflammation model.
[0020] FIGURE 7B is a bar graphs showing there was a decrease in IL- 12 (SRW sensitized mice) tear levels in the presence of ERB-002.
[0021] FIGURE 8A is a graph showing the effect of ERB-002 (referred to as ERB- 131) on weight loss of mice with dextran sulphate/indomethacin induced colitis.
[0022] FIGURE 8B is a graph showing the effect of ERB-002 (referred to as ERB-131) on diarrhea of mice with dextran sulfate/indomethacin induced colitis.
[0023] FIGURE 9A is a graph showing the effect of ERB-002 (referred to as ERB-131) on response to thermal hyperalgesia as measured by using a hot plate test.
[0024] FIGURE 9B is a graph showing the effect of ERB-002 (referred to as ERB-131) on inflammation as assessed by the formation of local edema from the treated paw.
[0025] FIGURE 1OA is a graph showing the effect of gabapentin on response to thermal hyperalgesia as measured by using a hot plate test.
[0026] FIGURE 1OB is a graph showing the effect of gabapentin on inflammation as assessed by the formation of local edema from the treated paw.
[0027] FIGURE 11 is a graph showing ERB-002 (referred to as ERB-131) does not alleviate formalin induced inflammatory pain.
[0028] FIGURE 12 are graphs showing ERB-002 (referred to as ERB-131) does does not affect Phase I or Phase II in the formalin test. FIGURE 12A shows the data from Phase 1 and FIGURE 12B shows the data from Phase II.
[0029] FIGURE 13 A is a graph showing the effect of ERB-002 (referred to as ERB-131) on hyperalgesia in the CFA model.
[0030] FIGURE 13B is a graph showing the effect of ERB-002 (referred to as ERB-131) on inflammation in the CFA model.
[0031] FIGURE 14 shows that ERB-002 (referred to as ERB-131) is a potent selective ERβ agonist. FIGURE 14A is a graph comparing the activity of ERB-002 with that of estrone at estrogen receptors ERa and ERβ. FIGURE 14B is a graph depicting the selectivity of ERb- 131 using R-SAT®.
[0032] FIGURE 15 shows the effect of ERB-002 (referred to as ERB-131) on the uterus size of sexually immature female rats at different doses: naϊve (Fig. 15A), vehicle (Fig. 15B), PPT (Fig. 15C), 10 mg/kg (Fig. 15D), 30 mg/kg (Fig. 15E), 100 mg/kg (Fig. 15F). Quantitative values are shown graphically in Fig. 15G.
[0033] FIGURE 16 is a graph showing that ERB-002 (referred to as ERB- 131) alleviates capsaicin-induced acute hyperalgesia.
[0034] FIGURE 17 shows that ERB-002 (referred to as ERB-131) broadly inhibits chemically-induced allodynia, when ERB-002 was injected 15 min prior to injection of the allodynia-inducing agents (Fig. 17A); effects of different doses of ERB- 002 were evaluated (Fig. 17B); and the estrogen receptor pan-antagonist ICI- 182740 was used to confirm that the effects of ERB-002 (Fig. 17C).
[0035] FIGURE 18A is a graph showing the effects of an acute dose of ERB- 002 (referred to as ERB-131) on inhibition of allodynia 30 min following injection.
[0036] FIGURE 18B is a graph showing the effects on inhibition of allodynia of different regimens using low doses of ERB-002.
[0037] FIGURE 19 is a graph showing hot plate latency of rats after injection with ERB-002 (referred to as ERB-131).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0038] In various embodiments, compounds having the formula (I) and methods for using these compounds for treating disorders related to estrogen receptors are provided:
Figure imgf000007_0001
(I)
In some embodiments, pharmaceutically acceptable salt or prodrugs of the compound of formula I are provided. In the compound of formula I: n is an integer selected from the group consisting of 3, 4, 5 and 6;
Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -CC=Z)R6, -CC=Z)OR6, -C(=Z)N(R6)2, -
Figure imgf000008_0001
-PC=O)(ORe)2, and -CH2OC(=O)R5;
R2, R2a, R2b, R2c are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR6, -NR6R63, -NRsNRsaRsb,
Figure imgf000008_0002
-CC=Z)OR6, -CC=Z)NR6R63, -NCRe)-CC=Z)R60, -N(R6)-C(=Z)NR6bR6a, -OCC=Z)R6,
Figure imgf000008_0003
-SR6; each R3 is separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, =0, and -OR6, or are separately absent to accommodate a double bond; two R3 groups are optionally bound together to form a substituted or unsubstituted C3-C9 cycloalkyl or C3-C9 heteroalicyclyl;
R2a is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
R4J, is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
R4, R43, R4b, Ric are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, nitro, halogen, sulfonyl, perhaloalkyl, -OR6, -NR6R63, -NR6NR63R6I,, -NR6N=CR63R*, -N(R6)C(R6a)=NR6b, -CN, -C(=Z)Rβ, -C(=Z)0R6,
Figure imgf000009_0001
-OCC=Z)R6, -N(Re)-SC=O)2R63, and -SR6;
R4J, and R4b are optionally bound together to form an aryl, heteroaryl, or heteroalicyclyl;
R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, halogen, -CN, -SR6, sulfonyl, -C(=O)NR6R6a, -CC=O)R6, -NR6R63, -COOR6, and perhaloalkyl;
Z is oxygen or sulfur; and
R6, R6a and R^ are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl.
[0039] In some embodiments, compounds are provided according to formula I but excluding the compounds selected from the group consisting of:
Figure imgf000009_0002
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000011_0001
Figure imgf000011_0002
Figure imgf000011_0003
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000012_0003
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000013_0003
Figure imgf000013_0004
Figure imgf000013_0005
Figure imgf000014_0001
[0040] In some embodiments of the compound of formula I: n is an integer selected from the group consisting of 3, 4, and 5;
Ri is selected from the group consisting of hydrogen, Ci-C4 straight chained or branched alkyl, Ci-C4 straight chained or branched alkenyl, Ci-C4 straight chained or branched perhaloalkyl, and substituted or unsubstituted aryl;
R2, R2a, R2b, R2c are separately selected from the group consisting of hydrogen, C1-Cs straight chained or branched alkyl, C1-Cs alkenyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR6, -C(O)R6, -C(O)OR6, -C(O)NR6R60, -N(Re)-C(O)R68, -N(Re)-S(O)2R68, -OC(O)R6, and -SR6; each R3 is separately selected from the group consisting of hydrogen, C1- C5 straight chained or branched alkyl, C1-Cs alkenyl, cycloalkyl, cycloalkenyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, =0, and -OR6, or each R3 is separately absent to accommodate a double bond;
R4, R43, Rib, R4C are separately selected from the group consisting hydrogen, C1-C5 straight chained or branched alkyl, C1-C5 alkenyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -OR6, -CN, -C(O)R6, -C(O)OR6,
Figure imgf000015_0001
Figure imgf000015_0002
and -SR6; and
R5 is selected from the group consisting of hydrogen, C1-C5 straight chained or branched alkyl, halogen, -CN, -SR6, sulfonyl, -OCF3, and perhaloalkyl. [0041] In other embodiments of the compound of formula I: n is 3;
Ri is selected from the group consisting of hydrogen, C1-C5 straight chained or branched alkyl, substituted or unsubstituted aryl;
R2, R2a, R2b, R2c are separately selected from the group consisting of hydrogen, C1-C5 straight chained or branched alkyl, F, Cl, Br, perhaloalkyl, -CN, -OR6, -C(O), and -SR6; each R3 is separately selected from the group consisting of hydrogen, C1- C5 straight chained or branched alkyl, C1-C5 alkenyl, cycloalkyl, halogen, perhaloalkyl, -CN, and -OR6, or each R3 is separately absent to accommodate a double bond; each R4, R43, Rn,, R40 is separately selected from the group consisting hydrogen, C1-C5 straight chained or branched alkyl, halogen, sulfonyl, perhaloalkyl, -OR6, -CN, ,
Figure imgf000015_0003
and -SR6; and Rs is selected from the group consisting of hydrogen, C1-Cs straight chained or branched alkyl, F, Cl, -CN, -SR6, -OCF3, and CF3. [0042] In some embodiments, the compound of formula I is selected from the group consisting of:
Figure imgf000016_0001
Figure imgf000016_0002
Figure imgf000016_0003
ERB-012 ERB-013 ERB-014 ERB-015
Figure imgf000016_0004
ERB-016 ERB-017
Figure imgf000017_0001
ERB-026 ERB-027 ERB-029
Figure imgf000017_0002
ERB-030 ERB-031 ERB-032 ERB-033
CF3
Figure imgf000017_0003
ERB-034 ERB-035 ERB-036
Figure imgf000017_0004
ERB-037 ERB-038 ERB-039 ERB-040
Figure imgf000018_0001
ERB-041 ERB-043 ERB-044
Figure imgf000018_0002
ERB-045
Figure imgf000018_0003
[0043] In various other embodiments, compounds having the formula (II) and methods for using these compounds for treating disorders related to estrogen receptors are provided:
Figure imgf000019_0001
(H)
[0044] In some embodiments, pharmaceutically acceptable salts or prodrugs of the compound of formula II are provided. In the compound of formula II: n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6;
Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cykloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -C(=Z)R5, -C(=Z)OR5, -CC=Z)N(Rs)2, -S(=O)2NR5aR5b, -P(=O)(OR5)2, and - CH2OC(=O)R5;
R2, R2a, R2b, R2c, and each R6 are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR5, -NR5R5a, -NR5NR5aR5b, -NR5N=CR5aR5b, -N(R5)C(R5a)=NR5b, -C(=Z)R5, -C(=Z)OR5, -C(=Z)NR5R5a, -N(R5)-C(=Z)R5a, -N(R5)-C(=Z)NR5bR5a, -OC(=Z)R5, -N(R5)-S(=O)2R5a, and -SR5; each Y is separately selected from the group consisting of methylene, methylene substituted with one or two R6 groups, sulphur, oxygen, unsubstituted nitrogen, nitrogen substituted with R5, and C=O; two Y groups are optionally bound together to form a single bond or a substituted or unsubstituted C1-C9 cycloalkyl or Ci- C9 heteroalicyclyl;
R2a is optionally bound to one Y group to form a substituted or unsubstituted C4- C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond; A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, substituted heteroalicyclyl, unsubstituted heteroalicyclyl, unsubstitued aryl, and substituted aryl;
A is optionally bound to one Y group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
Z is oxygen or sulfur; and each R5, Rsa and Rsb are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl; provided that when every Y is a substituted or unsubstituted methylene, then A is not a substituted or unsubstituted aryl.
[0045] In some embodiments, when A is a substituted aryl, it is not substituted at the para position.
[0046] In one embodiment of the compound of formula II, n is an integer selected from the group consisting of 3, 4, and 5; Ri is selected from the group consisting of hydrogen, C1-C4 straight chained or branched alkyl, C1-C4 straight chained or branched alkenyl, C1-C4 straight chained or branched perhaloalkyl, and substituted or unsubstituted aryl; R2, R23, R2b, R2c are separately selected from the group consisting of hydrogen, C1-Cs straight chained or branched alkyl, C1-Cs alkenyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR5, -C(O)R5, -C(O)OR5, -C(=O)NR5R5a, -N(R5)-C(O)R5a,
Figure imgf000020_0001
-0C(O)R5, and -SR5; each Y is separately selected from the group consisting of substituted or unsubstituted methylene, sulphur, oxygen, substituted or unsubstituted nitrogen or C=O; and A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, unsubstitued aryl, and substituted aryl that is unsubstituted at the para position.
[0047] In another embodiment of the compound of formula II, n is 3; Ri is selected from the group consisting of hydrogen, C1-Cs straight chained or branched alkyl, substituted or unsubstituted aryl; R2; R2a> R2b, R2c are separately selected from the group consisting of hydrogen, C1-Cs straight chained or branched alkyl, F, Cl, Br, perhaloalkyl, -CN, -OR5, -C(O), and -SR5; each Y is separately selected from the group consisting of substituted or unsubstituted methylene, oxygen, substituted or unsubstituted nitrogen, or C=O; and A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, unsubstitued aryl, and substituted aryl that is unsubstituted at the para position.
[0048] In various embodiments, the compound of formula II is selected from the group consisting of:
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000021_0003
Figure imgf000021_0004
or a pharmaceutically acceptable salt or prodrug thereof. Definitions
[0049] Unless otherwise specified, "R" group(s) such as, without limitation, R, Ra and Rb, is(are) independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl (bonded to the indicated group at a ring carbon atom) and heteroalicyclyl (likewise bonded to the indicated group at a ring carbon atom), as these groups are defined herein. If two "R" groups are covalently bonded to the same atom then they may be bound together so as to form a cycloalkyl or heteroalicyclyl group.
[0050] Unless otherwise indicated, when a substituent is deemed to be "optionally substituted," it is meant that the substituent is a group that may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. The protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, Protective Groups in Organic Synthesis, 3r Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein in its entirety.
[0051] As used herein, "Cm to Cn" in which "m" and "n" are integers refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group or the number of carbon atoms in the ring of a cycloalkyl or cycloalkenyl group. That is, the alkyl, alkenyl, alkynyl, ring of the cycloalkyl or ring of the cycloalkenyl can contain from "m" to "n", inclusive, carbon atoms. Thus, for example, a "Ci to C4 alkyl" group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3-, CH3CH2-, CH3CH2CH2-, CH3CH(CH3)-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3CH-. If no "m" and "n" are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl group, the broadest range described in these definitions is to be assumed.
[0052] As used herein, "aryl" refers to a carbocyclic (all carbon) ring or two or more fused rings (rings that share two adjacent carbon atoms) that have a fully delocalized pi-electron system. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene.
[0053] As used herein, "heteroaryl" refers to a ring or two or more fused rings that contain(s) one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur in the ring and that have a fully delocalized pi-electron system. Examples of heteroaryl rings include, but are not limited to, furan, thiophene, phthalazinone, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, triazole, thiadiazole, pyran, pyridine, pyridazine, pyrimidine, pyrazine and triazine.
[0054] As used herein, "alkyl" refers to a straight or branched chain fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group of this invention may comprise from 1 to 20 carbon atoms, that is, m = 1 and n = 20. An alkyl group herein may also be of medium size having 1 to 10 carbon atoms. An alkyl group herein may also be a lower alkyl having 1 to 5 carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec- butyl, tert-butyl, amyl, tert-amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
[0055] An alkyl group of this invention may be substituted or unsubstituted. When substituted, the substituent group(s) is(are) one or more group(s) independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S- sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NRaR and protected amino.
[0056] As used herein, "alkenyl" refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds. An alkenyl group of this invention may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution.
[0057] As used herein, "alkynyl" refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds. An alkynyl group of this invention may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution. [0058] As used herein, "acyl" refers to an "RC(=0)-" group with R as defined above.
[0059] As used herein, "cycloalkyl" refers to a completely saturated (no double bonds) hydrocarbon ring. Cycloalkyl groups of this invention may range from C3 to Cs. A cycloalkyl group may be unsubstituted or substituted. If substituted, the substituent(s) may be selected from those indicated above with regard to substitution of an alkyl group.
[0060] As used herein, "cycloalkenyl" refers to a cycloalkyl group that contains one or more double bonds in the ring although, if there is more than one, they cannot form a fully delocalized pi-electron system in the ring (otherwise the group would be "aryl," as defined herein). A cycloalkenyl group of this invention may unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution.
[0061] The term "alkylene" refers to an alkyl group, as defined here, which is a biradical and is connected to two other moieties. Thus, methylene (-CH2-), ethylene (- CH2CH2-), proylene (-CH2CH2CH2-), isopropylene (-CH2-CH(CH3)-), and isobutylene (- CH2-CH(CHs)-CH2-) are examples, without limitation, of an alkylene group. Similar, the term"cycloalkylene" refers to an cycloalkyl group, as defined here, which binds in an analogues way to two other moieties. If the alkyl and cycloalkyl groups contains unsaturated carbons, the terms "alkenylene" and "cycloalkenylene" are used.
[0062] As used herein, "heteroalicyclic" or heteroalicyclyl" refers to a ring or one or more fused rings having in the ring system one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. The rings may also contain one or more double bonds provided that they do not form a fully delocalized pi-electron system in the rings. Heteroalicyclyl groups of this invention may be unsubstituted or substituted. When substituted, the substituent(s) may be one or more groups independently selected from the group consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, alkyl, alkoxy, acyl, acyloxy, carboxy, protected carboxy, amino, protected amino, carboxamide, protected carboxamide, alkylsulfonamido and trifluoromethanesulfonamido.
[0063] An "O-carboxy" group refers to a "RC(=O)O-" group with R as defined above.
[0064] A "C-carboxy" group refers to a "-C(=O)R" group with R as defined above. [0065] An "acetyl" group refers to a CH3C(=O)- group.
[0066] A "trihalomethanesulfonyl" group refers to an "X3CSO2-" group wherein X is a halogen.
[0067] A "cyano" group refers to a "-CN" group.
[0068] An "isocyanato" group refers to an "-NCO" group.
[0069] A "thiocyanato" group refers to a "-CNS" group.
[0070] An "isothiocyanato" group refers to an " -NCS" group.
[0071] A "sulfinyl" group refers to an "-S(=O)-R" group with R as defined above.
[0072] A "sulfonyl" group refers to an "SO2R" group with R as defined above.
[0073] An "S-sulfonamido" group refers to a "-SO2NRaRb" group with Ra and R as defined above.
[0074] An "N-sulfonamido" group refers to a "RSO2N(R3)-" group with R and Ra as defined above.
[0075] A "trihalomethanesulfonamido" group refers to an "X3CSO2N(R)-" group with X as halogen and R as defined above.
[0076] An "O-carbamyl" group refers to a "-OC(=O)NRaRb" group with Ra and R as defined above.
[0077] An "N-carbamyl" group refers to an "ROC(=O)NRa-" group with Ra and R as defined above.
[0078] An "O-thiocarbamyl" group refers to a "-OC(=S)-NRaRb" group with Ra and Rb as defined above.
[0079] An "N-thiocarbamyl" group refers to an "ROC(=S)NRa-" group with Ra and R as defined above.
[0080] A "C-amido" group refers to a "-C(=O)NRaRb" group with Ra and Rb as defined above.
[0081] An "N-amido" group refers to a "RC(=O)NRa-" group with R and Ra as defined above.
[0082] The term "perhaloalkyl" refers to an alkyl group in which all the hydrogen atoms are replaced by halogen atoms.
[0083] As used herein, an "ester" refers to a "-C(=O)OR" group with R as defined above. [0084] As used herein, an "amide" refers to a "-C(=0)NRaRb" group with Ra and R as defined above.
[0085] Any unsubstituted or monosubstituted amine group on a compound herein can be converted to an amide, any hydroxyl group can be converted to an ester and any carboxyl group can be converted to either an amide or ester using techniques well- known to those skilled in the art (see, for example, Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999).
[0086] When two substituents are referred to herein as optionally binding together, it is meant that the groups may be joined to form a cycloalkyl, aryl, heteroaryl, or heteroalicyclyl group. For example, without limitation, if Ra and R of an NRaR group are indicated to be optionally bound together, it is meant that they are covalently bonded to one another at their terminal atoms to form a ring:
— < \
[0087] It is understood that, in any compound of this invention having one or more chiral centers, if an absolute stereochemistry is not expressely indicated, then each center may independently be R or S or a mixture thereof. In addition it is understood that, in any compound of this invention having one or more double bond(s) generating geometrical isomers that can be defined as E or Z each double bond may independently be E or Z a mixture thereof.
[0088] As used herein, "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to a patient to which it is administered and does not abrogate the biological activity and properties of the compound. Pharmaceutical salts can be obtained by reaction of a compound disclosed herein with an acid or base. Base-formed salts include, without limitation, ammonium salt (NH4 +); alkali metal, such as, without limitation, sodium or potassium, salts; alkaline earth, such as, without limitation, calcium or magnesium, salts; salts of organic bases such as, without limitation, dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine; and salts with the amino group of amino acids such as, without limitation, arginine and lysine. Useful acid-based salts include, without limitation, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, methanesulfonates, ethanesulfonates, p-toluenesulfonates and salicylates. [0089] A "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may decrease the rate of metabolic degradation for instance by decreasing O-glucuronidation and or O-sulfation. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound disclosed herein, which is administered as an ester (the "prodrug") to facilitate absorption over a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety. Synthesis
[0090] General synthetic routes to the compounds of this invention are shown in Schemes 1-5. The routes shown are illustrative only and are not intended, nor are they to be construed, to limit the scope of this invention in any manner whatsoever. Those skilled in the art will be able to recognize modifications of the disclosed synthesis and to devise alternate routes based on the disclosures herein; all such modifications and alternate routes are within the scope of this invention.
[0091] Synthetic routes for synthesizing the compounds of formula I include the following Schemes 1-6: SCHEME 1
Figure imgf000027_0001
Figure imgf000027_0002
SCHEME 2
Figure imgf000028_0001
Figure imgf000028_0002
SCHEME 3
Figure imgf000028_0003
SCHEME 4
Figure imgf000029_0001
SCHEME 6
Figure imgf000029_0002
Figure imgf000030_0001
+ acylating agent
Figure imgf000030_0002
Figure imgf000030_0004
Figure imgf000030_0003
Figure imgf000030_0005
[0092] In the above schemes, it is to be understood that the moiety:
Figure imgf000030_0006
ical to the moiety:
Figure imgf000030_0007
ribed above with respect to Formula I. [0093] Synthetic rounts for synthesizing the compounds of formula II include the following Schemes 7-17: SCHEME 7
Y ΛV n 1 ) LDA, THF, -78 0C n
J 2) PhNTf2, -78 °C to r.t.
Figure imgf000031_0001
SCHEME 8
R4-B(OH)2 +
Figure imgf000031_0002
Figure imgf000031_0003
SCHEME 9
Figure imgf000031_0004
SCHEME 10
Figure imgf000031_0005
SCHEME 11
Figure imgf000032_0001
nBuϋ, THF,
Figure imgf000032_0002
Figure imgf000032_0003
Figure imgf000032_0004
Figure imgf000032_0005
DMF, NaH
Figure imgf000032_0006
SCHEME 13 Lipase
Figure imgf000033_0001
SCHEME 14
Figure imgf000033_0002
Figure imgf000033_0003
SCHEME 15
C
Figure imgf000034_0001
Figure imgf000034_0002
SCHEME 16
Figure imgf000035_0001
-3
Figure imgf000035_0002
Figure imgf000035_0003
SCHEME 17
Figure imgf000036_0001
[0094] Also disclosed herein are methods of treating clinical manifestations in which modulation of the activity of the estrogen receptor function is beneficial; a method of treating or preventing inflammatory bowel syndrome, Crohn's disease, ulcerative proctitis or colitis; a method of treating or preventing prostatic hypertrophy, uterine leiomyomnas, breast carcinoma, endometrial carcinoma, polycystic ovary syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian carcinoma, melanoma, prostate carcinoma, colon carcinoma, or brain tumors including glioblastoma, astrocytoma, glioma, or meningioma; a method of treating or preventing prostatitis or interstitial cystitisl; a method of hormonal replacement therapy; a method of treating or preventing bone density loss including osteoporosis and osteopenia; a method of lowering cholesterol, triglycerides, or LDL levels; a method of treating or preventing discholesterolemia or dislipidemia; a method of treating or preventing cardiovascular disease, atherosclerosis, hypertension, peripheral vascular disease, restenosis or vasospasm; a method of treating impaired cognition or providing neuroprotection; a method of treating or preventing neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, Parkinson's disease or other dementias; a method of treating a spinal cord injury; a method of treating or preventing cognitive decline, stroke, or anxiety; a method of treating or preventing free radical induced disease states; a method of treating or preventing vaginal atrophy, vulvar atrophy, atrophic vaginitis, vaginal dryness, pruritus, dyspareunia, frequent urination, urinary incontinence, or urinary tract infections; a method of treating or preventing vasomotor symptoms including flushing or hot flashes; a method of preventing conception; a method of treating or preventing endometriosis; a method of treating or preventing arthritis, including but not limited to rheumatoid arthritis, osteoarthritis, or arthropathies; a method of treating or preventing psoriasis or dermatitis, a method of treating or preventing asthma or pleurisy; a method of treating or preventing multiple sclerosis, systemic lupus erthematosis, uveitis, sepsis, or hemmorhagic shock; a method of treating or preventing type II diabetes; a method for treating acute and chronic inflammation of any type; a method of treating or preventing lung disorders such as asthma, chronic obstructive pulmonary disease; a method of treating or preventing acute or chronic pain, including neuropathic pain; a method of treating or preventing ophthalmologic disorders including but not limited to glaucoma, dry eye, macular degeneration, and a method of modulating or specifically agonizing one or more Estrogen receptors where the methods comprise identifying a subject in need of treatment or prevention and administering to the subject a pharmaceutically effective amount of a compound of formula I or II.
