US 20140212486A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0212486A1 LEDERMAN et al. (43) Pub. Date: Jul. 31, 2014

(54) ISOMETHEPTENE ISOMER (52) U.S. Cl. CPC ...... C07C 211/21 (2013.01); C07C 59/285 (71) Applicant: TONIX PHARMACEUTICALS INC., (2013.01); A61 K3I/I31 (2013.01); A61 K NEW YORK, NY (US) 45/06 (2013.01); C07C 209/68 (2013.01); C07B 53/00 (2013.01); A61 K3I/I67 (72) Inventors: SETH LEDERMAN, NEW YORK, NY (2013.01); A61 K31/192 (2013.01); A61 K (US); BRUCE DAUGHERTY, 3 1/522 (2013.01); A61 K31/4152 (2013.01) MENDHAM, NJ (US); LELAND J. USPC ...... 424/463:564/509;562/582: 514/671; GERSHELL NEW YORK, NY (US); 424/464: 514/263.34: 514/.404 DARRYL RIDEOUT, PARKTON, MD (57) ABSTRACT DIEGO,(US); ANDREW CA (US) KAWASAKI, SAN The invention relates to a purified Isometheptene compound comprising the structure according to Formula (I), (73) Assignee: TONIX PHARMACEUTICALS INC., NEW YORK, NY (US) (6-methylamino-2-methylheptene)

(21)21) AppAppl. No.: 14/158,7359 21 NH (22) Filed: Jan. 17, 2014 Related U.S. Application Data or a hydrochloride, or a pharmaceutically acceptable addition (60) Provisional application No. 61/926,739, filed on Jan. salt thereof. In particular, the disclosure relates to the synthe 13, 2014, provisional application No. 61/814,664, sis, purification and characterization of an Isometheptene filed on Apr. 22, 2013, provisional application No. isomer mucate crystal 2, wherein the Isometheptene isomer 2 61/793.456, filed on Mar. 15, 2013, provisional appli- is stereochemically characterized as (R)-enantiomer, respec cation No. 61/754,281, filed on Jan. 18, 2013. tively. The Isometheptene (R)-enantiomeractivity indicates a selective centrally acting selective ligand for Imidazoline Publication Classification Subtype 1 (I.1) receptor sites; and more specifically, the dis closure provides an antihypertensive composition for treat (51) Int. Cl. ment of migraine and other neurovascular or neurogenic pain CD7C 2 II/2 (2006.01) from abdominal distress. (R)-Isometheptene enantiomer or A6 IK3I/3 (2006.01) isomer 2 may be an anti-hypertensive agent with lower side A6 IK 45/06 (2006.01) effects than the racemate form. Therefore (R)-Isometheptene A6 IK3 L/452 (2006.01) is believed to be effective against episodic tension-type head C07B 53/00 (2006.01) aches (ETTH). The (R)-Isometheptene enantiomer or isomer A6 IK3I/67 (2006.01) 2 is believed to effectively lower blood pressure, used alone or A6 IK3I/92 (2006.01) together with other headache ameliorating drugs. Methods of A6 IK3I/522 (2006.01) synthesis and treatment are described. Regarding (R)- C07C 59/285 (2006.01) Isometheptene crystals data of X-ray crystallography are pre CD7C 209/68 (2006.01) sented.

C23 Patent Application Publication Jul. 31, 2014 Sheet 1 of 24 US 2014/021248.6 A1

HPLC SOMER 2 ELS1A, Signal Voltage (MAR-2011-067WR111007491.D) s 7OO 6OO 500 400 300 200 1OO O O 5 1O 15 2O min

FIG. 1 Patent Application Publication Jul. 31, 2014 Sheet 2 of 24 US 2014/021248.6 A1

HPLC BLANK ELS1A, Signal Voltage (MAR-2011-067WCN(1603)BLK.D) mV 16OO 1400 1200 1 OOO 800 600 400 200

O 5 10 15 2O min

FIG.2 Patent Application Publication Jul. 31, 2014 Sheet 3 of 24 US 2014/021248.6 A1

Retention Time 300 300

200 200 o CC E 1OO 100

O O

O 5 10 15 20 25 30 35 40 45 Minutes CHIRAL HPLC ISOMER 2 FIG. 3 Patent Application Publication Jul. 31, 2014 Sheet 4 of 24 US 2014/021248.6 A1

CHIRAL HPLC RACEMIC SOMRTHPTENE

2000. Retention Time 2000

1500 1500

D 1000 1000 E 500 500

O --0 O 5 10 15 20 25 30 35 40 45 Minutes

FIG. 4 Patent Application Publication Jul. 31, 2014 Sheet 5 of 24 US 2014/021248.6 A1

LC OF SOMER 2

*Maximum Chromatogram of DADATAMAR-11WR111007491B.D, Signalld A mAU 500 400 300 200 100

O 1 2 3 4 5 6 min Patent Application Publication Jul. 31, 2014 Sheet 6 of 24 US 2014/0212486A1

ZNJEWNOSIHWNHI Patent Application Publication Jul. 31, 2014 Sheet 7 of 24 US 2014/021248.6 A1

MASS SPECTRUM OF SOMER 2

Intens. x107

1 go--- 6 Time (min)

AR11 1007491B.D.: TIC AIIMS

Intens.x107 +MS, 1.9min #78 1.O

0.8

O.6

0.4

0.2

O.O 125 150 175 200 225 250 250 300 m/z. FIG. 7 Patent Application Publication Jul. 31, 2014 Sheet 8 of 24 US 2014/021248.6 A1

HPLC OF SOMER 1 ELS1A, Signal Voltage (MAR-2011-08OWR111007807-1.D)

O 5 10 15 2O min

FIG. 8 Patent Application Publication Jul. 31, 2014 Sheet 9 of 24 US 2014/021248.6 A1

HPLC BLANK ISOMER 1 ELS1A, Signal Voltage (MAR-2011-08070N(1803)BLKD) mV 700 600 5OO 400 300 200 1 OO

O 5 10 15 2O min

F.G. 9

Patent Application Publication Jul. 31, 2014 Sheet 11 of 24 US 2014/021248.6 A1

CHRAL HPLC SOMER 1 800 800 Retention Time 600 600

400 400 CC E 200 200

O O O 5 1O 15 20 25 30 35 40 45 Minutes

FIG 11 Patent Application Publication Jul. 31, 2014 Sheet 12 of 24 US 2014/021248.6 A1

CHRAL HPLC OF RACEMIC SOMETHEPTENE

Retention Time 800 800

6OO 600

400 400

200 Patent Application Publication Jul. 31, 2014 Sheet 13 of 24 US 2014/021248.6 A1

Patent Application Publication Jul. 31, 2014 Sheet 14 of 24 US 2014/021248.6 A1

H NMR of (R)-isometheptene (R)-Mosher amide There is no (S)peak at 4.84 ppm.

CO) i? coco w OONO)o N. cow v. N. N. row rCNCNOOCOcy) was Ol?on CNV cocV wo Ocn w v. C. Cdo odoo cycNw-doon cold Vool w crocnwoodoococoncNwe doo odd c2c2 v V v v CoccoccNNNNNN rint (Occoncy Yooooooooooooo LLO LOOLOLLOOOOLOLOLOr riv rif r rve r rrr rrrrrr cycrococcydcoccy) cyco

1300FOCU.808A 7-57-Crude CDC13, H-1 NUMEGA 2-19-2013

s ce 5. 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5. 5.0 5.7 48 4.7 4.6 4.5 44 4.3 4.2 4.1 40 3.9 3.8 f1 (ppm) FIG. 14 Patent Application Publication Jul. 31, 2014 Sheet 15 of 24 US 2014/021248.6 A1

H NMR mixture of (R)- and (S)-isometheptene (R)-Mosheramide. nn door CNorward ld onw Cococoon rinivas we coco won Now WO w Cd on N.N. cocococo v - V - oooooooooon cocow rary sco canno?coso o NNNNNNNNNNN sus Losursrinus LSLs LSLs Louilors rrrrrrrrrrrrri. SSS-yl)-2sld-- ' ' ' ----iss N2-12s--- 1206FOCU,119B 7-14-lSomer 2 CDC13, H-1 NUMEGA 11-15-12

(S) (R)

H - I - st st se s

75 7.0 6.5 6.0 5.5 5.0 4.5 f1 (ppm) Patent Application Publication Jul. 31, 2014 Sheet 16 of 24 US 2014/021248.6 A1

::::::iii. 335i Patent Application Publication Jul. 31, 2014 Sheet 17 of 24 US 2014/021248.6 A1

Patent Application Publication Jul. 31, 2014 Sheet 18 of 24 US 2014/021248.6 A1

Patent Application Publication Jul. 31, 2014 Sheet 19 of 24 US 2014/021248.6 A1

Patent Application Publication Jul. 31, 2014 Sheet 20 of 24 US 2014/021248.6 A1

***

Patent Application Publication Jul. 31, 2014 Sheet 21 of 24 US 2014/021248.6 A1

Binding of Isometheptene to i-Imidazoline receptor

1OO o (S)-isometheptene Mucate, Ki= 1100 nM o (R)-isomethepene Mucate, Kil 18nM v isometheptene Mucate USP, Ki= 42nM 80

Log Compound), M FIG 21 Patent Application Publication Jul. 31, 2014 Sheet 22 of 24 US 2014/021248.6 A1

Binding of isometheptene to 2-Imidazoline receptor

o (S)-isometheptene Mucate, K=180 nM o (R)-isomethepene Mucate, Ki-62 nM v isometheptene Mucate USP, Ki= 93 nM 8 O

6 O

Log Compound), M FG, 22

Patent Application Publication Jul. 31, 2014 Sheet 24 of 24 US 2014/021248.6 A1

110 100 90 80 70 60 50 40 30 20 10

O m -11 - 10 -9 -8 -7 -6 -5 -4 -3 -2 log drug M

lodo-clonidine 104 V Amino-clonidine 227 O Guanabenz 1,410 O 2-BF 25,740 O ldaZOXan 71,070

F.G. 24 US 2014/021248.6 A1 Jul. 31, 2014

SOMETHEPTENESOMER ergotamines in the treatment of vascular headaches. Clin. Med. 1970. 77:33-36; Yuill, G. M., et al. A double-blind 0001. This patent application claims priority from Provi crossover trial of Isometheptene mucate compound and sional patent applications 61/926,739 filed Jan. 13, 2014: ergotamine in migraine. Br J Clin Pract. 1972 26(2):76-9. 61/814,664 filed Apr. 22, 2013: 61/793.456 filed Mar. 15, PMID:4552744: Diamond, S. Treatment of migraine with 2013; and 61/754,281 filed Jan. 18, 2013; which prior disclo Isometheptene, acetaminophen, and dichloralphenaZone Sures are incorporated in the present application. combination: A double-blind, crossover trial. Headache. 0002 All references cited in this specification, and their 1976 15(4):282-7. PMID: 1107267: Valdivia, L. F. Pharmaco references, are incorporated by reference herein in their logical analysis of the mechanisms involved in the tachy entirety where appropriate for teachings of additional or alter cardic and vasopressor responses to the antimigraine agent, native details, features, and/or technical background. Isometheptene, in pithed rats. Life Sciences 2004. 74:3223 3234; Freitag, F G. Comparative Study of a Combination of FIELD OF THE INVENTION Isometheptene Mucate, Dichloralphenazone With Acetami 0003. The invention relates to synthesis, separation and nophen and Sumatriptan Succinate in the Treatment of purification of racemic Isometheptene (6-methylamino-2- Migraine Headache. 2001 41(4):391-8. PMID: 11318886; methylheptene) or Isometheptene Mucate salt (in a 2:1 molar Gibbs TS. Health Care Utilization in Patients With Migraine: ratio) as well as enantiomers 1 and 2 thereof moreover, the Demographics and Patterns of Care in the Ambulatory Setting invention describes the use of each isomer alone or in com Headache. 2003 43(4):330-5. PMID: 12656703; Loder, E. bination with other ameliorative compounds for treatment of Fixed Drug Combinations for the Acute Treatment of various painful conditions such as tension or migraine head Migraine Place in Therapy CNS Drugs. 2005; 19(9):769-84. aches and menstrual cramps and various cognitive disorders PMID: 16142992; de Souza CD, et al. Efficacy and tolerabil Such as moderate cognitive impairment, presenile or senile ity of combined dipyrone, Isometheptene and caffeine in the dementia and cognitive disorders from drug treatments. treatment of mild-to-moderate primary headache episodes. 0004 More particularly, the invention provides the prepa Expert Rev Neurother. 2012. 12(2):159-67. doi:10.1586/ern. ration and structural characterization optical Isometheptene 11.193. PMID: 22288671). isomers 1 and 2, or (S)-enantiomer and (R)-enantiomer, 0006 Isometheptene (6-methylamino-2-methylheptene) respectively; in addition via X-ray of crystallized optical was first prepared according to the process described in U.S. Isometheptene mucate (R)-isomer. Pat. No. 2,230,753 (1941); and it is soluble in water in its hydrochloride or mucate salt form. “Isometheptene is a speci BACKGROUND fied stimulant listed by the World Anti-doping Agency 0005 Isometheptene is a non-ergot vasoconstrictor agent (WADA). Br. J. Pharmacol. 2008 June; 154(3):606-622. that has been used in the treatment of tension headache, 0007 More particularly, racemic Isometheptene is a well vascular headache and migraine headache either alone or as known aliphatic amine and includes its pharmacologically one or more than one active ingredients in various combina acceptable acid addition salts (i.e., 2:1 ratio of Isometheptene: tion drug products (CDPs) (Horton, BT, Petes, G A and mucate). It is a commercially available drug in its common Blumenthal, L S. A new product in the treatment of migraine: racemic form as a combination drug product (CDP) with A preliminary report, Proc. Staff Meet Mayo Clin. 1945.XX: acetaminophen (APAP) and dichloralphenazone (DCP) or as 241-248; Palmer, H. Toxicity of methylamino-iso-octene. a combination drug product with APAP and caffeine. In Bra Migraine headache, Clinics. 1945. Iv: 531-554; Walton, RP Zil, and other countries particularly in South America, and Preacher, CB, Federation Proc. 1946. v. 211; MacNeal, P. Isometheptene is available in combination with dipyrone (de S., and Davis, D.: The Use of methyl-iso-octenylamine in Souza, CD, et al. Efficacy and tolerability of combined dipy migraine. Ann Intern Med. 1947 26(4):526-7. PMID: rone, Isometheptene and caffeine in the treatment of mild-to 20292892; Pantalone AL and Thomas G. J. Methyl-iso-octe moderate primary headache episodes. Expert Rev Neurother. nylamine (octin) relief of headache following spinal tap. Curr 2012 12(2):159-67. doi: 10.1586/ern. 11.193. PMID: Res Anesth Analg. 1952 31(1):69-71. PMID: 14896.702: 22288671). It was available in Germany, among several Euro Peters G A and Zeller W. W. Evaluation of a new agent (me pean countries, under the trademark OCTIN from Knoll A. G. thyl-iso-octenylamine) in the treatment of vasodilating head (now part of Abbott). aches. Proc Staff Meet Mayo Clin. 1949 24(23):565-8. 0008. It is believed to be a cerebrovascular constrictor PMID:15396281; Seltzer, A.: The use of methyl-iso-octeny which action may reduce pressure on the pain producing lamine (Octin) in migraine and related headaches: prelimi areas Surrounding blood vessels. Isometheptene is usually nary report. Med Ann Dist Columbia. 1948 17(7):395-6. used in combination with other headache ameliorating drugs PMID: 1887 1972: Blumenthal, S., and Fuchs, M.: Headache such as APAP, caffeine and/or dichloralphenazone. Clinics I. Am. Pract. and Digest Treat. 1950. 10: 1012: Head 0009. Accordingly, the compositions containing ache Clinics II. Am. Pract. and Digest Treat. 1951. 2:163: Isometheptene and other active ingredients useful in this Ogden, H D., Headache Statistics II. Headache Patterns. J. invention may be administered either intravenously, parenter Allergy. 1952 23:458; Headache Clinics IV. Am. Pract. and ally, orally, transdermally, transmucosally, or as a nasal spray. Digest Treat. 1953. 4:31: Brazeau, P: Oxytocics, in Goodman Isometheptene (6-methylamino-2-methylheptene) was first L. S. Gilman, A. (eds): The Pharmacological Basis of Thera prepared according to the process described in U.S. Pat. No. peutics, ed 4. New York, The Macmillan Co. 1970. 893-907; 2,230,753 (1941); and it is soluble in water in its hydrochlo Ogden, H. D.: Controlled studies of a new agent in vascular ride or mucate salt form. “Isometheptene is a specified Stimu headache. Headache. 1963 3:29-31. PMID: 13939581; lant listed by the World Anti-doping Agency (WADA). Br. J. Young, H.: A study of a non-ergotamine agent in the office Pharmacol. 2008 June; 154(3):606-622. treatment of vascular headache. Ind Med. Surg. 1966 35(2): 0010 More particularly, racemic Isometheptene is a well 127-9. PMID:5323440; Johnson, D. E.: An alternative to known aliphatic amine and includes its pharmacologically US 2014/021248.6 A1 Jul. 31, 2014 acceptable acid addition salts (i.e., 2:1 ratio of Isometheptene: formulation with the same dosing instructions for migraine, mucate). It is a commercially available drug in its common tension and vascular headache as MigraTen. racemic form as a combination drug product (CDP) with (0019. One of the DESI IDA products marketed under the acetaminophen (APAP) and dichloralphenazone (DCP) or as trademark Midrin R, contains Isometheptene Mucate (65 a combination drug product with APAP and caffeine. In Bra mg); Dichloralphenazone (100mg) and Acetaminophen (325 Zil, and other countries particularly in South America, mg) and is indicated for relief of migraine headache with a Isometheptene is available in combination with dipyrone (de usual adult dosage of two capsules at once followed by one Souza, CD, et al. Efficacy and tolerability of combined dipy capsule every hour until relieved, administering up to 5 cap rone, Isometheptene and caffeine in the treatment of mild-to sules within a twelve hour period. Midrin R) is also indicated moderate primary headache episodes. Expert Rev Neurother. for relief of tension headache with a usual adult dosage of one 2012 12(2):159-67. doi: 10.1586/ern. 11.193. PMID:- or two capsules every four hours up to 8 capsules a day.” 22288671). It was available in Germany, among several Euro While the FDA has recognized that Isometheptene is safe and pean countries, under the trademark OCTIN from Knoll A. G. effective by the standards of DESI, various administrative (now part of Abbott). actions to compel DESI manufacturers to upgrade the status 0011. It is believed to be a cerebrovascular constrictor of DESI drugs to the level of NDA drugs have resulted in which action may reduce pressure on the pain producing ending the US Midrin production by Curaco since 2011; areas Surrounding blood vessels. Isometheptene is usually however, numerous other manufacturers have continued to used in combination with other headache ameliorating drugs manufacture and distribute similar products. such as APAP, caffeine and/or dichloralphenazone. 0020 Headache indications include several distinct and 0012. In the US, the IDA and ICA products are marketed overlapping conditions including primary and secondary as prescription drugs under the auspices of the Drug Efficacy headaches as described in the International Headache Society Study Implementation (DESI), because they were available (HIS) criteria (“Classification and diagnostic criteria for before the 1962 FDA reform (1938-1962), were evaluated by headache disorders, cranial neuralgias and facial pain. Head National Academy of Sciences/National Research Council to ache Classification Committee of the International Headache conduct Drug Efficacy Study and were deemed effective for Society”. Cephalalgia 8 Suppl 7: 1-96. 1988. PMID tension and vascular headache and possibly effective for 3048700.) In addition to the headaches classified by the HIS, migraine headache. headaches include vascular headache, which is not mentioned 0013. One DESI ICA product marketed as MigraTen(R) in the Headache classification of the International Headache contained: Society (IHS), although it is still used by many physicians, the 0014. It is believed to be a cerebrovascular constrictor US Food and Drug Administration and certain medical clas which action may reduce pressure on the pain producing sification systems. In the IHS classification, vascular head areas Surrounding blood vessels. Isometheptene is usually aches may be considered a cluster headache, migraine and used in combination with other headache ameliorating drugs toxic headache. such as APAP, caffeine and/or dichloralphenazone. 0021 Headache may be considered a cluster headache, 0.015 Accordingly, the compositions containing migraine and toxic headache. Isometheptene and other active ingredients useful in this 0022 Episodic tension-type headache (ETTH) is a sub invention may be administered either intravenously, parenter type of tension-type headache. Tension-type headaches ally, orally, transdermally, transmucosally, or as a nasal spray. (TTH) are the most common type of headaches among adults, 0016 For example, the Isometheptene mucate is com and as a result of high prevalence, imposes the greatest Socio monly administered as a mucate salt in a combination drug economic impact of any primary headache type (Bendtsen, product (CDP) containing Isometheptene, dichloral 2011; Crystal, 2010). The pain may render a sufferer unable phenazone (DCP), and acetaminophen (APAP) in which the to attend activities, force them to stay home from work, or combination product can be abbreviated IDA (IDA) or a impair their ability to function at work. According to Mayo different CDP containing Isometheptene, caffeine, and Clinic, tension headaches affect 90% of women and 70% of acetaminophen (APAP), which combination product can be men. According to Vos et al. (Vos, 2012) it is the most abbreviated ICA (ICA). common form of headache and affects 1.4 B people world 0017. In the US, the IDA and ICA products are marketed wide, approximately 20.8% of the population and affect as prescription drugs under the auspices of the Drug Efficacy women more than men (23% to 18% respectively). Study Implementation (DESI), because they were available 0023. A tension-type headache is classified into subtypes before the 1962 FDA reform (1938-1962), were evaluated by based on how often it occurs: infrequent ETTH (<1 day/ National Academy of Sciences/National Research Council to month on average), frequent ETTH (1-14 days/month on conduct Drug Efficacy Study and were deemed effective for average), or chronic TTH or CTTH (>15 days/month on tension and vascular headache and possibly effective for average) (International Headache Society, 2013). An ETTH migraine headache. (infrequent or frequent) may be described as a mild to mod 0018. One DESI ICA product marketed as MigraTen(R) erate constant band-like pain, tightness, or pressure around contained: Isometheptene mucate (65 mg); Caffeine (100 the forehead or back of the head and neck. ETTH may last mg): acetaminophen (APAP) (325 mg) in a capsule formula from 30 minutes to several days. ETTH usually begins gradu tion and was deemed possibly effective for relief of migraine ally, and often occurs in the middle of the day. The severity of headache in a regimen: 2 capsules at once; 1 capsule q1h (up a tension headache generally increases significantly with its to 5 caps/12 h period) and deemed effective for the relief of frequency. Because the symptoms of ETTH overlap with tension headache or vascular headache in a regimen of 1-2 other primary headache types, diagnosis is generally made, capsules q4h (up to 8 caps/day). Another ICA DESI product not only by inclusion, but also of exclusion of certain Symp on the market is branded as Prodrin R) and contains 130 mg of toms Such as nausea, exacerbation by physical exercise and Isometheptene, 20 mg of caffeine, 500 mg of APAP in a tablet occurrence of both photophobia and phonophobia (Sacco, US 2014/021248.6 A1 Jul. 31, 2014