[0095] Another embodiment is a method of identifying a compound that alleviates inflammation in a subject, comprising identifying a subject suffering from inflammation; providing the subject with at least one compound of Formula I or II, as defined herein; and determining if the at least one compound reduces inflammation in the subject.
[0096] Also disclosed herein is a method of reducing inflammation in a subject comprising identifying a subject in need of the reduction in inflammation; and administering to the subject a pharmaceutically effective amount of an ERβ agonist. [0097] In some embodiments, the inflammation to be treated is in the eye. In some of these embodiments, the inflammation results in lid edema, hyperemia, chemosis, or tearing. In other embodiments, inflammation is due to an ophthalmologic disorder selected from the group consisting of uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration, or is an ocular manifestation of a systemic inflammatory disease such as Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis.
[0098] In some embodiments, the inflammation is in the gastrointestinal tract. In some of these embodiments, the inflammation is colitis, or is caused by colitis.
[0099] In other embodiments, the inflammation is due to arthritis.
[00100] In some embodiments, the inflammation is acute, whereas in other embodiments, the inflammation is chronic.
[00101] Also disclosed herein is a method of treating allergic conjunctivitis in a subject comprising identifying a subject in need of such treatment; and administering to the subject a pharmaceutically effective amount of an ERβ agonist.
[00102] Also disclosed herein is a method of reducing IL-4 levels in a subject, comprising identifying a subject in need of reduction in IL-4 levels; and administering to the subject a pharmaceutically effective amount of an ERβ agonist.
[00103] Also disclosed herein is a method of reducing IFN-γ levels in a subject, comprising identifying a subject in need of reduction in IFN-γ levels; and administering to the subject a pharmaceutically effective amount of an ERβ agonist.
[00104] Also disclosed herein is a method of treating neuropathic pain in a subject, comprising identifying a subject in need of the treatment of neuropathic pain; and administering to the subject a pharmaceutically effective amount of an ERβ agonist.
[00105] Neuropathic pain is caused by abnormalities in the nerves, spinal cord, or brain and includes, without limitation, phantom limb pain, postherpetic neuralgia, reflex sympathetic dystrophy, causalgia, complex regional pain syndrome II, painful HIV-associated neuropathy, diabetic neuropathy. Neuropathic pain is also associated with many medical conditions including, without limitation, traumatic nerve injury, multiple sclerosis, stroke, syringomyelia, epilepsy, spinal cord injury, and cancer. The methods disclosed herein are useful in the treatment of all of the aforementioned manifestations of neuropathic pain. [00106] In some embodiments, the neuropathic pain is mechanical hyperalgesia. In other embodiments, the neuropathic pain is allydonia.
[00107] Also disclosed herein is a method of increasing IL- 12 levels in a subject, comprising identifying a subject in need of increase in IL-12 levels; and administering to the subject a pharmaceutically effective amount of an ERβ agonist.
[00108] In some embodiments, the ERβ agonist is a compound of formula I, as described herein. In some of these embodiments, the compound of formula I is selected from the group consisting of
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000041_0001
Figure imgf000041_0002
Figure imgf000041_0003
Figure imgf000041_0004
Figure imgf000042_0001
Figure imgf000042_0002
Figure imgf000042_0003
Figure imgf000043_0001
Figure imgf000043_0003
Figure imgf000043_0002
Figure imgf000044_0001
[00109] In another embodiment, the ERβ agonist is 4-(l- phenylcyclohexyl)phenol.
[00110] In other embodiments, the ERβ agonist is a compound of formula II, as described herein.
[00111] In some embodiments, the subject to be treated is a human.
[00112] In some embodiments, the ERβ agonist is administered topically, whereas in other embodiments, the ERβ agonist is administered intraperitoneally. In other embodiments, the ERβ agonist is administered orally.
[00113] The term "subject" refers to an animal, preferably a mammal, and most preferably a human, who is the object of treatment, observation or experiment. The mammal may be selected from the group consisting of mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, primates, such as monkeys, chimpanzees, and apes, and humans.
[00114] The term "therapeutically effective amount" is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. This response may occur in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and includes alleviation of the symptoms of the disease being treated.
[00115] Another embodiment is a method of identifying a compound which regulates activity of an Estrogen receptor by culturing cells that express the Estrogen receptors; incubating the cells with at least one compound of Formula I or II as defined herein; and determining any change in activity of the Estrogen receptors so as to identify a compound of Formula I or II which regulates activity of a Estrogen receptors.
[00116] In other embodiments, methods are provided for alleviating diseases by administering one or more compounds of Formula I or II. These methods include, but are not limited to methods such as: a method of treating clinical manifestations in which estrogen receptor function is altered; a method of treating or preventing inflammatory bowel syndrome, Crohn's disease, ulcerative proctitis or colitis; a method of treating or preventing prostatic hypertrophy, uterine leiomyomnas, breast carcinoma, endometrial carcinoma, polycystic ovary syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian carcinoma, melanoma, prostate carcinoma, colon carcinoma, brain tumors including but not limited to glioblastoma, astrocytoma, glioma, and meningioma; a method of treating or preventing prostatitis or interstitial cystitis; a method of hormonal replacement therapy; a method of treating or preventing bone density loss including but not limited to osteoporosis or osteopenia; a method of lowering cholesterol, triglycerides, or LDL levels; a method of treating or preventing discholesterolemia, or dislipidemia; a method of treating or preventing cardiovascular disease, atherosclerosis, hypertension, peripheral vascular disease, restenosis or vasospasm; a method of treating impaired cognition or providing neuroprotection; a method of treating neurodegenerative disorders, including but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease or other dementias; a method of treating a spinal cord injury; a method of treating or preventing cognitive decline, stroke, or anxiety; a method of treating or preventing free radical induced disease states; a method of treating or preventing vaginal atrophy, vulvar atrophy, atrophic vaginitis, vaginal dryness, pruritus, dyspareunia, frequent urination, urinary incontinence, or urinary tract infections; a method of treating or preventing vasomotor symptoms including but not limited to flushing or hot flashes; a method of preventing conception; a method of treating or preventing endometriosis; a method of treating or preventing arthritis, including but not limited to rheumatoid arthritis, osteoarthritis, arthropathies; a method of treating or preventing psoriasis or dermatitis; a method of treating or preventing asthma, or pleurisy; a method of treating or preventing multiple sclerosis, systemic lupus erthematosis, uveitis, sepsis, or hemmorhagic shock; a method of treating or preventing type II diabetes; a method for treating acute and chronic inflammation of any type; a method of treating or preventing lung disorders such as asthma or chronic obstructive pulmonary disease; a method of treating or preventing acute or chronic pain, including neuropathic pain; and a method of treating or preventing ophthalmologic disorders including but not limited to uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration, or is an ocular manifestation of a systemic inflammatory disease such as Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis. In other embodiments, methods of modulating, or specifically agonizing, the Estrogen receptors by administering an effective amount of a compound of Formula I or II are provided.
[00117] Another embodiment is a pharmaceutical composition comprising a compound of Formula I or II as described above, and a physiologically acceptable carrier, diluent, or excipient, or a combination thereof.
[00118] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
[00119] The term "carrier" defines a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.
[00120] The term "diluent" defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.
[00121] The term "physiologically acceptable" defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.
[00122] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990, which is hereby incorporated by reference in its entirety.
[00123] Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.
[00124] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into the area of pain or inflammation, often in a depot or sustained release formulation. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.
[00125] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. [00126] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., as disclosed in Remington's Pharmaceutical Sciences, cited above.
[00127] For injection, the agents disclosed herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[00128] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds disclosed herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with pharmaceutical combination disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[00129] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
[00130] Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
[00131] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[00132] For administration by inhalation, the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[00133] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[00134] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions.
[00135] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[00136] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
[00137] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[00138] A pharmaceutical carrier for the hydrophobic compounds disclosed herein is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase. A common co-solvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; and other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
[00139] Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acids or base forms.
[00140] Pharmaceutical compositions suitable for use in the methods disclosed herein include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[00141] The exact formulation, route of administration and dosage for the pharmaceutical compositions disclosed herein can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, which is hereby incorporated by reference in its entirety). Typically, the dose range of the composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient's body weight, or 1 to 500 mg/kg, or 10 to 500 mg/kg, or 50 to 100 mg/kg of the patient's body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. Where no human dosage is established, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
[00142] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 mg and 500 mg of each ingredient, preferably between 1 mg and 250 mg, e.g. 5 to 200 mg or an intravenous, subcutaneous, or intramuscular dose of each ingredient between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of each ingredient of the pharmaceutical compositions disclosed herein or a pharmaceutically acceptable salt thereof calculated as the free base, the composition being administered 1 to 4 times per day. Alternatively the compositions disclosed herein may be administered by continuous intravenous infusion, preferably at a dose of each ingredient up to 400 mg per day. Thus, the total daily dosage by oral administration of each ingredient will typically be in the range 1 to 2000 mg and the total daily dosage by parenteral administration will typically be in the range 0.1 to 400 mg. In some embodiments, the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
[00143] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety, which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
[00144] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
[00145] In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
[00146] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
[00147] The compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
[00148] It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure.
EXAMPLES
[00149] Embodiments of the present invention are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the invention. Example 1 - General analytical LC-MS procedure
[00150] Procedure 1 (API): The analysis was performed on a combined prep/analytical Waters/Micromass system consisting of a ZMD single quadropole mass spectrometer equipped with electro-spray ionization interface. The HPLC system consisted of a Waters 600 gradient pump with on-line degassing, a 2700 sample manager and a 996 PDA detector.
[00151] Separation was performed on an X-Terra MS C 18, 5 μm 4.6x50mm column. Buffer A: 1OmM ammonium acetate in water, buffer B: 1OmM ammonium acetate in acetonitrile/water 95/5. A gradient was run from 30%B to 100%B in 10 min, dwelling at 100%B for 1 min, and re-equilibrating for 6 min. The system was operated at 1 ml/min.
[00152] Procedure 2 (AP2): The analysis was performed on a combined prep/analytical Waters/Micromass system consisting of a ZMD single quadropole mass spectrometer equipped with electro-spray ionization interface. The HPLC system consisted of a Waters 600 gradient pump with on-line degassing, a 2700 sample manager and a 996 PDA detector.
[00153] Separation was performed on an X-Terra MS C 18, 5 μm 4.6x50mm column. Buffer A: 1OmM ammonium acetate in water, buffer B: 1OmM ammonium acetate in acetonitrile/water 95/5. A gradient was run from 30%B to 100%B in 7 min, dwelling at 100%B for 1 min, and re-equilibrating for 5.5 min. The system was operated at 1 ml/min. Example 2 - General gas chromatography (GC) procedure
[00154] GC method 50 was used. Method 50 starts at 500C and has a gradient of 20 °C/min until 250 0C then holds the temperature for 5 minutes. The analysis was performed on an Aglient 6850 series GC system with capillary S/SL inlet and FID with EPC installation. The column was a 10 m x 0.32 mm x 0.25 μm HP5 column. Example 3 - Synthesis of trifluoromethanesulfonates. general procedure 1 (GPl)
[00155] Trifluoromethanesulfonates were prepared according to literature procedure by McMurry and Scott (McMurry, J. E.; Scott, W. J., Tetrahedron letters, 1983, 979-982). ^isoPropyl-cyclehexenyl-l-trifluoromethanesulfonate
[00156] The title compound was prepared according to GPl from A- φropylcyclohexanone (10.0 g, 71 mmol). Crude yield: 14.1 g. 1H-NMR (400 MHz, CDCl3) d 5.78-5.69 (m, IH), 2.42-2.14 (m, 3H), 1.98-1.84 (m, 2H), 1.60-1.2 (m, 4H), 0.94-0.88 (m, 6H). 1-Cyclohexenyl-l- trifluoromethanesulfonate
[00157] The title compound was prepared according to GPl from cyclohexanone (9.8 g, 100 mmol). Crude yield: 14.0 g (83% pure by 1H-NMR). 1H-NMR (400 MHz, CDCl3) d 5.79-5.73 (m, IH), 2.36-2.28 (m, 2H), 2.23-2.14 (m, 2H), 1.83-1.75 (m, 2H), 1.66-1.56 (m, 2H). ^Trifluoromethyl-cyclohexenyl-l-trifluoromethanesulfonate
[00158] The title compound was prepared according to GPl from A- (trifluoromethyl)cyclohexanone (3.0 g, 18 mmol). Crude yield: 1.9 g. GC-FID Rt: 1.16 min (Method50) l-Cycloheptenyl-l-trifluoromethanesulfonate
[00159] The title compound was prepared according to GPl from cycloheptanone (2.2 g, 20 mmol). Crude yield: 3.7 g.
[00160] 1H-NMR (400 MHz, CDCl3) d 5.86-5.74 (m, IH), 2.51-2.40 (m, 2H), 2.14-2.06 (m, 2H), 1.77-1.49 (m, 6H).
Example 4 - Suzuki coupling, general procedure 2 (GP2)
[00161] The appropriate boronic acid (4.4 mmol) was dissolved in dry THF (20 mL) and cyclohexenyl triflate (4.0 mmol) and KF (13.2 mmol) was added. The solution was degassed and kept under Argon and PdCl2(dppf) (65.3 mg, 0.08 mmol) was added. The reaction was shaken overnight at rt after which time they were filtered through celite, rinsed with EtOAc and subjected to column chromatography (silica, hexane). 2, 6-Difluorophenyl cyclohexene
[00162] The title compound was prepared according to GP2. Yield: 551 mg (2.84 mmol, 71%). 1H-NMR (400 MHz, CDCl3) d 7.17-7.10 (m, IH), 6.88-6.81 (m, 2H), 5.80 (m, IH), 2.26-2.19 (m, 4H), 1.78-1.70 (m, 4H).GC Analysis: Rt = 2.55 min (Method 50), 97%. 2,5-Difluorophenyl cyclohexene.
[00163] The title compound was prepared according to GP2. Yield: 596 mg (3.07 mmol, 77%). 1H-NMR (400 MHz, CDCl3) d 6.88-6.81 (m, 2H), 6.78-6.73 (m, IH), 5.88 (m, IH), 2.26-2.23 (m, 2H), 2.13-2.09 (m, 2H), 1.68-1.64 (m, 2H), 1.61-1.57 (m, 2H). GC Analysis (method 50): Rt = 2.77 min, 91%. 2,4-Difluorophenyl cyclohexene
[00164] The title compound was prepared according to GP2. Yield: 290 mg (1.49 mmol, 37%),1H-NMR (400 MHz, CDCl3) d 7.12 (ddd, IH, J=8.6 Hz, 6.6 Hz, 6.6 Hz), 6.77-6.68 (m, 2H), 5.82 (m, IH), 2.29-2.25 (m, 2H), 2.16-2.11 (m, 2H), 1.73-1.59 (m, 4H). GC Analysis (method 50): Rt = 2.62 min, 98%. 5-Chloro-2-fluorophenyl cyclohexene
[00165] The title compound was prepared according to GP2. Yield: 701 mg (3.32 mmol, 83%). 1H-NMR (400 MHz, CDCl3) d 7.15 (dd, IH, J=6.65 Hz, 2.74 Hz), 7.06 (m, IH), 6.88 (dd, IH, J=10.27 Hz, 8.70 Hz), 5.90 (m, IH), 2.28-2.26 (m, 2H), 2.17- 2.13 (m, 2H), 1.71-1.67 (m, 2H), 1.65-1.60 (m, 2H). GC Analysis: Rt = 3.88 min, 94%.
Example 5 - Negishi coupling, general procedure 3 (GP3) l-(l-Cyclohexen-l-yl)-3-methoxy-benzene
[00166] In a dry and argon flushed two neck flask, tris(dibenzylidedeacetone) dipalladium (275 mg, 0.3 mmol) and tri-2-furylphosphine (278 mg, 1.2 mmol) was dissolved in N-methylpyrrolidone. Tetrabutyl ammonium iodide (2.21 g, 6.0 mmol) and 1-cyclohexenyl-l-trifluoromethanesulfonate (1.85 g, 6.0 mmol) were added to the reaction mixture followed by phenylzinc bromide(12 mL, 1.0 M, 12 mmol) and the reaction mixture was left stirring at room temperature over night. The reaction was quenched with saturated ammonium chloride solution. The product was filtered through celite, taken up in ethyl acetate and washed with brine, dried over Na2SO4, and concentrated in vacuo. The title compound was obtained and purified by flash chromatography (silica, 0-10% EtOAc in heptane). Yield: 700 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.23 (t, J = 7.8 Hz, IH), 7.04 (d, J = 7.8 Hz, IH), 6.98 (s, IH), 6.81 (d, J = 7.8 Hz, IH), 6.18-6.17 (m, IH), 3.82 (s, 3H), 2.45-2.42 (m, 2H), 2.24-2.21 (m, 2H), 1.83-1.79 (m, 2H), 1.72-1.68 (m, 2H). 4-(Trifluoromethyl)-l-cyclohexen-l-yl]-benzene
[00167] The title compound was prepared according to GP3 from A- (trifluoromethyl)-l-cyclohexenyl-l-trifluoromethanesulfonate (475 mg, 1.6 mmol) and phenylzinc bromide (3.2 mL, 1.0 M, 3.2 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 284 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.32- 7.15 (m, 5H), 6.01-5.98 (m, IH), 2.58-2.07 (m, 6H), 1.71-1.58 (m, IH). l-Fluoro-4-[4-(trifluoromethyl)-l-cyclohexen-l-yl]-benzene
[00168] The title compound was prepared according to GP3 from A- (trifluoromethyl)-l-cyclohexenyl-l-trifluoromethanesulfonate (475 mg, 1.6 mmol) and A- fluorophenylzinc bromide (3.2 mL, 1.0 M, 3.2 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 271 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.38- 7.32 (m, 2H), 7.07-6.98 (m, 2H), 6.02-5.99 (m, IH), 2.61-2.17 (m, 6H), 1.76-1.63 (m, IH). l-Fluoro-3-[4-(trifluoromethyl)-l-cyclohexen-l-yl]-benzene)
[00169] The title compound was prepared according to GP3 from A- (trifluoromethyl)-l-cyclohexenyl-l-trifluoromethanesulfonate (475 mg, 1.6 mmol) and 3- fluorophenylzinc bromide (3.2 mL, 1.0 M, 3.2 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 363 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.35- 6.92 (m, 4H), 6.15-6.11 (m, IH), 2.62-2.15 (m, 6H), 1.75-1.64 (m, IH). l-(Cyclohexen-l-yl)-2-fluorobenzene
[00170] The title compound was prepared according to GP3 from 1- cyclohexenyl-1-trifluoromethanesulfonate (1.84 g, 8.0 mmol) and 2-fluorophenylzinc bromide (16 mL, 1.0 M, 32 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 678 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.30-6.97 (m, 4H), 5.96- 5.90 (m, IH), 2.40-2.33 (m, 2H), 2.23-2.18 (m, 2H), 1.79-1.70 (m, 2H), 1.70-1.64 (m, 2H). 4-(l-Cyclohexea-l-yl)-l,2-diΩuoro-beazeae
[00171] The title compound was prepared according to GP3 from 1- cyclohexenyl- 1 -trifluoromethanesulfonate (1.84 g, 8.0 mmol) and 3,4-difluorophenylzinc bromide (32 mL, 0.5 M, 16 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 1.31 g. 1H-NMR (400 MHz, CDCl3) δ: 7.20-7.00 (m, 3H), 6.07-
6.00 (m, IH), 2.38-2.30 (m, 2H), 2.24-2.18 (m, 2H), 1.82-1.66 (m, 4H). l-(l-Cyclohexen-l-yl)-3,5-difluoro-benzene
[00172] The title compound was prepared according to GP3 from 1- cyclohexenyl- 1 -trifluoromethanesulfonate (1.84 g, 8.0 mmol) and 3,5-difluorophenylzinc bromide (32 mL, 0.5 M, 16 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 934 mg. GC-FID Rt: 2.96 min (Method50) l-Fluoro-2-[4-(l-i-propyl)-l-cyclohexen-l-yl]-benzene
[00173] The title compound was prepared according to GP3 from 4-(l-i- propyl)-l-cyclohexenyl-l -trifluoromethanesulfonate (2.18 g, 8.0 mmol) and 2- fluorophenylzinc iodide (32 mL, 0.5 M, 16.0 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 1.15 g. 1H-NMR (400 MHz, CDCl3) δ: 7.30-
7.01 (m, 4H), 5.97-5.93 (m, IH), 2.51-1.34 (m, 8H), 0.98-0.88 (m, 6H). l-Fluoro-3-[4-(l- i-propyl)-l-cyclohexen-l-yl]-benzene
[00174] The title compound was prepared according to GP3 from 4-(l-i- propyl)-l-cyclohexenyl-l -trifluoromethanesulfonate (2.18 g, 8.0 mmol) and 3- fluorophenylzinc iodide (32 mL, 0.5 M, 16.0 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 902 mg. 1H-NMR (400 MHz, CDCl3) δ: 7.35- 7.12 (m, 3H), 6.92-6.87 (m, IH), 6.18-6.12 (m, IH), 2.51-1.35 (m, 8H), 0.99-0.95 (m, 6H). l-Methoxy-3-[4-(l- i-propyl)-l-cyclohexen-l-yl]-benzene
[00175] The title compound was prepared according to GP3 from 4-(l-i- propyl)-l-cyclohexenyl-l -trifluoromethanesulfonate (2.18 g, 8.0 mmol) and 2- methoxyphenylzinc bromide (16 mL, 1.0 M, 16.0 mmol). The product was purified by flash chromatography (silica, heptane). Yield: 1.63 g. GC-FID Rt: 6.07 min (Method50)
Example 6 - Vinylaromatic Compounds, general procedure 4 (GP4)
[00176] The vinylaromatic compounds were prepared as exemplified below using cycloheptanone and phenylmagnesium chloride. 1-Phenylcycloheptene, General procedure 4 (GP4)
[00177] Mesitylmagnesium bromide (18.0 mL, 18.0 mmol, 1.0 M in THF) was added over 15 minutes to a solution of cycloheptanone (2.0 g, 17.8 mmol) and diphenyl chlorophosphate (1.1 eq.) in THF (5 mL) at 0 0C. The solution was stirred at 0 0C for 30 min, whereafter the solution was allowed to reach room temperature. After stirring the solution for 30 min, dichlorobis(triphenylphosphine)palladium (126 mg, 1 mol%) was added and the solution was warmed to 65 0C. Phenylmagnesium chloride (10.8 mL, 1.2 equivalents in THF) was added over 10 minutes, resulting in a gentle reflux of the solvent. After stirring at 65 0C for 30 minutes, the mixture was cooled to rt and poured into a mixture of 3 N HCl (30 mL) and pentane (30 mL). The phases were separated, and the aqueous portion was extracted with pentane (30 mL). The combined organic phase was washed sequentially with 3 N HCl (20 mL), 3 M NaOH (2 x 20 mL), and brine (20 mL), and dried over MgSO4. Evaporation of the solvent followed by distillation using a Kugelrohr apparatus (oven temperature 100-140 0C, 0.065 torr) yielded 1- phenylcycloheptene (1.29 g, 43%).
[00178] 1H-NMR (400 MHz, CDCl3) δ 7.37-7.20 (m, 5H), 6.11 (t, IH), 2.65 (m, 2H), 2.30 (m, 2H), 1.88 (m, 2H), 1.70 (m, 2H), 1.60 (m, 2H).
[00179] 13C-NMR (100 MHz, CDCl3) δ 145.2, 140.5, 130.5, 128.3 (2C), 126.5, 126.0 (2C), 33.0, 32.9, 29.0, 27.1, and 27.0.