2008). Once diagnosed and other conditions are ruled out, the adenosine (A1, A2, and A3) receptors, glutamate (NMDA, primary goal of treatment for ETTH is acute, abortive therapy AMPA, kainate, and metabotropic) receptors, dopamine (Bendtsen, 2011; Lenaerts, 2009), such as would be provided receptors, endothelin receptors, and female hormone (estro with TNX-201.hronic TTH occurs more frequently, has been gen and progesterone) receptors, plus some miscellaneous shown to be more refractory to Successful treatment, and receptors and ion channels. The anti-migraine pharmacologi shares many characteristics of a group of headaches called cal mechanism of action of Isometheptene is unknown, but Chronic Daily Headaches or CDH, including sometimes no Isometheptene is considered to be a sympathomimetic action more than one additional symptom of nausea, photophobia, leading to cranial vasoconstriction. As described above, orphonophobia (Crystal, 2010; International Headache Soci Isometheptene mucate is formulated combined in Midrin ety, 2013). As a result, accurate diagnosis is imperative, and with acetaminophen and dichloralphenaZone (a mild non the primary goal of treatment is headache prophylaxis, barbiturate sedative) wherein Isometheptene is known as including selected anti-depressants, serotonin-specific sympathomimetic agent acting as both an alpha and beta reuptake inhibitors, benzodiazepines, other anxiolytics, and adrenoreceptoragonist and a vasoconstrictor. If two capsules muscle relaxants (Bendtsen, 2011; Lenaerts, 2009). are taken near the onset of a mild migraine attack followed 0024. Although the mechanism of action for any headache within an hour by two or three capsules, the treatment con treatment is not yet fully understood, much work has been sisting of may be satisfactory. Although well tolerated, the performed and analysis published. For example, several dosage is usually not given more than two or three times a review articles (Solomon 1995, Gallagher 2002, Gibbs 2003, week. Mehrota 2008) provide information on the potential pharma 0030 Cramps are unpleasant, sometimes painful sensa cological mechanism of the medications that treat or prevent tions caused by involuntary muscle contraction or muscle migraine. over-shortening. Cramps can be symptoms of muscle spasm. 0025 Migraine is described as a paroxysmal disorder Cramps can be separated into Smooth muscle cramps and characterized by attacks of headache, nausea, vomiting, pho skeletal muscle cramps. Cramps related to internal (or vis tophobia, and phonophobia. ceral) organs are related to spasm of Smooth muscle or skel 0026. Migraine without aura often has a strict menstrual etal muscle or distension of organs by functional or patho relationship. In contrast to the first edition of The Interna logical disorders. Cramps of internal organs can be associated tional Classification of Headache Disorders, the second edi with visceral pain. Smooth muscle cramps are commonly tion gives criteria for A1.1.1 Pure menstrual migraine, and associated with menstruation or visceral disorders including A 1.1.2 Menstrually-related migraine, but lists these condi gastrointestinal and urinary disorders. In the female, Smooth tions in the appendix because of uncertainty over whether muscle cramps that are associated with menstruation, are they should be regarded as separate entities. called menstrual cramps and may also occur before and dur 0027 Migraine pathophysiology is believed by genetic ing a female menstrual cycle. Severe or persistent Smooth predisposition to involve leakage of ion channels in the brain muscle cramps may also be symptomatic of endometriosis or stem such that the decreased blood flow in the brain leads to other health problems. Moreover, Smooth muscle cramps can neuropeptide release from trigeminal nerves inducing dilata be associated with gastrointestinal disorders, including infec tion of cranial extracerebral blood vessel. This condition tious or autoimmune gastroenteritis and functional disorders stimulates the trigeminovascular system producing headache such as irritable bowel syndrome (IBS). Thus, smooth muscle associated phonophobia and photophobia as well as nausea cramps are associated with visceral pain in many conditions, and vomiting. A. Maassen VanDenBrink and K. Y. Chan including interstitial cystitis. An ideal treatment for cramps (2008) European J. Pharmacology, 585; 313-319. would have both analgesic and antispastic properties. 0028 Migraine affects people of all races and both sexes 0031 Skeletal muscle cramps are associated with muscle with women accounting for 79% (61% between 20 and 49 fatigue, low sodium, low potassium and certain drugs, includ years of age) of physician visits for migraines and Caucasians ing statins. Skeletal muscle cramps also include nocturnal leg for 91% of the physician visits. The pathogenesis of migraine cramps which are involuntary muscle contractions that occur headache involves a) the cranial blood vessels, b) the trigemi during the night or (less commonly) while resting. Nocturnal nal innervation of these vessels, and c) the reflex connection leg cramps are common in the elderly, teenagers or in women of the trigeminovascular system in the cranial parasympa during the late stages of pregnancy, and can vary in intensity thetic outflow. from mild to extremely painful. 0029. In addition to Isometheptene, medications for treat 0032 Menopause ing headache that are approved by the Food and Drug Admin 0033 Menopause is an event that typically (but not istration (FDA) include as single agents or in combination; always) occurs in women that can be defined as the permanent acetaminophen (APAP), aspirin (ASA), caffeine, barbiturates cessation of the primary functions of the ovaries and is func (e.g., butalbital), non-steroidal anti-inflammatory drugs tionally evident when there is a termination of periodic shed (NSAIDS) (e.g., diclofenac), ergot alkaloids (e.g. ergota ding of the uterine lining (known as menses). mine, dihydroergotamine), triptans (e.g., Sumatriptan, riza 0034 Hot and Cold Flushes triptan, naratriptan, Zolmitriptan, eletriptan, almotriptan, fro 0035 Hot and cold flushes (or flashes) are unpleasant sen Vatriptan, and avitriptan), anti-epileptics (e.g. topiramate) sations associated with vascular events. Hot flashes (also and toxins (ie, botulinum toxin). Some drugs that have been known as hot flushes, or night Sweats if they happen at night) used for headache include neuroleptics (e.g., thorazine, halo are a symptom which may have several other causes, but peridol). Potential pharmacological targets for the treatment which is often caused by the changing hormone levels that are of migraine include medications that target 5-hydrox characteristic of menopause. ytryptamine (5-HT) or serotonin receptor subtypes (5-HT 0036 Cognitive Disorders 7), adrenergic receptor Subtypes (C.1, C2, and B), calcitonin 0037 Cognitive disorders are a category of mental health gene-related peptide (CGRP1 and CGRP2) receptors, disorders that primarily affect learning, memory, perception, US 2014/021248.6 A1 Jul. 31, 2014 and problem solving, and include amnesia, dementia, and 0044 Schizophrenia delirium. Causes vary between the different types of disor 0045 Schizophrenia is a psychiatric diagnosis for a ders but most include damage to the memory portions of the thought disorder characterized by a breakdown of thought brain. Mild cognitive impairment (MCI, also known as incipi processes and by a deficit of typical emotional responses. ent dementia, or isolated memory impairment) is a brain Common symptoms include auditory hallucinations, para function syndrome involving the onset and evolution of cog noid or bizarre delusions, or disorganized speech and think nitive impairments beyond those expected based on the age ing. and education of the individual, but which are not significant 0046. Anxiety enough to interfere with their daily activities. MCI can be a 0047 Anxiety disorder is a blanket term covering several transitional stage between normal aging and dementia. different forms of a type of common psychiatric disorder Alzheimer's disease (AD) is the most common form of characterized by excessive rumination, worrying, uneasiness, dementia. AD is commonly diagnosed in people over 65 years apprehension and fear about future uncertainties either based of age, although the less-prevalent early-onset Alzheimer's on real or imagined events, which may affect both physical can occur much earlier. In the early stages, the most common symptom is difficulty in remembering recent events. As the and psychological health. disease advances, symptoms can include confusion, irritabil 0048 Epilepsy ity and aggression, mood Swings, trouble with language, and 0049 Epilepsy is a common and diverse set of chronic long-term memory loss. In later stages, AD patients often neurological disorders characterized by seizures. In many withdraw from family and society. Gradually, bodily func cases a cause cannot be identified; however, factors that are tions are lost, ultimately leading to death. associated include brain trauma, strokes, brain cancer, and 0038 Traumatic Brain Injury drug and alcohol misuse among others. 0039 Traumatic brain injury (TBI), also known as intrac 0050 Stress Disorders ranial injury, occurs when an external force traumatically 0051 Stress disorders are an increasingly recognized injures the brain. TBI can be classified based on severity, group of conditions relating to the body's reaction to stressful mechanism (closed or penetrating head injury), or other fea circumstances and in Some cases to decompensated reactions. tures (e.g., occurring in a specific location or over a wide “Hypothalamic hypophysiotropic factors' were proposed by spread area). Head injury usually refers to TBI, but is a G. W. Harris in the 1940s and substantial body of evidence has broader category because it can involve damage to structures confirmed that these factors may contribute to stress disorders other than the brain, such as the scalp and skull. (Chrousos, G.P. Stress and disorders of the stress system Nat. 0040 Neurotoxicity Rev. Endocrinol. 5,374-381 (2009); published online 2 Jun. 0041 Neurotoxicity: Neurotoxicity occurs when the 2009; doi:10.1038/nrendo.2009.106; Raisman, G. An urge to exposure to natural or artificial toxic Substances, which are explain the incomprehensible: Geoffrey Harris and the dis called neurotoxins, alters the normal activity of the nervous covery of the neural control of the pituitary gland. Ann. Rev. system in Such a way as to cause damage to nervous tissue. Neurosci. 1997: 20:533-566; Chrousos, G. P. & Gold, P. W. Neurotoxicity can result from exposure to Substances used in The concepts of stress and stress system disorders: overview chemotherapy, radiation treatment, drug therapies, certain of physical and behavioral homeostasis. JAMA 1992: 267: drug abuse, and organ transplants, as well as exposure to 1244-1252. Charmandari. e., Tsigos, C. & Chrousos, G. P. heavy metals, certain foods and food additives, pesticides, Neuroendocrinology of stress. Ann. Rev. Physiol. 2005. 67: industrial and/or cleaning solvents, cosmetics, and some 259-284.) naturally occurring Substances. Symptoms may appear 0052 Excess Sweating immediately after exposure or be delayed. They may include 0053 Hyperhidrosis or excess sweating, can either be gen limb weakness or numbness, loss of memory, vision, and/or eralized or localized to specific parts of the body. Hands, feet, intellect, uncontrollable obsessive and/or compulsive behav armpits, and the groin area are among the most active regions iors, delusions, headache, cognitive and behavioral problems of perspiration due to the relatively high concentration of and sexual dysfunction. Sweat glands. When excessive Sweating is localized it is 0042. Depression referred to as primary or focal hyperhidrosis. Generalized or 0.043 Anti-depressant, clinical depression, major depres secondary hyperhidrosis usually involves the body as a whole Sion, unipolar depression, unipolar disorder or recurrent and is the result of an underlying condition. Hyperhidrosis depression in the case of repeated episodes) is a psychiatric can also be classified depending by onset, either congenital or diagnosis for a mood disorder characterized by episodes of acquired. Primary or focal hyperhidrosis must be distin all-encompassing low mood accompanied by low self-esteem guished from secondary hyperhidrosis, which can start at any and loss of interest or pleasure in normally enjoyable activi point in life. The later form may be due to a disorder of the ties (anhedonia) and disturbed sleep (typically early morning thyroid or pituitary glands, diabetes mellitus, tumors, gout, awakening). MDD is a syndrome described by a cluster of menopause, certain drugs, or mercury poisoning. Hyper symptoms in the American Psychiatric Association’s diag hidrosis may also be divided into palmoplantar (symptomatic nostic manual, DSM-IV. The term “depression' is ambiguous Sweating of primarily the hands or feet), gustatory and gen and can be used to describe MDD, but is also used to describe eralized hyperhidrosis. other mood disorders or to lower mood states lacking clinical 0054 Symptoms Related to Drug Withdrawal significance. Bipolar disorder or bipolar affective disorder 0055 Withdrawal is the group of symptoms that occur (historically known as manic-depressive disorder or manic upon the abrupt discontinuation or decrease in intake of medi depression) is a psychiatric diagnosis for a mood disorder cations or recreational drugs. In order to experience the Symp characterized by episodes of a frenzied State known as mania toms of withdrawal, one must have first developed a physical (or hypomania), typically alternating with episodes of or mental dependence (often referred to as chemical depen depression described in DSM-IV. dency). Withdrawal happens after consuming one or more US 2014/021248.6 A1 Jul. 31, 2014

substances for a certain period of time, which is both dose 12 of these 35 papers provided some information on the dependent and varies based upon the drug consumed. mechanism of action of Isometheptene for the treatment of 0056. In view of these various aspects of migraine a wide migraine, the pharmacological profile of Isometheptene in variety of prospective targets for migraine treatment exists. animal models, and the pharmacokinetics and metabolism of 0057. As is presently understood, sympathomimetic drugs Isometheptene in animals and humans. None of these publi activate the sympathetic nervous system and mimic the effect cations provide information on the toxicological profile of of catecholamine Substances that act directly as agonists of Isometheptene in animals. one or more receptors that include alpha (C.1, C2) and beta (B) 0061 Moffett, Adrienne; Swash, M: Scott, D F (1972). adrenergic receptors. Natural endogenous catecholamines “Effect of tyramine in migraine: a double-blind study”. Jour include dopamine, norepinephrine (noradrenaline), and epi nal of Neurology, Neurosurgery, & Psychiatry with Practical nephrine (adrenaline). Of these catecholamine (or monoam Neurology 35, 4: 496-499. doi:10.1 136/innp.35.4496. ine) Substances, dopamine and norepinephrine are generally 0062 D’Andrea, G: Nordera, G P: Perini, F: Allais, G: considered neurotransmitters, and adrenaline is generally Granella, F (May 2007). “Biochemistry of neuromodulation considered a hormone, but these distinctions are vague in in primary headaches: focus on anomalies of tyrosine practice. Generally, sympathomimetic drugs act in several metabolism'. Neurological Sciences 28, Supplement 2: S94 direct or indirect ways including: causing the release of cat S96. doi:10.1007/s1OO72-007-0758-4. PMID 17508.188. echolamines from Synapses; inhibiting the reuptake of inhib 0063 Borowsky B. Adham N. Jones K A. Raddatz, R, iting the reuptake of catecholamines from Synapses; inhibit Artymyshyn R, Ogozalek K. L. Durkin MM, Lakhlani PP. ing the metabolism or degradation of catcholamines; Bonini JA, Pathirana S, et al. (2001). “Trace amines: identi interacting with adrenergic receptors directly as agonists; fication of a family of mammalian G protein-coupled recep and/or modulating the sensitivity or responsiveness of cat tors. Proc Natl AcadSci USA 98:8966-8971. echolamine responsive cells or systems. 0064. Imidazoline-I1 Receptors: 0058 Isometheptene has been shown to increase heart rate 0065. A receptor system that modulates sympathetic and diastolic blood pressure, which are properties associated effects has been recently identified and termed the imidazo with sympathomimetic agents. The heart rate increase has line receptors (I receptors). Three isoforms have been identi been shown to be blocked by propranolol, a non-selective fied: I1, I2, and I3. A putative natural ligand is agmatine (see beta (B) adrenergic receptor antagonist, while the diastolic below). blood pressure increase has been shown to be blocked by 0.066 Moxonidine is believed to be an agonist of the I1 prazosin, an alpha-1 (C.1) adrenergic receptor antagonist. receptor and the net effect of moxonidine is sympathoinhibi Valdivia, L. F., Cenurion, D., Perusquia, M., Arulmani, U. tory (Schafer), so that agonists of the I1 receptor generally are Saxena, P. R., and Villalom, C.M. “Pharmacological Analysis sympathoinhibitory and antagonists are sympathomimetic. of the Mechanisms Involved in the Tachycardic and Vasopres Schafer U. Burgdorf C, Engelhardt A. Raasch W. Kurz, T. Sor Responses to the Antimigraine Agent, Isometheptene, in Richardt G. Moxonidine displays a presynaptic alpha-2- Pithed Rats' Life Sciences 74: 3223-3234 (2004). Together adrenoceptor-dependent synergistic sympathoinhibitory these findings Suggest that racemic Isometheptene has sym action at imidazoline-I1 receptors. Ann NY AcadSci 2003: pathetic effects which are complex on heart rate and blood 1009: 265-269. pressure. However, it is unknown whether Isometheptene 0067. Clonidine is also a ligand of the I1/moxonidine interacts with alpha (C.)- or beta (B)-adrenergic receptors as an receptor and is sympathoinhibitory. However, Clonidine is agonist, and/or stimulates the release of catecholamines, and/ also a centrally acting alpha-2 agonist, which is believed to or modulates the sensitivity of catecholamine responsive cells account for many of its effects on sympathetic tone and on or systems. treatment effects. Central alpha-2 receptors are believed to be 0059. Some comparisons have been made of the pressor pre-synaptic and agonists to central alpha-2 receptors effects of Isometheptene and those of tyramine (4-hydrox decrease sympathetic outflow. Clonidine was developed and yphenethylamine; para-tyramine, mydrial or uteramin). initially FDA approved and used for the treatment of hyper Tyramine is a naturally occurring monoamine compound that tension but has since found other uses, including treatment of acts as a catecholamine releasing agent. Tyramine is present attention deficit hyperactivity disorder (ADHD) in an in a plants and animals. But high levels of tyramine are extended release form. Off-label uses, supported by less evi typically found in the diet from the metabolism of microor dence, Suggest effects on Some types of neuropathic pain, ganisms (decarboxylation of tyrosine) in cheese or wine. In opioid detoxification, sleep hyperhidrosis, anxiety and panic humans, tyramine is metabolized by monoamine oxidase disorder, insomnia, menopausal symptoms and as an anaes (MAO) and if MAO function is inhibited by the use of thetic drug. monoamine oxidase inhibitors (MAOIs), then dietary 0068 Clonidine is applied in conjunction with stimulants tyramine can raise blood pressure and in rare cases, precipi to treat ADHD because it may moderate ADHD-associated tate a hypertensive crisis, which is also referred to as the impulsive and oppositional behavior, and may reduce tics, "cheese effect”. Tyramine may act on a group of G protein which are involuntary movements in ADHD or Tourette syn coupled receptors, such as TA1 or TAAR1 that is expressed in drome. Clonidine is used to ease sympathetic withdrawal brain as well as peripheral tissues, including the kidneys. symptoms (tachycardia and hypertension) associated for 0060 A literature search was conducted to obtain infor example with the long-term use of narcotics, alcohol and mation on nonclinical pharmacology and safety (safety phar nicotine (Smoking). Moreover, Clonidine is applied to reduce macology, pharmacokinetic, and toxicology) of Isomethep Sweating, hot and cold flushes, and general restlessness in tenealone and in a CDP to supportan IND on Isometheptene. drug withdrawal and also menopause. The results of this literature search indicated that 35 papers 0069 Clonidine has also been studied to treat pain during that reference Isometheptene have been published since heart attack, postoperative and intractable pain as an oral or 1972. Upon review of the abstracts of these published papers, epidural agent. US 2014/021248.6 A1 Jul. 31, 2014

0070 Agmatine (4-aminobutyl)guanidine is believed to Vation of imidazoline I(2A) receptors in adrenal gland of rats. be an endogenous ligand for Il receptors and to function as an Neurosci Lett 2010; 468:297-9; Hwang SL, et al. Activation agonist. Agmatine was identified in herring sperm in 1910 by of imidazoline receptors in adrenal gland to lower plasma Albrecht Kossel. Kossel, Albrecht 1910. Uber das Agmatin. glucose in Streptozotocin-induced diabetic rats. Diabetologia Zeitschaft fir Physiologische Chemie 66: 257-261 2005:48:767-75; OZyazgan S, etal. The effect of agmatine on 0071 Agmatine has several properties of neurotransmit the vascular reactivity in streptozotocin-diabetic rats. Phar ters: it is synthesized in the brain, stored in synaptic vesicles, macol Res 2003: 48:133-8) and anti-oxidant (Battaglia V. accumulated by uptake, and released by membrane depolar Grancara S. Satriano J. Saccoccio S, Agostinelli E, Toninello ization. Agmatine is the decarboxylation product of the amino A. Agmatine prevents the Ca(2+)-dependent induction of acid arginine and inactivated by agmatinase. In addition to Il permeability transition in rat brain mitochondria. Amino receptors, agmatine binds to the C-adrenergic receptor Acids 2010: 38:431-7. Condello S, Curr M, Ferlazzo N, (where it has neither agonist nor antagonist effect) and blocks Caccamo D. Satriano J., Ientile R. Agmatine effects on mito NMDA receptors and other cation ligand-gated channels. chondrial membrane potential and NF-kB activation protect Functionally, agmatine inhibits nitric oxide synthase (NOS) against rotenoneinduced cell damage in human neuronal-like and induces the release of some peptide hormones. SH-SY5Y cells. J Neurochem 2010: 116:67-75; Demady D R, Jianmongkol S. Vuletich J L. Bender AT, Osawa Y. Agma 0072 Exogenous agmatine treatment has been shown in tine enhances the NADPH oxidase activity of neuronal NO animal studies to exert a variety of activities including: anti synthase and leads to oxidative inactivation of the enzyme. convulsant (Aricioglu, F., et al. Effect of agmatine on electri Mol Pharmacol 2001:59:24-9). Agmatine is metabolized by cally and chemically induced seizures in mice. Ann. N.Y. agmatinase (Mistry S K, et al. Cloning of human agnatinase. Acad. Sci., 2003. 1009:141-146), antineurotoxic (Halaris, A., An alternate path for polyamine synthesis induced in liver by Piletz, J. Agmatine: metabolic pathway and spectrum of hepatitis B virus. Am J Physiol Gastrointest Liver Physiol. activity in brain. CNS Drugs, 2007. 21:885-900), vasodila 200228202):G375-81), which is upregulated in hippocampal tory (Satriano, J. Agmatine: at the crossroads of the arginine interneurons of subjects with mood disorders (Bernstein, pathways. Ann. N.Y. Acad. Sci., 2003. 1009:34–43), neuro H-G et al. Agmatinase, an inactivator of the putative endog protective (Kuo, J. R. et al. Agmatine promoted angiogen enous antidepressant agmatine, is strongly upregulated in esis, neurogenesis, and inhibition of gliosis-reduced trau hippocampal interneurons of Subjects with mood disorders. matic brain injury in rats. J. Trauma 2011 71(4):E87-93. doi: Neuropharmacology 62 (2012) 237e246). 10.1097/TA.0b013e31820932e2: Lu W. et al. Agmatine inhibits morphine-induced memory impairment in the mouse 0073. Agmatinase is an enzyme encoded in human by a step-down inhibitory avoidance task. Pharmacol Biochem gene located on chromosome 1 p36, a gene locus which has Behav 2010; 97:256-61; McKay B E, et al. Learning and repeatedly been linked to bipolar disorder and major depres memory in agnatine-treated rats. Pharmacol Biochem sion and in some cases to schizophrenia (Fanous A. H. et al. Behavior 2002; 72:551-7; Zarifkar A, et al. Agmatine pre Genetic overlap of schizophrenia and bipolar disorder in a vents LPS-induced spatial memory impairment and hippoc high-density linkage Survey in the Portuguese Island popula ampal apoptosis. EurJ Pharmacol 2010; 634:134-8; Bhutada, tion. Am J Med Genet B Neuropsychiatr Genet. 2012 159B Petal Agmatine, an endogenous ligand of imidazoline recep (4):383-91. doi:10.1002/ajmg.b.32041. Epub 2012 Mar. 27. tor protects against memory impairment and biochemical PMID:22461138; McGuffin, P., et al. Whole genome linkage alterations in Streptozotocin-induced diabetic rats. Progress scan of recurrent depressive disorder from the depression in Neuro-Psychopharmacology & Biological Psychiatry 37 network study. Hum. Mol. Genet., 2005. 14:3337-3345; (2012) 96-105), anti-apoptotic (Wang, C. C., et al., Beneficial Tatemir, D., et al. Chromosomal fragile site expression in effect of agmatine on brain apoptosis, astrogliosis, and edema Turkish psychiatric patients. Psychiatry Res. 2006. 144: 197 after rat transient cerebral ischemia. BMC Pharmacol. 2010. 203: Demirhan, O., et al. The expression of folate sensitive 10:11), anxiolytic (Gong, Z. H., et al. Anxiolytic effect of fragile sites in patients with bipolar disorder. Yonsei Med. J. agmatine in rats and mice. Eur. J. Pharmacol. 2006. 550: 112 2009. 50:137-141; Diagnostic and Statistical Manual of Men 116), memory enhancing (Arteni NS, et al. Agmatine facili tal Disorders Revised 1987. DSM IIIR: American Psychiatric tates memory of an inhibitory avoidance task in adult rats. Association; Kaneva, R., et al. Bipolar disorder in the Bul Neurobiol Learn Mem 2002; 78:46.5-9.: Liu P and Bergin D garian Gypsies: genetic heterogeneity in a young founder H. Differential effects of i.c. v. microinfusion of agmatine on population. Am. J. Med. Genet. B Neuropsychiatr. Genet. spatial working and reference memory in the rat. Neuro 2009. 150B:191-201: Fullerton, J.M., et al. Two-dimensional science 2009; 159:951-61; Liu P and Collie ND. Behavioral genome scan identifies multiple genetic interactions in bipo effects of agmatine in naive rats are task- and delay depen lar affective disorder. Biol. Psychiatry, 2010. 67:478-486). dent. Neuroscience 2009; 163:82–96), antidepressant 0074. Several agonists of the I1 receptor have been iden (Zomkowski, A. D., et al. Agmatine produces antidepressant tified and studied. Moxonidine and Rilmenidine are consid like effects in two models of depression in mice. NeuroReport ered selective agonists at the imidazoline receptor Subtype 1 2002. 13:387-391; Aricioglu, F. and Altunbas, H. Is agmatine (I1). Moxonidine and Rilmenidine are prescription medica an endogenous anxiolytic/antidepressant agent? Ann. N.Y. tions for the treatment of hypertension outside the U.S. Ril Acad. Sci. 2003. 1009: 136-140. Li, Y. F., et al. Antidepres menidine is marketed under the brand names HYPERIUM, Sant-like effect of agmatine and its possible mechanism. Eur. Iterium and Tenaxum. The I1 receptor subtype is found in J. Pharmacol. 2003. 469:81-88; Halaris and Piletz, 2007 both the rostral ventro-lateral pressor and ventromedial (Ibid.); Taksande, B. G. et al. Antidepressant like effect of depressor areas of the medulla oblongata. Moxonidine there selective serotonin reuptake inhibitors involve modulation of fore causes a decrease in sympathetic nervous system activity imidazoline receptors by agmatine. Neuropharmacology and, therefore, a decrease in blood pressure. Compared to the 2009. 57: 415-424), anti-diabetic (Chang CH, et al. Increase older central-acting antihypertensives, moxonidine binds of beta-endorphin secretion by agmatine is induced by acti with much greater affinity to the Il-receptor than to the alpha US 2014/021248.6 A1 Jul. 31, 2014

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0104. Accordingly, the compositions containing induced canine external carotid vasoconstriction appeared to Isometheptene and other active ingredients useful in this be mediated by both indirect (a tyramine-like action) and invention may be administered either intravenously, parenter direct (acting at receptors) mechanisms that mainly involved ally, orally, transdermally, transmucosally, or as a nasal spray. alpha-2A-- and alpha-2C-adrenoceptors while the 0105 For example, the Isometheptene mucate is com involvement of alpha-1-adrenoceptors was rather limited monly administered as a mucate salt in a combination drug for this effect. product (CDP) containing Isometheptene, dichloral 0111. A third publication (Mehrotra 2006) investigated phenazone (DCP), and acetaminophen (APAP) in which the the in vitro effects of current and prospective anti-migraine combination product can be abbreviated IDA (IDA) or a drugs on porcine meningeal artery segments. Porcine different CDP containing Isometheptene, caffeine, and meningeal artery veins (n=5) were exposed to a single con acetaminophen (APAP), which combination product can be centration response curve of Isometheptene (about 0.01 to 50 abbreviated ICA (ICA). uM), either in the absence or presence of a particular antago 0106. In the US, the IDA and ICA products are marketed nist concentration. Results indicated that Isometheptene (and as prescription drugs under the auspices of the Drug Efficacy other antimigraine drugs) failed to contract the meningeal Study Implementation (DESI), because they were available artery while C.-CGRP (calcitonin gene related peptide) pro before the 1962 FDA reform (1938-1962), were evaluated by duced concentration-dependent relaxations that were antago National Academy of Sciences/National Research Council to nized by olcegepant (a CGRP receptor antagonist). Thus, the conduct Drug Efficacy Study and were deemed effective for porcine meningeal artery might not be involved in the vaso tension and vascular headache and possibly effective for constriction of the carotid vascular bed elicited by anti-mi migraine headache. graine drugs in anaesthetized pigs. 0107. One DESI ICA product marketed as MigraTen(R) 0112 Taken together, these nonclinical pharmacological contained: Isometheptene mucate (65 mg); Caffeine (100 results suggest that Isometheptene causes cranial vasocon mg): acetaminophen (APAP) (325 mg) in a capsule formula striction that may be effective in the treatment of migraine. tion and was deemed possibly effective for relief of migraine Furthermore, the non-clinical state of the art is summarized headache in a regimen: 2 capsules at once; 1 capsule q1h (up from the following reviews. to 5 caps/12 h period) and deemed effective for the relief of tension headache or vascular headache in a regimen of 1-2 0113 Solomon G. D. “The pharmacology of medications capsules q4h (up to 8 caps/day). Another ICA DESI product used in treating headache.” Semin. Pediatr. Neurol. (1995), on the market is branded as ProdrinRand contains 130 mg of 2(2):165-177. This review article discussed the pathogenesis Isometheptene, 20 mg of caffeine, 500 mg of APAP in a tablet of migraine and the various pharmaceutical agents, including formulation with the same dosing instructions for migraine, Isometheptene, used for treating migraine. Research on the tension and vascular headache as MigraTen. trigeminovascular system, serotonin receptors, and Substance 0108. One of the DESI IDA products marketed under the Phave provided clues to improving the pharmacotherapy of trademark Midrin R, contains Isometheptene Mucate (65 migraine, a common and disabling disease of uncertain mg); Dichloralphenazone (100 mg) and Acetaminophen (325 pathogenesis. mg) and is indicated for relief of migraine headache with a 0114 Selective serotonin agonists, nonsteroidal anti-in usual adult dosage of two capsules at once followed by one flammatory drugs (NSAIDs), Isometheptene mucate, and capsule every hour until relieved, administering up to 5 cap phenothiazines were listed as being useful to treat acute sules within a twelve hour period. MidrinR) is also indicated migraine attacks. Prophylactic agents for migraine were for relief of tension headache with a usual adult dosage of one listed to include beta-blockers, calcium channel blockers, or two capsules every four hours up to 8 capsules a day.” NSAIDs, antidepressants, and valproate. Isometheptene While the FDA has recognized that Isometheptene is safe and mucate, a sympathomimetic agent, was listed as being useful effective by the standards of DESI, various administrative for adults and adolescents with migraine. Actions involve actions to compel DESI manufacturers to upgrade the status indirect B- and C.-andrenoreceptor activity and direct C.-an of DESI drugs to the level of NDA drugs have resulted in drenoreceptor agonism (Valvidia et al. 2004). Available ending the US Midrin production by Curaco since 2011; preparations listed were Isometheptene with a mild sedative however, numerous other manufacturers have continued to and acetaminophen, or a CDP. The listed usual dosage and manufacture and distribute similar products. dosing regimen for adults and adolescents was 2 capsules at 0109. In the pharmacological literature references, effects onset, then 1 capsule every hour to a total of 5 capsules per of medication and treatment of migraine have been reported. day. For children from 8 to 12 years of age, the listed dosage 0110. Another publication (Willems 2001) relates to an and dosing regimen was 1 capsule initially, followed by 1 investigation of the external carotid vascular effects of capsule every hour to a limit of 3 capsules per day. Isometheptene (administered as 1 minute intra-arterial IA 0115 Gallagher RM and Cutrer F M. “Migraine: Diag infusions at 10,30, 100, and 300 g/min or about 1, 3, 10, and nosis, Management, and New Treatment Options.” Am J 30 ug/kg for a 10 kg dog) in Vagosympathectomized dogs. At Managed Care (2002), 8(3):S58-S73. This review article these dose levels, Isometheptene caused dose-dependent summarized the safety and tolerability of medications used to decreases in external carotid blood flow without affecting treat acute migraine attacks, reviewed the classification of blood pressure or heart rate. The vasoconstrictor responses to headaches and the causes of and diagnostic criteria for Isometheptene were attenuated in dogs pretreated with reser migraine, and discussed the clinical tolerability profiles and pine at 5,000 ug/kg and were also attenuated by rauwolscine therapeutic benefits of second-generation triptans. Mild to (apha-2-adrenoceptor antagonist) at 300 g/kg in untreated moderate migraines were listed as being often treated with dogs and were practically abolished in reserpine-pretreated aspirin, acetaminophen, NSAIDs, antiemetic drugs, or dogs. Results using selective antagonists for specific alpha Isometheptene (in combination with other active agents. Such 2-adrenoceptor Subtypes indicated that the Isometheptene as caffeine and acetaminophen). US 2014/021248.6 A1 Jul. 31, 2014

0116 Gibbs TS, Fleischer ABJr., Feldman SR, Sam MC, I0123. There is an urgent need for new and safer prescrip O'Donovan C. A. Health care utilization in patients with tion drugs for episodic tension-type headaches (ETTH). migraine: demographics and patterns of care in the ambula Although Isometheptene was approved as a DESI drug for tory setting. Headache (2003), 43(4):330–335. migraine headaches (OCTINR, Knoll) during the period of 0117 Spierings 1980 reported that Isometheptene admin 1938 to 1962, FDA withdrew approval of the OCTINR NDA istered intravenously IV at 0.25 mg/kg for each of 4 treat 6-420 in 1981 citing a lack of substantial evidence of effec ments for a maximum dose of 1.0 mg/kg) was compared to tiveness; however, it has been widely marketed as a combi saline (control) and shown to cause vasoconstriction in the nation ingredient drug product. Since the combination the arteriovenous anastomoses of anaesthetized cats (8/group). FDA considers combination drug products (CDPs) that con This review article indicated that migraine affects people of tain Isometheptene to be “identical, related or similar all races and both sexes. Women accounted for 79% (61% (“IRS) to the single-ingredient Isometheptene products between 20 and 49 years of age) of physician visits for reviewed under DESINotice 3265, it has declared Isomethep migraines and Caucasians for 91% of the physician visits. tene-containing drug products unapproved new drugs that The prevalence of headaches in preschool- and School entry cannot be marketed without approved NDA. Since (R)- age children varies from 5.9% to 37.7%. The most commonly Isometheptene binds I with much stronger affinity than (S)- used CDP for migraine over a 9-year study period (1990 to Isometheptene, delivering TNX-201 as a single (R)-isomer of 1998) was butalbital/aspirin/caffeine with 2.3 million pre Isometheptene may result in a better therapeutic than the Scriptions and the second most commonly used CDP was racemate. acetaminophen/dichloralphenaZone/Isometheptene mucate 0.124. It is therefore an object of the present invention to with 2.1 million prescriptions. develop a single isomeric form or enantiomer which can be identified on the basis of a specific receptor binding affinity. 0118. Mehrotra S, Gupta S, Chan KY, Villalon C M, 0.125. In particular, it is an object of the present invention Centurion D. Saxena PR, MaassenVanDenBrink A. “Current to isolate and characterize the enantiomer of Isometheptene and prospective pharmacological targets in relation to anti indicating the greater potential of relief for the sufferer from migraine action.” Naunyn Schmiedebergs Arch Pharmacol headache, cramps, menopause, flushes, cognitive impair (2008), 378(4):371-94. This review article indicated that ment, neurotoxins, TBI, anxiety disorders, depression, bipo migraine is a recurrent, incapacitating, neurovascular disor lar disorder, epilepsy, Schizophrenia, stress disorders, excess der characterized by unilateral and throbbing headaches asso Sweating and withdrawal symptoms. ciated with photophobia, phonophobia, nausea, and vomit I0126. More specifically, it is an object of the present inven ing. The pathogenesis of migraine headache was considered tion to treat episodic tension-type headaches (as more to involve three key factors, a) the cranial blood vessels, b) the recently classified by the International Headaches Society, trigeminal innervation of these vessels, and c) the reflex con 2013) by administering a composition containing an nection of the trigeminovascular system in the cranial para Isometheptene enantiomer. (R) Isometheptene was found to sympathetic outflow. bind more strongly to an Imidazoline Receptor Subtype 1 (I) 0119) Specific drugs (ergotamine, and dihydroergota than the opposite (S)-Isometheptene enantiomer. mine) used in the treatment of migraine interact with vascular receptors, a fact that has raised concerns about the cardiovas LIST OF ACRONYMS AND ABBREVIATIONS cular safety of these pharmaceutical agents. The various potential pharmacological targets for the treatment of O127 migraine were discussed. These targets included 5-hydrox ytryptamine (5-HT) receptor subtypes (5-HT1-7), adrenergic AE Adverse Event receptor Subtypes (C1, C2, and B), calcitonin gene-related API Active Pharmaceutical Ingredient peptide (CGRP1 and CGRP2) receptors, adenosine (A1, A2, CNS Central Nervous System and A3) receptors, glutamate (NMDA, AMPA, kainate, and CGRP Calcitonin Gene Related Peptide CDP Combination Drug Product metabotropic) receptors, dopamine receptors, endothelin CDH Chronic Daily Headache receptors, and female hormone (estrogen and progesterone) CFU Colony Forming Units receptors plus some miscellaneous receptors and ion chan CTTH Chronic Tension-Type Headache nels. DESI Drug Efficacy Study Implementation EAERS FDA Adverse Event Reporting System 0120 Reference is also made to Isometheptene treatment FDA Food and Drug Administration for relieving symptoms of acute pancreatitis in the afflicted GC, GLC Gas Liquid Chromatography GMP Good Manufacturing Practice patient in U.S. Pat. No. 4,522,827 (Knoll A G). HPLC High Performance Liquid Chromatography 0121. In studying the effect of Isometheptene, as an indi CA Formulation containing Isometheptene rectly acting non-ergot sympathomimetic with analgesic and (65 or 130 mg), caffeine (20 or 100 mg) and acetaminophen (325 or 500 mg) antispastic properties, on Secretin-cholecystokinin-stimu CH international Conference of Harmonization lated pancreatic and biliary outputs, Gullo et al. Dig. Dis. Sci. CH intracranial Hemorrhage (1985) reported that the drug inhibited only pancreatic out DA Formulation containing Isometheptene puts. Accordingly, the inhibitory effect on pancreatic secre (65 mg), dichloralphenaZone (100 mg) and acetaminophen (325 mg) tion, coupled with the analgesic action, Suggests a potential HCD international Headache Classification benefit of this drug in acute pancreatic pathology. HS international Headache Society MH Sometheptene mucate 0122) Moreover, as is known for many racemic com ND nvestigational New Drug pounds in medicine, the biology of only one single racemate RS dentical, Related, or Similar isomer was often found to be pharmacologically effective V intravenous with reduced significant side effects which may be due to the NMDA N-Methyl-D-aspartic acid nonspecific binding of the opposite racemate. US 2014/021248.6 A1 Jul. 31, 2014 10

-continued 0.135 An aspect of the invention now provides an (S)- Isometheptene mucate Salt according to Formula (II): NLT Not less than NMT Not more than

NOS Nitric oxide synthase PCV Post-partum cerebral vasculopathy PT Preferred term RRT Relative retention time SAE Serious Adverse Event SOC System Organ Class TDD Total daily dose TNX-201 TONIX oral dosage form containing TBD mg of (R)-Isometheptene mucate TTH Tension-Type Headache

SUMMARY 0.136 Another aspect of the invention provides an (R)- Isometheptene mucate Salt according to Formula (III): 0128 Aspects of the invention disclosed herein include synthesis and purification of the Isometheptene racemic com pound, having a structure according to Formula (I),

(6-methylamino-2-methylheptene)

N-rs or a hydrochloride, or a pharmaceutically acceptable addition 0.137 A special aspect of the invention provides a purified salt thereof. (R)-Isometheptene mucate salt in crystalline form. 0129. An aspect of the invention is found in the isolation 0.138. Further aspects of the invention disclosed herein and and purification of Isometheptene Stereoisomer 1 and stere copending reference applications include methods of synthe oisomer 2 which are stereochemically characterized as being sis and purification, isolation and characterization of com parative mechanism of action the Isometheptene. S-enantiomer and R-enantiomer, respectively. 0.139. An aspect of the invention also provides administer 0130. A special aspect shows an S-enantiomeric ing a therapeutic amount of a composition comprising an Isometheptene compound comprising the structure according Isometheptene (S)-enantiomer or (R)-enantiomer compound; to Formula (Ia), and a pharmaceutically acceptable carrier. 0140. This aspect of the invention provides such a compo sition, further including a therapeutic effect on headaches, (S)-Isometheptene cramping or cognitive disorders; in a particular aspect, the Isometheptene compounds can ameliorate or extinguish symptoms of headaches, including tension, vascular or migraine headaches, cramping or cognitive disorders. Sr.'s 0.141. This aspect of the invention provides a compound or composition, administered alone or in combination with one or more additional therapeutics, chemotherapeutic drugs, 0131 or a hydrochloride, or a pharmaceutically accept anti-proliferative agents, anti-inflammatory agents, anti-asth able addition salt thereof, and matic agents, anti-allergic agents, immunosuppressive 0132) an R-enantiomeric Isometheptene compound com agents, immunomodulatory agents, cardiovascular disease prising the structure according to Formula (Ib), treatmentagents, anti-diabetic agents, or blood disorder treat ment agents. 0142. An aspect of the invention provides a method, com (R)-Isometheptene prising administering to a patient an effective amount of a compound of Isometheptene (R)-enantiomer or (S)-enanti omer, or hydrochloride or addition salt thereof, or the com sN O position of Such compounds, so as to treat, reduce the severity Nr or even prevent the pain associated with the disease or con dition; wherein the disease or condition is one or more of inflammatory diseases or disorders, allergic diseases or dis 0.133 a hydrochloride, or a pharmaceutically acceptable orders, metabolic diseases, cardiovascular disease or disor addition salt thereof. ders, or a neurogenic vascular disease or painful condition 0134. An aspect of the invention provides each Isomethep associated therewith. tene isomer compound as a hydrochloride, a tartrate (1:1) or 0.143 An aspect of the composition including (R)- a mucate (2:1) salt. Isometheptene addition salt provides an ameliorative effect US 2014/021248.6 A1 Jul. 31, 2014

on headaches or cramping due to a vascular, neurovascular or salt combined with an active ingredient selected from neurogenic disorder or dysfunction during migraine epi acetaminophen, ibuprofen, aspirin, caffeine, dichloral sodes. phenaZone, naproxen, and Sumatriptan Succinate; and a phar 0144. An aspect of the composition including (R)- maceutically acceptable nontoxic carrier. Isometheptene addition salt provides an ameliorative effect 0156 Another separate aspect is found in the method of on headaches or cramping due to a vascular, neurovascular or treatment of migraine by administering a therapeutic amount neurogenic disorder or dysfunction during menstrual cycle of a composition comprising Isometheptene (S)-enantiomer episodes. mucate salt and a pharmaceutically acceptable carrier to a 0145 Another aspect of the (R)-Isometheptene treatment patient in need of Such treatment. would ease hypertension alleviating composition comprising 0157. A further method of treatment of migraine com a therapeutic amount of the (R)-Isometheptene enantiomer prises administering to a patient in need of such treatment, a compound or salt, and a pharmaceutically acceptable addi therapeutic amount of a composition comprising Isomethep tion salt thereof, and a nontoxic carrier. tene (R)-enantiomer mucate salt combined with an active 014.6 Another aspect is found in the effect of the compo ingredient selected from acetaminophen, ibuprofen, aspirin, sition of (S)-enantiomer of Isometheptene hydrochloride or caffeine, dichloralphenaZone, naproxen, and Sumatriptan pharmaceutically acceptable addition salt thereof, and one or Succinate; and a pharmaceutically acceptable carrier. more pharmaceutical Substances selected from acetami 0158. According to the present invention, a method of nophen, ibuprofen, aspirin, caffeine, dichloralphenaZone, nitric oxidase inhibition includes administering a therapeutic naproxen, and Sumatriptan Succinate; and a nontoxic carrier. amount of a composition comprising Isometheptene (R)- 0147 Another aspect of the invention provides the selec enantiomer mucate salt combined with an active ingredient tive I1 receptor binding effect in a composition, comprising selected from acetaminophen, ibuprofen, aspirin, caffeine, the therapeutic amount of (R)-Isometheptene hydrochloride dichloralphenaZone, naproxen, and Sumatriptan Succinate; or pharmaceutically acceptable addition salt thereof, and one and a pharmaceutically acceptable carrier. or more pharmaceutical Substances selected from acetami 0159. The inventive methods of treatment with any of the nophen, ibuprofen, aspirin, caffeine, dichloralphenaZone, Isometheptene compounds may include a composition com naproxen, and Sumatriptan Succinate; and a nontoxic carrier. prising peroral, parenteral. IV. Sublingual, inhalable, or trans 0148. The aforementioned ameliorative aspect of the dermal formulations; such a method may include a gel, cap invention further comprises a hypertension lowering effect. sule, tablet, delayed release pellet or tablet coating 0149. An advantageous aspect of the invention provides a formulation. treatment wherein a single dosage of (R)-Isometheptene 0160 An inventive method of treating hypertension by enantiomer hydrochloride or addition salt thereof is admin administering to a patient in need of Such treatment, a thera istered to a patient, optionally twice, three times or four times peutic amount of a composition comprising Isometheptene in a day, in capsules containing increasing amounts of 15 mg. (R)-enantiomer mucate salt combined with an active ingredi 30 mg. 32.5 mg, 50 mg, 65mg, 100 mg, 130 mg, 150 mg,300 ent selected from acetaminophen, ibuprofen, aspirin, caf mg or 500 mg. feine, dichloralphenaZone, naproxen, and Sumatriptan Succi 0150. The pharmaceutical composition also including nate; and a pharmaceutically acceptable carrier. Isometheptene mucate, Isometheptene bitartrate, or 0.161 Further aspects of the invention disclosed herein Isometheptene hydrochloride can be administered in the include methods of synthesis and purification, isolation and treatment of migraines and tension headaches, its effective characterization of comparative mechanism of action the ness believed based on its vasoconstricting properties. Isometheptene, as well as in vitro tests of the isolated 0151. Moreover, the pharmaceutical composition includ Isometheptene racemates, as centrally acting agonists; in par ing an Isometheptene salt, and an inert pharmaceutically ticular, the invention shows aspects of the different affinities acceptable excipient, can be further combined with one or of the (S)-enantiomer and (R)-enantiomer of Isometheptene more anti-tension compounds including paracetamol, dichlo to various CNS receptors. ralphenaZone, Sumatriptan Succinate, caffeine and either 0162. Other advantageous aspects of the invention provide acetaminophen, naproxen, aspirin or ibuprofen. a preparation of purified Isometheptene Stereoisomers 1 and 2 0152 The following methods address the tension head through chemical synthesis from chiral precursors, and fur aches, or migraine associated headaches, such as a method of ther stereochemically characterize the Isometheptene stere treatment of migraine comprising administering atherapeutic oisomers 1 and 2 as being an (S)-enantiomer and (R)-enanti composition containing (R)-Isometheptene enantiomer to a omer, respectively. human in need of Such treatment. 0163 A particularly advantageous aspect of the invention 0153. A method of treatment of migraine comprising is provided in that (R)-Isometheptene enantiomer is found to administering to a human in need of Such treatment a thera bind to the CNS moxonidine binding I1 about 60 fold more peutic composition containing the (S)-Isometheptene murate avidly than the (S)-Isometheptene enantiomer. salt in combination with Suitable pharmaceutical agents. 0164. An alternative aspect of the invention provides the 0154 Furthermore, according to the advantageous aspects purified (S)-Isometheptene enantiomer which interacts with of the invention, a method of treatment of hypertension com I1 about 60 fold less avidly than the R-Isometheptene enan prises administering a therapeutic amount of a composition tiomer. comprising Isometheptene (R)-enantiomer mucate salt and a 0.165 An advantageous aspect of the invention is found in pharmaceutically acceptable carrier to a patient in need of the selective or specific (R)-Isometheptene enantiomer activ Such treatment. ity may be favorably effective for the treatment of mild, 0155. In addition, a method of administering to a patient in moderate, or severe heart failure. need of treatment of hypertension, a therapeutic amount of a 0166 An advantageous aspect of the invention is found in composition comprising Isometheptene (R)-isomer mucate the selective or specific (R)-Isometheptene enantiomer activ US 2014/021248.6 A1 Jul. 31, 2014