[00180] 1-Phenylcycloheptene was also synthesized according to GP3
l-(3-Fluorophenyl)-cycloheptene
[00181] l-(3-Fluorophenyl)-cycloheptene was prepared according to GP4 and GP3 described above and isolated by column chromatography. 1H-NMR (400 MHz, CDCl3) δ 7.27-7.20 (m, IH), 7.10 (m, IH), 7.0 (m, IH), 6.95-6.85 (m, IH), 6.10 (t, IH, J = 8.0 Hz), 2.6 (m, 2H), 2.33-2.23 (m, 2H), 1.89-1.8 (m, 2H), 1.7-1.5 (m, 4H). 13C-NMR (100 MHz, CDCl3) δ 163.0 (d, J = 242 Hz), 147.7 (d, J = 20 Hz), 144.3, 131.7, 129.7 (d, J = 20 Hz), 121.5, 113.1 (d, J = 22 Hz), 112.7 (d, J = 22 Hz), 32.9, 32.8, 29.0, 27.0, 26.9. l-(2-Fluorophenyl)-cycloheptene
[00182] l-(2-Fluorophenyl)-cycloheptene was prepared according to GP3 described above and isolated by column chromatography. Rf = 0.85 (heptane). l-(4-Fluorophenyl)-cycloheptene
[00183] l-(4-Fluorophenyl)-cycloheptene was prepared according to GP3 and GP4 described above and isolated by column chromatography.
[00184] 1H-NMR (400 MHz, CDCl3) δ 7.35-7.25 (m, 2H), 7.00-6.90 (m, 2H), 6.05 (t, IH, J = 8.0 Hz), 2.60 (m, 2H), 2.33-2.23 (m, 2H), 1.90-1.80 (m, 2H), 1.70-1.50 (m, 4H).
[00185] 13C-NMR (100 MHz, CDCl3) δ 161.9 (d, J = 244 Hz), 144.2, 141.3 (d, J = 3 Hz), 130.5, 127.4 (d, 2C, J = 8 Hz), 115.0 (d, 2C, J = 21 Hz), 33.2, 32.9, 29.0, 27.1, 27.0. 1-Phenylcyclooctene
[00186] 1-Phenylcyclooctene was prepared according to GP3 and GP4 described above and isolated by Kugelrohr distillation (oven temperature 120-140 0C, 0.065 torr). Yield (1.5 g, 60%).
[00187] 1H-NMR (400 MHz, CDCl3) δ 7.44-7.39 (m, 2H), 7.33-7.18 (m, 3H), 6.01 (t, IH, J = 8.0 Hz), 2.67-2.61 (m, 2H), 2.34-2.26 (m, 2H), 1.70-1.50 (m, 8H). 13C- NMR (100 MHz, CDCl3) δ 143.4, 140.5, 128.4 (2C), 128.2, 126.6, 126.0 (2C), 30.2, 29.7, 28.7, 27.6, 27.1, 26.4. l-Methoxy-4-(l-phenyl-cyclohexyl)-benzene, procedure A
[00188] A mixture Of AuCl3 (7.6 mg, 0.025 mmol) and AgOTf (19.3 mg, 0.075 mmol) was stirred in dichloromethane (2 mL) for 30 min. Anisole (54 mg, 0.5 mmol) and 1 -Phenyl- 1-cyclohexene (158 mg, 1 mmol) were then added sequentially. The resulting mixture was stirred at room temperature overnight. Evaporation of the solvent under reduced pressure gave 130 mg of crude material. Flash chromatography (heptane: ethyl acetate 95:5) afforded 90 mg of a as a colorless oil. Rf =0.33 (heptane: ethyl acetate 95:5). 1H-NMR (400 MHz, CDCl3): 7.27-7.25 (m, 4H), 7.19 (d, 2H, J=8.8 Hz), 7.12 (m, IH), 6.81 (d, 2H, J=8.8 Hz), 3.77 (s, 3H), 2.30-2.20 (m, 4H), 1.62-1.44 (m, 6H). l-Methoxy-4-(l-phenyl-cyclohexyl)-benzene (B), procedure B
[00189] 4-(l-phenylcyclohexyl)phenol (20 mg, 0.08 mmol) was dissolved in DMF (2 mL). A suspension of NaH in oil (60%, 5 mg, 0.125 mmol) was added. After stirring for 5 minutes methyl iodide (0.05 mL; 0.8 mmol) was added. The reaction mixture was stirred for 2 h. (tic indicated full conversion of the starting material) then quenched with water (10 mL). Dichloromethane (10 mL) was added. The mixture was shaken and the organic phase separated off, dried (Na2SO4) and concentrated to syrup. The title product was afforded after work-up by flash-chromatography (eluent dichloromethane). Yield: 20 mg, quantitatively. LC-MS purity (UV/MS): 100/-, Rt 6.48 min. 1H NMR data were in accordance with the data written above.
Example 7 - General procedure 5 (GP5) 4-(l-Phenylcyclohexyl)phenol(ERB-002)
[00190] 1 -Phenyl- 1-cyclohexene (1 g, 6.3 mmol), phenol (1.5 g, 15.9 mmol) and BF3 H3PO4 (0.05 mL) were mixed and shaken at 8O0C overnight. Dichloromethane (30 mL) was added and the organic phase was washed with saturated NaHCθ3 (aq.) (2 x 10 mL), dried (Na2SO4) and concentrated. The title compound was crystallised from a mixture of methanol and water. Yield: 1040 mg. 1H NMR (400 MHz, CDCl3): δ 7.28- 7.25 (m, 4H), 7.16-7.10 (m, 3H), 6.75-6.70 (m, 2H), 4.51 (br. s, IH), 2.28-2.22 (m, 4H), 1.60-1.52 (m, 4H), 1.52-1.45 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 153.25, 149.15, 141.22, 128.63, 128.39, 127.29, 125.55, 115.21, 45.89, 37.50, 26.63, 23,14. LC-MS purity (UV/MS): 100/100%, Rt 5.07 min, M-I: 251.62. 4-(l-(2-Fluoro-phenylcyclohexyl)phenol(ERB-003)
[00191] The title compound was prepared according to GP5 from 1- (cyclohexen-l-yl)-2-fluorobenzene (400 mg, 1.99 mmol). Yield: 0.488 g white powder. 1H NMR (400 MHz, CDCl3): δ 7.40 (ddd, 2.3 Hz, 8.1 Hz, 8.3 Hz, IH), 7.18-7.06 (m, 3H), 6.88 (ddd, 2.3 Hz, 8.1 Hz, 12.7 Hz, IH), 6.74-6.69 (m, 2H), 4.51 (br. s, IH), 2.48-2.36 (m, 2H), 2.22-2.13 (m, 2H), 1.67-1.41 (m, 6H). LC-MS purity (UV/MS): 100/100%, Rt 4.98 min, M-I : 269.16. 4-(l-(3,5-Dffluoro-phenylcyclohexyl)phenol(ERB-008)
[00192] The title compound was prepared according to GP5 from 1- (cyclohexen-l-yl)-3,5-difluorobenzene (400 mg, 1.99 mmol). Yield: 330 mg white powder. LC-MS purity (UV/MS): 100/100%, Rt 5.08 min, M-I: 287.17. 4-(l-(3,4-DiΩuoro-phenylcyclohexyl)phenol(ERB-009)
[00193] The title compound was prepared according to GP5 from 1- (cyclohexen-l-yl)-3,4-difluorobenzene (400 mg, 1.99 mmol). Yield: 230 mg white powder. 1H NMR (400 MHz, CDCl3): δ 7.18-7.12 (m, 2H), 7.12-6.95 (m, 3H), 6.84-6.76 (m, 2H), 5.44 (br. s, IH), 2.40-2.05 (m, 4H), 1.67-1.30 (m, 6H). LC-MS purity (UV/MS): 100/98%, Rt 5.08 min, M-I: 287.22. 4-[l-(2,6-Difluoro-phenyl)-cyclohexyl]-phenol (ERB-010)
[00194] The title compound was prepared according to GP5 from 1- (cyclohexen-l-yl)-2,6-difluorobenzene (200 mg, 1.0 mmol). Yield: 218 mg white powder. 1H NMR (400 MHz, CDCl3): δ 7.24-7.18 (m, 2H), 7.18-6.98 (m, IH), 6.82-6.76 (m, 2H), 6.72-6.68 (m, 2H), 5.48 (br. s, IH), 2.85-2.76 (m, 2H), 1.95-1.85 (m, 2H), 1.76-1.64 (m, 2H), 1.64-1.34 (m, 4H). LC-MS purity (UV/MS): 100/100%, Rt 5.08 min, M-I : 287.60. 4-(l-Phenyl-[4-(trifluoromethyl)-cyclohexyl])-phenol(ERB-030)
[00195] The title compound was prepared according to GP5 from [4- (trifluoromethyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). Yield: 228 mg.
[00196] ERB-030: 1H NMR (400 MHz, CDCl3): δ 7.35-7.08 (m, 7H), 6.82- 6.77 (m, 2H), 4.80 (br. s, IH), 2.80-2.75 (m, 2H), 2.23-2.10 (m, IH), 1.99-1.82 (m, 4H), 1.62-1.55 (m, 2H). LC-MS purity (UV/MS): 100 /100 Rt 9.16 min, M-I : 319.19.
[00197] Isomer of ERB-030: 1H NMR (400 MHz, CDCl3): δ 7.38-7.15 (m, 5H), 7.10-6.98 (m, 2H), 6.72-6.64 (m, 2H), 4.67 (br. s, IH), 2.82-2.71 (m, 2H), 2.21-2.15 (m, IH), 2.00-1.82 (m, 4H), 1.62-1.50 (m, 2H). LC-MS purity (UV/MS): 100/ 100, Rt 9.21 min, M-I : 319.19. 4-(l-(4-Flourophenyl)-[4-(tήfluoromethyl)-cyclohexyl])-phenol (ERB-031)
[00198] The title compound was prepared according to GP5 from 4-fluoro-[4- (trifluoromethyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). Yield: 221 mg.
[00199] ERB-031: 1H NMR (400 MHz, CDCl3): δ 7.18-7.11 (m, 2H), 7.10- 7.07 (m, 2H), 6.92-6.87 (m, 2H), 6.83-6.80 (m, 2H), 4.80 (br. s, IH), 2.72-2.64 (m, 2H), 2.22-2.08 (m, IH), 1.95-1.85 (m, 4H), 1.59-1.47 (m, 2H). LC-MS purity (UV/MS): 100/100%, Rt 9.28 min, M-I : 337.17.
[00200] Isomer of ERB-031: 1H NMR (400 MHz, CDCl3): δ 7.32-7.24 (m, 2H), 7.08-6.96 (m, 2H), 6.72-6.66 (m, 2H), 4.73 (br. s, IH), 2.74-2.66 (m, 2H), 2.22-2.08 (m, IH), 1.96-1.86 (m, 4H), 1.56-1.44 (m, 2H). LC-MS purity (UV/MS): 100/100%, Rt 9.24 min, M-I : 337.17. 4-(l-(3-Flourophenyl)-[4-(tήfluoromethyl)-cyclohexyl])-phenol(ERB-032)
[00201] The title compound was prepared according to GP5 from 4-fluoro-[4- (trifluoromethyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). Yield: 221 mg. The diastereomers were separated by flash chromatography.
[00202] ERB-032: 1H NMR (400 MHz, CDCl3) δ 7.48-7.44 (m, IH), 7.24-7.13 (m, 2H), 7.08-7.03 (m, 2H), 6.96-6.90 (m, IH), 6.72-6.67 (m, 2H), 4.73 (br. s, IH), 2.94- 2.86 (m, 2H), 2.20-2.08 (m, IH), 1.98-1.90 (m, 2H), 1.88-1.78 (m, 2H), 1.60-1.48 (m, 2H). LC-MS purity (UV/MS): 100/100%, Rt 9.21 min, M-I: 337.17.
[00203] Isomer of ERB-032: 1H NMR (400 MHz, CDCl3): δ 7.26-7.21 (m, 2H), 7.18-7.11 (m, 2H), 7.05-7.00 (m, IH), 6.90-6.85 (m, IH), 6.80-6.76 (m, 2H), 4.78 (br. s, IH), 2.90-2.81 (m, 2H), 2.22-2.10 (m, IH), 2.10-1.99 (m, 2H), 1.90-1.84 (m, 2H), 1.62-1.48 (m, 2H). LC-MS purity (UV/MS): 100/100%, Rt 9.28 min, M-I : 337.17. 4-[l-(2-Fluoro-phenyl)-4-isopropyl-cyclohexyl]-phenol (ERB-039)
[00204] The title compound was prepared according to GP5 from l-fluoro-2- [4-(l-i-propyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). Yield: 180 mg. The diastereomers were separated by flash chromatography
[00205] ERB-039: 1H NMR (400 MHz, CDCl3): δ 7.68-7.52 (m, IH), 7.24- 7.10 (m, 2H), 7.10-7.04 (m, 2H), 6.95-6.86 (m, IH), 6.71-6.64 (m, 2H), 4.66 (br. s, IH), 2.84-2.75 (m, 3H), 1.90-1.73 (m, 4H), 1.40-1.25 (m, 3H), 0.83 (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/100%, Rt 6.10 min, M-I: 311.51.
[00206] Isomer of ERB-039: 1H NMR (400 MHz, CDCl3) δ 7.30-7.22 (m, 3H), 7.17-7.08 (m, IH), 7.07-7.00 (m, IH), 6.90-6.80 (m, IH), 6.78-6.74 (m, 2H), 4.58 (br. s, IH), 2.82-2.74 (m, 2H), 2.04-1.93 (m, 2H), 1.70-1.64 (m, 2H), 1.38-1.12 (m, 4H), 0.83 (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/86%, Rt 6.16 min, M-I : 311.52. 4-[l-(3-Fluoro-phenyl)-4-isopropyl-cyclohexyl]-phenol(ERB-037)
[00207] The title compound was prepared according to GP5 from l-fluoro-3- [4-(l-i-propyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). Yield: 150 mg. The diastereomers were separated by flash chromatography
[00208] ERB-037: 1H NMR (400 MHz, CDCl3): δ 7.24-7.18 (m, 2H), 7.18- 7.12 (m, IH), 6.96-6.92 (m, IH), 6.89-6.84 (m, IH), 6.82-6.74 (m, 3H), 4.66 (br. s, IH), 2.64-2.58 (m, 2H), 1.92-1.82 (m, 2H), 1.73-1.65 (m, 2H), 1.38-1.13 (m, 4H), 0.82 (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/ - , Rt 6.87 min, M-I: 311.
[00209] Isomer of ERB-037: 1H NMR (400 MHz, CDCl3) δ 7.29-7.20 (m, IH), 7.14-7.10 (m, IH), 7.08-7.00 (m, 3H), 6.88-6.81 (m, IH), 6.70-6.64 (m, 2H), 4.58 (br. s, IH), 2.66-2.58 (m, 2H), 1.94-1.82 (m, 2H), 1.75-1.67 (m, 2H), 1.37-1.24 (m, IH), 1.19- 1.10 (m, 3H), 0.82v (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/100%, Rt 6.16 min, M- 1 : 311. 4-[4-Isopropyl-l-(3-methoxy-phenyl)-cyclohexyl]-phenol (ERB-038)
[00210] The title compound was prepared according to GP5 from l-methoxy-3- [4-(l-i-propyl)-l-cyclohexen-l-yl] -benzene (200 mg, 1.0 mmol). The diastereomers were separated by flash chromatography
[00211] ERB-038: 1H NMR (400 MHz, CDCl3): δ 7.28-7.20 (m, 2H), 7.08- 7.02 (m, 2H), 6.96-6.92 (m, IH), 6.73-6.62 (m, 3H), 4.58 (br. s, IH), 3.80 (s, 3H), 2.68- 2.60 (m, 2H), 1.94-1.82 (m, 2H), 1.38-1.08 (m, 4H), 0.82 (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/ 100 , Rt 6.87 min, M-I: 323.
[00212] Isomer of ERB-038: 1H NMR (400 MHz, CDCl3) δ 7.29-7.20 (m, 3H), 7.19-7.10 (m, IH), 6.80-6.72 (m, 3H), 6.68-6.60 (m, IH), 4.60 (br. s, IH), 3.74 (s, 3H), 2.66-2.58 (m, 2H), 1.94-1.83 (m, 2H), 1.38-1.05 (m, 4H), 0.80 (d, 6H, 7 Hz). LC-MS purity (UV/MS): 100/100%, Rt 6.16 min, M-I: 323. 4-(l-Phenyl-cycloheptyl)-phenol(ERB-012)
[00213] The title compound was prepared according to GP5 with a yield of 40%-70%. LC-MS purity (UV/MS): 100/100%, Rt 5.35 min. 1H-NMR (400 MHz, CDCl3) δ 7.28-7.10 (m, 5H), 7.04 (d, 2H, J = 8.8 Hz), 6.70 (d, 2H, J = 8.8 Hz), 4.55 (s, IH), 2.35- 2.20 (m, 4H), 1.78-1.60 (m, 4H), 1.60-1.50 (m, 4H). 4-fl-(4-Fluoro-phenyl)-cycloheptyl]-phenol(ERB-013)
[00214] The title compound was prepared according to GP5 with a yield of 40%-70%. LC-MS purity (UV/MS): 100/100%, Rt 9.89 min. 1H-NMR (400 MHz, CDCl3) δ 7.15 (m, 2H), 7.01 (d, 2H, J = 8.7 Hz), 6.95 (m, 2H), 6.75 (d, 2H, J = 8.7 Hz), 4.55 (br. s, IH), 2.30-2.20 (m, 4H), 1.75-1.40 (m, 8H). 4-[l-(4-Fluoro-phenyl)-cycloheptyl]-benzene-l,2-diol(ERB-014)
[00215] The title compound was prepared according to GP5 with a yield of 40%-70%. LC-MS purity (UV/MS): 86/100%, Rt 9.05 min. 1H-NMR (400 MHz, CDCl3) δ 7.17-7.10 (m, 2H), 6.96-6.70 (m, 3H), 6.60 (m, 2H), 5.15-4.85 (m, 2-3H), 2.30-2.10 (m, 4H), 1.70-1.40 (m, 8H). 4-fl-(3-Fluoro-phenyl)-cycloheptyl]-phenol(ERB-015)
[00216] The title compound was prepared according to GP5 with a yield of 40%-70%. LC-MS purity (UV/MS): 99/93%, Rt 9.88 min. 1H-NMR (400 MHz, CDCl3) δ 7.30-6.68 (m, 4H), 7.12 (d, 2H, J = 8.8 Hz), 6.74 (d, 2H, J = 8.8 Hz), 4.55 (s, IH), 2.26 (m, IH), 2.0 (m, IH), 1.72-1.50 (m, 8H), 1.30-1.10 (m, 2H). 4-[l-(2-Fluoro-phenyl)-cycloheptyl]-phenol(ERB-016)
[00217] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.44 (m, IH), 7.22-7.10 (m, 2H), 7.02 (d, 2H, J = 8.8 Hz), 6.92-6.86 (m, IH), 6.70 (d, 2H, J = 8.8 Hz), 4.51 (s, IH), 2.42-2.26 (m, 4H), 1.82-1.52 (m, 8H). 13C-NMR (100 MHz, CDCl3) δ 161.7 (d, J = 249 Hz), 153.2, 143.2, 137.4 (d, J = 11 Hz), 128.0 (d, J = 5 Hz), 127.9 (d, J = 9 Hz), 127.7 (2C), 123.5 (d, J = 3 Hz), 116.8 (d, J = 24 Hz), 114.9 (2C), 48.7 (d, J = 2 Hz), 39.4 (d, J = 2 Hz), 30.6, 24.6. 4-(l-Phenyl-cyclooctyl)-phenol(ERB-017)
[00218] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.26-7.10 (m, 5H), 7.09 (d, 2H, J = 8.8 Hz), 6.71 (d, 2H, J = 8.8 Hz), 2.34-2.28 (m, 4H), 1.68-1.54 (m, 6H), 1.46-1.38 (m, 4H). 4-(l-Phenyl-cycloheptyl)-benzene-l,2-diol(ERB-035)
[00219] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.28-7.10 (m, 5H), 6.76-6.72 (m, IH), 6.67- 6.63 (m, 2H), 4.90 (bs, IH), 2.30-2.10 (m, 4H), 1.8-1.4 (m, 8H). 2-Methyl-4-fl-(3-Fluoro-phenyl)-cycloheptyl]-phenol(ERB-036)
[00220] The title compound was prepared according to GP5 with a yield of 40%-70%. LC-MS purity (UV/MS): 94/100%, Rt 10.37 min. 4-(l-(2,4-Difluoro-phenyl)-cyclohexyl)-phenol (ERB-OIl)
[00221] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.34 (m, IH), 7.12 (d, 2H, J = 8.4 Hz), 6.82 (m, IH), 6.72 (d, 2H, J = 8.4 Hz), 6.64 (m, IH), 4.50 (s, IH), 2.36 (m, 2H), 2.18 (m, 2H), 1.66-1.40 (m, 6H). 4-(l-(2,5-Difluoro-phenyl)-cyclohexyl)-phenol(ERB-044)
[00222] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.14 (d, 2H, J = 8.8 Hz), 7.14-7.06 (m, IH), 6.83 (m, 2H), 6.73 (d, 2H, J = 8.0 Hz), 4.50 (s, IH), 2.35 (m, 2H), 2.20 (m, 2H), 1.66-1.40 (m, 6H). 4-(l-(2-Fluoro-5-chloro-phenyl)-cyclohexyl)-phenol(ERB-045)
[00223] The title compound was prepared according to GP5 with a yield of 40%-70%. 1H-NMR (400 MHz, CDCl3) δ 7.38 (dd, IH, J = 8.0 Hz, 4.4 Hz), 7.17-7.09 (m, IH), 7.14 (d, 2H, J = 8.4 Hz), 6.86-6.74 (m, IH), 6.73 (d, 2H, J = 8.4 Hz), 4.58 (s, IH), 2.33 (m, 2H), 2.21 (m, 2H), 1.54 (m, 6H). Example 8 - tert-Butyl l-((4-('l-phenylcvclohexyl)phenoxy)carbonyl)-2- methylpropylcarbamate
[00224] To a stirred solution at room temperature of 4-(l- phenylcyclohexyl)phenol (311 mg, 1.23 mmol) in dry THF (2 mL) was added BocValOH (295 mg, 1.36 mmol) dissolved in THF (2 mL). A solution of DIC (231 μL; 1.48 mmol) in THF (2 mL) was added drop wise, which caused precipitation after a few minutes. After 5 min DMAP (166 mg; 1.36 mmol) was added and stirring was continued for 19h. The reaction mixture was concentrated in vacuo and purified by flash chromatography (eluent: EtOAc (0-10%) in heptane) affording 517 mg (1.14 mmol; 93%) of colourless oil. LC-MS purity (UV/MS): 100/100%, Rt 6.77 min, M+18: 469.53.
Example 9 - 4-(l-Phenylcvclohexyl)phenyl 2-amino-3-methylbutanoate
[00225] To a stirred solution at room temperature of tert-butyl l-((4-(l- phenylcyclohexyl)phenoxy)carbonyl)-2-methylpropylcarbamate (142 mg, 0.31 mmol) in dry DCM (3 mL) was added TFA (300 μL), which caused gas evolution. Stirring was continued. After Ih the reaction mixture was concentrated in vacuo to afford 96 mg (0.21mmol; 67%) of colourless oil. LC-MS purity (UV/MS): 92/97%, Rt 5.45 min, M+l : 352.49. 1H NMR (400 MHz, CDCl3): 11.12 (br s), 7.76 (br s), 7.29 (d, 2H, J = 8.8 Hz), 7.27 (d, 4H, J = 4.8 Hz), 7.13-7.16 (m, IH), 6.95 (d, 2H, J = 8.8 Hz), 6.16(d, IH, J = 4.0 Hz), 2.40-2.48 (m, IH), 2.18-2.33 (m, 4H), 1.50-1.58 (m, 6H), 1.05-1.08 (m, 6H).