ity may be favorably effective for the treatment of mild, 0177 Such an aspect of the invention provides a method of moderate, or cognitive dysfunction. treatment wherein the therapeutic composition comprises: a tablet formulation comprising about 20 mg-150 mg of 0167 One aspect of treatment includes providing the Isometheptene (R)-enantiomer, 20 mg of caffeine, and 325 or patient in need of the treatment with a therapeutic dose of 500 mg of acetaminophen, (or 200 mg, 400 mg. or 800 mg about 10-30 mg (R)-Isomethepteneenantiomer, once or twice ibuprofen). daily. Another aspect of the (R)-Isometheptene enantiomer 0.178 Alternatively, the method of treatment of claim 7. may be provided by the therapeutic dose of about 30-40 mg. wherein the therapeutic composition comprises: once or twice daily. One aspect of the (R)-Isometheptene 0179 a capsule formulation comprising about 30 to about 90 mg of Isometheptene (R)-enantiomer, about 100 mg of enantiomer may be provided by the therapeutic dose of about caffeine, and 325 mg of acetaminophen (or 200 mg. 400 mg. 50-100 mg. once or twice daily. One aspect of the (R)- or 800 mg ibuprofen). Isometheptene enantiomer may be provided by the therapeu 0180. The method of treatment of neurogenic discomfort, tic dose of about 50-500 mg. three or four doses every six to wherein the therapeutic composition comprises: eight hours daily. One aspect of the (R)-Isometheptene enan an injectable solution comprising about 30 mg to about 100 tiomer may be provided by the therapeutic dose, in controlled mg of Isometheptene (R)-enantiomer, 20 mg of caffeine, and release preparations, of about 50-500 mg. once or twice daily. 150 mg of acetaminophen (or 200 mg. 400, or 800 mg ibu profen), and physiological saline Solution. 0168 Another aspect of the invention is found in the thera 0181 Another aspect of the invention provides a method peutic use of CDP compositions including a combination of for producing a composition which includes the steps of the (R)-Isometheptene enantiomer with other analgesic mixing a therapeutically effective amount of (R)-metheptene agents (FDC). Such as caffeine, acetaminophen, Sumatriptan mucate salt with an anti-inflammatory Substance selected Succinate, aspirin, ibuprofen, naproxen and dichloral from the group consisting of acetaminophen, ibuprofen, aspi phenaZone. rin, dichloralphenaZone, naproxen, and Sumatriptan Succi 0169. An alternate aspect of the invention is provided by a nate; and a nontoxic carrier. composition combining the (S)-Isometheptene enantiomer 0182. It is known that the chiral forms of methamphet molecule with other analgesic agents (FDC). Such as caffeine, amine, amphetamine and MMDA show that chirality in acetaminophen, Sumatriptan Succinate, aspirin, ibuprofen, related molecules can have profound effects on their biologi naproxen and dichloralphenaZone. cal activity and disposition in the body. 0183 In vitro tests of the isolated Isometheptene race 0170 The invention provides these useful aspects: mates, as centrally acting agonists, show aspects of the dif ferent affinities of the (S)-enantiomer and (R)-enantiomer of 0171 A method of treatment of hypertension including Isometheptene to various CNS receptors. administering a therapeutic amount of a composition com 0184. A distinct aspect of the invention includes the dis prising Isometheptene (R)-enantiomer mucate salt and a covery that the (R)-Isometheptene enantiomer compound is pharmaceutically acceptable carrier to a patient in need of capable of selectively or specifically binding to Imidazoline Such treatment. Subtype 1 (I) receptor. 0172 A method of treatment of hypertension including 0185. Another aspect includes a composition comprising administering to a patient in need of Such treatment, a thera an (R)-enantiomer of Isometheptene or pharmaceutically peutic amount of a composition comprising an (R)- acceptable addition salt thereof, and a nontoxic carrier. Non Isometheptene mucate Salt combined with an active ingredi toxic carriers may include alpha-cyclodextrin, beta cyclodex ent selected from acetaminophen, ibuprofen, aspirin, trin, hydroxyethyl-beta-cyclodextrin, and hydroxyethyl-al caffeine, dichloralphenaZone, naproxen, and Sumatriptan pha-cyclodextrin. Succinate; and a pharmaceutically acceptable carrier. 0186. An aspect of the invention also provides administer ing a therapeutic amount of a composition comprising an 0173 Further aspect includes a method to ameliorate or (R)-enantiomeric salt compound; and a pharmaceutically eliminate discomfort of a patient Suffering from cramps, hot acceptable carrier. and cold flushes, cognitive disorders, TBI, neurotoxicity, 0187. An aspect of the invention provides (R)-Isomethep depression, Schizophrenia, anxiety, epilepsy, stress disorders, tene mucate for treatment of episodic tension-type headaches excess Sweating, or symptoms related to drug withdrawal. (ETTH). 0.174. A method of treatment of migraine including 0188 Another aspect of the invention provides a method administering a therapeutic amount of a composition com for producing a composition which includes steps of mixing prising an (S)-Isometheptene mucate salt and a pharmaceu a therapeutically effective amount of (R)-Isometheptene tically acceptable carrier to a patient in need of Such treat mucate salt with an anti-inflammatory Substance selected ment. from the group consisting of acetaminophen, ibuprofen, aspi rin, dichloralphenaZone, naproxen, and Sumatriptan Succi 0.175. A method of treatment of migraine including nate; and a nontoxic carrier. administering to a patient in need of Such treatment, a thera 0189 Another aspect of the invention provides (R)- peutic amount of a composition comprising Isometheptene Isometheptene mucate for treatment of episodic tension-type (R)-enantiomer mucate salt combined with an active ingredi headaches including infrequent ETTH, frequent ETTH, or ent selected from acetaminophen, ibuprofen, aspirin, caf chronic TTH or CTTH. feine, dichloralphenaZone, naproxen, and Sumatriptan Succi 0190. An aspect of the invention provides also a method of nate; and a pharmaceutically acceptable carrier. making the compound comprising the steps of: 0176 An aspect of the invention is provided by the use of (a) combining (S)-methyloxirane with 3-methyl-2-buten-1- (R)-Isometheptene enantiomer for inhibiting nitric oxidase y1 magnesium chloride in THF in the presence of Copper (NO). iodide, US 2014/021248.6 A1 Jul. 31, 2014

(b) neutralizing, extracting with ether, drying with magne (0205 FIG. 13 illustrates a LC/MS data of Isometheptene sium sulfate, removing solvent, and distilling the (S)-6-Me (S)-enantiomer. thyl-hept-5-en-2-ol (0206 FIG. 14 illustrates an H1 NMR of (R)-Isomethep (c) reacting (S)-6-Methyl-hept-5-en-2-ol with methanesulfo tene (R)-Mosher amide, showing no (S)-peak at 4.84 ppm, nyl chloride and DIEA in anhydrous DCM; and (d) cooling the reaction mixture to about 5°C.; 0207 FIG. 15 illustrates separation of H1 NMR mixture of (e) adding mesyl chloride dropwise; (R)- and (S)-Isometheptene (R)-Mosher amide. (f) diluting the reaction mixture after 18 hours at RT: 0208 FIG. 16 illustrates (R)-Isometheptene mucate crys (g) washing said mixture with water, brine and drying over tal structure showing intermolecular hydrogen bonds; MgSO; (0209 FIG. 17 illustrates the twofold crystal structure of (h) concentrate to oily product, (S)-Methanesulfonic acid (R)-Isometheptene mucate salt X-ray, demonstrating a greater 1,5-dimethyl-hex-4-enyl ester, wherein the 1H NMR in amount of disorder at the dimethyl end than at the amine end; CDC1 at 500 MHz identifies the parameters: 5.08 ppm 1 Hm, 0210 FIG. 18 illustrates the crystal structure of (R)- 4.80 ppm 1 Hm, 2.99 ppm 3 Hs, 2.08 ppm 2 Hm, 1.75 ppm 1 Isometheptene mucate X-ray, including hydrogen atoms; and Hm, 1.69 ppm 3 Hs, 1.6241 Hm, 1.613 Hs, 1.43 ppm 3 Ha: 0211 FIG. 19 illustrates the crystal structure of (R)- (i) reacting (S)-Methanesulfonic acid 1,5-dimethyl-hex-4- Isometheptene mucate X-ray, not including hydrogen atoms; enyl ester with N.N-dimethyl-acetamide (170 mL), and 40% 0212 FIG. 20 illustrates the effects of Isometheptene methylamine in water in a sealed, heavy-walled reaction ves mucate and its Isomers 1 and 2 on (A) mean, (B) systolic, (C) sel at 50° C.; diastolic arterial blood pressure and (D) heart rate, following (k) adding diethyl ether and washing said mixture with water i.V. administration in the rat; and brine, drying over MgSO4, and filtering to remove the 0213 FIG. 21 illustrates the I. Imidazoline receptor bind solvent; ing of (S)-Isometheptene Mucate, (R) Isomentheptene (1) removing the ether in vacuo (-25 mmHg) to yield (R)- Mucate, and Isometheptene Mucate; Isometheptene in the free base form; and 0214 FIG. 22 illustrates the I. Imidazoline receptor bind (m) adding a pharmacologically acceptable acid (such as ing of (S)-Isometheptene Mucate, (R) Isomentheptene hydrochloric, mucic, tartaric) to form a pharmacologically Mucate, and Isometheptene Mucate; acceptable salt. 0215 FIG. 23 illustrates the TAAR1 cAMP Biosensor 0191) A useful aspect of the present invention provides a Assay of (A) Isometheptene Mucate Isomer 1, (B) method for producing a composition including the steps of Isometheptene Mucate Isomer 2, (C) Isometheptene Mucate mixing a therapeutically effective amount of (R)-metheptene USP (D) p-Tyramine; and mucate salt with an anti-inflammatory Substance selected 0216 FIG. 24 illustrates the I receptor binding assay from the group consisting of acetaminophen, ibuprofen, aspi comparison of several reference drugs. rin, dichloralphenaZone, naproxen, and Sumatriptan Succi DETAILED DESCRIPTION nate; and a nontoxic carrier. 0217. In general, Isometheptene is known as a sympatho BRIEF DESCRIPTION OF THE DRAWINGS mimetic agent, often used in combination with dichloral phenaZone and acetaminophen in the treatment of migraine 0.192 In the detailed description reference is made to the and tension headache. However, in embodiments of this illustrative figures listed below. invention, there is illustrated the synthesis, purification and 0193 FIG. 1 illustrates high performance liquid chroma both structural and biological characterization, in addition to tography (HPLC) elution of a purified Isometheptene isomer the racemic Isometheptene, of both (R)- and (S)-Isomethep 2. tene racemates. (0194 FIG. 2 illustrates an HPLC sample elution of a blank 0218 Aspects of the invention provide distinct binding from a chiral pack column; affinities of the twin enantiomers to CNS receptors. Certain (0195 FIG. 3 illustrates a chiral HPLC sample elution of examples of molecules with related structures show how the purified Isometheptene isomer 2: activity of enantiomers is unpredictable, including 3.4-meth 0.196 FIG. 4 illustrates a chiral pack HPLC eluting a race ylenedioxy-N-methylamphetamine (MMDA). For example, mic mixture of Isometheptene into peak 1 and peak 2 peak; (R)-amphetamine (which is optically Levo- or (-) amphet 0.197 FIG. 5 illustrates a liquid chromatogram of the amine) has a chain of four contiguous carbons with a chiral Isometheptene (R)-enantiomer, carbon in an alpha position relative to a methyl-amino group (0198 FIG. 6 illustrates an 1H NMR of the Isometheptene that shares an absolute configuration with (R)-Isomethep (R)-enantiomer; tene. (S)-amphetamine (which is optically Dextro- or (+) (0199 FIG. 7 illustrates mass spectrum (MS) of amphetamine) has a chain of four contiguous carbons with a Isometheptene (R)-enantiomer, chiral carbon in an alpha position relative to a methyl-amino (0200 FIG. 8 illustrates an HPLC of Isometheptene (S)- group that shares an absolute configuration with (S)- enantiomer, Isometheptene. 0201 FIG.9 illustrates an HPLC of a blank of Isomethep 0219. As a background of similar activity, amphetamines tene isomer; are psychoactive stimulants of central nervous system (CNS) (0202 FIG. 10 illustrates a 1H NMR of (S)-enantiomer; which as a mixture includes amphetamine, dextroamphet 0203 FIG. 11 illustrates a Chiral HPLC of (S)-enanti amine, and methamphetamine. Since amphetamine includes Omer, isomers called dextroamphetamine and levoamphetamine, 0204 FIG. 12 illustrates a second Chiral HPLC of wherein pure dextroamphetamine has been found more Isometheptene racemic mixture resolved in Peak 1 and Peak potent than the amphetamine mixture. Medications contain 2. ing the amphetamines include AdderallC), DexedrineC), Dex US 2014/021248.6 A1 Jul. 31, 2014 troStatC), and DesoxynC) for treatment of narcolepsy, obesity, 0223. Another aspect includes a composition comprising and attention deficit/hyperactivity disorder. Amphetamines an (S)- or (R)-enantiomer of Isometheptene or pharmaceuti can be modified to emphasize specific actions, which include cally acceptable addition salt thereof, and a nontoxic carrier. CNS stimulation, cardiovascular actions, or appetite Suppres Nontoxic carriers may include alpha-cyclodextrin, beta sant. Both methylphenidate and amphetamine compounds cyclodextrin, hydroxyethyl-beta-cyclodextrin, and hydroxy have been in Schedule II of the Controlled Substances Act ethyl-alpha-cyclodextrin as possible nontoxic carriers here. since 1971. In medical use, there is controversy over whether 0224. The Isometheptene can be solubilized by binding the benefits of amphetamines for ADHD and weight loss into the cyclodextrin cavity, which might improve rapidity of outweigh the drugs harmful side effects. However, it is onset and bioavailability. An abstract for “Cyclodextrins as known that amphetamines appear Successful in treating nar pharmaceutical solubilizers.” Adv Drug Deliv Rev. 2007 Jul. colepsy. Diethylproprion, fenfluramine, methylphenidate 30; 59(7):645-66. Epub 2007 May 29, can be found at: http:// (commonly known as the prescription drugs RitalinC or Con www.ncbi.nlm.nih.gov/pubmed/17601630 certaC), and phenmetrazine are drugs imitating amphet 0225. The identification of the I1 receptor as the amines. Substances legally available over-the-counter Isometheptene receptor reveals a new molecular target for include discovery that the intravenous injection of amphet ETTH therapeutics. In particular the data described below amines (particularly methamphetamine) produced enhanced have shown that (R)-Isometheptene binds I1 with much stron euphoric effects with a more rapid onset than oral adminis ger affinity than (S)-Isometheptene. Together, these data indi tration. Although structurally similar to amphetamine, meth cate that (R)-Isometheptene, as a single isomer may be an amphetamine has more pronounced effects on the CNS. improved treatment for ETTH relative to racemic Isomethep 0220. As another example, (R)-methamphetamine (which tene. is optically Levo- or (-) methamphetamine) has four contigu 0226. The structural formula (I) of racemic Isometheptene ous carbons and a methylamino-group with a chiral alpha is disclosed: carbon in the same absolute configuration as (R)-Isomethep tene. In addition, (S)-methamphetamine (which is optically Dextro- or (+) methamphetamine) has four contiguous car bons and a methylamino-group with a chiral alpha-carbon in the same absolute configuration as (S)-Isometheptene. (R)- methamphetamine is a sympathomimetic vasoconstrictor N- N which is the active ingredient used in some over-the-counter nasal decongestants including the US formulation of Vicks.(R) IUPAC name: 3,6-dimethylhept-5-en-2-amine. Vapor Inhaler. 0227 Isometheptene is a monounsaturated aliphatic sec 0221 (S)-methamphetamine is a stimulant and addictive ondary amine. Alternatively named compound formulas drug, street named “crystal meth’, which is controlled by the include 6-methylamino-2-methylheptene, N,6-Dimethyl-5- US Drug Enforcement Administration (DEA). (S)-metham hepten-2-amine, or 6-methylamino-2-methyl-heptene. phetamine is a stimulant by virtue of interacting with the 0228. According to this invention, as shown below, the norepinephrine transporter and other receptors. As yet Isometheptene isomers have now been stereostructurally another example, (R)-3,4-methylenedioxy-N-methylam purified and their stereochemistry characterized. Thus, it has phetamine (R-MMDA) (which is optically Levo- or now been established that Isometheptene Isomer 1 is an (S)- (-)MMDA) has four contiguous carbons and a methylamino enantiomer or (S)-Isometheptene, and Isometheptene Isomer group with a chiral alpha-carbon in the same absolute con 2 is an (R)-enantiomer, or (R)-Isometheptene. figuration as (R)-Isometheptene. S-MMDA (which is opti 0229. More particularly, the purified Isometheptene race cally Dextro- or (+) MMDA) has four contiguous carbons and mic compound consists of approximately equal parts, isomer a methylamino-group with a chiral alpha-carbon in the same 1 and isomer 2, respectively, having the structural formulae absolute configuration as (S)-Isometheptene. Given as the (Ia) and (Ib) respectively; racemate MDMA has a half-life of around 8 hours. For example, the area under the blood plasma concentration ver sus time curve (AUC) was two to four times higher for (R)- MDMA than the (S)-MDMA after a 40 mg oral dose in human volunteers. Likewise, the plasma half-life of (R)- MDMA was significantly longer than for (S)-MDMA Sr.'s and (S)-Isometheptene (5.8+2.2 hours vs 3.6+0.9 hours). While (R)-MDMA has H longer half-life, (S)-MDMA is more effective in eliciting N 5-HT, NE, and DA release. (R)-MDMA is more selective for 5-HT and NE release and has only a small effect on DA release. Chizh BA, Headley PM. NMDA antagonists and N-rs neuropathic pain multiple drug targets and multiple uses. (R)-Isometheptene Curr Pharm Des. 2005; 11:2977-2994. 0222. It is known that the chiral forms of methamphet 0230 Disclosed in the embodiments of this invention are amine, amphetamine and MMDA show that chirality is the (R) and (S) type enantiomers of the racemic compound, related molecules can have profound effects on their biologi Isometheptene, or a pharmaceutical composition thereof, and cal activity and disposition in the body. An aspect of the a process of preparing it, for use in a method for treatment of invention includes the (R)-Isometheptene enantiomer com patients Suffering from migraine; in addition, the compound pound being capable of selectively or specifically binding to may be effectively combined with other suitable pharmaceu the I1 receptor. tical agents. US 2014/021248.6 A1 Jul. 31, 2014

0231 Aspects of the invention provide distinct binding tified as trans-2-methyl-6-methylamino-2-hepten-1-ol (Tay affinities of the twin enantiomers to CNS receptors. lor 1977). Another metabolite, cis-2-Methyl-6-methylamino 0232 Further aspects of the invention disclosed herein 2-hepten-1-ol, was identified as a minor urinary metabolite of include methods of synthesis and purification, isolation and Isometheptene in the rat. characterization of comparative mechanism of action the Isometheptene, as well as in vitro tests of the isolated 0242. According to this invention, Isometheptene is a Isometheptene racemates, as centrally acting agonists; in par racemic compound which has now been separated into its ticular, the invention shows aspects of the different affinities twin isomer racemates 1 and 2 and characterized as pure of the (S)-enantiomer and (R)-enantiomer of Isometheptene (S)-enantiomer and (R)-enantiomer, respectively. to various CNS receptors. 0243 Isometheptene isomers 1 and 2 can be produced 0233. Other advantageous aspects of the invention provide according to either of two alternative synthetic schemes A and purified Isometheptene stereoisomers 1 and 2 through chemi B, outlined below: cal synthesis from chiral precursors, and further stereochemi cally characterized the Isometheptene Stereoisomers 1 and 2 as (S)-enantiomer and (R)-enantiomer, respectively. Scheme A: 0234. A particularly advantageous aspect of the invention is provided in that (R)-Isometheptene enantiomer is found to bind to the CNS moxonidine binding I 1 receptor about 60 fold Sr- -e-Step 1 more avidly than the (S)-Isometheptene enantiomer. The (R) O Isometheptenebinds to the I1 receptor with a 60-fold increase 1 in potency to that of (S) isomentheptene. See Table 1. 21 0235. The I1 receptor has been found to be the receptor for O Step 2 the central hypertensive agent moxonidine. Besides central S. 1. N hypertension, moXonidine has been efficacious in the treat S ment of metabolic syndrome, which includes dyslipidemia, insulin resistance, glucose intolerance, hypertension and cen tral obesity. Moxonidine has also been considered as a treat 2 ment for heart failure. Reference: Edwards, L. P. Brown Bryan, T. A. McLean, L., and Ernsberger, P. 21 "Pharmacological Properties of the Central Antihypertensive O Step 3 Agent, Moxonidine.” Cardiovasc. Ther. 30: 199-208 (2012). ss-1 0236 Moxonidine is believed to function as an agonist of the I1 receptor. Known imidazoline drugs with a postulated high affinity to the I1 receptor are AGN192403, rillmenidine, moXonidine and clonidine. Specific ligands of the I1 receptor 3 are 2-BFI, BU239. The putative natural ligand for imidazo Separate two isomers line I(1), I(2) and I(3) receptors is agmatine. Idazoxan is 21 believed to function as antagonist of the I1 and I2 receptors. 0237 Radwanska A, Dlugokecka J. Wasilewski R. Kalis Zan R.J. Physiol Pharmacol. 2009 March; 60(1): 131-42. Test ing conception of engagement of imidazoline receptors in imidazoline drugs effects on isolated ratheart atria. 0238 Idazoxan is believed to function as antagonist. Ida Zoxan (INN) is both a selective O2 adrenergic receptor antagonist, and an antagonist for the imidazoline receptor. Idoxane was investigated as an adjunctive treatment in 2 Isomers. Each 30-50 g Schizophrenia because it may treat the “negative' symptoms of schizophrenia. These effects were associated with the O2 adrenergic receptor antagonist properties. 0239 Bousquet, P: Bruban, V: Schann, S: Greney, H: Ehrhardt, J D: Dontenwill, M: Feldman, J (1999). “Participa tion of imidazoline receptors and alpha(2-)-adrenoceptors in the central hypotensive effects of imidazoline-like drugs'. Annals of the New York Academy of Sciences 881: 272-8. doi:10.11111.1749-6632. 1999.tb09369.x. PMID 10415925. 0240. The potential human metabolic pathway for genera tion of 2-methyl-6-methylamino-2-hepten-1-ol from Isometheptene has been described (Lyris 2005). 0241. Accordingly, Isometheptene is first demethylated to N-desmethyl-Isometheptene (which was also detected in urine samples collected during this study). Further, the N-des methyl-Isometheptene metabolite undergoes acid hydrolysis to form 2-methyl-6-methylamino-2-hepten-1-ol. The major urinary metabolite of (+/-)-Isometheptene in the rat was iden US 2014/021248.6 A1 Jul. 31, 2014 16

-continued synthesis thereof by determining the area under the respective 21 curves in the chromatogram produced. O NH Step 3 0250. According to another aspect of the invention a Ns 1. method for isolation and purification of salt intermediate is provided, comprising recrystallizing the crude products or intermediates thereof from a solvent or a mixture of solvents. uk This process can be in addition to the method described 3 above, and the anion exchange resin column step. Separate two isomers Stereoselective Synthesis of Isometheptene and Determination Of Isometheptene Stereochemistry by Use of Mosher's Amides 0251

DIEA, CHCl2

1 2 Isomers. Each 30-50 g V O 0244. The existence of separately active (S)-Isomethep \, tene enantiomer and (R)-Isometheptene enantiomer Suggests xn that the metabolism of Isometheptene isomers in rats may be 2 Stereospecific. Synthesis Formation of (R)-Methanesulfonic acid 1.5-dim 0245. The present disclosure relates to synthesis and puri ethyl-hex-4-enyl ester from (R)-6-Methyl-hept-5-en fication of the Isometheptene racemic compound, and further, 2-ol (1) and methanesulfonyl chloride to separation, purification and stereochemistry of its (S)- and (R) enantiomers, thereof, as described below. Large scale 0252 (R)-Methanesulfonic acid 1,5-dimethyl-hex-4-enyl syntheses of the (R)-(-)- and (S)-(+)-6-methyl-5-hepten-2-ol ester (2). (R)-6-Methyl-hept-5-en-2-ol (1) (2.26 g. 17.6 (sulcatol) from ethyl (S)-(-)-lactate have been described mmol) and DIEA (5.52 mL, 31.7 mmol) were dissolved in (Can. J. Chem. Vol. 57, 1979). anhydrous DCM (24 mL), the reaction mixture was cooled to 0246 Such a method can further involve purification of 5oC, mesyl chloride (1.64 mL. 21.1 mmol) was added drop the salt using at least one purification technique. Such as wise, and the reaction mixture was allowed to slowly warm to chromatography or recrystallization. The chromatography RT. After 18 h at RT the reaction mixture was diluted with can be reverse-phase chromatography or regular phase chro DCM (60 mL), washed with water (3x35 mL), brine (35 mL), matography. In some embodiments, the regular phase chro dried with MgSO4, solvent was concentrated to give a dark matography can use alumina or silica gel. According to oil which was purified by SiO2 flash chromatography using another embodiment of the invention, a method for synthesis, Hexane-EtOAc to give the title compound as an oil (3.225 g, isolation and purification of the compounds is provided, 86%). H1 NMR: see supporting information. TLC, Hexane including passing the crude reaction products through a chro DCM-EtOAc, 40:40:20, 2 Rf 0.70 (developed with KMnO4 matography column and collecting the particular compound stain). 1H NNMR in CDC13 (500MHz): 5.08 ppm 1 Hm, 4.80 which elutes at the appropriate retention time. ppm 1 Hm, 2.99 ppm 3 Hs, 2.08 ppm 2 Hm, 1.75 ppm 1 Hm, 0247. A method for isolation and purification of the novel 1.69 ppm 3 Hs, 1.624 1 Hm, 1.613 Hs, 1.43 ppm 3 Ha. compounds is provided, comprising passing the crude reac tion products through a chromatography column and collect DMAC, ing the particular compound which elutes at the appropriate O 50 C., retention time. \, -O N 48 h. 0248. According to another embodiment of the invention, 1 \ Me-NH HO a method for analyzing stereoisomers is provided. The O method involves conducting high performance liquid chro matography (HPLC) and applying specific compounds of 2 Formulae (Ia, Ib, and to the chromatography column as a standard. The method preferably involves applying both - No. r types of stereoisomers as standards to determine relative retention/elution times. 0249. The foregoing HPLC can be used to determine the relative amount of stereoisomer and the intermediates of the US 2014/021248.6 A1 Jul. 31, 2014 17

Formation of (5) Isometheptene from extracts were combined, dried with MgSO4, solvent was (R)-Methanesulfonic acid 1,5-dimethyl-hex-4-enyl concentrated, to give an oil which was purified by SiO2 flash ester and methylamine chromatography using Hexane-EtOAc to give the title com poundas an oil (80 mg, 40%). H1 NMR in CDC13 (500 mHz): 0253 (S)-Isometheptene (3): (R)-Methanesulfonic acid 1H NMR in CDC13: 7.54 ppm (2H m), 7.37 ppm(3 Hm), 5.08 1,5-dimethyl-hex-4-enyl ester (2) (1.00 g, 4.85 mmol) was ppm 1 Hm, 4.835 ppm 1 Hm, 3.68 ppm 3 Hs, 2.45 ppm 3 Hs, dissolved in N,N-dimethyl-acetamide (24 mL), 40% methy 1.82 ppm 1 Hm, 1.92 ppm 1 Hm, 1.68 ppm 3 Hs, 1.56 ppm 3 lamine in water (17.11 mL, 194 mmol) was added at RT using Hs, 1.41 ppm 2 Hm, 1.118 ppm 3 Ha (J–6.5 Hz). TLC, a heavy-walled reaction vessel (150 mL) with teflon screw DCM-7 N NH3-MeOH, 90:10, 3 Rf, 0.45. Hexane-EtOAc, cap. The reaction vessel was sealed, stirred, and heated at 50 85:15, 5 Rf 0.45 (developed with KMnO4 stain). C for 66 h after which the reaction mixture was cooled to 5o Determination of Isometheptene Stereochemistry by C, and the system was opened to monitor the reaction by TLC. Use of Mosher's amides The reaction mixture was diluted with Et2O (200 mL), 0256 The twin enantiomers of Isometheptene were shaken, the organic layer was washed with water (4x50 mL), assayed for their individual activities while the stereochem brine (50 mL), dried with MgSO4, and solvent was concen istry of the enantiomers were unknown. Therefore, the Mosh trated to give the titled compound as an oil (606 mg, 88%). H1 er's amides of these Isometheptene enantiomers were pre NMR in CDC13 (500 mHz): 1H NNMR in CDC13:5.10 ppm pared, then the H1 NMR were analyzed and compared to that 1 Hit (J=6.8 Hz), 2.53 ppm 1 Hm, 2.40 ppm 3 Hs, 2.00 ppm 2 of compound 5 with known stereochemistry. H1 NMR were Hm, 1.68 ppm3 Hs, 1.61 ppm3 Hs, 1.49 ppm 1 Hm, 1.31 ppm taken of the individual compounds and of mixtures of com 1 Hm, 1.04 ppm 3 Ha (J–6.5 Hz). pounds as well. Inspection of the chemical shifts for the alpha 0254 TLC sample was prepared by taking an aliquot (100 proton (approx. 4.8 ppm) (attached to chiral C of Isomethep uL) of reaction mixture, diluting with EtOAc (100 uL), wash tene) and methyl protons (approx. 1.1 ppm) attached to chiral ing with brine (500 ul), TLC organic layer. TLC, Hexane C revealed that isomer 1 has (S) stereochemistry and isomer EtOAc, 75:25, 2Rf.0.35. DCM-7NNH3-MeOH,90:10,3Rf, 2 has (R) stereochemistry. 0.45 (developed with KMnO4 stain).

C = (S)-isometheptene-(R)-Mosher's amide A = isomer 1 isometheptene-(R)-Mosher's amide (R)-isometheptene-(R)-Mosher's amide B = isomer 2 isometheptene-(R)-Mosher's amide

0257 Structures of the Mosher's amides of Isometheptene used in the NMR assignment of stereochemistry (see Table Formation of (S) Isometheptene (R)-Mosher amide A).

Synthesis of (S)-Isometheptene (R)-Mosher amide (5) 0255 (S)-Isometheptene (3) (79 mg 0.56 mmol) was dis solved in anhydrous DCM (5.6 mL), DMAP (10 mg, 0.082 mmol), DIEA (0.156 mL, 0.90 mmol) were added, and the reaction mixture was cooled to 5 C. Mosher's (S)-acid chlo ride (0.126 mL, 0.67 mmol) was added dropwise at 5 C and the reaction was allowed to warm to RT. After 16 h at RT additional DIEA (0.156 mL, 0.90 mmol), then Mosher's (S)- acid chloride (0.126 mL, 0.67 mmol) were added dropwise at RT and the reaction mixture was stirred at RT for 16 h. The CHN alpha proton reaction was quenched with saturated NH4C1 (28 mL), the c-methylprotons mixture was extracted with Et2O (3x28 mL), the organic Jul. 31, 2014

TABLE A 3.95 ppm 1 Hs, 3.2 ppm 1 Hm, 2.66 ppm 3 Hs, 2.09 ppm 2 Hm, 1.75 ppm 1 Hm, 1.69 ppm3 Hs, 1.62 ppm3 Hs, 1.58 ppm HNMR HNMR 1 Hm, 1.29 ppm 3 Ha (J–7 Hz). MS: mass calc for C9H19N, Amide CHN alpha proton C-methyl proton 141.15. Found: 142.4 (M+H). A = isometheptene isomer 1 4.838 ppm 1.113 ppm (R)-Mosher's amide Synthesis of Isometheptene Mucate-Isomer-2 B = isometheptene isomer 2 4.757 ppm 1.028 ppm (R)- Mosher's amide (R-Isomer) C = (S)-isometheptene-(R)- 4.835 ppm 1.118 ppm Mosher's amide 0261 Mucic acid (628 mg, 3.0 mmol) was suspended in distilled water (40 mL) at RT. Isometheptene-isomer-2 (3.04 g, 21.5 mmol) was dissolved in Et2O (57 mL) and added dropwise via addition funnel to the mucic acid at RT while Location in Molecule and HNMR Chemical Shifts to stirring to give dissolution of the mucic acid suspension. The Determine the Stereochemistry of Isometheptene biphasic solution was stirred at RT for 16 h, the aqueous layer Isomer 1-(R)-Mosher's Amide and Isometheptene was separated, extracted with toluene (5x80 mL) (the final Isomer 2-(R)-Mosher's Amide by Comparison to extraction with toluene was stood for 30 min before the aque (S)-Isometheptene-(R)-Mosher's Amide ous layer was separated), then the aqueous layer was evapo 0258 rated in vacuo (1 mm Hg) at 35°–40°C. to give a white solid which was dissolved in MeCH (8 mL) by gently heating to give a solution. CHCN (80 mL) was added slowly dropwise via addition funnel at RT to the stirred MeOH solution to give a suspension which was stirred at RT for about 30 min. The solids were filtered, washed with CHCN thrice, air-dried, ---> then dried in vacuo (0.1 mm Hg) at RT for 16 h to give the (S)-isometheptene titled compound as a white (1.390 mg, 70%). The molar ratio isomer 1 isometheptene of Isometheptene to mucic acid in the salt was 2:1, based on the NMR spectrum: 1H NMR (500 MHz, in D.O), 1H NNMR in CDC1:5.16 ppm 1 Hit (J=6.8 Hz), 4.25ppm 1 Hs, 3.95 ppm Sr- N 1 Hs, 3.2 ppm 1 Hm, 2.66 ppm 3 Hs, 2.09 ppm 2 Hm, 1.75 (R)-isometheptene ppm 1 Hm, 1.69 ppm 3 Hs, 1.62 ppm 3 Hs, 1.58 ppm 1 Hm, isomer 2 isometheptene 1.29 ppm 3 Ha (J–7 Hz). MS: mass calc for CHIN, 141.15. Found: 142.4 (M+H). (S)-6-Methyl-hept-5-en-2-ol Assignment of (S) and (R) Stereostructures of Isometheptene Isomer 1 and Isometheptene Isomer 2, Based on the HNMR Studies of Mosher's Amides. Synthesis of the (R)-Mosher Ester of (S)-Sulcatol 0259. The following description is directed to the method 0262 of stereospecific synthesis and structural characterization of Isometheptene Isomer 1 ((S)-enantiomer) and Isometheptene Isomer 2 ((R)-enantiomer). O CuI, THF, O C., it, "A \ r He1 h, O C.-RT Mg Synthesis of Isometheptene Mucate-Isomer-1 H C1 (S-Isomer) HO 0260 Mucic acid (840 mg. 4.0 mmol) was suspended in distilled water (40 mL) at RT. Isometheptene-isomer-1 (1.356 r g, 9.6 mmol) was dissolved in Et2O (19 mL) and added putative (S) dropwise via addition funnel to the mucic acid at RT while (S)-6-Methyl-hept-5-en-2-ol stirring to give dissolution of the mucic acid suspension. The ((S)-Sulcatol) biphasic solution was stirred at RT for 16 h, the aqueous layer was separated, extracted with toluene (5x80 mL) (the final extraction with toluene was stood for 30 min before the aque 0263 3-Methylbut-2-enylmagnesium chloride, 0.50M in ous layer was separated), then the aqueous layer was evapo THF (100 mL, 50 mmol) was cooled to 0°C., CuI (952 mg, rated in vacuo (1 mm Hg) at 35°–40°C. to give a white solid 5.0 mmol), then (S)-2-Methyl-oxirane (1.752 mL. 25.0 which was dissolved in MeCH (8 mL) by gently heating to mmol) in anhydrous THF (12 mL) was slowly added to the give a solution. CHCN (80 mL) was added slowly dropwise stirred reaction mixture via Syringe pump at a rate of 16 mL/h via addition funnel at RT to the stirred MeOH solution to give over 45 minto give a dark suspension which was stirred at 0° a suspension which was stirred at RT for about 30 min. The C. under an Aratm for 1 h, then allowed to slowly warm to RT solids were filtered, washed with CHCN thrice, air-dried, over several h. After stirring at RT for 18 h the reaction then dried in vacuo (0.1 mm Hg) at RT for 16 h to give the mixture was poured into sat. aqueous NH4C1 (75 mL), the titled compound as a white solid (1.390 mg, 70%). The molar mixture was extracted with EtO (3x100 mL), the organic ratio of Isometheptene to mucic acid in the salt was 2:1, based extracts were combined, washed with water (100 mL), brine on the NMR spectrum: 1H NMR (500 MHz), in DO: 1H (100 mL), dried with MgSO, and solvent was evaporated in NNMR in CDC1: 5.16 ppm 1 Hit (J–6.8 Hz), 4.25 ppm 1 Hs, vacuo (30 mm Hg) at 25°C. to give an oil which was purified US 2014/021248.6 A1 Jul. 31, 2014

by SiO flash chromatography to give the title compound as -continued an oil in 2 fractions (1.6 g., 0.6 g. 69%). H NMR (CDC1).

N-- putative (S) (S)-6-Methyl-hept-5-en-2-ol r ((S)-Sulcatol) (S)-6-Methyl-hept-5-en-2-ol. Small Scale, e.g., ca. 1 g Final Product 0267. 3-Methylbut-2-enylmagnesium chloride, 0.50M in THF (100 mL, 50 mmol) was cooled to 0°C., CuI (952 mg, 5.0 mmol), then (S)-2-methyl-oxirane (1.752 mL. 25.0 mmol) in anhydrous THF (12 mL) was slowly added to the stirred reaction mixture via Syringe pump at a rate of 16 mL/h over 45 minto give a dark suspension which was stirred at 0° C. under an Argon atm for 1 h, then allowed to slowly warm to RT over several h. After stirring at RT for 18 h the reaction mixture was poured into sat aqueous NH4C1 (75 mL), the mixture was extracted with EtO (3x100 mL), the organic extracts were combined, washed with water (100 mL), brine (100 mL), dried with MgSO, and solvent was evaporated in (R)-Mosher ester of (S)-Sulcatol. (R)-3,3,3-Trif. vacuo (30 mm Hg) at 25°C. to give an oil which was purified luoro-2-methoxy-2-phenyl-propionic acid (S)-1,5- by SiO flash chromatography to give the title compound as dimethyl-hex-4-enyl ester.} and Determination of an oil in 2 fractions (2.2g, 69%). H NMR (CDC1): 5.14 (m, Chiral Purity of Synthesized (S)-Sulcatol Via (R)- 1H), 3.82 (m. 1H), 3.08 (m, 1H), 1.69 (s.3H), 1.62 (s. 2H), Mosher Ester(S)-Sulcatol 1.48 (m, 2H), 1.19 (d. 3H, J=6 Hz). 0264 (S)-6-Methyl-hept-5-en-2-ol (0.088 mL, 0.58 0268 Below is the description of a method of synthesis of mmol) was dissolved in anhydrous DCM (6 mL), DMAP a larger amount of S-Sulcatol (i.e. 13.68 g). (10.6 mg, 0.087 mmol), DIEA (0.192 mL, 1.10 mmol) were 0269. 3-Methylbut-2-enylmagnesium chloride, 0.50M in added, then (S)-3,3,3-Trifluoro-2-methoxy-2-phenyl-propio THF (760 mL, 380 mmol) was transferred to a 2 liter, flame nyl chloride (0.152 mL, 0.813 mmol) was added dropwise at dried round-bottom flask. After cooling to 4°C., CuI (7.62g, RT while stirring. After 18 h at RT N,N-Dimethyl-1,3-pro 40 mmol) was added to give a dark Suspension, and (S)-2- panediamine (0.146 mL, 1.16 mmol) was added at RT and the methyl-oxirane 99% (14 mL, 200 mmol) in anhydrous THF reaction mixture was stirred at RT for 20 min. The mixture was diluted with EtO(60 mL), washed with 1 Naqueous HCl (12 mL) was slowly added to the stirred reaction mixture via (20 mL), 2 MNaCO (20 mL), brine (20 mL), dried with Syringe pump at a rate of 110 mL/h at 4°C. to give a dark MgSO solvent was evaporated in vacuo at 30°C. to give an suspension which was allowed to slowly warm to RT in the oil which was coevaporated with CC1 three times to remove ice bath. After stirring at RT for 16h, TLC and NMR showed residual EtO to give an oil which was further dried in vacuo (S)-sulcatol (S)-6-Methyl-hept-5-en-2-ol} and a more polar, (0.1 mm Hg) for 1 h to give an oil (242 mg). H NMR more volatile side product. The reaction mixture was poured (CDC1): see supporting information. LRMS: mass calcu into sat. aqueous NH4C1 (600 mL) to give a blue aqueous lated for CHFOs: 344.16. found: 366.8 (M+ Suspension and an organic layer with pH 9. This mixture was 0265 Another useful method has been found as follows: stirred for 30 minutes, the mixture was extracted three times with EtO (800 mL, 400 mL, and 400 mL). The organic Stereoselective Synthesis of (R)-Isometheptene and extracts were combined, washed with water (200 mL), brine (R)-Isometheptene Mucate (200 mL), dried with MgSO and stripped of solvent in vacuo (30 mm Hg) at 35°C. to give 31.8grams of a yellow oil which 0266 was purified three times by distillation in vacuo at 15-20 mm Hg. The higher boiling fractions, bp. 80° C., contained the (S)-sulcatol. The yield of (S)-sulcatol was 13.68 g. H NMR O CuI, THF, O C., (CDC1): 5.14 (m. 1H), 3.81 (m. 1H), 2.08 (m. 1H), 1.69 (s. litt ...A \ r 1 h, O C.-RT Mg 3H), 1.62 (s. 2H), 1.48 (m, 2H), 1.19 (d. 3H, J=6 Hz). TLC in H C1 3:1 hexane/ethyl acetate:sulcatol: Rf 0.35, impurity: Rf 0.3, visualized with KMnO and charring. US 2014/021248.6 A1 Jul. 31, 2014 20

(S)-Sulcatol to give the title compound as a crude oil (241 mg). H NMR (CDC1): 7.54 (m,2H), 7.40 (m,3H), 5.14 (m, 1H), 5.08 (m, 1H), 3.55 (s, 3H), 2.02 (m, 2H), 1.74 (m, 1H), 1.68 (s, 3H), 1.60 (m, 1H), 1.57 (s, 3H), 1.27 (d. 3H, J=6.5 Hz). Determination of Chiral Purity of Synthesized (S)-Sulcatol Via (R)-Mosher Ester of (S)-Sulcatol. 0272 Inspection of regions 5.02-5.08 ppm (chemical shifts of protons bonded to chiral carbon) for (R)-Mosher ester of (S)-Sulcatol and (R)-Mosher ester of (R)-Sulcatol indicated chiral purity of (S)-6-Methyl-hept-5-en-2-ol to be >95%.

C (R)-Mosher ester of (S)-Sulcatol. ((R)-3,3,3-Trif O EO -- luoro-2-methoxy-2-phenyl-propionic acid (S)-1,5- N-- DIEA, CH2Cl2 dimethyl-hex-4-enyl ester (0270 (S)-6-Methyl-hept-5-en-2-ol (0.088 mL, 0.58 21 O Ns/ mmol) was dissolved in anhydrous DCM (6 mL), DMAP (10.6 mg, 0.087 mmol), DIEA (0.192 mL, 1.10 mmol) were N-- / N added, then (S)-3,3,3-Trifluoro-2-methoxy-2-phenyl-propio nyl chloride (0.152 mL, 0.813 mmol) was added dropwise at RT while stirring. After 18 h at RT, N.N-Dimethyl-1,3-pro panediamine (0.146 mL, 1.16 mmol) was added at RT; and (S)-Methanesulfonic acid 1,5-dimethyl-hex-4-enyl the reaction mixture was stirred at RT for 20 min. The mixture ester was diluted with EtO(60 mL), washed with 1 Naqueous HCl (20 mL), 2 MNaCO (20 mL), brine (20 mL), dried with 0273 (S)-6-Methyl-hept-5-en-2-ol (2.64 g. 20.58 mmol) MgSO solvent was evaporated in vacuo at 30°C. to give an and DIEA (6.45 mL, 37.0 mmol) were dissolved in anhydrous oil which was coevaporated with CC1 three times to remove DCM (28 mL), the reaction mixture was cooled to 5° C. residual EtO to give an oil which was further dried in vacuo mesyl chloride (1.91 mL. 24.7 mmol) was added dropwise, (0.1 mmHg) for 1 h to give the title compound as a crude oil and the reaction mixture was allowed to slowly warm to RT. (242 mg). H NMR (CDC1): 7.55 (m, 2H), 7.40 (m, 3H), After 18 h at RT the reaction mixture was diluted with DCM 5.15 (m, 1H), 5.03 (m, 1H), 3.57 (s.3H), 1.92 (m, 2H), 1.70 (100 mL), washed with water (3x35 mL), brine (35 mL), (m. 1H), 1.65 (s.3H), 1.55 (m. 1H), 1.51 (s.3H), 1.34 (d. 3H, dried with MgSO4, solvent was concentrated to give a dark J=6.5 Hz). LRMS: mass calculated for CHFOs: 344.16. oil which was purified by SiO2 flash chromatography using found: 366.8 (M+Na"). Hexane-EtOAc to give the title compound as an oil (3.213 g, 76%). TLC, Hexane-DCM-EtOAc, 40:40:20, 2 R,0.70 (de veloped with KMnO4 stain). H NMR (CDC1): 5.08 (m, 1H), 4.80 (m, 1H), 2.99 (s.3H), 2.10 (m, 2H), 1.75 (m, 1H), 1.69 (s.3H), 1.62 (m, 1H), 1.61 (s, 3H), 1.43 (d. 3H, J=6.5 Hz). 0274 Optionally, for larger scale preparations, the dark oil may be used in the next step (reaction with methylamine) without silica chromatography, or dissolved in Hexane DCM-EtOAc, 40:40:20, filtered through silica to remove baseline impurities, and stripped of solvent. 0275 Below is the description of a method of synthesis of a larger amount of (S)-Methanesulfonic acid 1,5-dimethyl hexyl-enyl ester (19.8 g). 0276. The (S)-sulcatol (13.68 g) from the aforegoing sec tion was converted to (S) Methanesulfonic acid 1,5-dimethyl hexyl-4-enyl ester as described above. However, instead of by chromatography the product was purified by filtering a meth (R)-Mosher ester of (R)-Sulcatol. (R)-3,3,3-Trif. ylene chloride Solution through silica gel in a filtration funnel luoro-2-methoxy-2-phenyl-propionic acid (R)-1,5- (6 cm heightx9.5 cm inside diameter, 60 A 220-240 mesh dimethyl-hex-4-enyl ester.} slica) and removing under vacuum. The yield of (S)-Meth 0271 The title compound was prepared using the proce anesulfonic acid 1.5-dimethyl-hexyl-4-enyl ester was 19.8g. dure described for the preparation of (R)-Mosher ester of This material showed no side products by NMR. US 2014/021248.6 A1 Jul. 31, 2014

Elemental analyses calculated for CHN: C%, 58.51; H%, DMAC, 9.82: N 96, 5.69. found: C96, 58.21: H%, 9.95; N 96, 5.54. O 50 C., On / e- 48 h. 0279 Optionally, the Isometheptene mucate can be fur N-- WN M NH HO -> ther purified by recrystallization from solvents and solvent O combinations such as acetone/methanol, acetone/DMSO, acetonitrile/DMSO, acetonitrile/MeOH, DMSO, n-butanol, N- N or isopropanol. (R)-Isometheptene 0277 Optionally, the (R)-Isometheptene free base could be further purified by distillation. Thus, the chemical and 0280 (S)-Methanesulfonic acid 1,5-dimethyl-hex-4-enyl optical purity of the product could optionally be further ester (2) (3.21 g, 15.56 mmol) was dissolved in N,N-dim improved by recrystallization of the salt formed with a chiral ethyl-acetamide (55 mL), 40% methylamine in water (54.9 carboxylic acid (e.g. D-tartaric acid, mandelic acid, O.O- mL, 622 mmol) was added at RT using a heavy-walled reac dibenzoyl-D-tartaric acid, N-acetyl-L-phenylalanine) fol tion vessel (150 mL) with teflon screw cap. The reaction lowed by treatment with aqueous NaOH and extraction with vessel was sealed, stirred, and heated at 50° C. for 64 h after ether to re-isolate the (R)-Isometheptene free base. which the reaction mixture was cooled to 5° C., and the system was opened to monitor the reaction by TLC. The