Example 10 - 2-(2-tert-Butoxycarbonylamino-3-methyl-butyrylamino)-3-methyl-butyric acid 4-(l-phenyl-cvclohexyl)-phenyl ester
[00226] To a stirred mixture at room temperature of PS-carbodiimide (1.2 g; 1.32 mmol) and 4-(cyclohexyl-phenyl)methyl-hydroxybenzene (152 mg, 0.60 mmol) in dry THF (2 mL) was added BocValValOH (285 mg, 0.90 mmol) dissolved in THF (2mL). After 5 min, DMAP (81 mg; 0.66 mmol) was added and stirring was continued for 47 h. The reaction mixture was filtered and concentrated in vacuo and purified by CC using EtOAc (0-25%) in heptane affording 297 mg (0.54 mmol; 90%) of colourless oil. LCMS purity (UV/MS): 89/100%, Rt 7.61 min, M+l: 551.53. Example 11 - 2-(2-Amino-3-methyl-butyrylamino)-3-methyl-butyric acid 4-(l-phenyl- cvclohexyD-phenyl ester
[00227] To a stirred solution at room temperature of 4'-(cyclohexyl)- (phenyl)methyl- 1 '-(2-tert-butoxycarbonylamino-2-[2-methyl]ethyl)acetoxybenzene (297 mg, 0.54 mmol) in dry DCM (3 mL) was added TFA (300 μL), which caused gas evolution. Stirring was continued. After Ih the reaction mixture was concentrated in vacuo to afford 240 mg (0.43 mmol; 79%) of colourless oil as a mixture of isomers. LCMS purity (UV/MS): 100/98%, Rt 4.64/4.95 min, M+l: 451.56. 1H NMR (400 MHz, CDCl3): 8.52 (br s), 7.63 (br s), 7.26-7.29 (m, 13H), 7.12-7.16 (m, IH), 7.06 (d, IH, J = 7.6Hz), 6.95 (d, IH, J = 8.8Hz), 6.92 (d, IH, J = 8.8Hz), 4.71-4.74 (m, IH), 4.60-4.63 (m, IH), 4.17-4.21 (m, 2H), 2.44 (m, IH), 2.16-2.41 (m, 12H), 1.47-1.57 (m, 12H), 0.98-1.05 (m, 24H).
Example 12 - 2-Iodo-4-(l-phenyl-cyclohexyl)-phenol (ERB-026)
[00228] 4-(l-Phenyl-cyclohexyl)-phenol (600 mg, 2.38 mmol) was dissolved in acetic acid (10 mL). N-iodosuccinimid (537 mg, 2.38 mmol) was added. The mixture was stirred for 4 h., concentrated and worked-up on the combiflash (1O g column, 0-10% ethyl acetate in heptane). 718 mg (80%) of the desired product, 90 mg (7.5 %) of 2,6-diiodo-4- (l-phenyl-cyclohexyl)-phenol and 20 mg (3%) of starting material were isolated.
[00229] 1H ΝMR (400MHz, CDCl3): 7.58 (d, IH, J = 2.4 Hz), 7.34-7.24 (m, 4H), 7.19-7.14 (m, IH), 7.12 (dd, IH, J = 2.4 Hz, J = 8.6 Hz), 6.88 (d, IH, J = 8.6Hz), 5.18 (br. s, IH), 2.23-2.15 (m, 4H), 1.62-1.44 (m, 6H).
Example 13 - 2-Iodo-l-methoxy-4-(l-phenyl-cvclohexyl)-benzene
[00230] NaH (60% in mineral oil, 60 mg, 1.50 mmol) was added an ice-cooled solution of 2-iodo-4-(l-phenyl-cyclohexyl)-phenol (400 mg, 1.06 mmol) in DMF (10 mL). Methyl iodide (125 μL, 2.0 mmol) was added. The reaction temperature was allowed to reach room temperature. The mixture was stirred for 2 h, then quenched with water 10 mL and extracted with dichloromethane (2 x 20 mL). The combined organic phase was dried (Na2SO4) and concentrated in vacuo. The residue was taken up in water (20 mL) and extracted with dichloromethane. The organic phase was dried (Na2SO4) and concentrated in vacuo. xH-nmr of the syrup (430 mg) indicated full conversion to the methyl ether. 1H NMR (400MHz, CDCl3): 7.71 (d, IH, J = 2.1 Hz), 7.32-7.25 (m, 4H), 7.18 (dd, IH, J = 2.1 Hz, J = 8.8 Hz), 7.17-7.13 (m, IH), 6.72 (d, IH, J = 8.8Hz), 3.83 (s, 3H), 2.32-2.18 (m, 4H), 1.62-1.44 (m, 6H).
Example 14 - 2-Fluoro-l-methoxy-4-(l -phenyl -cyclohexyl)-benzene
[00231] A solution of w-butyl lithium in hexane (1 mL, 1.6 M, 1.6 mmol) was added dropwise to a solution of 2-iodo-l -methoxy-4-(l -phenyl -cyclohexyl)-benzene (200 mg, 0.51 mmol) and N-fluoro-benzenesulfonimide (320 mg, 1.02 mmol) in dry THF (5 mL) at -78 0C under an argon atmosphere. The mixtures was stirred at -78°C for 2 h. A GC run after Ih and 2 h indicated no further conversion of the starting material. A saturated solution of NH4Cl (10 mL) was added. The mixture was extracted with dichloromethane (2 x 20 mL), the combined organic phase was dried (TS^SO4) and concentrated in vacuo and worked-up by flash-chromatography. The desired product was isolated in an approx. 75% purity according to a 1H nmr spectrum of the isolated mixture. The mixture was used without further purification in the next reaction.
[00232] 1H NMR (400 MHz, CDCl3): 7.32-7.24 (m, 4H), 7.20-7.11 (m, IH), 7.04-6.95 (m, IH), 7.02-6.94 (m, 2H), 6.89-6.84 (m, IH), 3.85 (s, 3H), 2.32-2.18 (m, 4H), 1.61-1.50 (m, 6H).
Example 15 - 2-Fluoro-4-(l-phenyl-cvclohexyl)-phenol (ERB-006)
[00233] A solution of the crude 2-fluoro-l-methoxy-4-(l-phenyl-cyclohexyl)- benzene in dichloromethane (100 mg, 0.25 mmol, 2 mL) was added to a solution of borane tribromide in dichloromethane (1 M, 0.25 ml, 025 mmol) at -78°C under an argon atmosphere and stirred at r.t for 3 h. Water (10 mL) was added and the mixture was extracted with dichloromethane (2 x 10 mL). The combined organic phase was dried (Na2SO4) and concentrated to a syrup. Work-up by flash-chromatography (eluent: 30 — > 80% dichloromethane in heptane) gave the title product in a yield of 50 mg.
[00234] LC-MS purity (UV/MS): 100/100%, Rt 5.15/5.19 min, M-I : 269.6. 1H NMR 7.31-7.24 (m, 5H), 7.17-7.12 (m, IH), 6.96 (ddd, IH, J = 2.0 Hz, J = 12.9 Hz, J = 14.7 Hz.), 6.91 (dd, IH, J = 8.4 Hz, J = 17.1 Hz), 4.98 (br. s, IH), 2.28-2.18 (m, 4H), 1.61-1.48 (m, 6H).
Example 16 - l-Methoxy-4-(l-phenyl-cvclohexyl)-benzene, procedure A [00235] A mixture Of AuCl3 (7.6 mg, 0.025 mmol) and AgOTf (19.3 mg, 0.075 mmol) was stirred in dichloromethane (2 mL) for 30 min. Anisole (54 mg, 0.5 mmol) and 1 -Phenyl- 1-cyclohexene (158 mg, 1 mmol) were then added sequentially. The resulting mixture was stirred at room temperature overnight. Evaporation of the solvent under reduced pressure gave 130 mg of crude material. Flash chromatography (heptane: ethyl acetate 95:5) afforded 90 mg of a as a colorless oil. Rf =0.33 (heptane: ethyl acetate 95:5). 1H-NMR (400 MHz, CDCl3) δ 7.27-7.25 (m, 4H), 7.19 (d, 2H, J=8.8 Hz), 7.12 (m, IH), 6.81 (d, 2H, J=8.8 Hz), 3.77 (s, 3H), 2.30-2.20 (m, 4H), 1.62-1.44 (m, 6H). 13C-NMR (100 MHz, CDCl3) δ 157.4, 149.2, 140.9, 128,4 (two carbons), 128.4 (two carbons), 127.3 (two carbons), 125.5, 113.8 (two carbons), 55.4, 45.9, 37.5 (two carbons), 26.7, 23.2 (two carbons).
Example 17 - l-Methoxy-4-(l-phenyl-cvclohexyl)-benzene (B), procedure B
[00236] 4-(l-phenylcyclohexyl)phenol (20 mg, 0.08 mmol) was dissolved in DMF (2 mL). A suspension of NaH in oil (60%, 5 mg, 0.125 mmol) was added. After stirring for 5 minutes methyl iodide (0.05 mL; 0.8 mmol) was added. The reaction mixture was stirred for 2 h. (tic indicated full conversion of the starting material) then quenched with water (10 mL). Dichloromethane (10 mL) was added. The mixture was shaken and the organic phase separated off, dried (Na2SO4) and concentrated to syrup. The title product was afforded after work-up by flash-chromatography (eluent dichloromethane). Yield: 20 mg, quantitatively. LC-MS purity (UV/MS): 100/-, Rt 6.48 min. 1H NMR data were in accordance with the data written above.
Example 18 - Acetic acid 4-(l-phenyl-cvclohexyl)-phenyl ester
[00237] 4-(l-phenylcyclohexyl)phenol (100 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL). Pyridine (1 mL) and the acylating reagent were added (1.60 mmol, 5 equiv.). The reaction mixture was stirred for 2 h. at room temperature (tic indicated full conversion of the starting material) then quenched with water (10 mL). Dichloromethane (10 mL) was added. The mixture was shaken and the organic phase separated off, dried (Na2SO4) and concentrated to syrup. The acylated product was afforded after flushing the product through a block of silica (eluent dichloromethane) Yield 120 mg, isolated as a crystalline product. 1H-NMR (400 MHz, CDCl3) δ 7.30-7.24 (m, 6H), 7.17-7.10 (m, IH), 7.01-6.96 (m, 2H), 2.36-2.19 (m, 7H), 1.62-1.45 (m, 6H). Example 19 - 2,2-Dimethyl-propionic acid 4-(l-phenyl-cvclohexyl)-phenyl ester
[00238] 4-(l-phenylcyclohexyl)phenol (100 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL). Pyridine (1 mL) and the acylating reagent were added (1.60 mmol, 5 equiv.). The reaction mixture was stirred for 2 h. at room temperature (tic indicated full conversion of the starting material) then quenched with water (10 mL). Dichloromethane (10 mL) was added. The mixture was shaken and the organic phase separated off, dried (Na2SO4) and concentrated to syrup. The acylated product was afforded after flushing the product through a block of silica (eluent dichloromethane) Yield: 110 mg, isolated as crystalline product 1H-NMR (400 MHz, CDCl3) δ 7.29-7.23 (m, 6H), 7.16-7.10 (m, IH), 6.98-6.93 (m, 2H), 2.34-2.20 (m, 4H), 1.61-1.45 (m, 6H), 1.33 (s, 9H).
Example 20 - Dimethyl-sulfamic acid 4-(l-phenyl-cyclohexyl)-phenyl ester
[00239] DMAP (20 mg, 0.16 mmol) and dimethylcarbamoylchloride (568 mg, 4.0 mmol) was added sequentially to a mixture of 4-(l-phenylcyclohexyl)phenol (200 mg, 0.79 mmol) and Et3N (400 mg, 4.0 mmol) in CH2CI2 (10 mL). The resulting mixture was stirred at room temperature overnight. The solution was further washed with 2M HCL (aq) (20 mL), water (20 mL), saturated NaHCO3 (20 mL) and water (20 mL) and dried over Na2SO4. Evaporation of the solvent under reduced pressure gave 180 mg of crude material. Flash chromatography (heptane: ethyl acetate 95:5) afforded 120 mg (42%) of a as a slightly yellowish oil. Rf =0.21 (heptane: ethyl acetate 90: 10). 1H-NMR (400 MHz, CDCl3) δ 7.30-7.12 (m, 7H), 7.17 (d, 2H, J=9.2 Hz), 2.94(s, 6H), 2.34-2.18 (m, 4H), 1.66-1.44 (m, 6H). 13C-NMR (100 MHz, CDCl3) δ 148.2, 148.0, 147.7, 128,8 (two carbons), 128.5 (two carbons), 127.4 (two carbons), 125.8, 121.4 (two carbons), 46.3, 39.0 (two carbons), 37.4 (two carbons), 26.5, 23.0 (two carbons).
Example 21 - Phosphoric acid dibenzyl ester 4-(l-phenyl-cyclohexyl)-phenyl ester
[00240] lH-tetrazole (6 g of a 3% solution in CH3CN, 2.6 mmol) was added to a stirred solution of 4-(l-phenylcyclohexyl)phenol (200 mg, 0.79 mmol) and dibenzyl- N,N-diisopropyl phosphoramidite (575 mg, 1.7 mmol) in CH2CI2 (10 mL). After 30 min, the mixture was cooled to 0 0C and m-CPBA (570 mg, 2.3 mmol, 70%) was added. The mixture was stirred for 40 min, washed with 10% aqueous Νa2S2θ3 and NaHCO3 (saturated), dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure. Flash chromatography (heptane: ethyl acetate 80:20) afforded 90 mg of a as a colorless oil. Rf =0.58 (heptane: ethyl acetate 50:50). 1H-NMR (400 MHz, CDCl3) δ 7.34- 7.10 (m, 15H), 7.19 (d, 2H, J=8.8 Hz), 7.04 (d, 2H, J=8.8 Hz), 5.04 (d, 2H, J=8.4 Hz), 5.04 (d, 2H, J=8.4 Hz), 2.32-2.18 (m, 4H), 1.60-1.46 (m, 6H). 13C-NMR (100 MHz, CDCl3) δ 151.6, 148.4 (d, J=15 Hz), 145.7, 135.8 (two carbons, d, J=8.6 Hz), 128,8 (two carbons), 128.8 (four carbons), 128.7 (two carbons), 128.5 (two carbons), 128.2 (four carbons), 127.3 (two carbons), 125.7, 119.8 (two carbons, d, J=4.9 Hz), 70.1 (two carbons, d, J=5.7 Hz), 46.1, 37.4 (two carbons), 26.5, 23.1 (two carbons).
Example 22 - Phosphoric acid mono-r4-(l-phenyl-cvclohexyl)-phenyl1ester
[00241] A mixture of phosphoric acid dibenzyl ester 4-(l-phenyl-cyclohexyl)- phenyl ester (133 mg, 2.6 mmol) and 10% palladium on carbon (100 mg) in EtOAc (10 mL) was stirred under H2 (one atmosphere) for two hours. The mixture was filtered through a pad of Celite, and the solvent was evaporated under reduced pressure to yield 79 mg (92%) of a colourless semisolid. Rf =0.22 (CHCl3 :MeOH:H2O 65:25:4). 1H-NMR (400 MHz, CDCl3) δ 7.40-6.90 (m, 9H), 2.30-2.00 (m, 4H), 1.60-1.30 (m, 6H).
Example 23 - (4-Benzyloxy-phenyl)-phenyl-methanone
[00242] NaH (60% in mineral oil, 1.2 g, 30 mmol) was added to an ice-cooled solution of (4-hydroxy-phenyl)-phenyl-methanone (5 g, 25.2 mmol) in DMF (25 mL). Stirred for 20 minutes at room temperature. Benzyl bromide (6.1 ml, 50 mmol) was added at 00C. The reaction temperature was raised to room temperature after Ih. Stirred 2 h. Water (20 mL) was added and aqueous phase was extracted dichloromethane (2 x 30 mL). The combined organic phase was concentrated and purified by flash chromatography (eluent: dichloromethane). 1H nmr revealed the product was pure. Yield: 7.0 g, 96%
[00243] 1H NMR (400 MHz, CDCl3): 7.83 (d, 2H, J = 8.4 Hz), 7.77 (d, 2H, J = 7.2 Hz), 7.58-7.52 (m, IH), 7.50-7.32 (m, 6H), 7.03 (d, J = 8.4 Hz), 5.17 (s, 2H).
Example 24 - l-(l-Allyloxy-l-phenyl-but-3-enyl)-4-benzyloxy-benzene
[00244] A solution of allylmagnesium bromide in diethyl ether (1 M, 15 ml) was added to an ice water cooled solution of the (4-benzyloxy-phenyl)-phenyl-methanone (4.0 g, 13.87 mol) in THF (20 mL). The mixture was stirred for 1 h at r.t, and then quenched with water (20 mL). The aqueous phase was extracted with dichloromethane (2 x 30 ml). The combined organic phase was dried (Na2SO4) and concentrated to syrup. The crude was dissolved in DMF (10 mL) cooled in an ice-water bath. NaH (600 mg, appr. 15 mmol) was added and the mixture was stirred for 1Ah. Allylbromide (1.25 mL, 15.0 mmol) was added and the mixture was stirred at 400C for 1 h. Water (20 mL) was added and aqueous phase was extracted dichloromethane (2 x 30 mL). The combined organic phase was concentrated. Purification using the combiflash (1O g, column, 0-10 ethyl acetate in heptane) gave the diene in an yield of 4.2 g (81%).
[00245] 1H NMR (400 MHz, CDCl3): 7.53-7.26 (m, 12H), 6.98 (d, 2H, J = 8.6 Hz), 6.05-5.94 (m, IH), 5.78-5.65 (m, IH), 5.47-5.39 (m, IH), 5.24-5.03 (m, 5H), 3.84- 3.80 (m, 2H), 3.22-3.17 (m, 2H).
Example 25 - 2-(4-Benzyloxy-phenyl)-2-phenyl-3.6-dihydro-2H-pyran
[00246] l-(l-Allyloxy-l-phenyl-but-3-enyl)-4-benzyloxy-benzene (4.2 g, 10.1 mmol) was dissolved in dichloromethane (50 mL) and bis(tricyclohexylphosphine)benzylidene ruthenium (IV) dichloride (25 mg, 0.03 mmol) was added. Stirred at r.t. for 1 h. TLC indicated full conversion of the starting material. A solution of methylamine in THF (1 M, 1 mL) was added. The solution was eluted through a block of silica. The silica was washed with a solution of dichloromethane and ethyl acetate (1 : 1, 100 mL). The combined organic phase was concentrated to slightly coloured syrup (4.1 g, quantitatively).
[00247] 1H NMR (400 MHz, CDCl3): 7.49-7.24 (m, 12H), 6.95 (d, 2H, J = 8.8 Hz), 6.05-5.98 (m, IH), 5.72-5.65 (m, IH), 4.16-4.00 (m, 2H), 2.90-2.76 (m, 2H).
Example 26 - 4-(2-Phenyl-tetrahvdro-pyran-2-yl)-phenol
[00248] 2-(4-Benzyloxy-phenyl)-2-phenyl-3,6-dihydro-2H-pyran (4.1 g) was dissolved in methanol (25 mL) and ethyl acetate (5 mL). 5% Palladium on carbon (10 mg) was added. Nitrogen was bubbled through the solution. Ammonium acetate (4 g) was added. The mixture was stirred for 6 h, filtered through a block of celite and concentrated to a solid. NMR indicated full conversion to the title product (3.1 g, 12.2 mmol). [00249] 1H NMR (400 MHz, CDCl3): 7.39-7.16 (m, 7H), 6.78-6.73 (m, 2H), 3.71 (t, 2H, J = 5.4 Hz), 2.26 (ddd, 2H, J = 2.6 Hz, J = 5.4 Hz, 9.2 Hz), 1.76-1.70 (2H, m), 1.64-1.57 (m, 2H).
Example 27 - Acetic acid 4-(2-phenyl-tetrahydro-pyran-2-yl)-phenyl ester
[00250] 4-(2-Phenyl-tetrahydro-pyran-2-yl)-phenol (500 mg, 2 mmol) was dissolved in pyridine (10 mL). Acetic anhydride (1 mL) was added at drop wise at 00C. The reaction temperature was allowed to raise to room temperature. After 1 h the reaction was quenched the addition of water (5 mL). Dichloromethane (20 mL) was added. The organic phase was washed with aqueous HCl (1 M, 2 x 10 mL), brine (10 mL), dried (Na2SO4) and concentrated in vacuo. Purified on a short column (eluent: dichloromethane). Yield: 560 mg.
[00251] 1H NMR (400 MHz, CDCl3): 7.39-7.66 (m, 2H), 7.32-7.19 (m, 5H), 7.02-7.00 (m, 2H), 3.73-3.69 (m, 2H), 2.25 (s, 3H), 1.73 (t, 2H, J = 6 Hz), 1.65-1.57 (2H, m), 1.30-1.25 (m, 2H).
Example 28 - Enzymatic Resolution
[00252] Acetic acid 4-(2-phenyl-tetrahydro-pyran-2-yl)-phenyl ester (50 mg, 0.17 mmol) was dissolved in a mixture of isopropylether (0.5 mL) and THF (0.2 mL). Phosphate buffer (100 mmol, pH = 1, 2 mL) and Amono Lipase AK, from Pseudomonas Fluorescens (50 mg) were added. Stirred at room temperature for 2 h. The mixture was filtered, dichloromethane (10 mL) was added. The organic phase was washed with water (10 mL), dried (Na2SO4) and concentrated in vacuo. Chiral LC-MS indicated that 4-(2- phenyl-tetrahydro-pyran-2-yl)-phenol was formed in an ee of 71%, the absolute configuration of the enantiomer was not determined.
Example 29 - 4-Phenyl-3.6-dihvdro-2H-pyridine-l-carboxylic acid 9H-fluoren-9- ylmethyl ester
[00253] 4-Phenyl-l,2,3,6-tetrahydropyridine (200 mg, 1.26 mmol) was dissolved in dry acetonitrile (5 mL ) and cooled in ice-water bath. N-(9- Fluoroenylmethoxycarbonyloxy)-succinimide (475 mg, 1.4 mmol) was added. Stirred over night at room temperature. TLC indicated full conversion of the starting material. An aqueous solution of saturated NaHCO3 (5 mL) was added and the mixture was extracted with dichloromethane (2 x 20 mL). The combined organic phase was dried (TS^SO4) and concentrated in vacuo. The product was isolated after column chromatography (gradient dichloromethane — > ethylacetate). Yield: 390 mg. The product was pure according to 1H nmr.
[00254] 1H NMR (400 MHz, CDCl3): 7.79 (d, 2H, J = 7.4 Hz), 7.63 (d, 2H, J = 7.4 Hz), 7.44-7.25 (m, 9H), 6.07 (br. s, IH), 4.49 (d, 2H, J = 6.9 Hz), 4.30 (t, IH, J = 6.9 Hz), 4.16 (br. s, 2H), 3.72 (br. s, 2H), 2.55 (br. s, 2H).
Example 30 - 4-(4-Hvdroxy-phenyl)-4-phenyl-piperidine- 1 -carboxylic acid 9H-fluoren-9- ylmethyl ester
[00255] 4-Phenyl-3,6-dihydro-2H-pyridine-l-carboxylic acid 9H-fluoren-9- ylmethyl ester (200 mg, 0.52 mmol) was treated with a drop of BF3*Η3PO4 and phenol (200 mg, 2.1 mmol) at 600C for 6h. Water (10 mL) was added and aqueous phase was extracted dichloromethane (2 x 10 mL). The combined organic phase was concentrated. 1H NMR indicated full conversion of the starting material. The desired product was isolated after flash chromatography. Yield: 270 mg. LC/MS purity (UV/MS): 96/91%, Rt 7.32 min. M+1: 432.
[00256] 1H NMR (400 MHz, CDCl3): 7.76 (d, 2H, J = 7.2 Hz), 7.58 (d, 2H, J = 7.2 Hz), 7.42-7.15 (m, 9H), 7.13-7.08 (m, 2H), 6.80-6.75 (m, 2H), 4.45 (d, 2H, J = 6.7 Hz), 4.23 (t, IH, J = 6.7 Hz), 3.52 (br. s, 4H), 2.30 (br. s., 4H).