H reaction mixture was diluted with EtO(320 mL), shaken, the organic layer was washed with water (4x100 mL), brine (100 mL), dried with MgSO4, and solvent was concentrated to give --> 2 the titled compound as an oil (1.974g.90%). TLC sample was prepared by taking an aliquot (100 uL) of reaction mixture, diluting with EtOAc (100 uL), washing with brine (500 uI), TLC organic layer. TLC, Hexane-EtOAc, 75:25, product, R, 0.35. DCM-7 N. NH3-MeOH, 90:10, 3 R, 0.45 (developed with KMnO, stain). H NMR (CDC1): 5.10 (m. 1H), 2.52 (m. 1H), 2.40 (s.3H), 1.99 (m, 2H), 1.68 (s.3H), 1.61 (s.3H), (R)-Isometheptene Mucate Salt, See Table A 1.49 (m, 2H), 1.31 (m. 1H), 1.04 (d. 3H, J=6.0 z). (R)-Isometheptene Mucate TABLE A

0278 Mucic acid (1.224g, 5.82 mmol) was suspended in (R)-Isometheptene Mucate Drug Substance distilled water (58 mL) at RT. (R)-Isometheptene (1.974 g. 13.98 mmol) was dissolved in EtO (28 mL) and added drop Common API name: (R)-Isometheptene mucate wise via addition funnel to the mucic acid at RT while stirring Chemical name: (R)-N,6-dimethylhept-5-en-2-amine to give dissolution of the mucic acid Suspension. The biphasic solution was stirred at RT for 16 h, the aqueous layer was tetrahydroxyhexanedioate) separated, extracted with toluene (3x50 mL) (the final extrac Physical state: Crystalline solid tion with toluene was stood for 30 min before the aqueous Molecular weight: 464.59 layer was separated), then the aqueous layer was evaporated Molecular formula: C22H4N2Os in vacuo (1 mm Hg) at 35-40°C. to give a white solid which was dissolved in MeCH (12 mL) by gently heating to give a Structure: solution which was allowed to cool to RT. CH3CN (65 mL) was added slowly dropwise via addition funnel at RT to the stirred MeOH solution to give a white suspension which was stirred at RT for about 30 min. The solids were filtered, washed with CH3CN thrice, air-dried, then dried in vacuo (0.1 mm Hg) at RT for 16 h to give the titled compound as a white solid (2.531 g, 88%). H NMR (DO) 5.17 (m. 1H), 4.24 (s, 1H), 3.93 (s, 1H), 3.21 (m. 1H), 2.66 (s.3H), 2.09 (m, 1H), 1.76 (m. 1H), 1.69 (s.3H), 1.62 (s, 3H), 1.57 (m, 1H), 1.29 (d,3H, J=6.5 Hz). CNMR (DO) 178.4, 133.8, 1214, 70.86, 70.57, 54.2, 31.3, 28.8, 23.9, 22.3, 16.0, 14.1. LRMS: mass calculated for CHN, 141.15. found: 142.3 (M--H)". US 2014/021248.6 A1 Jul. 31, 2014 22

Synthetic Scheme for (R)-Isometheptene Mucate -continued H 0281 ---> (R)-Isometheptene Step 1 Chemical Formula: CoHoN Step 4 1. CuI, tetrahydrofuran Molecular Weight: 141.25 1. Water, ether -es 2. Ammonium 2. Separate aqueous; O N chloride, ether He wash with toluene "A -- MgCl 3.. Uoncentrate,C trat 3. Remove water H distill (S)-2-Methyloxirane 3-Methylbut-2-enyl Magnesium Chloride Chemical Formula: C5H9CIMg Mucic Acid Molecular Weight: 128.88 Chemical Formula: C6H10O8 Molecular Weight:210.14 N-- (S)-6-Methyl-hept-5-en-2-ol (S-sulcatol) Chemical Formula: C8H16O Molecular Weight: 128.21 ---> HO HOIII Step 2 1. Diisopropylethylamine, methylene chloride, mesyl chloride Hip (R)-Isometheptene Mucate 2. Water, brine Chemical Formula: C24H48N2O8 N-- 3. Filtration through Molecular Weight: 492.65 (S)-6-Methyl-hept-5-en-2-ol silica gel (S-Sulcatol) Chemical Formula: C8H16O Molecular Weight: 128.21 Synthesis of (R)-Enantiomeric Isometheptene Salt 0282. A method of making the compound of claim 3, comprising the steps of (a) combining (S)-methyloxirane with 3-methyl-2-buten-1- ylmagnesiurn chloride in THF in the presence of Copper iodide; (S)-6-Methylhept-5-en-2-yl (b) neutralizing, extracting with ether, drying with magne Methanesulfonate sium sulfate, removing solvent, and distilling the (S)-6-Me Chemical Formula: CoH18O3S thyl-hept-5-en-2-ol; Molecular Weight: 206.30 (c) reacting (S)-6-Methyl-hept-5-en-2-ol with methanesulfo nyl chloride and DIEA in anhydrous DCM; Step 3 O (d) cooling the reaction mixture to about 5°C.; | 1. N,N-dimethylacetamide, (e) adding mesyl chloride dropwise; 40% methylaminefwater (f) diluting the reaction mixture after 18 hours at RT: 2. Ether; water; brine (g) washing said mixture with water, brine and drying over MgSO; (S)-6-Methylhept-5-en-2-yl (h) concentrate to oily product, (S)-Methanesulfonic acid Methanesulfonate 1,5-dimethyl-hex-4-enyl ester, wherein the 1H NMR in Chemical Formula: CoH18O3S CDC1 at 500 MHz identifies the parameters: 5.08 ppm 1 Hm, Molecular Weight: 206.30 4.80 ppm 1 Hm, 2.99 ppm 3 Hs, 2.08 ppm 2 Hm, 1.75 ppm 1 Hm, 1.69 ppm3 Hs, 1.6241 Hm, 1.613 Hs, 1.43 ppm 3 Hal.: H (i) reacting (S)-Methanesulfonic acid 1,5-dimethyl-hex-4- enyl ester with N,N-dimethyl-acetamide (170 mL), and 40% --> methylamine in water in a sealed, heavy-walled reaction ves (R)-Isometheptene sel at 50° C.; Chemical Formula: CoHoN (k) adding diethyl ether and washing said mixture with water Molecular Weight: 141.25 and brine, drying over MgSO4, and filtering to remove the solvent; (1) removing the ether in vacuo (-25 mmHg) to yield (R)- Isometheptene in the free base form; and US 2014/021248.6 A1 Jul. 31, 2014

(m) adding a pharmacologically acceptable acid, comprising TLC, DCM-7 NNH3-MeOH,90:10, (R)-isometheptene, R, hydrochloric, mucic, or tartaric, to form a pharmacologically 0.45. TLC, Hexane-EtOAc, 85:15, (R)-Isometheptene (R)- acceptable salt. Mosher amide, R, 0.45 (developed with KMnO, stain). H NMR (CDC1): 7.51 (m, 2H), 7.38 (m, 3H), 5.10 (m, 1H), Synthesis of Isometheptene Mucate-Isomer-2 ((R) 4.76 (m. 1H), 3.71 (s.3H), 2.43 (s.3H), 1.95 (m, 2H), 1.68 (s, Isomer) 3H), 1.58 (s.3H), 1.50 (m. 1H), 1.45 (m. 1H), 1.03 (d,3H, J=7 0283 Mucic acid (628 mg, 3.0 mmol) was suspended in Hz). distilled water (40 mL) at room temperature (RT). Isomethep Determination of Chiral Purity of Synthesized tene-isomer-2 (3.04 g. 21.5 mmol) was dissolved in Et2O(57 (R)-Isometheptene Via (R)-Mosher Amide of mL) and added dropwise via addition funnel to the mucic acid (R)-Isometheptene at RT while stirring to give dissolution of the mucic acid suspension. The biphasic solution was stirred at RT for 16 h, 0288 Inspection of regions 4.9-4.7 ppm (chemical shifts the aqueous layer was separated, extracted with toluene of protons bonded to chiral carbon) for (R)-Mosher amide of (5x80 mL) (the final extraction with toluene was stood for 30 (S)-Isometheptene (4.84 ppm) and (R)-Mosheramide of (R)- min before the aqueous layer was separated), then the aque Isometheptene (4.76 ppm) indicated chiral purity of (R)- ous layer was evaporated in vacuo (1 mmHg) at 35°–40°C. to Isometheptene to be >98%. give a white solid which was dissolved in MeCH (8 mL) by (0289 Polarimetry gently heating to give a solution. CHCN (80 mL) was added 0290 (R) Isometheptene mucate (synthesized as slowly dropwise via addition funnel at RT to the stirred described) has an optical rotation CD=+6.0 in water at room MeOH solution to give a suspension which was stirred at RT temperature, and isomer 1 Isometheptene mucate has an for about 30 min. The solids were filtered, washed with C.D=-6.6 in water at room temperature. This is consistent CHCN thrice, air-dried, then dried in vacuo (0.1 mm Hg) at with isomer 1 being the S isomer, and provides further sup RT for 16 h to give the titled compound as a white (1.390 mg. port for the contention that isomer 1 is (S) and isomer 2 is (R). 70%). The molar ratio of Isometheptene to mucic acid in the The experimental procedure of the above summarized evi salt was 2:1, based on the NMR spectrum: 1H NMR (500 dence is as follows: MHz, in DO), 1H NNMR in CDC1: 5.16 ppm 1 Hit (J–6.8 0291 Polarimeter was calibrated with glucose (2.845 g in HZ), 4.25 ppm 1 Hs, 3.95 ppm 1 Hs, 3.2 ppm 1 Hm, 2.66 ppm 100 ml, 1 dim path length, reading +1.50) C=+52.7, 3 Hs, 2.09 ppm 2 Hm, 1.75 ppm 1 Hm, 1.69 ppm 3 Hs, 1.62 Temperature-ambient; ppm 3 Hs, 1.58 ppm 1 Hm, 1.29 ppm 3 Ha (J–7 Hz). MS: 0292 199.3 mg of (R) Isometheptene mucate (isomer 2) mass calc for CHIN, 141.15. Found: 142.4 (M--H). dissolved in 10 ml water. 0284 Another useful method has been found as follows: 0293 Readings=+0.10 and +0.14 (average=+0.12), path length=1 dim, Stereoselective Synthesis of (R)-Isometheptene and 0294 C=+6.0 (R)-Isometheptene Mucate 0295 205.6 mg of (S) Isometheptene mucate (isomer 1) dissolved in 10 ml water. 0285 0296 Readings=-0.15 and -0.12 (average=-0.135), path length=1 dim, O CuI, THF, O C., 0297 C)=-6.6. it...A \ r 1 h, O C.-RT M X-Ray Crystal Structure of (R)-Isomentheptene H -" Mucate Hemi-Hydrate OH 0298. According to the invention a crystals of (R)- Isometheptene mucate anhydrous were obtained by evapora N-- tion of a solution in water at ambient temperature and tested putative (S) by X-ray diffraction. The X-ray structure was consistent with (S)-6-Methyl-hept-5-en-2-ol the R stereochemistry assigned to Isomer 2 through the afor ((S)-Sulcatol) described NMR experiment. 0299 Moreover, elemental analysis of (R)-Isometheptene 0286 (R)-Isometheptene (R)-Mosher Amide. mucate precipitated from methanol through addition of aceto 0287 (R)-Isometheptene (76 mg, 0.54 mmol) (derived nitrile showed presence of 0.5 equivalent of HO in the crys from (R)-Isometheptene mucate) was dissolved in anhydrous tal. By contrast, however, X-ray crystallography found no DCM (5.4 mL), DMAP (10 mg, 0.082 mmol), DIEA (0.178 water molecule present in the crystals grown in water. mL, 1.02 mmol) were added, and Mosher's (S)-acid chloride 0300. According to further analysis the X-ray crystal of (0.141 mL, 0.753 mmol) was added dropwise at RT while (R)-Isometheptene mucate revealed that the two different stirring. After 18 hat RT N,N-Dimethyl-1,3-propanediamine conformations of the (R)-Isometheptene cation in Isomethep (0.135 mL, 1.08 mmol) was added at RT and the reaction tene mucate salt overlap. In fact, the analysis showed a Sub mixture was stirred at RT for 20 min. The mixture was diluted stantially disordered (poor overlap) hydrophobic dimethyla with EtO (27 mL), washed with 1 Naqueous HCl (10 mL), lkene part of the molecule but highly ordered (good overlap) sat NaHCO (10 mL), brine (10 mL), dried with MgSO near the cation where the mucate anion appears to dock. solvent was evaporated in vacuo at 30°C. to give an oil which (0301 Method of X-Ray Diffraction Experiment: was coevaporated with CC13 times to remove residual EtO 0302 All reflection intensities were measured at 100(2) K to give an oil which was further dried in vacuo (0.1 mm Hg) using a SuperNova diffractometer (equipped with Atlas for 1 h to give the title compound as a crude oil (212 mg). detector) with Mo KO. radiation ( 0.71073 A) under the US 2014/021248.6 A1 Jul. 31, 2014 24 program CrysAllispro (Version 1.171.36.24 Agilent Tech (0309 Biological Activity nologies, 2012). The program CrysAlisPro (Version 1.171. 0310. As presently described, the racemic, enantiomer (S) 36.24 Agilent Technologies, 2012) was used to refine the cell and (R)-Isometheptene compound has now been purified and dimensions. Data reduction was done using the program tested. All molecules were found having distinctly different Crys AlisBro (Version 1.171.36.24 Agilent Technologies, affinities for certain CNS target receptors, e.g., imidazoline-1 2012). The structure was solved with the program SHELXS subtype 1 (I.1) and Imidazoline-2 subtype (I2) (Table 1). The 2013 (Sheldrick, 2013) and was refined on P with SHELXL receptor I1 is presently known to bind the drug, moxonidine, 2013 (Sheldrick, 2013). Analytical numeric absorption cor which is a centrally acting antihypertensive therapeutic com rections based on a multifaceted crystal model were applied pound with low activation of adrenergic receptors. As evi using CrysAlisPro (Version 1.171.36.24 Agilent Technolo denced in recent assays, Isomer 1 and Isomer 2 bind differ gies, 2012). ently to Imidazoline receptor 2 (I2). 0311. It has now been discovered that the purified 0303. The temperature of the data collection was con Isometheptene isomer 2 (or (R)-Isometheptene enantiomer) trolled using the system Cryojet (manufactured by Oxford has a significant binding affinity for the Il-Imidazoline sites Instruments). The H atoms were placed at calculated posi (Table 1), which is also the selective receptor of moxonidine, tions using the instructions AFIX 13, AFIX 23, AFIX 43 or a centrally acting antihypertensive agent. Present understand AFIX 137 with isotropic displacement parameters having ing of the I1-imidazoline receptor is that of a cell-surface values 1.2 or 1.5 times Ueq of the attached C or Natoms. receptor mediating cellular responses, participating in cardio 0304. As shown in FIG. 16, the Isometheptene species are vascular control by the CNS, and providing a potential thera H-bond donors via two N H . . . O(mucate) to two mucate peutic target for multiple cardiovascular and metabolic dis species. The mucate species form 2-D H-bonded plane that orders. The antihypertensive agents with an imidazoline or are parallel to (100); those intermolecular hydrogen bond related structure, all show high affinity for I1-imidazoline interactions are most likely found along the largest crystal sites (affinity constant Ki-10 nM). The second-generation faces. Each Isometheptene species are H-bond donors via two agents, moxonidine and rillmenidine have reduced sedative N—H. ... O (mucate) to two mucate species. The mucate side-effects, and show lower affinity (higher Ki) at C2-adren species form 2-D H-bonded plane that are parallel to (100) ergic receptors relative to the first-generation agents with (those intermolecular hydrogen bond interactions are most pronounced sedative side-effects, including clonidine, gua likely found along the largest crystal faces). nabenz, guanfacine, and C.-methylnorepinephrine (the active metabolite of C.-methyldopa). 0305 The (R)-Isometheptene mucate crystal structure is 0312 Screening at 10uM with racemic Isometheptene did partly disordered (FIG. 17). One of the two crystallographi not show meaningful binding (i.e. >70% inhibition) at several cally independent Isometheptene cations is found to be dis receptors on Screening which include C.1-, O2A- or B-adren ordered over two orientations (the occupancy factor of the ergic receptors, serotonin receptors. Of the receptors that major component of the disorder refines to 0.520(4)). The were positive on Screening, racemic Isometheptene at 10 LM absolute configuration was established by the structure deter inhibited ligand binding to the O2B-adrenergic receptor of mination of a compound containing a chiral reference mol 76%. Further testing the purified S-Isometheptene and ecule of known absolute configuration and was confirmed by R-Isometheptene isomers at 10M in this assay, showed they anomalous-dispersion effects in diffraction measurements on inhibited ligand binding to the C2B-adrenergic receptor by the crystal. Although the Flack parameter refines to 0.1(3), 29% and 57%, respectively. Further testing of (S)-Isomethep both Hooft and Parsons parameters refine to -0.08(4) and tene and (R)-Isometheptene by equilibrium binding showed -0.05(4), respectively. similar binding of C.2B-adrenergic receptor with Kis of 2700 0306 Taking reference to FIG. 18, the (R)-Isometheptene nM and 2300 nM, respectively. Mucate X-ray structure is illustrated by displacement plot for 0313 For pharmacologic effects on C.2B-adrenergic (R)-Isometheptenemucate (with hydrogenatoms). The struc receptor, we focus on ligands that interact with Kis-100 nM, ture refines to about R1 equal to 5%. The chiral atoms of the so this level of interaction is low. Of the receptors that were Isometheptene species are in the (R)-configuration, consis positive on Screening, racemic Isometheptene at 10M inhib tent with Mosher's amide NMR data. ited ligand binding to the Sigma receptor (non-selective, meaning either Sigma 1 or Sigma 2 receptor) of 62%. Further 0307 FIG. 19 represents a displacement plot for (R)- testing the purified (S)-Isometheptene and (R)-Isomethep Isometheptene mucate (showing only C, N, and O, no hydro tene isomers at 10 uM in this assay, showed they inhibited genatoms). The structure refines to about R1 equal to 5%. The ligand binding to the non-selective Sigma receptor by 87% chiral atoms of the Isometheptene species are in the (R)- and 86%, respectively. Further testing of (S)-Isometheptene configuration, consistent with the Mosher's amide NMR and R-Isometheptene by equilibrium binding showed they data. bound Sigma-1 receptor with Ki’s of only 2100 nM and 1900 0308 Fw–492.64, colorless plate, 0.56x0.26x0.08 mm, nM, respectively; and the Sigma-2 receptor with Kis of only monoclinic, C2 (no. 5), a 36.3410(7), b=7.88266(12), c=9. 5200 nM and 5600 nM, respectively. For pharmacologic 82344(18) A, B=94.0983(17), V=2806.86(9) A, Z=4, effects on Sigma receptors, we focus on ligands that interact D-1.166 g cm, u=0.708 mm', abs. corr, range: 0.764-0. with Ki’s 2500 nM, so this level of interaction for both iso 955. 311 14 Reflections were measured up to a resolution of mers of Isometheptene on Sigma-2 is of potential biological (sin0/), 0.62 A'.5499 Reflections were unique (R-0. interest. Of the receptors that were positive on screening, 0287), of which 5295 were observed D2O.(I)]. 403 Param racemic Isometheptene at 10 uM inhibited ligand binding to eters were refined using 304 restraints. R1/wR2 D2O (1): the Imidazoline 1 (I.1) receptor of 95%. Further testing of 0.0498/0.1458. R1/wR2 all refl.: 0.0522/0.1486. S=1.054. S-Isometheptene and R-Isometheptene by equilibrium bind Residual electron density found between -0.28 and 0.38 e ing showed they bound I1 receptor with Ki’s of 1100 nMand A-3 18 nM, respectively. For pharmacologic effects, we focus on US 2014/021248.6 A1 Jul. 31, 2014 ligands that interact with Ki’s <100 nM, so the binding of daily, capsules containing increasing amounts of 15 mg. 30 R-Isometheptene to I2 is physicologically significant. Note mg, 50 mg, 100 mg, 150 mg, 300 mg or 500 mg. that Imidazoline-I1 and Imidazoline-I2 binding have not been 0319 Arterial Blood Pressure (BP) and Heart Rate Effects associated with headache treatments to our knowledge. In Vivo (FIG. 20) 0314. Many racemic compounds consist of individual (R)- 0320 A study in rat described below shows a comparison or (S)-enantiomers which exhibit different binding activities. of the effects of Isometheptene Mucate USP and its Isomers It has now been discovered that as shown in the affinity on arterial blood pressure and heart rate following i.v. admin measurements of the isolated Isometheptene isomers at Table istration in the anesthetized rat. 1, the (R)-enantiomer of Isometheptene may have a structural 0321) Rats were anesthetized with InactinR (sodium domain better suited for binding with greater affinity or avid thiobutabarbital 100 to 150 mg/kg i.p.) and prepared for the ity to a specific CNS target receptor than the opposite race recording of the following parameters: mate or (S)-enantiomer. The purified racemic Isometheptene 0322 (A) Mean, (B) systolic and (C) diastolic arterial compound has been tested as two racemate molecules with blood pressure (mmHg), via a pressure transducer intro different affinities for certain CNS acceptors, e.g., imidazo duced into the left carotid artery; line-1 (I.1) and imidazoline-2 (I2) (see Table 1). As mentioned 0323 (D) Heart rate (beats/min), derived from pulse above, the I1 receptor is described particularly to bind the blood pressure. drug, moxonidine, which is a centrally acting antihyperten 0324. The parameters measured were allowed to stabilize sive therapeutic compound. for a period of at least 20 minutes before the test substance was administered. TABLE 1. 0325 Measurements were performed before and then 30 seconds, 1, 2, 5, 10 and 15 minutes after the end of each i.v. BINDING AFFINITIES OF ISOMETHEPTENE bolus administration. INVARIOUS CNS RECEPTORS 0326 Results are expressed in absolute values and as Inhibition (a) 10 % Ki(nM maximal change from pre-dose. 0327 Six (6) rats were studied per group. Isomer Isomer Isomer Isomer 0328. Each test substance was evaluated at 3 doses (0.05, Racemic 1 2 1 2 0.5 and 5 mg/kg), administered i.v. as a 30-second bolus, at 5-HT1A 37 intervals of 15 minutes, and compared with a time-matched 5-HT7 46 13 31 vehicle control group. Adrenergic a2B 76 29 57 2700 2300 Adrenergic a2C 41 O O 0329. The experiment therefore included 4 groups. Imidazoline-1 95 1100 18 0330 Inter-group comparison on maximal absolute (I1) changes from pre-dose (each test Substance versus vehicle, Imidazoline-2 84 86 1100 1SOO and each isomer versus racemate) was performed using an (I2) Sigma 62 87 86 Unpaired Student's t test. (non-select) 0331. The results are illustrated in the diagrams, FIG. 20 Sigma 1 2100 1900 A-D. Sigma 2 S200 S600 Results: In view of the ambiguous physiological effects of tests 0332 Isometheptene Mucate USP (0.05, 0.5 and 5 involving the racemic Isometheptene compound or composi mg/kg), administered as a 30-secondi.V. bolus to anesthetized tion thereof, it is believed that the effect of its single active rats, only induced a slight but dose-dependent hypertension (R)-Isometheptene enantiomer mucate (Isomer 2) will be from 0.5 mg/kg when comparing the amplitude of the more specific with fewer side effects. Accordingly, a single response to that observed in the time-matched vehicle control (R)-isomer of Isometheptene will resultina better therapeutic group. The increase occurred shortly following the adminis than the racemate. This will eliminate the potential cardio tration and reached a maximum of 4.0+0.6 mmHg from pre vascular effect from the (S)-isomer without affecting the anti dose for mean BP at 0.05 mg/kg (NS), 10.5-0.9 mmHg from ETTH activity that predominantly resides in (R)-Isomethep pre-dose at 0.5 mg/kg (NS) and 11.7+2.8 mmHg from pre tene. dose at 5 mg/kg (p<0.05). Isometheptene Mucate USP 0315. Therefore, treatment of patients suffering from induced a clear and rapid dose-dependent tachycardia that ETTH (migraine) may be studied by applying increasing reached a maximum of 19.0+4.3 bpm from respective pre doses of Isometheptene (R)-enantiomer in order to determine dose at 0.05 mg/kg (NS), 40.8+4.0 bpm at 0.5 mg/kg (p<0. the efficacy of the composition in a pharmacological profile 001) and 57.8+8.6 bpm at 5 mg/kg (p<0.001). of the induced cardiovascular responses and side effects on 0333) Isometheptene Mucate Isomer 1 (0.05, 0.5 and 5 heart rates. mg/kg), administered as a 30-secondi.V. bolus to anesthetized 0316 For example, (R)-Isometheptene mucate isomer as rats, induced a dose-dependent hypertension, but also only an active ingredient of a CDP with caffeine and acetami from 0.5 mg/kg when comparing the amplitude of the nophen as the other active ingredients is due to be evaluated response to that observed in the time-matched vehicle control for the treatment of migraine. group. The increase occurred shortly following the adminis 0317. The (R)-Isometheptene isomer mucate can be tration and reached a maximum of 2.7+0.8 mmHg max from administered in the form of hard gelatin capsules without pre-dose for mean BP at 0.05 mg/kg (NS), 10.3+4.0 mmHg excipients added. max from pre-dose at 0.5 mg/kg (NS) and 34.7+7.6 mmHg 0318. In order to determine the relieving effect of the max from pre-dose at 5 mg/kg (p<0.01). Isometheptene single (R)-Isometheptene mucate isomer, a patient in need of Mucate Isomer 1 also caused a clear and rapid dose-depen Such treatment is administered, optionally twice or four times dent tachycardia that reached a maximum of 23.8+3.2 bpm US 2014/021248.6 A1 Jul. 31, 2014 26 from respective pre-dose at 0.05 mg/kg (p<0.01), 70.39.1 tors and the corresponding vasopressor responses were medi bpm at 0.5 mg/kg (p<0.001) and 76.5-8.2 bpm at 5 mg/kg ated by a predominantly indirect (tyramine-like action) as (p<0.001). well as a minor direct (C.-adrenoceptors) sympathomimetic 0334) Isometheptene Mucate Isomer 2 (0.05, 0.5 and 5 mechanism. mg/kg), administered as a 30-secondi.V. bolus to anesthetized 0342. We suggest that Isometheptene (R)-enantiomer rats, induced a slight but dose-dependent hypertension, but treatment, administered orally either alone or in a CDP. may only from 0.5 mg/kg when comparing the amplitude of the have a lower potential adverse effects to the cardiovascular response to that observed in the time-matched vehicle control system. Thus, we set out to determine the molecular target of group. The increase occurred shortly following the adminis Isometheptene by Screening for binding to a broad set of tration and reached a maximum of 3.0+0.9 mmHg max from targets, which included receptors, enzymes, channels and pre-dose for mean BP at 0.05 mg/kg (NS), 7.5-1.5 mmHg transporters. Isometheptene does not display significant bind max from pre-dose for mean BP at 0.5 mg/kg (NS) and ing to any of the adrenergic receptors studied. Of the targets 19.2+4.4 mmHg max from pre-dose at 5 mg/kg (p<0.01). studied, Isometheptene bound the I1 receptor. On screening, Isometheptene Mucate Isomer 2 also caused a clear and rapid 10 uM racemic Isometheptene showed >95% inhibition of dose-dependent tachycardia that reached a maximum of ligand binding. A full binding profile of Isometheptene 24.7+5.6 bpm from respective pre-dose at 0.05 mg/kg (p<0. mucate and the (R)- and (S)-Isometheptene mucate enanti 05), 49.5+9.2bpm at 0.5 mg/kg (p<0.001) and 72.2+5.9 bpm. omers on the I1 receptor is shown below. As shown in the at 5 mg/kg (p<0.001). figure below, (R)-Isometheptene showed strong binding 0335 Although the maximum increase in arterial blood (K-18 nM), while (S)-Isometheptene showed very weak pressure observed following the administration of binding (K-1 100 nM) (Molderings, 1993). Consistent with Isometheptene Mucate Isomer 1 at 5 mg/kg was higher than the approximately 50:50 ratios of the isomers in racemic that observed with Isometheptene mucate Isomer 2 (34.7+7.6 Isometheptene mucate, binding studies showed racemic mm Hg versus 19.24.4 mmHg), there was no statistical Isometheptene possessed approximately half the potency of difference between the two isomers. A trend for systolic the pure (R)-isomer (K. 42 nM), see Table 1. blood pressure was however observed (p=0.0762). The tachy 0343. The identification of I1 as the Isometheptene recep cardia observed following Isometheptene Mucate Isomer 1 tor reveals a new molecular target for ETTH therapeutics and and Isomer 2 administration was not statistically different. also indicates that (R)-Isometheptene as a single isomer is an 0336. These results suggest that Isometheptene Mucate improved treatment for ETTH relative to racemic Isomethep (the racemate), Isometheptene Mucate Isomer 1, and tene. The I1 receptor was not previously known to be a target Isometheptene Mucate Isomer 2 (0.05, 0.5 and 5 mg/kg), for ETTH therapeutics. The putative natural ligand for I1 is administered as abolus of 30-secondi.v.bolus to anesthetized agmatine and pharmacologic ligands of I1 include monoxi rats, induced a dose-dependent hypertension. Both Isomer 1 dine and clonidine (Head, 2006; Molderings, 1997). The and Isomer 2 induced a more pronounced hypertension than activities of agmatine, moxonidine and clonidine can be used that induced by the racemate, and Isomer 1 clearly induced to understand the function of the I1 receptor. The I1 receptor (although not statistically significant) a more pronounced subtype is found in both the rostral ventro-lateral pressor and hypertension than Isomer 2. The racemate and the two iso ventromedial depressor areas of the medulla oblongata. The mers induced a dose-dependent tachycardia that was not sta discovery that (R)-Isometheptene binds the I1 receptor is the tistically different, although that observed following the first known link of this receptor to therapeutics for ETTH. administration of 0.5 mg/kg was slightly higher with isomer 0344) Many racemic compounds consist of (R)- or (S)- 1. enantiomers which are mirror images of each other exhibiting 0337 Safety of the Isometheptene (R)-Enantiomer different binding activities. In fact, the receptor binding affin 0338. The pharmacology, pharmacokinetics, and toxicol ity measurements differ between the two isolated Isomethep ogy of Isometheptene enantiomer alone or as a CDP contain tene enantiomers as shown at Table 1, Suggesting perhaps that ing, e.g., Isometheptene (R)-enantiomer, caffeine, and the two enantiomers of the racemic compound have different acetaminophen are important aspects for characterizing the structural domains. Initial measurements revealed for efficacy of the inventive compound. example, that Isomer 2 (which is now identified as the (R)- 0339 No published papers were identified that provided Isometheptene enantiomer) appears to bind with greater information on the safety pharmacology profiles of racemic (about 60 fold) affinity or avidity to a specific CNS target Isometheptene alone or as an active ingredient in a CDP. receptor than its opposite racemate isomer 1 or (S)- 0340 Treatment with (R)-Isometheptene includes a daily Isometheptene enantiomer). oral dose or twice daily ranging from about 10 mg to about 50 0345. As presently described, the racemic Isometheptene mg, taken alone, or combined with other active ingredients compound has now been purified and tested as two racemate Such as a mild sedative, and a decongestant or proton pump molecules with distinctly different affinities for certain CNS inhibitor, such as Ibuprofen or Celebrex, at a dosage of about target receptors, e.g., imidazoline-1 (I1) and imidazoline-2 100 mg to about 900 mg. (I2) (see Table 1). The I1 receptor is presently known to bind 0341 One published report (Valdivia 2004) investigated the drug, moxonidine, which is a centrally acting antihyper the cardiovascular responses induced by Isometheptene (IV tensive therapeutic compound with low activation of adren at consecutive doses of 0.03, 0.1.0.3, 1, and 3 mg/kg with 5 to ergic receptors. As evidenced in the above assays, Isomer 1 15 minutes between doses) in male Wistar rats. Isomethep and Isomer 2 bind differently to the I2 receptor. tene alone produced dose-dependent increases in heart rate 0346. It has now been discovered that the purified and diastolic blood pressure. Using co-administration of vari Isometheptene isomer 2 (or (R) Isometheptene enantiomer) ous receptor antagonists, the Isometheptene-induced tachy has a significant binding affinity for I 1 (Table 1), which is also cardic responses in rats apparently involved only an indirect the selective receptor of moxonidine, a centrally acting anti (tyramine-like action) mechanism mediated by C.-adrenocep hypertensive agent. Present understanding of the I1 receptor US 2014/021248.6 A1 Jul. 31, 2014 27 is that of a cell-surface receptor mediating cellular responses, above, the I1 receptor is described particularly to bind the participating in cardiovascular control by the CNS, and pro drug, moxonidine, which is a centrally acting antihyperten viding a potential therapeutic target for multiple cardiovas sive therapeutic compound. cular and metabolic disorders. The antihypertensive agents 0350. The diagram shown in FIG. 21, the results of an with an imidazoline or related structure, all show high affinity experiment in which (S)-Isometheptene Mucate salt, (R)- for the I1 receptor (affinity constant Ki-10 nM). The second Isometheptene Mucate salt, and Isometheptene Mucate salt generation agents, moxonidine and rilmenidine have reduced USP (racemic) were studied for binding to the I1 receptor. sedative side-effects, and show lower affinity (higher Ki) at 0351. The diagram in FIG. 22 shows the results of an C2-adrenergic receptors relative to the first-generation agents experiment in which (S)-Isometheptene Mucate salt, (R)- with pronounced sedative side-effects, including clonidine, Isometheptene Mucate salt, and Isometheptene Mucate salt guanabenz, guanfacine, and C.-methylnorepinephrine (the USP (racemic) were studied for binding to the I2 receptor. active metabolite of C.-methyldopa). 0352 Surprisingly, further activities of the optical 0347 Screening at 10M with racemic Isometheptene did Isometheptene isomers have been found directed to the not show meaningful binding (i.e. >70% inhibition) at several TAAR1 (trace amine associated receptor) receptor which is receptors on Screening which include C.1-, O2A- or B-adren expressed in the CNS and belongs to the G-Protein Coupled ergic receptors, serotonin receptors. Of the receptors that Receptor (GPCR) receptor family. TAAR1 is activated by were positive on Screening, racemic Isometheptene at 10 LM biogenic amines such as tyramine, a molecule found in trace inhibited ligand binding to the O2B-adrenergic receptor of levels in the CNS Borowsky et al., PNAS 98: 8966-8971 76%. Further testing the purified (S)-Isometheptene and (R)- (2001). Biogenic amines present in food have been shown to Isometheptene isomers at 10 uM in this assay, showed they result in hypertensive crisis when taken with MAO inhibitors inhibited ligand binding to the C2B-adrenergic receptor by Maguire et al., Pharmacol. Rev. 61: 1-8 (2009). 29% and 57%, respectively. Further testing of (S)-Isomethep 0353 To measure interactions with TAAR1, cell line sta tene and (R)-Isometheptene by equilibrium binding showed bly expressing a recombinant human TAAR1 receptor were similar binding of C.2B-adrenergic receptor with Kis of 2700 studied in the cAMP Biosensor Assay (DiscoveRx, Free nM and 2300 nM, respectively. mont, Calif.) since TAAR1 is coupled to G-proteins that signal through cAMP. The (S)- and (R)-Isometheptene iso 0348 For pharmacologic effects on C.2B-adrenergic mers at a final concentration of 10 uM activate TAAR1 dif receptor, we focus on ligands that interact with Kis-100 nM, ferentially, as shown on the table below, wherein (S)- so this level of interaction is low. Of the receptors that were Isometheptene activates more effectively than the positive on Screening, racemic Isometheptene at 10LM inhib R-Isometheptene. These activities may be linked to side ited ligand binding to the Sigma receptor (non-selective, effects of racemic Isometheptene, due to the interaction of meaning either Sigma 1 or Sigma 2 receptor) of 62%. Further S-Isometheptene to the TAAR1 tyramine receptor. testing the purified (S)-Isometheptene and (R)-Isomethep tene isomers at 10 uM in this assay, showed they inhibited ligand binding to the non-selective Sigma receptor by 87% Compound Efficacy and 86%, respectively. Further testing of (S)-Isometheptene and (R)-Isometheptene by equilibrium binding showed they (S)-Isometheptene free base 49.4% bound Sigma-1 receptor with Ki’s of only 2100 nM and 1900 (R)-Isometheptene free base 25.6% nM, respectively; and the Sigma-2 receptor with Kis of only 5200 nM and 5600 nM, respectively. For pharmacologic 0354. Therefore, the lower TAAR1 activation of the effects on Sigma receptors, we focus on ligands that interact Isometheptene (R)-enantiomer indicates lower side effects with Ki’s 2500 nM, so this level of interaction for both iso with its use. mers of Isometheptene on Sigma-2 is of potential biological 0355 Isometheptene Mucate USP. Isometheptene Mucate interest. Of the receptors that were positive on Screening, isomer 1, Isometheptene Mucate isomer 2 and p-Tyramine racemic Isometheptene at 10 uM inhibited ligand binding to (control) in the TAAR1 cAMP Biosensor Assay of FIG. 23 as the Imidazoline 1 (I.1) receptor of 95%. Further testing of shown by the following diagrams (A), (B), (C), and (D). The (S)-Isometheptene and (R)-Isometheptene by equilibrium data is shown as an agonist dose response curve, normalized binding showed they bound I1 receptor with Kis of 1100 nM to the maximal and minimal response observed in the pres and 18 nM, respectively. For pharmacologic effects, we focus ence of control ligand and vehicle respectively on ligands that interact with Kis-100 nM, so the binding of 0356. The TAAR1 cAMP accumulation assay was also (R)-Isometheptene to I1 is physicologically significant. Note used to study the activity of (S)- and (R)-Isometheptene as that I 1 - and I2-receptor binding have not been associated with mucate salts. In the figure shown (FIG. 23), (A) Isomer 1 is headache treatments to our knowledge. (S) and (B) Isomer 2 is (R) Isometheptene. The SIsomethep 0349. Many racemic compounds consist of individual (R)- tene mucate salt (ECs 21.12 uM) activates TAAR1 more or (S)-enantiomers which exhibit different binding activities. potently than R-Isometheptene mucate salt (ECs. 29.45uM), It has now been discovered that as shown in the affinity which is consistent with the Isometheptene isomer free base measurements of the isolated Isometheptene isomers at Table activation of TAAR1 described above. 1, the (R)-enantiomer of Isometheptene may have a structural 0357 Mechanism of Action: domain best suited for binding with greater affinity or avidity 0358. The (R)-enantiomer of Isometheptene (Isomer 2) is to a specific CNS target receptor than the opposite racemate found to bind to the CNSI1 receptor, with comparatively high or enantiomer. The presently described racemic compound, affinity (Table 1); with its binding avidity being about 60 fold Isometheptene, has now been purified and tested as two iso greater than that of Isometheptene S-enantiomer (Isomer 1). meric molecules with different affinities for certain CNS However, the compound appears to bind only weakly to the acceptors, e.g., and I2-receptor (see Table 1). As mentioned adrenergic alpha-2B receptor. US 2014/021248.6 A1 Jul. 31, 2014 28

This neuronal I1 receptor, is found in both the rostral ventro retention time) and Peak 2 (35.100 retention time) using the lateral pressor and ventromedial depressor areas of the chiral pack IC 250x4.6 mm, 5 microns, with a solvent gradi medulla oblongata. The same receptor also binds a selective ent of MPA-nHexane:MPB-IPA::90:10, at a flow rate of about agonist, moXonidine, which is a centrally-acting antihyper 0.8 ml/min., and observed at a wavelength of 205 nm with tensive causing a decrease in Sympathetic nervous system column temperature of 30 C. Peak 2 is designated as contain activity and blood pressure. Therefore, like moxonidine, on ing the Isometheptene enantiomer 2 and Peak 1 is believed to the basis of its specific binding to the (I) I1 receptor, the contain Isometheptene enantiomer 1. Isometheptene (R)-enantiomer (Isomer 2) may similarly pro 0367 Similarly, the racemic mixture was resolved by mote sodium excretion, improve insulin resistance and glu chiral HPLC at FIG. 12 on the same chiral Pack IC as cose tolerance and protect against hypertensive target organ described above. damage. Such as kidney disease and cardiac hypertrophy. A 0368 FIG. 5 shows a chromatogram of LC-Isomer 2 further aspect may be found in beta blockers inhibiting the wherein the purified enantiomer 2 occupies 99.639% of the metastatic cascade of tumor cells. Further activities of the eluted peak. optical Isometheptene isomers have been found directed to 0369 FIG. 6 shows 1H NMR of Isomer 2 demonstrating the TAAR1 (trace amine associated receptor) receptor which the purity of the molecule. is expressed in the CNS and belongs in the GPCR receptor 0370 FIG. 7 depicts a mass spectrum analysis of Isomer 2 family. TAAR1 is activated by biogenic amines such as indicating the purity of the sample. tyramine, a molecule found in trace levels in the CNS 0371. The HPLC Isomer 1 (or enantiomer 1) of the puri Borowsky et al., PNAS 98: 8966-8971 (2001). Biogenic fied Isometheptene isomer shown in FIG.8 was eluted from a amines present in food have been shown to result in hyper reverse phase Zorbax SB-CN column: 25 mmx4.6 mm, 5 tensive crisis when taken with MAO inhibitors Maguire etal, microM; at a flow of 1.0 ml/minute with a solvent of MPB Pharmacol. Rev. 61: 1-8 (2009). CAN MPA-0.1%TFA in water. Max Plot. The Signal voltage 0359. Therefore, the lower TAAR1 activation of the (R)- is 8.433-8.773 minutes RT. Isometheptene enantiomer indicates lower side effects with 0372. The chromatogram of purified Isometheptene Iso its use. mer 1 is shown in FIG. 11, as eluted from the chiral pack IC 0360. Several comparative studies of acute migraine treat 250x4.6 mm, 5 microns, with a solvent gradient of MPA ment using pharmaceutical compositions of Isometheptene nHexane:MPB-IPA::90:10, at a flow rate of about 0.8 alone, and Isometheptene containing fixed drug combina ml/min., and observed at a wavelength of 205 nm with col tions (FDCs) have been reported (E. Loder Review, CNS umn temperature of 30 C. The Isometheptene Peak 1 eluted at DRUGS 2005: 19(9):780). 29.637 minutes, distinct from a putative Peak 2 time of 33.353 0361 Simultaneous administration of more than one drug minutes. in acute treatment of migraine is common because, in general, 0373 FIG. 13 discloses a liquid chromatography/mass treatment with only one the agent has not been as Successful. spectroscopy analysis (LC/MS) of purified Isomer 1 wherein This observation holds true mostly for Isometheptene or com the chromatogram of Isomer 1 indicates an area of 95.186%. binations with other drugs regardless of the type of adminis 0374 Finally, the HPLC RT blank of Isomer 2 on Zorbax tration Such as parenteral ori.V. column (FIG. 2), and similarly the HPLC RT of the blank of 0362 Considering the several stages and processes Isomer 1 at FIG. 9 show absence of any distinct isomer involved in migraine pathophysiology, adjustable poly material. therapy is preferred for the treatment of dopamine antagonist, an anti-inflammatory agent plus a serotoninagonist, a dopam Pharmaceutical Preparations. ine antagonist plus a serotoninagonist, a dopamine antagonist 0375. The pharmaceutical preparations of the invention plus an anti-inflammatory agent plus a serotonin agonist. embrace a variety of forms, including, but not limited to, a Such fixed combinations (FDCs) contain standard quantities composition that is enteric coated, a composition that is a of the combined drugs in a tablet, injectable solution, nasal controlled release or Sustained release formulation, a compo spray or Suppository. sition that is a solution, a composition that is a topical formu 0363 Taking reference to the appended figures which lation, a composition that is a Suppository, a composition that depict comparative chromatography of the twin Isomethep is a transdermal patch, a composition that is lyophilized, a tene enantiomer forms as illustrative examples that are not composition that is in an inhaler, a compositions that is in a deemed in any way as limiting the present invention: prefilled Syringe, a composition that is in a nasal spray device, 0364. The HPLC Isomer 2 (or (R)-Enantiomer) of and the like. The composition can be for oral administration, Isometheptene-is shown in FIG. 1 as eluted from a reverse parenteral administration, mucosal administration, nasal phase Zorbax SB-CN column: 25mmx4.6 mm, 5 microM; at administration, topical administration, ocular administration, a flow of 1.0 ml/minute with a solvent of MPB-CAN MPA local administration, rectal, intrathecal, etc. If parenteral, the 0.1% TFA in water. Max Plot. The signal voltage is recorded administration can be subcutaneous, intravenous, intrader at 8.470-8.809 minutes RT. mal, intraperitoneal, intrathecal, etc. The pharmaceutical 0365. The chromatogram of purified Isometheptene Iso preparation may be in a packaged unit dosage or multi-unit mer 2 is shown in FIG.3 as eluted from the chiral pack IC dosage. Routes of administration of the compounds in a phar 250x4.6 mm, 5 microns, with a solvent gradient of MPA maceutically acceptable form may include, without limita nHexane:MPB-IPA::90:10, at a flow rate of about 0.8 tion, parenteral, Subcutaneous, intramuscular, intravenous, ml/min., and observed at a wavelength of 205 nm with col intrarticular, intrabronchial, intraabdominal, intracapsular, umn temperature of 30° C. Accordingly, the Isometheptene intracartilaginous, intracavitary, intracelial, intracerebellar, peak 2 eluted at 35.610 minutes. intracerebroVentricular, intracolic, intracervical, intragastric, 0366. The Chiral HPLC Racemic Isometheptene mixture intrahepatic, intramyocardial, intraosteal, intrapelvic, intrap is shown at FIG. 4 as resolved in Peak 1 (31.370 minutes ericardiac, intraperitoneal, intrapleural, intraprostatic, intra US 2014/021248.6 A1 Jul. 31, 2014 29 pulmonary, intrarectal, intrarenal, intraretinal, intraspinal, ceutical efficacy or stability. Carrier formulations suitable for intrasynovial, intrathoracic, intrauterine, intravesical, bolus, oral administration, for Suppositories, and for parenteral vaginal, rectal, buccal, Sublingual, intranasal, and transder administration, etc., can be found in Remington's Pharma mal. ceutical Sciences, Mack Publishing Company, Easton, Pa. 0376. According to yet another embodiment of the inven 0381 Aqueous formulations may include a chelating tion, a pharmaceutical preparation containing a compound of agent, a buffering agent, an anti-oxidant and, optionally, an the present invention, additive salt or intermediate, in a lyo isotonicity agent. In an embodiment, the formulation is pH philized formulation is prepared by combining a cryoprotec adjusted to between 3.0 and 3.5. tive agent. Such as mannitol, with the same. The resulting 0382 Chelating agents include, for example, but are not preparation may also contain any one of any combination of limited to ethylenediaminetetraacetic acid (EDTA) as a free or all of a buffering agent, an antioxidant, and an isotonicity acid, salt or various combinations and derivatives thereof, agent. citric acid and derivatives thereof, niacinamide and deriva 0377. In various embodiments, the pharmaceutical agent tives thereof, sodium desoxycholate and derivatives thereof, is an antispasmodic agent, a steroidal or non-steroidal anti and L-glutamic acid, N,N-diacetic acid and derivatives inflammatory agent, a 5HT agonist, a 5HT antagonist, a thereof. 5HT antagonist, a 5HT agonist, a bulk-forming agent, an 0383 Buffering agents include but are not limited to, citric alpha2-adrenergic agonist, a mineral oil, a fiber or a hemato acid, Sodium citrate, Sodium acetate, acetic acid, sodium poietic stimulating agent. phosphate and phosphoric acid, sodium ascorbate, tartaric 0378 More particularly, depending on the disease or con acid, maleic acid, glycine, Sodium lactate, lactic acid, ascor dition to be treated or prevented, one or more additional bic acid, Imidazoline, sodium bicarbonate and carbonic acid, therapeutic drugs, compounds, reagents, or agents, which are Sodium Succinate and Succinic acid, histidine, and sodium normally or typically administered to treat or prevent the benzoate and benzoic acid, or combinations thereof. disease or condition, may also be administered with the com 0384 Antioxidants include, for example, an ascorbic acid pounds of this invention, or may also be present in the com derivative, butylated hydroxy anisole, butylated hydroxy positions of this invention. It will be appreciated that addi toluene, alkyl gallate, Sodium meta-bisulfite, sodium tional therapeutic agents that are normally or typically bisulfite, sodium dithionite, sodium thioglycolate acid, administered to treat or prevent a given disease or condition sodium formaldehyde sulfoxylate, tocopheral and derivatives are termed 'appropriate for the disease or condition being thereof, monothioglycerol, or Sodium Sulfite or combinations treated'. thereof. In one embodiment, the antioxidant is monothioglyc 0379. In another embodiment, other agents, compounds, erol. drugs, or reagents are suitable for administering in combina 0385 Illustrative isotonicity agents include, but are not tion with the compounds of the present invention, including limited to, Sodium chloride, mannitol, lactose, dextrose, glyc without limitation, anti-inflammatory agents, e.g., non-ste erol, or sorbitol, or combinations thereof. roidal anti-inflammatory drugs (NSAIDs), corticosteroids, 0386 Preservatives that can be used with the present com TNF blockers or inhibitors, IL-RA, azathioprine, cyclophos positions include without limitation benzyl alcohol, para phamide, Sulfasalazine; agents; cardiovascular disease treat bens, thimerosal, chlorobutanol and preferably benzalko ment agents, e.g., ACE inhibitors, beta-blockers, diuretics, nium chloride. Typically, the preservative will be present in a nitrates, calcium channel blockers, statins; diabetes treatment composition in a concentration of up to about 2% by weight. agents, e.g., insulin, glitaZones, Sulfonylureas. The amount of The exact concentration of the preservative, however, will additional therapeutic agent, compound, drug, or reagent vary depending upon the intended use and can be easily present in the compositions of the invention, or administered ascertained by one skilled in the art. in conjunction with the compounds of the invention, are no 0387. The compounds of the invention can be prepared in more than the amount which would normally be administered lyophilized compositions, typically in the presence of a cryo in a composition comprising that therapeutic agent, com protecting agent such as mannitol, or lactose, Sucrose, poly pound, drug, or reagent as the only active agent. As a guide, ethylene glycol, and polyvinyl pyrrolidines. Cryoprotecting the amount of additional therapeutic agent, compound, drug, agents which result in a reconstitution pH of 6.0 or less are or reagent in a composition according to the present invention desired. The invention therefore provides a lyophilized prepa will range from about 40%-100% of the amount normally ration of the therapeutic agent(s) of the invention. The prepa present in a composition comprising that agent, compound, ration can contain a cryoprotecting agent, such as mannitol or drug, or reagent as the only therapeutically active agent. lactose, which is preferably neutral or acidic in water. 0380. The pharmaceutical preparations of the present 0388 Oral, parenteral and suppository formulations of invention may include, or be diluted into, a pharmaceutically agents are well known and commercially available. The acceptable carrier. The term “pharmaceutically-acceptable therapeutic compound(s) of the invention can be added to carrier as used herein means one or more compatible solid, Such well known formulations. One or more compounds of gel, or liquid fillers, diluents or encapsulating Substances the invention can be mixed together in Solution or semi-solid which are suitable for administration to a human or other Solution in Such formulations, provided in a Suspension mammal Such as a non-human primate, a dog, cat, horse, cow, within Such formulations, or contained in particles within sheep, pig, or goat. The term “carrier denotes an organic or Such formulations. inorganic ingredient, natural or synthetic, with which the 0389. As used herein, “pharmaceutically acceptable' active ingredient is combined to facilitate the application. The refers to those compounds, materials, compositions, and/or carriers are capable of being commingled with the composi dosage forms that are, within the scope of Sound medical tions, compounds and preparations of the present invention, judgment, Suitable for contact with the tissues of human and with each other, in a manner Such that there is no inter beings and animals without a resulting or excessive toxicity, action which would substantially impair the desired pharma irritation, allergic response, or other problem complications US 2014/021248.6 A1 Jul. 31, 2014 30 commensurate with a reasonable benefit/risk ratio. As used pound or agent could be released immediately in the stomach, herein, “pharmaceutically acceptable salts' refer to deriva throughout the gastrointestinal tract or only in the intestine. tives of the disclosed compounds wherein the parent com 0391 The materials useful for achieving these different pound is modified by making acid or base salts thereof. release profiles are well known to those of ordinary skill in the Examples of pharmaceutically acceptable salts include, but art. Immediate release is obtainable by conventional tablets are not limited to, mineral or organic acid salts of basic with binders which dissolve in the stomach. Coatings which residues, such as amines, alkali or organic salts of acidic dissolve at the pH of the stomach, or which dissolve at residues, such as carboxylic acids, and the like. The pharma elevated temperatures, will achieve the same purpose. ceutically acceptable salts include the conventional non-toxic Release only in the intestine is achieved using conventional salts or the quaternary ammonium salts of the parent com enteric coatings such as pH sensitive coatings which dissolve pound formed, for example, from non-toxic inorganic or in the pH environment of the intestine (but not the stomach), organic acids. For example. Such conventional non-toxic salts or coatings that dissolve over time. Release throughout the include those derived from inorganic acids such as hydro gastrointestinal tract is achieved by using Sustained-release chloric, hydrobromic, Sulfuric, Sulfamic, phosphoric, nitric materials and/or combinations of the immediate release sys and the like; and the salts prepared from organic acids such as tems and Sustained and/or delayed intentional release systems acetic, propionic, Succinic, glycolic, Stearic, lactic, malic, (e.g., pellets which dissolve at different pHs). tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phe 0392. In the event that it is desirable to release the thera nylacetic, glutamic, benzoic, Salicylic, Sulfanilic, 2-acetoxy peutic compound(s) of the invention first, the therapeutic benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane compound(s) of the invention could be coated on the Surface disulfonic, oxalic, isethionic, and the like. These physiologi of the controlled release formulation in any pharmaceutically cally acceptable salts are prepared by methods known in the acceptable carrier Suitable for Such coatings and for permit art, e.g., by dissolving the free amine bases with an excess of ting the release of the therapeutic compound(s) of the inven the acid in aqueous alcohol, or neutralizing a free carboxylic tion, Such as in a temperature sensitive pharmaceutically acid with an alkali metal base Such as a hydroxide, or with an acceptable carrier routinely used for controlled release. Other amine. Certain acidic or basic compounds of the present coatings, which dissolve when placed in the body, are well invention may exist as Zwitterions. All forms of the com known to those of ordinary skill in the art. pounds, including free acid, free base and Zwitterions, are 0393. The therapeutic compound(s) of the invention also contemplated to be within the scope of the present invention. may be mixed throughout a controlled release formulation, It is well known in the art that compounds containing both whereby it is released before, after, or simultaneously with amino and carboxyl groups often exist in equilibrium with another agent. The therapeutic compound(s) of the invention their Zwitterionic forms. Thus, any of the compounds may be free, that is, solubilized within the material of the described herein that contain, for example, both amino and formulation. The therapeutic compound(s) of the invention carboxyl groups, also include reference to their correspond also may be in the form of vesicles, such as wax-coated ing Zwitterions. micropellets dispersed throughout the material of the formu 0390 A product containing therapeutic compound(s) of lation. The coated pellets can be fashioned to immediately the invention and, optionally, one or more other active agents release the therapeutic compound(s) of the invention based on can be configured as an oral dosage. The oral dosage may be temperature, pH, or the like. The pellets also can be config a liquid, a semisolid or a solid. The oral dosage may be ured so as to delay the release of the therapeutic compound(s) configured to release the therapeutic compound(s) of the of the invention, allowing the other agent a period of time to invention before, after or simultaneously with the other agent. act before the therapeutic compound(s) of the invention exerts The oral dosage may be configured to have the therapeutic its effects. The therapeutic compound(s) of the invention also compound(s) of the invention and the other agents release can be configured, e.g., as pellets, to release the therapeutic completely in the stomach, release partially in the stomach compound(s) of the invention in virtually any Sustained and partially in the intestine, in the intestine, in the colon, release pattern, including patterns exhibiting first order partially in the stomach, or wholly in the colon. The oral release kinetics or sigmoidal order release kinetics using dosage also may be configured whereby the release of the materials of the prior art and well known to those of ordinary therapeutic compound(s) of the invention is confined to the skill in the art. stomach or intestine while the release of the other active agent 0394 The therapeutic compound(s) of the invention also is not so confined or is confined differently from the thera can be contained within a core within the controlled release peutic compound(s) of the invention. For example, the thera formulation. The core may have any one or any combination peutic compound(s) of the invention may be an enterically of the properties described above in connection with the coated core or pellets contained within a pill or capsule that pellets. The therapeutic compound(s) of the invention may releases the other agent first and releases the therapeutic be, for example, in a core coated with a material, dispersed compound(s) of the invention only after the therapeutic com throughout a material, coated onto a material or adsorbed into pound(s) of the invention passes through the stomach and into or throughout a material. the intestine. The therapeutic compound(s) of the invention 0395. It should be understood that the pellets or core may also can be in a Sustained release material, whereby the thera be of virtually any type. They may be drug coated with a peutic compound(s) of the invention is released throughout release material, drug interspersed throughout material, drug the gastrointestinal tract and the other agent is released on the adsorbed into a material, and so on. The material may be same or a different schedule. The same objective for thera erodible or nonerodible. peutic compound(s) of the invention release can be achieved 0396 The therapeutic compound(s) of the invention may with immediate release of therapeutic compound(s) of the be provided in particles. Particles as used herein means nano invention combined with enteric coated therapeutic com or microparticles (or in Some instances larger) which can pound(s) of the invention. In these instances, the other com consist in whole or in part of the therapeutic compound(s) of US 2014/021248.6 A1 Jul. 31, 2014