Example 31 - 4-(4-Phenyl-piperidin-4-yl)-phenol
[00257] 4-(4-Hydroxy-phenyl)-4-phenyl-piperidine-l -carboxylic acid 9H- fluoren-9-ylmethyl ester (270 mg, 0.57 mmol) was treated with a solution of piperidine in DMF (20%, 5 mL) for 2 h. TLC indicated full conversion of the starting material. Concentrated in vacuo to a solid. Worked-up by flash chromatography. LC-MS indicated formation of a complex mixture. The title product was isolated in an analytical amount. LC/MS purity (UV/MS): 90/90%, Rt 3.15 min. M+l : 254
[00258] 1H NMR (400 MHz, CD3OD): 7.30-7.29 (m, 4H), 7.19-7.13 (m, 3H), 6.78-6.74 (m, 2H), 4.58 (br s, 3 H), 3.21 (t, 4H, J = 5.8 Hz), 2.66-2.62 (m, 4H).
Example 32 - 4-(4-Phenyl-tetrahvdro-pyran-4-yl)-phenol [00259] 4-Phenyl-tetrahydro-pyram-4-ol (20 mg) and phenol (100 mg) were melted and FeCl3 (few crystals) was added. The mixture was stirred at 500C for 1A h. GC- MS indicated full conversion to the desired compound. TLC revealed formation of a compound with an Rf value just below phenol (eluent: dichloromethane Phenol, Rf = 0.28, product, Rf = 0.26).
[00260] Dichloromethane (10 mL) was added. The organic phase was washed with water (10 mL), dried (Na2SO4) and concentrated in vacuo. Purified by flash chromatography (eluent 5% ethyl acetate in dichloromethane — > 25% ethyl acetate in dichloromethane). Yield: 32 mg. LC/MS purity (UV/MS): 100/100%, Rt 2.98 min. M-I : 253
[00261] 1H NMR (400 MHz, CD3OD): 7.29-7.22 (m, 4H), 7.14-7.07 (m, 3H), 6.72-6.67 (m, 2H), 3.71 (br. t, 4H, J = 5.4 Hz), 2.39 (br. t, 4H, J = 5.4 Hz).
Example 33 - 2-(l-Phenyl-cvclohexyl)-thiophene
[00262] Phenyl cyclohexene (50 mg) and thiophene (100 mg) were dissolved in 33% HBr in acetic acid (0.5 mL). The mixture was stirred at room temperature over night. TLC indicated full conversion of the starting material. Dichloromethane (10 mL) was added. The organic phase was washed with saturated NaHCO3 (3 x 10 mL), dried Na2SO4 and concentrated in vacuo. The desired product was afforded after flash chromatography (eluent: dichloromethane). Yield: 39 mg. LC/MS purity (UV/MS): 100/90%, Rt 6.22 min. M+l : 243
[00263] 1H NMR (400 MHz, CD3Cl): 7.29-7.17 (m, 4H), 7.12-7.07 (m, IH), 7.06 (dd, IH, J = 5.0 Hz, J = 1.0 Hz), 6.83 (dd, IH, J = 5.0 Hz, J = 3.5 Hz, IH), 6.71 (dd, J = 3.5 Hz, J = 1.0 Hz, IH), 2.35-2.24 (m, 4H), 1.62-1.35 (m, 6H).
Example 34 - 4-(l-Pyridin-3-yl-cvclohexyl)-phenol
[00264] The pyridine-boronic acid (1 g, 8.1 mmol), 1-cyclohexenyl-l- trifluoromethanesulfonate the triflate (2 g, 8.1 mmol) (TLC indicated that the triflate was partly decomposed (estimated purity 30%)) and potassium fluoride (200 mg) were suspended in dioxane. Argon was bubbled through the mixture. The palladium catalyst (50 mg) was added and the reaction mixture was stirred at 1000C for 20 h. The mixture was stirred at 1000C for 1 day. Dichloromethane (20 mL) and aqueous HCl (1 n, 20 mL) were added. The organic phase was separated off. The aqueous phase was neutralised with saturated NaHCO3 (pH 7-8) and extracted with dichloromethane (2 x 30 mL). The combined organic phase was dried (Na2SO4) and concentrated in vacuo. The desired product was obtained after purification with flash chromatography. Yield: 150 mg syrup.
[00265] The syrup (75 mg) was dissolved in phenol (200 mg). Triflic acid (1 drop) was added. Stirred at 800C over night. The desired product was afforded after purification by flash chromatography. Yield: 25 mg. LC/MS purity (UV/MS): 100/100%, Rt 3.20 min. M+ 1: 254
[00266] 1H NMR (400 MHz, CD3Cl): 8.49 (d, J = 2.2 Hz, IH), 8.36 (dd, J = 4.9 Hz, J = 1.5 Hz, IH), 7.62 (ddd, IH, J = 8.0 Hz, J = 2.2 Hz, J = 1.5 Hz), 7.24 (dd, IH, J = 8.0 Hz, J = 4.9 Hz, IH), 7.10-7.06 (m, 2H), 6.76-6.72 (m, 2H), 2.33-2.16 (m, 4H), 1.64- 1.46 (m, 6H).
Example 35 - l-(4-Hydroxy-phenyl)-cvclohexanecarbonitrile
[00267] l-(4-Methoxyphenyl)-l-cyclohexanexanecarbonitrile (100 mg, 0.46 mmol) was dissolved in dichloromethane (5 mL) and a solution of boron tribromide in dichloromethane (IM; 0.5 mmol, 0.5 mL) was added dropwice. Stirred at room temperature for Ih, concentrated. 1H NMR the reaction had proceeded 20%. The syrup was dissolved in dichloromethane and a solution of boron tribromide in dichloromethane (1 mL, 1.0 mmol) was added. The solution was stirred for 24h, concentrated and worked- up by flash chromatography. Yield: 82 mg. . LC/MS purity (UV/MS): 100/90%, Rt 6.0 min. M- 1 : 200
[00268] 1H NMR (400 MHz, CD3Cl): 7.34-7.29 (m, 2H), 6.87-6.83 (m, 2H), 2.16-2.10 (m, 2H), 1.90-1.66 (m, 8H).
Example 36 - Thiophene-3-yl cvclohexene
[00269] 3-Thiophene boronic acid (128 mg, 1.00 mmol), cyclohexenyl triflate (230.2 mg, 1.00 mmol) was dissolved in Et2O (4 mL) and 2M Na2CO3 (1 mL). The mixture was degassed and Pd(PPh3)4 (0.05 mmol) added. After stirring at room temperature for 4h the mixture was filtered through celite, rinsed with Et2O, dried (Na2SO4) and subjected to column chromatography (silica, pentane) to yield 150mg of a volatile liquid. GC-MS: M+ = 164.
[00270] 1H-NMR (400 MHz, CDCl3): 7.26-7.23 (m, 2H), 7.09-7.07 (m, IH), 6.19-6.17 (m, IH), 2.43-2.38 (m, 2H), 2.22-2.18 (m, 2H), 1.81-1.75 (m, 2H), 1.69-1.63 (m, 2H). 13C-NMR (125 MHz, CDCl3): 144.3, 132.1, 125.4, 124.9, 124.1, 118.0, 27.5, 25.8, 23.1, 22.5.
Example 37 - l-(4-Hydroxyphenyl)-thiophene-3-yl cyclohexane
[00271] Thiophene-3-yl cyclohexene (72.3 mg, 0.44 mmol), and phenol (82.8 mg, 0.88 mmol) was mixed and gently heated until uniform and BF3 115H3PO4 (4 uL, 0.044 mmol) was added. The mixture was heated at 70 0C overnight and the dark residue was diluted with EtOAc, washed with sat. NaHCO3, dried and concentrated. Column chromatography (silica, 0-20% EtOAc) gave a mixture of isomers and the desired product could be isolated by preparative TLC (DCM eluent). Yield: 3.7 mg, off-white/pale yellow solid. GC-MS: M+=258.
[00272] 1H-NMR (400 MHz, CDCl3): 7.20 (dd, J=5.08Hz, 2.93Hz, IH), 7.13 (d, J=8.80Hz, 2H), 6.92 (dd, J=2.93Hz, 1.37Hz, IH), 6.87 (dd, J=5.08Hz, 1.37Hz, IH), 6.74 (d, J=8.80Hz, 2H), 2.21-2.17 (m, 4H), 1.57-1.44 (m, 6H).
Example 38 - Receptor Selection and Amplification Technology Assay
[00273] The functional receptor assay, Receptor Selection and Amplification Technology (R-SAT™), was used with minor modifications from the procedure described in U.S. Patent No. 5,707,798, which is hereby incorporated by reference in its entirety, to screen compounds for efficacy at the Estrogen receptors alpha and beta (ERa, ERβ). NIH3T3 cells were grown in roller bottles to 70-80% confluence. Cells were then transfected for 12-16 h with plasmid DNAs using Polyfect (Qiagen Inc.) as per the manufacturer's protocol. R-SAT assays were typically performed by transfecting 30 ug/bottle of receptor and 50 ug/bottle of β-galactosidase plasmid DNA. All receptor and helper constructs used were in mammalian expression vectors. Helpers are defined as signaling molecules that modulate both ligand-dependent and/or ligand-independent function of the ER receptors, typically co-activators and kinases. NIH3T3 cells were transfected for 12-16 h, then trypsinized and frozen in DMSO. Frozen cells were later thawed, plated at 10,000-40,000 cells per well of a 96 well plate containing 4-(l-Phenyl- cyclohexyl)-phenol. Cells were then grown in a humidified atmosphere with 5% ambient CO2 for five days. Media was then removed from the plates and marker gene activity was measured by the addition of the β-galactosidase substrate o-nitrophenyl β-D- galactopyranoside (ONPG, in PBS with 5% NP-40). The resulting colorimetric reaction was measured in a spectrophotometric plate reader (Titertek Inc.) at 420 nM. All data were analyzed using the computer program XLFit (IDBSm).
[00274] These experiments provided a molecular profile, or fingerprint, for each agent tested at the human Estrogen receptors. As can be seen in Table 1 and Figure 1, 4-(l -Phenyl -cyclohexyl)-phenol (ERB-002) selectively activates Estrogen beta receptor (ERβ) relative to the Estrogen alpha receptor (ERa).
TABLE 1
Figure imgf000077_0001
Efficacy is relative to the reference ligand Estrone.
Example 39 - CFA induced arthritis rat model assay
[00275] Naϊve, male Sprague-Dawley rats (225 - 250 g; n = 6 per group) served as subjects. Response latencies to a noxious thermal stimulus were measured using the 52°C hot plate test. After obtaining baseline responses, 0.1 ml of Freund's complete adjuvant (CFA) or vehicle (inactivated CFA (iCFA)) was injected into the dorsal surface of the left hind paw. Response latencies were again measured 4 days following CFA (or iCFA) administration, a time point when thermal hyperalgesia is stable. A significant decrease in the hot plate latency was interpreted as the presence of thermal hyperalgesia. Following testing, thickness of both hind paws were measured (using a micrometer) in order to quantify possible edema formation at the injection site. Various doses of 4-(l-Phenyl-cyclohexyl)-phenol (ERB-002) (1.0, 3.0 or 10 mg/kg) or vehicle (DMSO) were administered (s.c.) following testing on Day 4, and then daily following testing for a period of 3 days. Figure 2 illustrates the dose dependent reversal of thermal hyperalgesia in this model. Figure 3 illustrates the dose dependent reversal of edema in this model.
Example 40 - Uterotrophic in vivo assay
[00276] The effect of ERB-002 on uterine weight was assessed based on the previously published method of Harris et al, Endocrin, 2003, 143:4172, which is hereby incorporated by reference in its entirety. Naϊve, female Sprague-Dawley rats (30 - 40 g; n = 6 per group) served as subjects. Rats received daily subcutaneous injections of vehicle (100% DMSO), PPT (1.0 mg/rat) ), a reportedly selective ERa agonist (Stauffer, 2000, J Med Chem 43:4934) or various doses of ERB-002 (10, 30 or 100 mg/kg) for a total of 3 days. Approximately 24 hours after the final injection, the rats were sacrificed, the uteri removed, trimmed of adhesions, fluid expelled and then weighed. Uterine weight was normalized as a percentage of total body weight by the following formula: %TBW = [(uterus weight ( in mg) / 1000) / (body weight (in g))] * 100. Figure 4 illustrates that ERB-002 does not display uterotrophic properties in vivo in immature female rats.
Example 41 - The Effect of ERβ2 Agonists (ERB-002) in the Dry Eye Blower Model
[00277] The blower model treatment used for this experiment includes running fans directly at mouse cages with wire screens, along with scopolamine injections subcutaneously TID for the total of four days. The timeline for desiccating treatment was for four days before tissues were collected. Mice used for this experiment were BALB/c Wild type female at the approximate age range of 6-8 weeks old. Tear production was measured for all animals before and after blower treatment. All animals' tear were collected before and after blower treatment for the evaluation of inflammation and signaling markers using the Luminex IS 100 system. The tear collection process was as follows:
• Prepared 0.1% BSA Assay Buffer (Beadlyte Cytokine Assay Buffer from Upstate Cat. # 43-002 + Bovine Albumin Serum (BSA) Sigma Cat # A2153- 50GM)
• Added 6 μL of Assay Buffer to each 0.5 mL micro tube (Sarstedt Catalog # 72.730.106) and placed tubes on ice • Collected 1 μL of tear per eye, both eyes from each mouse
• Collected 4 μL total from 2 mice in same group per micro tube
• Stored micro tubes in -80 0C freezer
[00278] At tissue collection time, tissues, including OD, OS eyes, and right and left lacrimal glands, were retrieved. Compounds, including ERB-002 and vehicle, were administered BID for the total of 4 days. Two administration routes were used: topical and intra-peritoneal (IP). Two tested concentrations via IP were at 1 mg/kg/dose and 10 mg/kg/dose. Note that the animal group, which was dosed with higher concentration of ERB-002 (10 mg/kg/dose) was immediately altered to lower dosage (5 mg/kg/dose) to eliminate observed adverse effects to subjects. For topical, ERB-002 applied concentration was 1 mg/mL at 5 μL/eye/dose for both eyes.
[00279] The following groups were studied: 1) control group, which were not blower treated, nor were drug or vehicle treated (n=5); 2) Group 1, blower treated but were not drug or vehicle treated (n=5); 3) Group 2, blower treated, received vehicle (PEG300) IP BID (n=4); 4) Group 3, blower treated, received ERB-002 1 mg/kg IP (n=3); 5) Group 4, blower treated, received ERB-002 5 mg/kg IP (n=5); 6) Group 5, blower treated, received vehicle (PEG300) topically (n=5); 7) Group 6, blower treated, received ERB-002 1 mg/mL (5 μL/eye) topically (n=5).
[00280] Tear production was measured in all groups. The decrease in the percentage of tear production from baseline was normalized with respect to the control group. The results are shown in Table 2.
TABLE 2
Figure imgf000079_0001
[00281] The ERβ agonist (ERB-002) given IP at 1 mg/kg or 5 mg/kg BID to mice for the 4 days of blower treatment had a decrease in tear production (81% and 73%, respectively) from baseline as compared to a 43% decrease of blower only treatment or a 60% decrease in tear production of blower with PEG300 (vehicle) treatment. Topical ERB-002 (1 mg/mL, BID, 5 μL/eye) had no effect over topical PEG300.
[00282] Luminex analysis of mouse tear samples collected on the morning of day 5 detected a decrease in IFN-γ tear levels compared to the blower only mice in the presence of 1 mg/kg and 5 mg/kg IP ERB-002 (Figure 5A (n=5 unless marked otherwise; AC74131 is another designator for ERB-002)). Topical ERB-002 had no effect on IFN-γ levels as compared to the blower only group. Interestingly, tears from blower treated mice lacked detectable levels of IL-4 (Figure 5B (n=5 unless marked otherwise)). IL-4 levels were increased in the tears of blower mice treated with 1 mg/kg of ERB-002 administered IP. Topical ERB-002 resulted in increased IL-4 levels (2 pg/mL). ERβ agonists in some models have been reported to increase TH2 cytokine production.
[00283] A protective effect of ERB-002 on the mucosa in the blower model was observed. Control mice had an average of 98.5 goblet cells in the conjunctival fornix area. Blower treated and IP vehicle treated animals had an average of 33.7 and 58.8 goblet cells in the conjunctival fornix respectively. Treatment with 1 mg/kg or 5 mg/kg ERB-002 IP resulted in an increase in goblet cell numbers as compared to the vehicle control (80.8 and 87.75 goblet cells, respectively).
[00284] Conclusions: Luminex analysis of tears suggests that IFN-γ (THl) levels are decreased and IL-4 (TH2) levels are increased in the presence of ERB-002. A mucosal protective effect of systemic ERB-002was observed in the dry eye model.
Example 42 - Effect of ERβ Agonist (ERB-002) in the Mouse Multi-Hit Allergic Conjunctivitis Model (TH2)
[00285] ERB-002 was applied to the mouse model of multi-hit allergic conjunctivitis using BALB/c background female mice with age range at approximately 6- 8 weeks old. Animals in all groups, with the exception of the control group, were sensitized with Short Ragweed (SRW) in Alum, via left hind paw injection of 50 μL per mouse, ten days prior to the secondary challenge. One day before the animals were subjected to secondary challenge, mice were pre-dosed with ERB-002 and vehicle at different concentrations and administration routes. For intra-peritoneal, the tested concentrations were 100 μg/kg/dose/mouse and 500 μg/kg/dose/mouse at BID. For topical administration, the concentration was 5 μg/5 μL/dose/eye at BID for both eyes. The animals were dosed with drug and vehicle for a total of 8 days while they were topically challenged with SRW/PBS for 7 days. Eye pictures were obtained at various time points, which occurred before sensitization, after 1st SRW/PBS challenge, and after 7th challenge. Clinical scoring of the subjects' eyes was recorded by two operators after the 1 st and 7th challenge.
[00286] Immediately before tissue collection session, which was one day after the 7th challenge, tears from all animals were collected as follows:
• Prepared 0.1% BSA Assay Buffer (Beadlyte Cytokine Assay Buffer from Upstate Cat. # 43-002 + Bovine Albumin Serum (BSA) Sigma Cat # A2153- 50GM)
• Added 6 μL of Assay Buffer to each 0.5 mL micro tube (Sarstedt Catalog # 72.730.106) and placed tubes on ice
• Collected 1 μL of tear per eye, both eyes from each mouse
• Collected 4 μL total from 2 mice in same group per micro tube
• Stored micro tubes in -80 0C freezer
[00287] At the tissue collection session, the following specimens were obtained: blood, OD & OS eyes, and right & left lacrimal glands.
[00288] Mice in each group (n=5) were given a score in the following categories: lid edema, hyperemia, chemosis, and tearing. Scores were given as follows: 0 = absent; 1 = minimal; 2 = mild; 3 = moderate; and 4 = severe.
[00289] The scores were averaged for the mice in each group. The results are shown in Figures 6A-6D (AC74131 is another designator for ERB-002). As determined by clinical pictures and scoring of eyes for lid edema, hyperemia, chemosis, and tearing, 100 μg/kg and 500 μg/kg IP and 1 mg/mL topical administration of ERB-002 significantly reduced these allergic inflammatory disease phenotypes as compared to SRW sensitized and multi-hit challenged animals in the absence or presence of PEG300 IP or topically. Thus, ERB-002 given IP or topically significantly decreased lid edema, hyperemia, chemosis, and tearing.
[00290] SRW sensitized and multi-hit challenged animals had increased tear levels of IL-4, as shown in Figure 8A (n=5 for all groups) (PEG300 is vehicle). In the figures, "S+C" refers to the group of mice who received SRW sensitization and challenge. However, there was no increase in IL- 12 levels in these mice, as shown in Figure 8B (n=5 for all groups) (PEG300 is vehicle). ERB-002 (100 μg/kg IP) treatment of SRW sensitized and multi-hit challenged mice reduced IL-4 levels in mouse tears to basal levels (10 pg/mL). IL- 12 tear levels were reduced by 75% in the presence of topical ERB-002. Thus, ERB-002 reduced IL- 12 levels in the tears of SRW multi-hit mice. IL- 12 induces IFN-γ production. IFN-γ is required for upregulation of VCAM-I on endothelial cells. VCAM-I binds to T cells and eosinophils and permits their access to the site of inflammation.
Example 43 - Effect of ERβ2 Agonist (ERB-002) in Rodent Models of Neuropathic Pain
[00291] ERB-002 was tested in models in which sensory nerve pathways are sensitized by chemical or surgical insults, resulting in either spontaneous pain (capsaicin model) or pain sensation to normally non-noxious tactile stimuli (chemical allodynia and nerve ligation models). The pain sensitivity takes anywhere from 15 min to 1 week to fully develop. These models are distinct from models of acute nociceptive pain in which there is an immediate pain response to a noxious stimulus such as heat or inflammatory mediators.
[00292] ERB-002 was effective over a wide dose range (0.5 mg/kg - 10 mg/kg) while the non-selective estrogen receptor agonist, β-estradiol, was not active even at a high dose of 10 mg/kg. The estrogen receptor antagonist ICI 182,780 could block the actions of ERB-002 (see mouse allodynia model data), demonstrating that its actions are estrogen receptor mediated.
Intrathecal Injections in Mice:
[00293] Male Black6-C57 mice (20-30 grams) were intrathecally injected according to the method devised by Hylden and Wilcox (Eur J Pharmacol, 67: 313-316, 1980). A sterile 30-gauge 1A inch needle attached to a microsyringe was inserted between the L5 and L6 vertebrae. The mouse was held firmly by the pelvic girdle in one hand, while the syringe was held in the other hand at an angle of approximately 200 above the vertebral column. The needle was inserted into the tissue to one side of the L6 spinous process so that it slipped into the groove between the spinous and transverse processes. The needle angle was then decreased to about 100 and slowly advanced forward into the intervertebral space. (A pop was felt at this point along with a visible serpentine tail movement allowing the investigator to assure of being in the correct position.) Compounds were slowly injected in the subarachnoid space of conscious mice in a volume of 5 μL. Extensive studies with dye and radiolabeled compounds confirmed that the distribution of these intrathecal compounds remained concentrated near the injection site and never extended rostral of the thoracic segments and were consistently intradural.
Mouse Allodvnia Studies:
[00294] Studies on Allodynia were carried out essentially according to the method of Yaksh and Harty (1998). All compounds were injected intrathecally in 5 μL volume or IP in a 1 mL/kg volume, as described above. The test compounds were injected IP 15 min prior to injection of the allodynia- inducing agents (300 ng/kg IP sulprostone in DMSO; 100 ng/kg IP phenylephrine (PE) in H2O; 100 ng IT N-methyl D- aspartate (NMDA) in DMSO). The mice were then assessed for allodynia once every 5 minutes over a 15-50 min period post injection of allodynic agent by light stroking of the flank with a paintbrush. The allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape. Data was expressed as the average total score for each group. (Each animal can have a maximum score of 16 over the 50- min period.) Each group of mice comprised 5-6 animals per group. The allodynic agents typically caused a pain score of 14 and the vehicle controls typically caused a pain score of 4-5.
[00295] The results fo the experiments are shown in the following tables.
Figure imgf000083_0001
Figure imgf000083_0002
Figure imgf000084_0002
All test estrogen receptor compounds were administered in a 50% DMSO vehicle.
Chung Surgery:
[00296] Male sprague-dawley rats (100-120 grams) were anesthetized through inhalation of an isoflourane/oxygen mix. The surgical site was shaved and prepared with betadine. An incision was made from the thoracic vertebra XIII toward down the sacrum. The muscle was separated from the spinal vertebra (left side) at the L4 - S2 levels. The L6 vertebra was located then the transverse process was carefully removed with a small rongeur to visually identify the L4 - L6 spinal nerves. The L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread. A complete hemostasis was confirmed, then the wound was sutured. The duration of the surgery was approximately 20 minutes. A small amount of antibiotic ointment was applied to the incised area and the animals were transferred to a plastic recovery cage under a regulated heat-temperature lamp. Animals were not n any topical or local anesthetics post-operatively because they would inhibit the development of the pain syndrome, which was the phenomenon to be studied.
Chung Testing:
[00297] Allodynia was assessed by applying a light tactile stimulus (Von Frey hairs) to the affected surgical paw. Von Frey hairs were applied in an up-down manner depending on the response (Dixon et al 1980) until the 50% threshold was established. The Von Frey hairs were applied to the plantar surface of the surgical paw with just enough force to bend them. A positive response was recorded if the paw was sharply withdrawn. Eight VonFrey hairs used 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18.
Von Frey Analysis:
[00298] Von Frey analysis was performed using the following equation:
. . Λ 11 , . , ^
% Allodynia reversal 100
Figure imgf000084_0001
Mean + SEM:
Mean = average of allodynia reversals (n=6)
SEM = STDEV/Vn
[00299] ERB-002 and β-estradiol were dosed IP (in 50% DMSO) using 3 different regimens at indicated concentrations:
1. a single dose followed 30 min later by assessment of allodynia to assess acute drug effect
2. a single dose followed 6 hr later by assessment of allodynia to determine prolonged effect
3. three doses given at 3 hr intervals followed 30 min later by assessment of allodynia to determine continuous effect
[00300] β-Estradiol was not active using either regimen 1 or regimen 3. ERB- 002 was active with a single 10 mg/kg dose assessed 30 min later (regimen 1; 91±6% allodynia reversal) and with a lower 0.5 mg/kg dose given 3 times over 6 hrs and assessed 30 min after the last dose (regimen 3; 72±10% allodynia reversal).
Capsaicin Model
[00301] A solution of 0.3% caspacin was prepared as follows. Caspaicin (1 g) was discsolved in 2 mL of ethanol. To this solution, 10 mL of a cocktail of 0.7 mL TWEEN 80 and 9.3 mL of saline was slowly added while stirring on a hot plate until the volume is reduced to 10 mL. The solution is then dissoved in enough ethanol to result in a 0.3% solution.
[00302] The animals were allowed to acclimate for 20 minutes prior to testing. Baseline data were taken for 3 days before the study was run. For baselines, the plantar surface of the left paw was poked with the 5.07 Von Frey Hair ten times in succession for a series of 3 trials each day (approximately 10 min between each trial). Animals that did not respond by withdrawing their paws at all or respond an average of 4 or higher times/trial were excluded. After the baseline data were obtained on the day of the study, the test compound or vehicle was injected by IP, IV or oral routes (1 ml/kg volume), 15 minutes before the capsaicin injection. Capsaicin (0.3%) was injected into the plantar surface of the left hind paw in a 10 μL volume and the left paw was then tested in the manner described above at 15, 30 and 60 minutes post capsaicin. [00303] A single 10 mg/kg IP dose of ERB-002 (in 50% DMSO vehicle) completely prevented the tactile hyperalgesia occurring 30 min after capsaicin injection.
Example 44 - Effect of ERβ Agonist (ERB-002) in Rodent Models of Colitis
[00304] 8 week-old mice (n=10 per group) were injected intra-peritoneally with 5% dextran sulfate, 4 mg/kg indomethacin, to induce severe intestinal epithelial injury. One group of mice received ERB-002, injected concomittently (10 mg/kg), whereas another group of mice received vehicle only. Mice were then monitored daily for body weight, diarrhea, food/water intake, and alertness. Table 3 shows the results of the comparison between the two groups.
TABLE 3
Figure imgf000086_0001
[00305] The observed body weight loss and diarrhea in each mouse was given a score according to the severity of the symptom. The scores were as follows: 1 = minimal; 2 = mild; 3 = moderate; and 4 = severe. The results comparing the control group (vehicle) with the treatment group (ERB-002) are shown in Figures 9A (weight loss) and 9B (diarrhea). In the figures, ERB-002 is referred to as ERB-131.
Example 45 - Protection Against Inflammatory Pain by a Selective Estrogen Receptor β- Agonist
Introduction
[00306] Among the plethora of physiological functions involving estrogens is their ability to modulate inflammatory as well as nociceptive processes. For instance, non selective estrogens such as 17β-estradiol display anti-inflammatory properties in certain animal models (Josefsson et al. 1992; Jansson and Holmdahl 1998; Miyamoto et al. 1999; Cuzzocrea et al. 2001). More recently, animal studies have emphasized that the sole activation of estrogen receptor beta is sufficient to alleviate inflammation in a number of in vivo paradigms (Harris 2006).
[00307] In animal models of inflammatory pain, estrogens have been ascribed both pro- and anti-nociceptive properties. For example, in the formalin test, female rats display increased flinching responses, when compared to males (Gaumond et al. 2002). In contrast, estradiol administration in female rats attenuates both inflammatory and pain behaviors in different models (Kuba et al. 2005; Mannino et al. 2006).
[00308] Estrogens classically mediate their actions through two distinct estrogen receptors (ERs): ER alpha (NR3A1, ESRl, ERa) and ER beta (NR3A2, ESR2, ERβ), which act as inducible transcription factors. Expression studies indicate that estrogen receptors are expressed in a number of tissues and cells that are implicated in inflammation and pain sensation. For instance, both estrogen receptors are expressed in classical immunocompetent cells such as peripheral macrophages, leukocytes and microglial cells (Baker et al. 2004; Ghisletti et al. 2005; Stygar et al. 2006). Estrogen receptors are also present in immune-like dorsal root ganglia cells including endothelial and dendritic cells (Evans et al. 2002; Nalbandian and Kovats 2005). Expression of ERs is also evident in DRG sensory neurons, with neurons expressing either or both subtypes (Papka and Storey- Workley 2002).
[00309] Because recent work has highlighted a role for ERβ agonism in inhibiting inflammatory (Harris 2006) and nociceptive states (Piu et al. 2007), we investigated the role of selective activation of ERβ in influencing inflammatory pain states.
Materials and Methods: Animals
[00310] Naϊve, male Sprague-Dawley rats (225 - 250 g; n = 6 per group except for formalin testing where n = 11) served as subjects for the present experiments. All animal studies were conducted in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals.
Carrageenan model of acute inflammatory pain
[00311] Acute inflammatory pain was produced by injecting 0.1 ml of 2% λ- carrageenan (Sigma, St. Louis, MO) into the left hind paw of rats. Three hours after carrageenan injection, rats were tested for their responsiveness to noxious thermal stimuli. Test compounds were administered 3 hr following carrageenan injection. Rats were tested at various time-points following compounds administration for up to 2 hours. Response latencies to a noxious thermal stimulus were measured using the 52 0C hot plate test. Additionally, paw thickness was measured, using a micrometer, immediately following testing as a measure of potential anti-inflammatory activity.
Formalin model of persistent pain
[00312] To quantify formalin paw-injection- induced flinching/licking behavior, an automated nociception analyzing system was used (Yaksh et al. 2001). Briefly, a C- shaped metal band was placed on the left hind paws of rats approximately 16 hr prior to testing. After acclimation in test chambers for 40 min, rats were injected subcutaneously into the dorsal surface of the banded paw with 20 μl of 5% formalin solution using a 30- gauge needle. Data collection was initiated immediately after the animal was placed inside the test chamber. Nociceptive behavior was quantified by automatically counting incidences of spontaneous flinching/shaking of the injected paw (Yaksh et al. 2001). The flinches were counted over 1 -min intervals for 60 min and are presented as total number of flinches per 5 min or per phase (phase 1 = 0-10 min; phase 2 = 10.1-60 min).
Complete Freund's Adjuvant model of chronic inflammatory pain
[00313] Response latencies to a noxious thermal stimulus were measured using the 52°C hot plate test. After obtaining baseline responses, 0.1 ml of Freund's complete adjuvant (CFA) or vehicle (iCFA: inactivated CFA) was injected into the dorsal surface of the left hind paw. Response latencies were again measured 4 days following CFA (or iCFA) administration, a time point when thermal hyperalgesia is stable. A significant decrease in the hot plate latency was interpreted as the presence of thermal hyperalgesia. Following testing, thickness of both hind paws were measured (using a micrometer) in order to quantify possible edema formation at the injection site. Test compounds were administered sub-cutaneously following testing on Day 4, and then dosed once daily following testing for an additional period of 3 days.
[00314] The hot plate latency was determined by placing rats in a plexiglass enclosure on a thermostatically controlled metal plate maintained at 52°C (Columbus Instruments, Columbus, OH). The time elapsed until the rat demonstrated an obvious nociceptive response (i.e., licking/elevating the hind paw) was measured. Animals were tested before and at various time-points following drug administration. A significant (p ≤ 0.05) reduction in the hot plate latency was interpreted as the presence of thermal hypersensitivity. A cut-off time of 25 sec was employed in order to prevent tissue damage.
Results
[00315] Characteristics of ERb-131. A class of non-steroidal ERβ selective agonists, of which ERB-131 (also referred to herein as ERB-002) represents a prototype lead have been previously reported (Olsson et al. 2005; Piu et al. 2007). The biochemical characteristics of ERB-131 are summarized in Table 4, where the biochemical characteristics of ERB-131 are presented. Data are from two functional assays (R-SAT® and Luciferase) and binding assays. The activity of ERB-131 is compared to estrone, a reference non selective estrogen ligand and to two relatively selective ERβ agonists, genistein and daidzein. Data is reported as potency (EC50, K;, nM : average ± standard deviation) and N = number of replicates. Summarized from Piu et al. 2007.
TABLE 4 ERB- 131 is a selective ERb agonist
R-SAT Luciferase Binding
ER alpha ER beta ER alpha ER beta ER alpha ER beta
EC50 N EC50 N EC50 N EC50 N Ki N Ki N
ERb-131 3160 ± 1640 86 33 ± 21 8S 15800 ± net 1 21 ± 15 8 17940 ± πd 1 50 ± 30 3
Estrone 0 131 0 08 154 0 15 ± 0 08 174 0 56 ± 0 31 29 041 ± 022 51 008 ± 0 03 2 079 ± 0 47 3
Genistein 25t0 ± 2500 5 29 ± 12 5 500 ± 100 6 18 ± B 8 79 ± πd 1 3 ± nd 1
Daidzein 3980 ± 190 5 250 ± 180 5 740 ± 140 6 54 ± 35 6
[00316] In the cellular proliferation assay R-SAT®, ERb-131 displays potent agonism at ERβ (EC50 = 33 ± 21 nM), and weak agonism at ERa (EC50 = 3160 ± 1640 nM). In comparison, the natural estrogen ligands genistein and daidzein exhibit potencies at ERβ of 29 ± 12 nM and 250 ± 180 nM, respectively. ERB-131 presents properties of a classical nuclear receptor ligand as evidenced by its ability to modulate ERβ-dependent transcriptional activity and to physically interact with the ERb receptor. In these assays, and consistent with the R-SAT® data, ERB- 131 binds with high affinity to ERβ (K; = 50 ± 30 nM) and stimulates transcription through a synthetic ER response element (EC50 = 21 ± 15 nM), while weakly interacting at ERa (K; = 17940 nM, EC50 = 15800 nM). In addition, no significant activities at up to 10 μM were found at other nuclear receptors, including the steroid hormone receptors (Piu et al. 2007). Therefore, ERB- 131 constitutes a potent ERβ agonist with a selectivity over ERa and other nuclear receptors of over 100 fold. Furthermore, in an in vivo bioassay of ERa activity (rat uterotrophic paradigm), ERB-131 was found to lack any significant ERa activity in vivo at doses as high as 100 mg/kg after several days of exposure (Piu et al. 2007).
[00317] Inflammatory pain. To understand the contribution of ERb agonism in inflammatory and nociceptive processes, ERB-131 was evaluated in animal models of acute and chronic inflammatory pain.
[00318] Carrageenan model of acute inflammatory pain. ERB-131 was administered at the doses of 1, 3 and 10 mg/kg based on previous studies showing that ERB-131 is efficacious in animal models of neuropathic pain at doses ranging from 1-10 mg/kg (Piu et al. 2007). Thermal hyperalgesia was assessed using the hot plate latency test. Rats were injected once with carrageenan (2%) to induce a stable state of inflammation and thermal hyperalgesia. Three hours later, ERB-131 was injected (1, 3, 10 mg/kg) and its effects followed for a period of 2 hrs. Response to thermal hyperalgesia was measured using the 52 0C hot plate test and evaluated at 30, 60, 90 and 120 min post ERB-131 injections. Latencies, expressed in seconds (sec), were defined as the time needed for the animal to physically remove the treated paw from the hot surface. Hyperalgesia and inflammation measures are presented as AVG ± STD. P values were calculated compared to vehicle treated animals, using a two-tailed unpaired t-test. The data are shown in Fig. 9A and Table 5. Legends are: sham + vehicle (filled square, dotted line), sham + 10 mg/kg ERB-131 (filled diamond, dotted line), carrageenan + vehicle (filled circle, dotted line), carrageenan + 1 mg/kg ERB-131 (filled square, plain line), carrageenan + 3 mg/kg ERB-131 (filled circle, plain line), carrageenan + 10 mg/kg ERB- 131 (filled diamond, plain line). Base = naϊve response latency; PIB = Post inflammatory latency, determined 3 hours after injection of carrageenan or sham.
TABLE 5 Activity of ERB-131 in the carrageenan model Vehicle ERb-131 (1) ERb-131 (3) ERb-131 (10)
Hyperalgesia latency (sec) 6.9 ± 0.2 7.1 ± 0.3 7.0 ± 0.4 7.1 ± 0.3 p value 0.591 0 787 0 5S1
Edema paw width {%} 105 7 ± 1 S 140.8 ± 4 2 1450 ± 1 5 145 a ± 1.7 p value 0.156 0296 0.416
[00319] Carrageenan treated animals had a significantly decreased latency compared to vehicle treated animals (p value < 0.0001), with values averaging 6.9 ± 0.2 sec vs 11.0 ± 0.3 sec, respectively. In addition, the effect of carrageenan was stable over the 2hr course of the experiment. At none of the doses tested was ERB-131 capable of alleviating hyperalgesia. The hot plate latencies did not significantly differ from the carrageenan animal group: 7.1 ± 0.3 (p value = 0.591), 7.0 ± 0.3 (p value = 0.787) and 7.1 ± 0.3 sec (p value = 0.591) at 1, 3, 10 mg/kg ERB-131 vs carrageenan treated rats, respectively. Further, rats injected with carrageenan presented a significant edema compared to vehicle treated animals: 148.8 ± 3.1 % vs 105.7 ± 1.8 % (p value < 0.0001), respectively.
[00320] Inflammation was assessed by the formation of local edema from the treated paw. The edema was quantified at the end of the experiment, i.e. at 120 min. The paw width was normalized to the change seen in the contralateral (untreated) paw. The data are shown in Fig. 9B and Table 5.
[00321] Administration of ERB-131 did not improve the inflammatory response, at either doses tested. The edema values were 140.8 ± 4.2 % (p value = 0.156), 145.0 ± 1.5 % (p value = 0.296) and 145.8 ± 1.7 % (p value = 0.416) for the doses of ERB-131 of 1, 3, 10 mg/kg, respectively.
[00322] To ensure that the effects induced by carrageenan were reversible, the pain suppressor gabapentin (10, 30, 100 mg/kg i.p.) was used as a positive control. Gabapentin dose-dependently alleviated hyperalgesia with an ED50 of approximately 43 mg/kg (p value < 0.0001). Hyperalgesia was assessed using the 52°C hot plate test, measured at 60, 120, 180, 240 and 300 min. The data are shown in Fig. 1OA, where Legends are: sham + vehicle (filled square, dotted line), carrageenan + vehicle (filled circle, dotted line), carrageenan + 10 mg/kg gabapentin (filled square, plain line), carrageenan + 30 mg/kg gabapentin (filled circle, plain line), carrageenan + 100 mg/kg gabapentin (filled diamond, plain line). Base = naϊve response latency; PIB = Post inflammatory latency, determined 3 hours after injection of carrageenan or sham. The anti-hyperalgesic effect was transient, peaking after one hour and decreasing within 3-4 hours, as previously reported (Urban et al. 2005). Gabapentin failed to alleviate edema at either doses tested (p value = 0.289). Edema was determined at the outcome of the experiment (t = 300 min), normalize relative to the untreated contralateral paw. The data are shown in Fig. 1OB. The results are consistent with published literature. Therefore, the lack of efficacy of ERB- 131 in the carrageenan model is not the result of an irreversible effect of carrageenan, but rather a true reflection of ERβ activity in a reverse paradigm of carrageenan induced inflammatory pain.
[00323] Formalin model of persistent inflammatory pain. Formalin measures pain sensations to a continuous noxious stimulus and results in a characteristic biphasic response, with a transient Phase I (10 min) relating to behaviors associated with acute pain and nociception and a longer-lasting Phase II (> 60 min) corresponding to inflammatory pain. As expected, formalin induced significant pain with a biphasic modality, the phases I and II averaging 222 ± 21 and 1293 ± 110 flinches, respectively. However, ERB-131 was unable to reduce the number of flinches at all doses tested. The Phase I values for ERB- 131 were 245 ± 24, 284 ± 14 and 282 ± 19 at 1, 3 and 10 mg/kg respectively, not statistically different from vehicle treated animals (p value = 0.101). The phase II flinches values averaged 1050 ± 97, 1081 ± 132 and 1110 ± 104 respectively, and thus were not differenr from vehicle (p value = 0.422). Therefore, ERB- 131 does not alleviate the acute and persistent inflammatory pain states induced by formalin injection. Formalin treated animals were injected with vehicle or ERB-131 at 1, 3 and 10 mg/kg as described in the methods. Data was pooled into Phase I and Phase II. Phase I represents data from 5 and 10 min time points. Phase II incorporates remaining timepoints (from 15 to 60 min). P values were calculated using a one-way ANOVA test. Figs. 11-12 and Table 6 show the data. Legends are: formalin + vehicle (filled circle, dotted line), formalin + 1 mg/kg ERb-131 (filled square, plain line), formalin + 3 mg/kg ERB-131 (filled circle, plain line), formalin + 10 mg/kg ERb-131 (filled diamond, plain line).
TABLE 6 Activity of ERB-131 in the formalin model Phase I Phase Il
AVG STD AVG STD
Vehicle 222 21 1293 110
ERb-131 1 mg/kg 245 24 1050 97
3 mcf/kg 284 16 1081 132
16 mcj/fcg 282 19 1110 104
p value 0.101 0.422
[00324] For the data shown on Fig. 12A, data from 5 and 10 min timepoints was pooled and reported as Phase I. The number of flinches per 5 min interval represents cumulative data. For the data shown on Fig. 12B, data from 15-60 min timepoints was pooled and reported as Phase II. Data is thus a cumulative representation of 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min timepoints.
[00325] CFA model of chronic inflammatory pain. The Complete Freund's Adjuvant (CFA) rat model is a well characterized model, producing inflammation as well as chronic pain primarily in the form of hyperalgesia. Rats were injected with Complete Freund's Adjuvant (CFA) to induce a stable state of inflammation and thermal hyperalgesia. Subsequently, control (iCFA: inactivated CFA) or CFA animals were treated with ERB-131 once daily, injected s.c. Response to thermal hyperalgesia was measured daily from day 4 to day 8. Latencies for paw withdrawal are expressed in seconds (sec). The assessment of therrmal hyperalgesia is presented in Fig. 13A and Table 7. Rats were treated as described in the methods. Hyperalgesia and inflammation measures are presented . Data is summarized as AVG ± STD. P values were calculated compared to vehicle treated animals, using a two-tailed unpaired t-test.
TABLE 7 Activity of ERB-131 in the CFA model
Vehicle ERb-131 (1) ERb-131 {3} ERb-131 (10)
Hyperalgesia latency (sec) 6.9 ± 0.3 8.1 ± 0.3 9.0 ± 0.4 9.7 ± 04 v value 0018 0002 < 0.001
Edema paw width (%) 149.2 ± 2 5 133.4 ± 2 1 131 0 ± 2 3 121 B ± 2 8 p value < 0.001 < 0.001 < 0.0001 [00326] Treatment with inactivated CFA (iCFA) did not alter the sensitivity to the noxious thermal stimulus across the period of testing, as expected . Rats that received CFA demonstrated a significant reduction in hot plate latency. The hot plate latency for the iCFA-treated group (sham) was 11.5 ± 0.2 sec as compared to 6.7 ± 0.1 sec for the CFA-treated group (p value = < 0.0001). Daily administration of ERb-131 produced a dose-dependent reversal of CFA-induced thermal hyperalgesia. Rats that received 1, 3 or 10 mg/kg ERB-131 demonstrated latencies that were significantly elevated above those obtained in the CFA-treated controls, indicating a strong reversal of the hyperalgesic state induced by CFA. The latency values at 1 and 3 mg/kg peaked at 8.4 ± 0.2 (p value = 0.001) and 9.7 ± 0.3 sec (p value < 0.0001), respectively. The latency obtained for the ERB-131 10 mg/kg group was 10.2 ± 0.4 sec, values relatively similar to those seen in the iCFA-treated group, 11.2 ± 0.2 sec. The ED50 for ERB-131 for the thermal hyperalgesia endpoint was approximately 3 mg/kg. Inflammation was assessed by the formation of local edema from the treated paw. The edema was quantified daily from day 4 on using a micrometer. The paw width was normalized to the change seen in the contralateral (untreated) paw. Administration of CFA into the hind paws of rats produced significant edema formation (Fig. 13B and Table 7). (Legends are: iCFA + vehicle (filled square, dotted line), iCFA + 10 mg/kg ERB-131 (filled diamond, dotted line), CFA + vehicle (filled circle, dotted line), CFA + 1 mg/kg ERB-131 (filled square, plain line), CFA + 3 mg/kg ERB-131 (filled circle, plain line), CFA + 10 mg/kg ERB-131 (filled diamond, plain line).) iCFA resulted in a 19.4 ± 1.4 % increase in paw thickness relative to the contralateral paw (non-injected paw), whereas CFA triggered an increase of 53 ± 1.3 % (p value < 0.0001). Systemic administration of ERB-131 produced a dose-dependent reversal of CFA-induced edema. Rats that received 1, 3 or 10 mg/kg displayed paw widths at day 8 of 130.6 ± 2.1 % (p value = 0.012), 127.7 ± 1.7 % (p value = 0.003) and 117.4 ± 3.1 % (p value < 0.0001), respectively. The edema values for the lOmg/kg ERB- 131 group were similar to that of the iCFA group: 121.8 ± 2.8 % vs 120.6 ± 2.4 % (p value = 0.752). The ED50 for ERB-131 in the inflammation assay was approximately 1 mg/kg.
Discussion
[00327] Using a variety of animal models, we demonstrated that the selective ERβ agonist ERB-131 modulates inflammation and associated nociceptive states. While ERB-131 did not affect acute nociception, it was effective in alleviating chronic inflammatory pain. These findings are particularly interesting in light of the confusing literature pertaining to non-selective estrogens and their effects on pain.
[00328] In the carrageenan model of acute pain, ERB-131 had no effect on measures of inflammation and pain sensation. The lack of efficacy was not a consequence of irreversible effects of carrageenan as the pain suppressor gabapentin dose-dependently inhibited thermal hyperalgesia but not edema, consistent with published literature. Similar to ERB-131, the selective ERb agonist ERB-041 did not inhibit edema in a reverse paradigm of the carrageenan model (Leventhal et al. 2006), and only partially resolved the thermal hyperalgesia (reversal ~ 50% at 30 mg/kg). In contrast, pre-administration of the non selective ligand estradiol attenuates inflammation in carrageenan-induced pleuresy (Cuzzocrea et al. 2001), suggesting that estradiol acts in a prophylactic manner. In the formalin model of persistent pain, v was unable to inhibit inflammatory pain in contrast to non selective estrogens which selectively inhibit Phase II (Kuba et al. 2005; Mannino et al. 2006). It is thus apparent that the sole activation of ERb is not sufficient to promote anti-hyperalgesic effects in models of acute and persistent pain.
[00329] Interestingly, the actions of estrogens (both genomic and non-genomic) involve the inhibition of NF-kB, a master switch of inflammation. NF-kB can be activated by a large variety of stimuli and, in response, regulates a multitude of inflammatory genes including cytokines, chemokines, adhesion molecules and acute phase proteins. By targeting NF-kB, estrogens can influence inflammatory processes, and consequently pain perception (Ghisletti et al. 2005; Harkonen and Vaananen 2006). Proinflammatory cytokines act at many levels to increase pain sensitivity, directly on sensory neurons (Fukuoka et al. 1994; Opree and Kress 2000; Watkins and Maier 2005) and indirectly through activation of signaling cascades (DeLeo and Yezierski 2001 ; Sommer and Kress 2004). Consistent with such findings, the magnitude of pain sensation was found to directly correlate with the number of macrophages present at the site of injury in amimal models (Cui et al. 2000; Liu et al. 2000). A strong correlation between high cytokine content in nerve biopsies and pain sensation was also reported in human patients suffering of various neuropathies (Lindenlaub and Sommer 2003). Since ERβ agonism consistently promote anti-inflammatory activities (Harris 2006 and our unpublished observations), it is thus likely that selective ERβ activation alleviates pain in models of chronic inflammatory pain through, in part, targeting inflammatory pathways. This is further supported by the fact that the estrogen receptor beta is expressed in a number of classical immunocompetent (Baker et al. 2004; Ghisletti et al. 2005; Stygar et al. 2006) and immune-like DRG cells (Evans et al. 2002; Nalbandian and Kovats 2005) that are in proximity of sensory neurons or recruited to the site of injury. These various immunocompetent cells have been reported to relay immune activation from a peripheral site of injury and impact DRG sensory function (McMahon et al. 2005; Watkins and Maier 2005).
[00330] It is particularly interesting that estrogens, through activation of ERb, can modulate directly inflammation as well as nociception. In light of the fact that inflammatory processes can also directly influence the initiation and maintenance of pain states (reviewed in Watkins and Maier 2005), it is suggested that ERβ activation presents a unique advantage in resolving noxious pain states. Moreover, because inflammation is a critical event in the establishment of neuropathic pain, it would follow that ERβ activation could be a novel and effective way of addressing neuropathies, consistent with our recent findings that selective activation of ERβ using ERB-131 is extremely effective in alleviating allodynia and hyperlagesia in various
Example 46 - Broad Modulation of Neuropathic Pain States by a Selective Estrogen Receptor β Agonist
Introduction
[00331] Estrogens regulate a large spectrum of neuronal functions, including pain perception. However, the effects of estrogens in various animal models of pain, including neuropathic pain, are often conflicting. In particular, estrogens can display pro- or anti-nociceptive effects, depending on the animal model considered (Tsao et al. 1999; Liu and Gintzler 2000; Shir et al. 2002; Evrard and Balthazart 2004; Hucho et al. 2006). In the human population, clinical trials examining the impact of soy diet (rich in isoflavones and phytoestrogens) on various pain endpoints reached different conclusions (McFadyen et al. 2000; Albert et al. 2002; Ingram et al. 2002). A Phase I study of methoxyestradiol, a catabolite of the estrogen pathway with affinity for both ER subtypes, showed significant improvement in bone pain of breast cancer patients (Lakhani et al. 2003). A recent study of young healthy women inversely correlated their estrogen levels to pain sensitivity (Smith et al. 2006), while the MORGEN study, a cross-sectional analysis of over 10,000 Dutch women aged 20-59 years, positively correlated factors increasing estrogen levels with a higher incidence of chronic lower back pain (Wijnhoven et al. 2006).
[00332] The effects of estrogens are classically mediated by two nuclear hormone receptors, ER alpha (NR3A1, ESRl, ERa) and ER beta (NR3A2, ESR2, ERβ), that belong to the superfamily of ligand-activated transcription factors. Differences between the two receptors are evident on multiple levels. First, there is a moderate conservation in the amino acid sequence of the ligand binding domain (~ 50%) and significant differences throughout the regions involved in the transcriptional activity (Kuiper et al. 1996; Tremblay et al. 1997). Consistent with this, ERa displays a more pronounced transcriptional ability than its counterpart (Mosselman et al. 1996), and ERβ has been reported to decrease ERa transcriptional potential (Lindberg et al. 2003). This finding has led to the description of a yin/yang balance of estrogen function, by which ERβ, through the formation of heterodimers ERα/ERβ, can antagonize ERa function (Weihua et al. 2003). Second, both ERs differ in their spatial and temporal tissue distribution. Both ER subtypes are synthesized by autonomic and sensory neurons (Papka et al. 2001). However, in the dorsal root ganglia (DRG) of adult rats, ERβ mRNAs are widely expressed in sensory neurons, while ERa expression is mostly restricted to small size sensory neurons (Taleghany et al. 1999). Immunoreactivity studies also revealed the presence of DRG neurons that express both ERa and ERβ, while others express either subtype (Papka and Storey- Workley 2002). Temporal regulation was also evident in long term studies of ovariectomized rats. Estrogen treatment downregulated ERa mRNA levels while upregulating ERβ (Taleghany et al. 1999). Similarly, 17β-estradiol enhanced regeneration of the sciatic nerve after crush injury, in part through accumulation of both ER proteins in motor neurons and regenerating neurites of the lumbar spinal cord (Islamov et al. 2003).
Materials and Methods R-SAT® Assays
[00333] R-SAT® (Receptor Selection and Amplification Technology) is a cell- based functional assay that allows one to monitor receptor-dependent proliferative responses and has been described elsewhere. The technology has been validated for a number of receptors including GPCRs (Brauner-Osborne and Brann 1996), RTKs (Burstein et al. 1998), cytokine receptors (Piu et al. 2002) and nuclear receptors (Piu et al. 2005; Piu et al. 2006). This process is achieved by partial cellular transformation via loss of contact inhibition and growth factor dependency. Monitoring is achieved by transfecting the cells with a β-galactosidase reporter gene vector whose expression is under a constitutively active promoter. Briefly, NIH-3T3 fibroblasts were plated overnight in 96-wells plates in DMEM 10 % calf serum (Hyclone) and grown to 60-70 % confluency prior to transfection. Transient transfections were performed using Polyfect (Qiagen) according to manufacturer's instructions. Typically a transfection mix would consist of the receptor and the β-galactosidase expression vectors. Sixteen hours post- transfection, cells were incubated with different doses of ligand in DMEM containing 30% Ultraculture (Hyclone) and 0.4% calf serum (Hyclone) to generate a dose response curve. After 5 days, plates were developed by adding onto the washed cells a solution containing the β-galactosidase substrate o-nitrophenyl-d-galacto pyranoside ONPG (in phosphate-buffered saline with 5% Nonidet P-40 detergent) as described (Piu et al. 2002). Plates were read using a microplate reader at 420 nm. Data from R-SAT® assays were fit to the equation: r = A + B(x/(x + c)), where A = minimum response, B = maximum response minus minimum response, c = EC50, r = response, and x = concentration of ligand. Curves were generated using the curve fitting softwares Excel Fit and GraphPad Prism (San Diego, CA).
Luciferase reporter gene assay
[00334] HEK293 cells were grown to 70% confluency in DMEM containing 10 % calf serum (Hyclone) prior to transfection. On the day of transfection (day 1), expression vectors for ERa or ERβ were cotransfected along a construct containing a synthetic 3*ERE upstream of the luciferase gene (Panomics), using Polyfect (Qiagen) per manufacturer's recommendations. Sixteen hours post-transfection, cells were incubated in serum free DMEM. On day 3, cells were incubated with the test compounds for 48 hours in serum free DMEM. Cells extracts were then obtained by lyzing and the Luciferase activity measured, all of these steps performed using a commercially available kit (Promega).
Binding assay
[00335] HEK293T cells were transiently transfected for 48 hours with expression vectors encoding ERa or ERβ, before being serum starved for 4-6 hours. Cells were then harvested by scraping in ice-cold PBS and subsequently lysed using a cold buffer containing 1OmM Tris pH 7.4, 1 mM EDTA, 1 mM DTT before being subjected to polytron twice for 10 seconds. Cytosolic extracts were isolated by centrifugation at 15000 g for 30 minutes at 4 0C. Competitive binding of the test compounds or the estradiol control was performed on 2OuL extract by overnight incubation at 4 0C in a total volume of 100 μL containing 1 nM 3H-estradiol (Perkin Elmer, Boston, MA). The bound fraction was separated from the unbound one by the addition of 100 μL of Dextran coated charcoal, incubated for 10 minutes and subsequent centrifugation at 1000 rpm for 10 minutes. Samples (100 μL per sample) were then analyzed by liquid scintillation. Data was then analyzed using the curve fitting software GraphPad Prism.
Animal Studies
[00336] All animal studies were conducted in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals.
Rat uterotrophic assay
[00337] Sexually immature female Sprague-Dawley rats (30^0 g; n = 6 per group) served as subjects. Rats received daily subcutaneous injections of vehicle (100% DMSO), (l,3,5-tris(4-hydroxyphenyl)-4-propyl-lH-pyrazole (PPT, 1 mg/kg) or various doses of ERB-131 (10, 30 or 100 mg/kg) for a total of 3 days. Approximately 24 hours after the final injection, the rats were sacrificed, the uteri removed, trimmed of adhesions, fluid expelled and then weighed.
Capsaicin-induced mechanical hyperalgesia
[00338] Mice were allowed to acclimate for 20 minutes prior to testing. Baseline measures of nociceptive response were taken for 3 consecutive days before the administration of test compounds. For baseline measures, the plantar surface of the left paw was poked with the 5.07 Von Frey Hair ten times in succession for one trial with 3 trials each day (approximately 10 min between each trial). Animals that did not respond by withdrawing their paws or responded an average of 4 or higher times/trial were excluded. After the baselines were completed on the day of the study, the test compounds or vehicle were injected by the intraperitoneal (i.p.) route, 15 minutes before the capsaicin injection (n=5 per group). Capsaicin (0.3%) was injected into the plantar surface of the left hind paw in a 10 μL volume and the left paw was then tested in the manner described above at 15, 30 and 60 minutes post-capsaicin.
L5/L6 Spinal nerve ligation model
[00339] Spinal nerve ligation was done as originally described by Kim and Chung (Kim and Chung 1992). Male Sprague-Dawley rats (200-225 grams, n=6 per group) were anesthetized through inhalation of an isoflourane/oxygen mix. An incision was made from the thoracic vertebra XIII toward the sacrum. The muscle was separated from the spinal vertebra (left side) at the L4 - S2 levels. The transverse process was carefully removed with a small rongeur to visually identify the L4 - L6 spinal nerves. The L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread. Following suturing, the animals recovered in a plastic cage under a regulated heat-temperature lamp. Animals were not given topical or local anesthetics post-operatively because they would have inhibited the development of the pain syndrome.
[00340] Allodynia was assessed at least one week after surgery by applying a light tactile stimulus (Von Frey hairs) to the affected surgical paw. Eight calibrated Von Frey filaments (Stoelting, Wood Dale, IL) were used in logarithmically spaced increments ranging from 0.41 to 15 gm (4-150 mN). Each filament was applied perpendicularly to the plantar surface of the ligated paw of rats kept in suspended wire- mesh cages. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength ("up and down" method). A positive response was recorded if the paw was sharply withdrawn. Von Frey hairs were applied in an up-down manner depending on the response until the 50% threshold was established (Dixon 1980). Data was converted using the following formula: % Allodynia reversal = ((PoDT - PrDT) / (15-PrDT))*100, where PoDT is Post Drug Threshold and PrDT is Pre Drug Threshold. Three different regimen were investigated: an acute drug effect (regimen 1) in which a single dose was administered followed 30 min later by assessment of allodynia, a prolonged drug effect (regimen 2) in which a single dose was injected followed 6 hr later by assessment of allodynia, and a continuous drug effect (regimen 3) in which three doses were given at 3 hr intervals followed 30 min later by assessment of allodynia.
Chemically-induced tactile allodvnia [00341] These studies were carried out in male Black6-C57 mice (20-30 grams, n=8 per group) essentially according to the method of Yaksh and Harty (Yaksh and Harty 1988). All compounds were injected intrathecally (i.t.) in 5 μL volume or i.p. in a 1 mL/kg volume. The test compounds were injected 15 min prior to injection of the allodynia-inducing agents: 300 ng/kg i.p. sulprostone (S) in DMSO; 100 ng/kg i.p. phenylephrine (PE) in H2O; 100 ng i.t. N-methyl D-aspartate (N) in DMSO. The mice were then assessed for allodynia once every 5 minutes over a 15-50 min period post injection of the allodynic agent by light stroking of the flank with a paintbrush. The allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape. Data is expressed as the average total score for each group (Each animal can have a maximum score of 16 over the 50-min period). The sensitizing agents typically elicit pain scores of 14 while the vehicle controls typically show pain score of 4-5.
[00342] Intrathecal Injections in Mice - Mice were injected intrathecally according to the method devised by Hylden and Wilcox (Hylden and Wilcox 1980). A sterile 30-gauge Vi inch needle attached to a microsyringe is inserted between the L5 and L6 vertebrae. The mouse is held firmly by the pelvic girdle in one hand, while the syringe is held in the other hand at an angle of approximately 200° above the vertebral column. The needle is inserted into the tissue to one side of the L6 spinous process so that it slips into the groove between the spinous and transverse processes. The needle angle is then decreased to about 100° and slowly advanced forward into the intervertebral space (A pop is felt at this point along with a visible serpentine tail movement allowing the investigator to verify being in the correct position). Test compounds were slowly injected in the subarachnoid space of conscious mice in a volume of 5 μL.
Thermal escape model
[00343] Naϊve, male Sprague-Dawley rats (225 - 250 g; n = 6 per group) were injected subcutaneously (s.c) with the test compounds. Following administration, response to a noxious thermal stimulus were measured using the 52 0C hot plate test. Latencies were followed over a period of 2 hours. Latencies, expressed in seconds (sec), were defined as the time needed for the animal to physically remove the treated paw from the hot surface.
Results
[00344] There thus might be different contributions of ERa and ERβ in pain modalities that could explain the opposite findings of the use of non selective estrogens in various animal and human studies. We sought to investigate how the selective activation of the ERβ subtype would affect pain perception in various animal models of neuropathic pain. Here we report on the characterization of a novel class of non steroidal subtype selective ERβ agonists, and the contribution of ERβ to pain modalities.
[00345] Characterization of ERB- 131. ERB-131 is an ERβ agonist identified using the functional cell-based assay R-SAT® (Receptor Selection and Amplification Technology) and is representative of a novel class of non steroidal ERβ ligands (Olsson et al. 2005). ERB-131 displays a strong affinity for the ERβ receptor (pEC50 7.5 ± 0.4) versus ERa (pECso 5.5 ± 0.3) as shown in R-SAT®. The activity of ERB-131 was compared to that of several estrogen ligands. Estrone is a non-selective agonist, while genistein, daidzein and 5a-androstan 3b, 17b diol are to various degrees selective towards ERβ. Compounds were assessed in a binding assay and two functional assays, the cell based R-SAT® and a transcriptional reporter gene assay (Luciferase). Full dose response experiments were performed multiple times in triplicate. Results represent the average ± standard deviation (AVG ± STD) of all experiments. All functional data are reported as efficacy (% Eff) and potency (pEC50 = - Log [EC50]). Binding data is reported as PK1 = - log [K1]. Estrone was defined as the reference compound, and its activity defined as 100% efficacy. N = number of replicates. The results are shown in Fig. 14A (estrone (filled square), ERB- 131 (filled triangle)) and Table 8.
TABLE 8 Characterization of ERB- 131
Figure imgf000102_0001
[00346] For the data shown in Figure 14A, The activity of ERB-131 was compared to estrone, a reference non-selective estrogen ligand. Compounds were evaluated for activity at estrogen receptors ERa and ERβ. Dose response curves were generated using R-SAT®. Data is reported as efficacy (%eff) normalized to estrone. Results are a representative experiment performed in triplicates. Estrone (filled square), ERB-131 (filled triangle). ERB-131 behaves as a full agonist at ERβ and a partial agonist at ERa.
[00347] No significant activities at up to 10 μM were found at other nuclear receptors, including the steroid hormone receptors, when assessed functionally using R- SAT®. The selectivity of ERB-131 was assessed using R-SAT®. A significant number of nuclear receptors were tested including all of the steroid receptors (AR, ERa, ERβ, GR, MR, PR) and other liganded nuclear receptors. Data is presented as fold response over vehicle treated cells, and is representative of several experiments done in triplicate. The data is shown in Fig. 14B. Further, ERB-131 displays greater partial agonism properties at ERa (40-60% efficacy) than at ERβ (70-80% efficacy). To assess whether ERB-131 displays properties of a classical nuclear receptor ligand and to expand upon the R-SAT® data, a transcriptional reporter gene assay was performed (Table 8). ERB-131 stimulated the transcriptional activity of ERβ and to a lesser extent that of ERa (pEC50 7.7 ± 0.5 vs 4.8 ± 0.0, respectively). Similarly to what was observed in R-SAT®, ERB-131 demonstrated partial agonism at both ERa and ERβ subtypes (% efficacy 58 ± 0.0 vs 80 ± 21, respectively). Additionally, a direct physical interaction between ERB-131 and the ER receptors was evident from competitive binding assays (Table 8). ERB-131 displayed an affinity of 50 nM towards ERβ (pKi 7.3 ± 0.5) and only of 7900 nM towards ERa (PK1 5.1 ± 0.0). Thus, ERB-131 is more selective towards ERβ (100-800 X) than the purportedly ERβ selective phytoestrogens genistein and daidzein.
[00348] Classical estrogenic function. ERB-131 was evaluated in a rat uterotrophic paradigm, a model of classic estrogenic actions. Because of the relative lack of ERβ expression in the uterus, this bioassay is deeemed to represent a sensitive way to assess ERa activity in vivo. Sexually immature female rats were administered s.c. daily for 3 days with vehicle, PPT a selective ERa agonist (1 mg/kg) or ERB-131 at different doses. On the fourth day, the uterus was harvested and its wet weight measured. Pictures depicting the morphologies of the uteri are presented in Figure 15: naϊve (Fig. 15A), vehicle (Fig. 15B), PPT (Fig. 15C), ERB-131 at 10 (Fig. 15D), 30 (Fig. 15E), 100 mg/kg (Fig. 15F). Quantitative values are shown graphically in Fig. 15G (*** = p < 0.001). Administration of the selective ERa agonist propyl pyrazole triol (PPT) to sexually immature female rats caused a 4-fold increase in the wet weight of the uterus relative to vehicle treated animals. (Fig. 15; compare 15C and 15B). The uterus weight increased significantly from 34 ± 2 mg to 125 ± 6 mg (p value < 0.0001). In contrast, treatment with ERB-131 at doses as high as 100 mg/kg s.c. only had a marginal effect on uterine weight (Fig. 15D, 15E, 15F). For instance, at the 100 mg/kg dose, the uterus weight was not statistically different from that of vehicle treated animals: 39 ± 2 mg (p value = 0.108). Thus, consistent with the in vitro findings, ERB-131 was found to lack any significant ERa activity in vivo.
[00349] ERβ and hyperalgesia and allodynia. The selective contribution of ERβ agonism, using the small molecule selective ERβ agonist ERB-131, was investigated in a variety of animal models of hyperalgesia and allodynia, symptoms that are characteristic of neuropathic pain.
[00350] Capsaicin-induced hyperalgesia. Intradermal injection of capsaicin (0.3%) triggered within 15 min a marked mechanical hyperalgesia. Acute treatment of mice with capsaicin triggers mechanical hyperalgesia. Hyperalgesia was followed overtime by monitoring the number of flinches in reaction to the poking of the paw with Von Frey Hair. Vehicle or ERB-131 (10mg/kg) were injected i.p. 15 min prior to the injection of capsaicin (0.3%). * = p < 0.05, ** = p < 0.001. (Fig. 16). This response was stable for over an hour, reaching a maximum after 30 min of 4.2 ± 0.8 flinches. ERB-131 (10 mg/kg i.p.) was administered 15 min prior to capsaicin, and its effects on hyperalgesia monitored over time (Fig. 16). Strikingly, ERB-131 completely abolished the capsaicin- induced mechanical hyperalgesia. At 30 min of treatment, the number of flinches presented by ERB-131 was 0.7 ± 0.2, significantly different from the vehicle treated animals (p value = 0.002). The beneficial effects of ERB-131 remained significant for the duration of the experiment (over an hour): 0.9 ± 0.2 flinches vs 2.1 ± 0.4 for vehicle treated animals (p value = 0.023) at t = 1 hour.
[00351] Chemically induced tactile allodynia. Three pain sensitizers that evoke an allodynic response by activating different pathways were chosen: sulprostone, phenylephrine and NMDA (Yaksh and Harty 1988; Gil et al. submitted). The vehicle controls caused a baseline pain score of 4.4 ± 0.9. Transient tactile allodynia was induced by injection of the pain sensitizers sulprostone (300 ng/kg i.p.) phenylephrine (100 ng/kg i.p) and NMDA (100 ng Lt). Mice were then assessed for allodynia every 5 minutes over a 15-50 min period by light stroking of the flank with a paintbrush. The allodynia response was ranked as follows: 0, no response; 1, mild squeaking with attempts to move away from the paintbrush; 2, vigorous squeaking, biting at the paintbrush and strong efforts to escape. The allodynic agents typically cause a pain score of 14 (out of a maximum of 16) and the vehicle controls typically cause a pain score of 4- 5. The results are shown in Fig. 17A, where ERB-131 (10 mg/kg i.p.) was injected 15 min prior to injection of the allodynia- inducing agents and the pain score of treated animals quantified. S: Sulprostone; PE: Phenylephrine; N: NMDA.. Treatment with the three agents produced marked and significant allodynic responses. Administration of sulprostone resulted in a pain score of 14.0 ± 0.7 (p value < 0.0001), phenylephrine in a score of 11.0 ± 0.6 (p value = 0.0001) and NMDA in a score of 14.0 ± 1.0 (p value < 0.0001). We then investigated whether ERB-131 could antagonize the effects of these pain sensitizers. ERB-131 (10 mg/kg) was administered 15 min prior to the injection of the pain sensitizers. ERB-131 reversed the tactile allodynia induced by sulprostone (4.2 ± 0.6; p value < 0.0001), phenylephrine (3.5 ± 0.2; p value < 0.0001 ) and NMDA (4.5 ± 0.8; p value < 0.0001). ERB-131 treatment restored the pain thresholds of these animals to levels comparable to vehicle treated animals. A dose response of ERB-131 (Fig. 17B), where effects of different doses of ERB-131 (1 and 10 mg/kg) were evaluated in the sulprostone induced allodynia, indicated that while it is fully effective at reversing sulprostone-induced tactile allodynia at 10 mg/kg, it was completely ineffective at the lower dose of 1 mg/kg: 13.9 ± 0.5 (p value = 0.910). Further, to demonstrate that the anti- allodynic effects of ERB-131 were dependent upon activation of the ERβ receptors, an experiment was devised in which animals were concomittantly treated with ICI-182780, an estrogen receptor antagonist. ICI- 182780 is a potent "pure" antiestrogen with antagonist activities at both ERa and ERβ receptors, and devoid of partial agonist properties. As shown in Figure 17C, ICI-182780 had only marginal effects on sulprostone-induced allodynia on its own (11.7 ± 0.8 vs 13.9 ± 0.7). The estrogen receptor pan-antagonist ICI- 182740 was used to confirm that the effects of ERB-131 on sulprostone-induced allodynia were dependent on activation of estrogen receptors. ICI- 182740 (0.1 mg/kg i.p.) was co-administered with ERB-131. ICI = ICI-182740. *** = p<0.001. When co-administered, ICI-182780 reversed the anti-allodynic effects of ERB- 131 following sulprostone administration, the pain score being restored from 4.2 ± 0.6 to 13.1 ± 0.9. Taken altogether, these results indicate that ERB-131 mediates its broad anti- allodynic effects against chemical insults through activation of the estrogen receptor ERβ. [00352] Spinal nerve ligation. The L5/L6 spinal nerve ligation model constitutes a well-established and characterized assay of peripheral neuropathy. Various drug administration regimens were tested to evaluate acute, prolonged and continuous drug responses of ERB- 131. In the acute drug effect regimen, previously reported pain suppressors such as gabapentin (an orphan drug, 30 mg/kg), morphine (an opiate analgesic, 15 mg/kg) and amitriptyline (a tricyclic antidepressant, 1 mg/kg) all produced significant anti-allodynic effects. Tactile allodynia was established by surgical ligation of L5-L6 spinal nerves in rats. Allodynia was assessed by applying a light tactile stimulus (Von Frey Hair) to the affected paw. The results are shown in Figure 18. The effects of an acute dose of ERB-131 (1 or 10 mg/kg i.p.) were evaluated 30 min following injection. The standard pain reducers morphine (15 mg/kg i.p.), gabapentin (10 mg/kg i.p.), and amintryptiline (30 mg/kg i.p.) were used for comparison purposes (Fig. 18A). For instance, morphine partially reversed allodynia by 46.3 ± 9.4 % (p value = 0.0008) relative to vehicle treated animals (1.6 ± 1.5 %). Similarly, gabapentin and amitryptiline were partially effective, displaying reversals of 73.4 ± 9.6 % (p value < 0.0001) and 70.1 ± 8.3 % (p value < 0.0001), respectively. Different regimen were investigated using low doses of ERb-131 (0.5 mg/kg). In regimen 1, the acute effects of a single dose of ERB- 131 on allodynia was examined 30min following injection. Prolonged drug effect was evaluated in Regimen 2, in which a single dose was administered followed 6 hr later by assessment of allodynia. (Fig. 18B) In Regimen 3, the continuous drug effects of ERB- 131 were examined by giving 3 doses of ERB-131 at 3 hr intervals followed 30 min later by assaying allodynia. *** =p<0.001. At low doses (0.5 and 1 mg/kg i.p.), ERB-131 was ineffective in inhibiting allodynia (Figs. 18A, 18B). The percentage reversal values were 1.5 ± 1.2 and 0.6 ± 1.1 %, respectively. However, at the 10 mg/kg dose, ERB-131 almost completely reversed the tactile allodynia experienced by the surgically ligated animals (91.0 ± 6.0 %). The magnitude of the anti-allodynic effects mediated by ERB-131 was significantly stronger than the ones exhibited by the reference pain suppressors tested, with p values ranging from 0.003 to < 0.0001. In a regimen of prolonged drug effect, a low dose of ERB-131 (0.5 mg/kg) did not reverse the allodynia experienced by the surgically ligated animals. In contrast, the same low ERB-131 dosing markedly inhibited allodynia in a regimen of continuous drug effect. In that regimen (Fig. 5B), ERB-131 produced a significant reversal of 76.0 ± 6.0 % (p value < 0.0001). [00353] Normal pain threshold. As ERB-131 broadly modulates neuropathic pain behavior in various models of hyperalgesia and allodynia, we investigated whether ERB- 131 would affect the pain threshold of normal rats. Naive rats were injected with vehicle or ERB-131 (10 mg/kg s.c.) and their responses to a noxious stimulus (hot plate 52°C) followed over time. Their hot plate latency, expressed in seconds (sec), were defined as the time needed for the animal to physically remove the treated paw from the hot surface, was followed overtime. The results are shown in Figure 19. The 10 mg/kg dose was chosen as it consistently alleviated neuropathic pain in the various animal models. Vehicle treated animals exhibited a hot plate latency of 8.8 ± 0.9 sec, which did not vary over the 2 hour course of the testing (Fig. 19). Rats treated with ERB-131 displayed an average latency of 8.5 ± 0.5 sec, a value that did not differ statistically from the vehicle treated group (p value = 0.777). Thus, administration of ERB-131 did not modify the pain threshold of normal animals.
Discussion
[00354] A novel class of non-steroidal ERβ agonists has been identified, of which ERB-131 represents a prototype lead. ERB-131 constitutes a potent ERβ agonist with a potency of 20-50 nM, and a high degree of selectivity versus other nuclear receptors, especially the related ERa (>100X). Biochemical characterization indicated that ERB-131 displays classical properties of a nuclear receptor ligand, including the ability to directly modulate transcription through direct binding and activation of ERβ. In vivo, ERB-131 lacks any significant ability to stimulate ERa activity, even at doses as high at 100 mg/kg. In sharp contrast, ERB-131 at doses ranging from 0.5 to 10 mg/kg, efficiently reverses allodynia and hyperalgesia in several models of altered pain sensation consequent to chemical and surgical insults. To our knowledge, this work is the first demonstration of a role for selective ERβ activation in the alleviation of neuropathic pain.
[00355] Non selective estrogens have been reported to exhibit pro- and antinociceptive properties. Normal male rats treated acutely with estrogen exhibit dose- dependent mechanical hyperalgesia (Hucho et al. 2006). Similarly, estradiol administration to quails increases pain sensitivity to a noxious thermal stimulus (Evrard and Balthazart 2004). In contrast, our results indicate that the selective ERβ agonist ERB- 131 does not affect the pain threshold of normal animals. Further, treatment with ERB- 131 inhibits allodynia induced by several chemicals, namely sulprostone, phenylephrine and NMDA. On the other hand, estrogen attenuates substance P induced-antinociception in the NMDA model (Nag and Mokha 2004; Claiborne et al. 2006). In models of surgical insult, treatment of ovariectomized rats with estradiol following sciatic nerve resection results in decreased autotomy (Tsao et al. 1999). Similarly, ERB- 131 reverses tactile allodynia resulting from spinal nerve ligation. Thus, we propose that the lack of selectivity of estrogen ligands most likely explain these discrepancies. Indeed, the studies described herein highlight the fact that administration of ERB-131 significantly and consistently alleviated altered pain sensation in a variety of animal models associated with nerve injury and hyperexcitability. Furthermore, natural ligands with a relative selectivity towards ERβ, such as isoflavones, consistently alleviate pain sensation in animal models (Shir et al. 2002). In addition, ERβ agonism protects from inflammatory pain in vivo, as we and others demonstrated (Gardell et al. 2007; Leventhal et al. 2006).
[00356] Neuropathic pain occurs as a result of a peripheral nerve injury and is associated with functional and biochemical changes, not only at the peripheral site of insult but also in higher order neurons of the spinal cord and the brain. While the mechanisms of actions by which estrogens modulate neuropathic pain have remained largely unanswered, there appears to be a contribution from both central and peripheral components. Peripherally, within the dorsal root ganglia, estrogens activate non-genomic (Chaban et al. 2003; Purves-Tyson and Keast 2004; Hucho et al. 2006), genomic (Amandusson et al. 1999; Mowa et al. 2003; Shimozawa et al. 2006) and paracrine (Evrard and Balthazart 2004) regulatory pathways. These functional changes affect the activity of various pain effectors and result additionally in survival and regeneration of spinal neurons (Patrone et al. 1999; Islamov et al. 2003). Centrally, estrogens trigger neurochemical changes that modulate pain responses. For instance, estradiol influences opioid neurotransmission through the mu opioid receptor (Smith et al. 2006). By activating ERβ-bearing inhibitory neurons, estrogen also modulates GABAergic neurons that innervate BDNF expressing neurons (Blurton- Jones and Tuszynski 2006). Whether activation of both peripheral and central sensory neurons is required for the antinociceptive properties of ERβ still remains an open question. Interestingly, ERB- 131 displays significant CNS penetration (FP unpublished). In addition, ERB- 131 alleviated pain sensation triggered by the various chemicals that act either centrally or peripherally. Both sulprostone and phenylephrine act through peripheral activation of sensory neurons of the dorsal root ganglia (Minami et al. 1994; Lee et al. 2000), while activation of the NMDA glutamate receptors in the spinal dorsal horn is essential for central sensitization (Riedel and Neeck 2001).
[00357] The findings that acute administration of ERB-131 could very rapidly alleviate pain are quite intriguing, raising the question of the contribution of rapid non- genomic actions. Non-genomic effects of estrogens represent an integral aspect of estrogen function, culmulating in the activation of various signal transduction pathways, in particular the MAP kinases, the PI3K/Akt, the NF -kB and calcium systems (Warner and Gustafsson 2006). Several mechanisms have been proposed to explain the rapid effects of estrogens. For instance, classical estrogen receptors can associate with the plasma membrane through interactions with adapter proteins such as Src (Song et al. 2005). Alternatively, a putative brain-specific membrane associated ER-X receptor has been proposed (Toran-Allerand et al. 2002). More recently, the orphan GPCR receptor GPR30 has been reported to bind estrogen with high affinity (Filardo et al. 2002). However, a recent study using selective SiRNAs concluded that non-genomic actions of estrogen can be explained by the sole presence of the classical estrogen receptors (Pedram et al. 2006). Therefore, the rapid effects seen with ERB-131 on measures of pain sensation most likely rely on activation of the classical ERβ. In support, the pan ER antagonist ICI-182780 antagonized the anti-allodynic effects of ERB-131. In addition to non-genomic actions, ERB-131 also activated genomic pathways as was evident from the prolonged drug regimen data in the spinal nerve ligation model.
[00358] The selective activation of ERβ by ERB-131 appears sufficient to provide beneficial effects in animal models of symptoms associated with neuropathic pain. The robustness of ERB-131 effects across a variety of neuropathic pain models suggests a strong potential and clinical utility of ERβ selective agonists in treating neuropathic pain disorders in humans. Additionally, the lack of ERa and thus of "classical" estrogenic actions could provide for optimal efficacy and enhanced safety profile.
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[00437] The following applications are incorporated herein by reference in their entirety, including any drawings: U.S. Application No. 11/269,913 filed November 8, 2005, entitled "COMPOUNDS WITH ACTIVITY AT ESTROGEN RECEPTORS"; U.S. Application No. 11/120,397, filed May 3, 2005, entitled "COMPOUNDS WITH ACTIVITY AT ESTROGEN RECEPTORS"; and U.S. Provisional Application No. 60/568,332, filed May 4, 2004, entitled "IDENTIFICATION OF COMPOUNDS WITH ACTIVITY ON ESTROGEN RECEPTORS".

Claims

WHAT IS CLAIMED IS:
1. Use of an estrogen receptor β (ERβ) agonist for the preparation of a medicament for affecting a response in a subject, wherein the response is selected from the group consisting of reducing inflammation, treating neuropathic pain, treating allergic conjunctivitis, reducing IL-4 levels, decreasing IFN-γ levels, treating dry eye, and increasing IL- 12 levels.
2. The use of claim 1, wherein the ERβ agonist is a compound of formula I:
Figure imgf000118_0001
(I) or a pharmaceutically acceptable salt or prodrug thereof, wherein: n is an integer selected from the group consisting of 3, 4, 5 and 6;
Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -CC=Z)R6, -CC=Z)OR6, -C(=Z)N(R6)2, -
Figure imgf000118_0002
-P(=O)(OR6)2, and -CH2OC(=O)R5;
R2, R2a, R2b, R2c are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR6, -NR6R63, -NReNReaReb, -NR6N=CR63R6^ -N(R6)C(R6a)=NR6b, -CC=Z)R6, -CC=Z)OR6, -N(R6)-C(=Z)NR6bR6a, -OCC=Z)R6,
Figure imgf000118_0003
each R3 is separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, =0, and -ORe, or are separately absent to accommodate a double bond; two R3 groups are optionally bound together to form a substituted or unsubstituted C3-C9 cycloalkyl or C3-C9 heteroalicyclyl;
R2a is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
R4a is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
R4, R4a, R41,, R4c are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, nitro, halogen, sulfonyl, perhaloalkyl, -OR6, -NR6R63, -NR6NR63RSb, -NR6N=CR63R61,, -N(R6)C(R6a)=NR6b, -CN, -CC=Z)R6, -CC=Z)OR6, -CC=Z)NR6R63, -SC=Z)NR6R63, -N(Re)-CC=Z)R63, -N(R6)-C(=Z)NR6bR6a, -OCC=Z)R6, -N(Re)-SC=O)2R63, and -SR6;
R43 and R4b are optionally bound together to form an aryl, heteroaryl, or heteroalicyclyl;
R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, halogen, -CN, -SR6, sulfonyl, -C(=O)NR6R6a, -C(=0)R6, -NR6R63, -COOR6, and perhaloalkyl;
Z is oxygen or sulfur; and
R6, R63 and R^ are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl.
3. The use of claim 2, wherein the compound is selected from the group consisting of
Figure imgf000120_0001
Figure imgf000120_0002
Figure imgf000121_0001
Figure imgf000121_0002
Figure imgf000122_0001
Figure imgf000122_0002
Figure imgf000122_0003
Figure imgf000123_0001
Figure imgf000123_0002
Figure imgf000123_0003
Figure imgf000124_0001
Figure imgf000124_0003
Figure imgf000124_0002
Figure imgf000125_0001
4. The use of claim 2, wherein the compound is 4-(l- phenylcyclohexyl)phenol.
5. The use of claim 1, wherein the inflammation is in the eye.
6. The use of claim 5, wherein the inflammation results in lid edema, hyperemia, chemosis, or tearing.
7. The use of claim 5, wherein the inflammation is due to an ophthalmologic disorder selected from the group consisting of uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration, or is an ocular manifestation of a systemic inflammatory disease selected from Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis.
8. The use of claim 1, wherein the inflammation is in the gastrointestinal tract.
9. The use of claim 1, wherein the inflammation is acute or chronic inflammation.
10. The use of claim 1, wherein the inflammation is due to arthritis.
11. The use of claim 1, wherein the subject is a human.
12. The use of claim 1, wherein the ERβ agonist is a selective ERβ agonist.
13. The use of claim 1, wherein the ERβ agonist is administered topically.
14. The use of claim 1, wherein the ERβ agonist is administered intraperitoneally.
15. The use of claim 1, wherein the ERβ agonist is administered systemically.
16. The use of claim 15, wherein the ERβ agonist is administered orally.
17. The use of claim 1, wherein the neuropathic pain is hyperalgesia.
18. The use of claim 17, wherein the hyperalgesia is tactile hyperalgesia.
19. The use of claim 1, wherein the neuropathic pain is allodynia.
20. The use of claim 1, wherein the neuropathic pain is selected from the group of phantom limb pain, postherpetic neuralgia, reflex sympathetic dystrophy, causalgia, complex regional pain syndrome II, painful HIV-associated neuropathy, and diabetic neuropathy.
21. The use of claim 1, wherein the neuropathic pain is associated with a medical condition selected from the group of traumatic nerve injury, multiple sclerosis, stroke, syringomyelia, epilepsy, spinal cord injury, and cancer.
22. The use of claim 1, wherein the ERβ agonist is a compound of formula II:
Figure imgf000126_0001
(H) or a pharmaceutically acceptable salt or prodrug thereof, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6; Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cykloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -C(=Z)R5, -C(=Z)OR5, -C(=Z)N(R5)2, -S(=O)2NR5aR5b, -P(=O)(OR5)2, and - CH2OC(=O)R5;
R2, R2a, R2b, R2c, and each Re are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR5, -NR5R5a, -NR5NR5aR5b, -NR5N=CR5aR5b, -N(R5)C(R5a)=NR5b, -C(=Z)R5, -C(=Z)0R5, -C(=Z)NR5R5a, -N(R5)-C(=Z)R5a, -N(R5)-C(=Z)NR5bR5a, -0C(=Z)R5, -N(R5)-S(=O)2R5a, and -SR5; each Y is separately selected from the group consisting of methylene, methylene substituted with one or two Re groups, sulphur, oxygen, unsubstituted nitrogen, nitrogen substituted with R5, and C=O; two Y groups are optionally bound together to form a single bond or a substituted or unsubstituted C1-C9 cycloalkyl or Ci- C9 heteroalicyclyl;
R2a is optionally bound to one Y group to form a substituted or unsubstituted C4- C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, substituted heteroalicyclyl, unsubstituted heteroalicyclyl, unsubstitued aryl, and substituted aryl;
A is optionally bound to one Y group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
Z is oxygen or sulfur; and each R5; R5a and R5b are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl; provided that when every Y is a substituted or unsubstituted methylene, then A is not a substituted or unsubstituted aryl.
23. A method of reducing inflammation, treating neuropathic pain, treating allergic conjunctivitis, reducing IL-4 levels, decreasing IFN-γ levels, treating dry eye, or increasing IL- 12 levels in a subject comprising: identifying a subject in need of the reducing inflammation, treating neuropathic pain, treating allergic conjunctivitis, reducing IL-4 levels, decreasing IFN-γ levels, treating dry eye, or increasing IL- 12 levels; and administering to the subject a pharmaceutically effective amount of an estrogen receptor β (ERβ) agonist.
24. The method of claim 23, wherein the ERβ agonist is a compound of formula I:
Figure imgf000128_0001
(I) or a pharmaceutically acceptable salt or prodrug thereof, wherein: n is an integer selected from the group consisting of 3, 4, 5 and 6;
Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -CC=Z)R6, -CC=Z)OR6, -CC=Z)N(Re)2, -
Figure imgf000128_0002
R2, R2a, R2b, R2c are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR6, -NR6R63, -NReNReaReb, -NR6N=CR68R61,, -N(R6)C(R^)=NR*, -CC=Z)R6, -CC=Z)OR6, -CC=Z)NR6R63, -N(Re)-CC=Z)R60, -N(R6)-C(=Z)NR6bR6a, -OCC=Z)R6,
Figure imgf000129_0001
-SR6; each R3 is separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, =0, and -OR6, or are separately absent to accommodate a double bond; two R3 groups are optionally bound together to form a substituted or unsubstituted C3-C9 cycloalkyl or C3-C9 heteroalicyclyl;
R2a is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
R4J, is optionally bound to one R3 group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
R4, R4a, R41,, R4c are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, nitro, halogen, sulfonyl, perhaloalkyl, -OR6, -NR6R63, -NR6NR63R6,,, -NR6N=CR63R61,, -N(R6)C(R6a)=NR6b, -CN, -CC=Z)R6, -CC=Z)OR6, -CC=Z)NR6R63, -SC=Z)NR6R63, -N(Re)-CC=Z)R63, -N(R6)-C(=Z)NR6bR6a, -OCC=Z)R6, -N(Re)-SC=O)2R63, and -SR6;
R43 and R4b are optionally bound together to form an aryl, heteroaryl, or heteroalicyclyl;
R5 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, halogen, -CN, -SR6, sulfonyl, -C(=O)NR6R6a, -C(=0)R6, -NR6R63, -COOR6, and perhaloalkyl;
Z is oxygen or sulfur; and
R6, R63 and R^ are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl.
25. The method of claim 24, wherein the compound is selected from the group consisting of
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000131_0002
Figure imgf000132_0001
Figure imgf000132_0002
Figure imgf000132_0003
Figure imgf000132_0004
Figure imgf000133_0001
Figure imgf000133_0002
Figure imgf000133_0003
Figure imgf000134_0001
Figure imgf000134_0003
Figure imgf000134_0002
Figure imgf000135_0001
26. The method of claim 24, wherein the compound is 4-(l- phenylcyclohexyl)phenol.
27. The method of claim 23, wherein the inflammation is in the eye.
28. The method of claim 27, wherein the inflammation results in lid edema, hyperemia, chemosis, or tearing.
29. The method of claim 27, wherein the inflammation is due to an ophthalmologic disorder selected from the group consisting of uveitis, blepharitis, meibonian gland disease, glaucoma, dry eye, or macular degeneration, or is an ocular manifestation of a systemic inflammatory disease selected from Sjogrens Syndrome, ocular sicatricial pemphygoid and Lupus erythmatosis.
30. The method of claim 23, wherein the inflammation is in the gastrointestinal tract.
31. The method of claim 23, wherein the inflammation is acute or chronic inflammation.
32. The method of claim 23, wherein the inflammation is due to arthritis.
33. The method of claim 23, wherein the subject is a human.
34. The method of claim 23, wherein the ERβ agonist is a selective ERβ agonist.
35. The method of claim 23, wherein the ERβ agonist is administered topically.
36. The method of claim 23, wherein the ERβ agonist is administered intraperitoneally.
37. The method of claim 23, wherein the ERβ agonist is administered systemically.
38. The method of claim 23, wherein the ERβ agonist is administered orally.
39. The method of claim 23, wherein the neuropathic pain is hyperalgesia.
40. The method of claim 22, wherein the hyperalgesia is tactile hyperalgesia.
41. The method of claim 23, wherein the neuropathic pain is allodynia.
42. The method of claim 23, wherein the neuropathic pain is selected from the group of phantom limb pain, postherpetic neuralgia, reflex sympathetic dystrophy, causalgia, complex regional pain syndrome II, painful HIV-associated neuropathy, and diabetic neuropathy.
43. The method of claim 23, wherein the neuropathic pain is associated with a medical condition selected from the group of traumatic nerve injury, multiple sclerosis, stroke, syringomyelia, epilepsy, spinal cord injury, and cancer.
44. A method of reducing inflammation in a subject, treating neuropathic pain in a subject, treating allergic conjunctivitis in a subject, reducing IL-4 levels in a subject, decreasing IFN-γ levels in a subject, increasing IL-12 levels in a subject, or treating dry eye in a subject comprising: identifying a subject in need thereof; and administering to the subject a pharmaceutically effective amount of an estrogen receptor β (ERβ) agonist, wherein the ERβ agonist is a compound of formula II,
Figure imgf000137_0001
(H) or a pharmaceutically acceptable salt or prodrug thereof, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6;
Ri is selected from the group consisting of hydrogen, Ci-Cs straight chained or branched alkyl, Ci-Cs straight chained or branched alkenyl, cycloalkyl, cykloalkenyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalicyclyl, sulphonyl, Ci-Cs straight chained or branched perhaloalkyl, -C(=Z)R5, -C(=Z)OR5, -C(=Z)N(R5)2, -S(=O)2NR5aR5b, -P(=O)(OR5)2, and - CH2OC(=O)R5;
R2, R2a, R2b, R2c, and each R6 are separately selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalicyclyl, hydroxy, halogen, sulfonyl, perhaloalkyl, -CN, -OR5, -NR5R5a, -NR5NR5aR5b, -NR5N=CR5aR5b, -N(R5)C(R5a)=NR5b, -C(=Z)R5, -C(=Z)0R5, -C(=Z)NR5R5a, -N(R5)-C(=Z)R5a, -N(R5)-C(=Z)NR5bR5a, -0C(=Z)R5, -N(R5)-S(=O)2R5a, and -SR5; each Y is separately selected from the group consisting of methylene, methylene substituted with one or two R6 groups, sulphur, oxygen, unsubstituted nitrogen, nitrogen substituted with R5, and C=O; two Y groups are optionally bound together to form a single bond or a substituted or unsubstituted C1-C9 cycloalkyl or Ci- C9 heteroalicyclyl;
R2a is optionally bound to one Y group to form a substituted or unsubstituted C4- C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl; any bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond;
A is selected from the group consisting of substituted heteroaryl, unsubstituted heteroaryl, substituted heteroalicyclyl, unsubstituted heteroalicyclyl, unsubstitued aryl, and substituted aryl; A is optionally bound to one Y group to form a substituted or unsubstituted C4-C9 heteroalicyclic, C4-C9 cycloalkyl, or C4-C9 cycloalkenyl;
Z is oxygen or sulfur; and each Rs, Rsa and Rsb are separately selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heteroalicyclyl; provided that when every Y is a substituted or unsubstituted methylene, then A is not a substituted or unsubstituted aryl.
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US8470872B2 (en) 2007-10-26 2013-06-25 Acadia Pharmaceuticals Inc. Compounds with activity at estrogen receptors
EP2554532A1 (en) * 2011-08-01 2013-02-06 Acadia Pharmaceuticals Inc. Diphenyl substituted cyclohexane derivatives, useful as modulators of the estrogen receptors beta
WO2013017619A1 (en) * 2011-08-01 2013-02-07 Acadia Pharmaceuticals Inc. Diphenyl substituted cyclohexane derivatives, useful as modulators of the estrogen receptors beta
US9061981B2 (en) 2011-08-01 2015-06-23 Acadia Pharmaceuticals Inc. Diphenyl substituted cyclohexane derivatives, useful as modulators of the estrogen receptors beta
US9126999B2 (en) 2012-05-31 2015-09-08 Eisai R&D Management Co., Ltd. Tetrahydropyrazolopyrimidine compounds
US9446046B2 (en) 2012-05-31 2016-09-20 Eisai R&D Management Co., Ltd. Tetrahydropyrazolopyrimidine compounds
US9850242B2 (en) 2012-05-31 2017-12-26 Eisai R&D Management Co., Ltd Tetrahydropyrazolopyrimidine compounds
US10640500B2 (en) 2012-05-31 2020-05-05 Eisai R&D Management Co., Ltd. Tetrahydropyrazolopyrimidine compounds
US11130758B2 (en) 2012-05-31 2021-09-28 Eisai R&D Management Co., Ltd. Tetrahydropyrazolopyrimidine compounds
US9974776B2 (en) 2013-12-05 2018-05-22 Karo Pharma Ab Estrogen receptor beta agonists for use in treating mesothelioma
US20220380378A1 (en) * 2021-04-22 2022-12-01 Essa Pharma, Inc. Androgen receptor modulators and methods for their use

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