the invention or the other agents as described herein. The many reports on Such "enteric coatings have been made. In particles may contain the therapeutic compound(s)/agent(s) general, an enteric coating is one which passes through the in a core Surrounded by a coating, including, but not limited stomach without releasing Substantial amounts of drug in the to, an enteric coating. The therapeutic agent(s) also may be stomach (i.e., less than 10% release, 5% release and even 1% dispersed throughout the particles. The therapeutic agent(s) release in the stomach) and Sufficiently disintegrating in the also may be adsorbed into the particles. The particles may be intestinal tract (by contact with approximately neutral or of any order release kinetics, including Zero order release, alkaline intestine juices) to allow the transport (active or first order release, second order release, delayed release, Sus passive) of the active agent through the walls of the intestinal tained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the tract. therapeutic agent(s), any of those materials routinely used in 04.01 Various in vitro tests for determining whether or not the art of pharmacy and medicine, including, but not limited a coating is classified as an enteric coating have been pub to, erodible, nonerodible, biodegradable, or nonbiodegrad lished in the pharmacopoeia of various countries. A coating able material or combinations thereof. The particles may be which remains intact for at least 2 hours, in contact with microcapsules which contain the antagonistina Solution or in artificial gastric juices such as HCl of pH 1 at 36 to 38°C. and a semi-solid State. The particles may be of virtually any shape. thereafter disintegrates within 30 minutes in artificial intesti 0397 Both non-biodegradable and biodegradable poly nal juices such as a KHPO buffered solution of pH 6.8 is one meric materials can be used in the manufacture of particles for example. One such well known system is EUDRAGIT mate delivering the therapeutic agent(s). Such polymers may be rial, commercially available and reported on by Boehringer, natural or synthetic polymers. The polymer is selected based Manchester University, Saale Co., and the like. Enteric coat on the period of time over which release is desired. Bioadhe ings are discussed further below. sive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. 0402. A timed release system is represented by Time Ero Hubell in Macromolecules, (1993)26:581-587, the teachings sion System (TES) by Fujisawa Pharmaceutical Co., Ltd. and of which are incorporated herein. These include polyhyalu Pulsincap by R. P. Scherer. According to these systems, the ronic acids, casein, gelatin, glutin, polyanhydrides, poly site of drug release is decided by the time of transit of a acrylic acid, alginate, chitosan, poly(methyl methacrylates), preparation in the gastrointestinal tract. Since the transit of a poly(ethyl methacrylates), poly(butylmethacrylate), poly preparation in the gastrointestinal tract is largely influenced (isobutyl methacrylate), poly(hexylmethacrylate), poly(iso by the gastric emptying time, some time release systems are decyl methacrylate), poly(lauryl methacrylate), poly(phenyl also enterically coated. methacrylate), poly(methyl acrylate), poly(isopropyl acry 0403 Systems making use of the enterobacteria can be late), poly(isobutyl acrylate), and poly(octadecyl acrylate). classified into those utilizing degradation of aZoaromatic 0398. The therapeutic agent(s) may be contained in a con polymers by an azo reductase produced from enterobacteria trolled release formulation or controlled release systems. The as reported by a group at Ohio University (M. Saffran, et al., term “controlled release' is intended to refer to any drug Science, Vol. 233: 1081 (1986)) and a group at Utah Univer containing formulation in which the manner and profile of sity (J. Kopecek, et al., Pharmaceutical Research, 9(12), drug release from the formulation are controlled. This refers 1540-1545 (1992)); and those utilizing degradation of to immediate as well as nonimmediate release formulations, polysaccharides by beta-galactosidase of enterobacteria as with nonimmediate release formulations including but not reported by a group a Hebrew University (unexamined pub limited to Sustained release and delayed release formulations. lished Japanese patent application No. 5-50863 based on a The term “sustained release' (also referred to as “extended PCT application) and a group at Freiberg University (K. H. release') is used in its conventional sense to refer to a drug Bauer et al., Pharmaceutical Research, 10(10), S218 (1993)). formulation that provides for gradual release of a drug overan In addition, the system using chitosan degradable by chitosa extended period of time, and that preferably, although not nase by Teikoku Seiyaku K.K. (unexamined published Japa necessarily, results in Substantially constant blood levels of a nese patent application No. 4-2 17924 and unexamined pub drug over an extended time period. The term “delayed lished Japanese patent application No. 4-225922) is also release' is used in its conventional sense to refer to a drug included. formulation in which there is a time delay between adminis 04.04 The enteric coating is typically, although not neces tration of the formulation and the release of the drug there sarily, a polymeric material. Preferred enteric coating mate from. “Delayed release' may or may not involve gradual rials comprise bioerodible, gradually hydrolyzable and/or release of drug over an extended period of time, and thus may gradually water-soluble polymers. The "coating weight, or or may not be “sustained release.” These formulations may be relative amount of coating material per capsule, generally for any mode of administration. dictates the time interval between ingestion and drug release. 0399. Delivery systems specific for the gastrointestinal Any coating should be applied to a Sufficient thickness Such tract are roughly divided into three types: the first is a delayed that the entire coating does not dissolve in the gastrointestinal release system designed to release a drug in response to, for fluids at pH below about 5, but does dissolve at pH about 5 and example, a change in pH; the second is a timed-release system above. It is expected that any anionic polymer exhibiting a designed to release a drug after a predetermined time; and the pH-dependent solubility profile can be used as an enteric third is a microflora enzyme system making use of the abun coating in the practice of the present invention. The selection dant enterobacteria in the lower part of the gastrointestinal of the specific enteric coating material will depend on the tract (e.g., in a colonic site-directed release formulation). following properties: resistance to dissolution and disintegra 0400. An example of a delayed release system is one that tion in the stomach; impermeability to gastric fluids and drug/ uses, for example, an acrylic or cellulosic coating material carrier/enzyme while in the stomach; ability to dissolve or and dissolves on pH change. Because of ease of preparation, disintegrate rapidly at the target intestine site; physical and US 2014/021248.6 A1 Jul. 31, 2014 32 chemical stability during storage; non-toxicity; ease of appli plished at Some generally predictable location. The enteric cation as a coating (Substrate friendly); and economical prac coating also prevents exposure of the therapeutic agent and ticality. carrier to the epithelial and mucosal tissue of the buccal 04.05 Suitable enteric coating materials include, but are cavity, pharynx, esophagus, and stomach, and to the enzymes not limited to: cellulosic polymers such as cellulose acetate associated with these tissues. The enteric coating therefore phthalate, cellulose acetate trimellitate, hydroxypropylm helps to protect the active agent, carrier and a patients inter ethyl cellulose phthalate, hydroxypropylmethyl cellulose nal tissue from any adverse event prior to drug release at the Succinate and carboxymethylcellulose sodium; acrylic acid desired site of delivery. Furthermore, the coated material of polymers and copolymers, preferably formed from acrylic the present invention allows optimization of drug absorption, acid, methacrylic acid, methyl acrylate, ammonium methy active agent protection, and safety. Multiple enteric coatings lacrylate, ethyl acrylate, methyl methacrylate and/or ethyl targeted to release the active agent at various regions in the methacrylate (e.g., those copolymers sold under the trade gastrointestinal tract would enable even more effective and name EUDRAGIT); vinyl polymers and copolymers such as Sustained improved delivery throughout the gastrointestinal polyvinyl acetate, polyvinylacetate phthalate, vinylacetate tract. crotonic acid copolymer, and ethylene-vinyl acetate copoly 0408. The coating can, and usually does, contain a plasti mers; and shellac (purified lac). Combinations of different cizer to prevent the formation of pores and cracks that would coating materials may also be used. Well known enteric coat permit the penetration of the gastric fluids. Suitable plasticiz ing material for use herein are those acrylic acid polymers and ers include, but are not limited to, triethylcitrate (Citroflex 2), copolymers available under the trade name EUDRAGIT from triacetin (glyceryl triacetate), acetyl triethylcitrate (Citroflec Rohm Pharma (Germany). The EUDRAGIT series E. L. S. A2), Carbowax 400 (polyethylene glycol 400), diethyl phtha RL, RS and NE copolymers are available as solubilized in late, tributyl citrate, acetylated monoglycerides, glycerol, organic solvent, as an aqueous dispersion, or as a dry powder. fatty acid esters, propylene glycol, and dibutyl phthalate. In The EUDRAGIT series RL, NE, and RS copolymers are particular, a coating comprised of an anionic carboxylic insoluble in the gastrointestinal tract but are permeable and acrylic polymer will usually contain approximately 10% to are used primarily for extended release. The EUDRAGIT 25% by weight of a plasticizer, particularly dibutyl phthalate, series E. copolymers dissolve in the stomach. The polyethylene glycol, triethylcitrate and triacetin. The coating EUDRAGIT series L., L-30D and (S) copolymers are can also contain other coating excipients such as detackifiers, insoluble in stomach and dissolve in the intestine, and are thus antifoaming agents, lubricants (e.g., magnesium Stearate). most preferred herein. and stabilizers (e.g., hydroxypropylcellulose, acids and 0406 A particular methacrylic copolymer is EUDRAGIT bases) to solubilize or disperse the coating material, and to L. particularly L-30D and EUDRAGIT L 100-55. In improve coating performance and the coated product. EUDRAGIT L-30D, the ratio of free carboxyl groups to ester 04.09. The coating can be applied to particles of the thera groups is approximately 1:1. Further, the copolymer is known peutic agent(s), tablets of the therapeutic agent(s), capsules to be insoluble in gastrointestinal fluids having pH below 5.5, containing the therapeutic agent(s) and the like, using con generally 1.5-5.5, i.e., the pH generally present in the fluid of ventional coating methods and equipment. For example, an the upper gastrointestinal tract, but readily soluble or partially enteric coating can be applied to a capsule using a coating soluble at pH above 5.5, i.e., the pH generally present in the pan, an airless spray technique, fluidized bed coating equip fluid of lower gastrointestinal tract. Another particular meth ment, or the like. Detailed information concerning materials, acrylic acid polymer is EUDRAGITS, which differs from equipment and processes for preparing coated dosage forms EUDRAGIT L-30D in that the ratio of free carboxyl groups to may be found in Pharmaceutical Dosage Forms: Tablets, eds. ester groups is approximately 1:2. EUDRAGIT (S) is Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and insoluble at pH below 5.5, but unlike EUDRAGIT L-30D, is in Ansel et al., Pharmaceutical Dosage Forms and Drug poorly soluble in gastrointestinal fluids having a pH in the Delivery Systems, 6th Ed. (Media, PA: Williams & Wilkins, range of 5.5 to 7.0, such as in the small intestine. This copoly 1995). The coating thickness, as noted above, must be suffi mer is soluble at pH 7.0 and above, i.e., the pH generally cient to ensure that the oral dosage form remains intact until found in the colon. EUDRAGIT (S) can be used alone as a the desired site of topical delivery in the lower intestinal tract coating to provide drug delivery in the large intestine. Alter is reached. natively, EUDRAGIT S, being poorly soluble in intestinal 0410. In another embodiment, drug dosage forms are pro fluids below pH 7, can be used in combination with vided that comprise an enterically coated, osmotically acti EUDRAGITL-30D, soluble in intestinal fluids above pH 5.5, vated device housing a formulation of the invention. In this in order to provide a delayed release composition which can embodiment, the drug-containing formulation is encapsu be formulated to deliver the active agent to various segments lated in a semipermeable membrane or barrier containing a of the intestinal tract. The more EUDRAGIT L-30D used, the small orifice. As known in the art with respect to so-called more proximal release and delivery begins, and the more “osmotic pump' drug delivery devices, the semipermeable EUDRAGIT (S) used, the more distal release and delivery membrane allows passage of water in either direction, but not begins. It will be appreciated by those skilled in the art that drug. Therefore, when the device is exposed to aqueous flu both EUDRAGIT L-30D and EUDRAGIT (S) can be ids, water will flow into the device due to the osmotic pressure replaced with other pharmaceutically acceptable polymers differential between the interior and exterior of the device. As having similar pH solubility characteristics. In certain water flows into the device, the drug-containing formulation embodiments of the invention, the preferred enteric coating is in the interior will be “pumped out through the orifice. The ACRYL-EZETM (methacrylic acid co-polymer type C; Col rate of drug release will be equivalent to the inflow rate of orcon, West Point, Pa.). water times the drug concentration. The rate of water influx 0407. The enteric coating provides for controlled release and drug efflux can be controlled by the composition and size of the active agent, Such that drug release can be accom of the orifice of the device. Suitable materials for the semi US 2014/021248.6 A1 Jul. 31, 2014

permeable membrane include, but are not limited to, polyvi meable laminated capsule is then dried using conventional nyl alcohol, polyvinyl chloride, semipermeable polyethylene techniques. Then, an orifice having a desired diameter (e.g., glycols, semipermeable polyurethanes, semipermeable about 0.99 mm) is provided through the semipermeable lami polyamides, semipermeable Sulfonated polystyrenes and nated capsule wall, using, for example, mechanical drilling, polystyrene derivatives; semipermeable poly(sodium styre laser drilling, mechanical rupturing, or erosion of an erodible nesulfonate), semipermeable poly(vinylbenzyltrimethylam element such as a gelatin plug. The osmotically activated monium chloride), and cellulosic polymers such as cellulose device may then be enterically coated as previously acetate, cellulose diacetate, cellulose triacetate, cellulose pro described. For osmotically activated devices containing a pionate, cellulose acetate propionate, cellulose acetate Solid carrier rather than a liquid or semi-solid carrier, the butyrate, cellulose trivalerate, cellulose trilimate, cellulose interior capsule is optional; that is, the semipermeable mem tripalmitate, cellulose trioctanoate, cellulose tripropionate, brane may be formed directly around the carrier-drug com cellulose disuccinate, cellulose dipalmitate, cellulose dicy position. However, preferred carriers for use in the drug late, cellulose acetate Succinate, cellulose propionate Succi containing formulation of the osmotically activated device nate, cellulose acetate octanoate, cellulose Valerate palmitate, are solutions, Suspensions, liquids, immiscible liquids, emul cellulose acetate heptanate, cellulose acetaldehyde dimethyl sions, Sols, colloids, and oils. Particularly preferred carriers acetal, cellulose acetate ethylcarbamate, cellulose acetate include, but are not limited to, those used for enterically methylcarbamate, cellulose dimethylaminoacetate and ethyl coated capsules containing liquid or semisolid drug formula cellulose. tions. 0411. In another embodiment, drug dosage forms are pro 0415 Cellulose coatings include those of cellulose acetate vided that comprise a Sustained release coated device housing phthalate and trimellitate; methacrylic acid copolymers, e.g. a formulation of the invention. In this embodiment, the drug copolymers derived from methylacrylic acid and esters containing formulation is encapsulated in a Sustained release thereof, containing at least 40% methylacrylic acid; and espe membrane or film. The membrane may be semipermeable, as cially hydroxypropyl methylcellulosephthalate. Methylacry described above. A semipermeable membrane allows for the lates include those of molecular weight above 100,000 dal passage of waterinside the coated device to dissolve the drug. tons based on, e.g. methylacrylate and methyl or ethyl The dissolved drug solution diffuses out through the semiper methylacrylate in a ratio of about 1:1. Typical products meable membrane. The rate of drug release depends upon the include Eudragit L, e.g. L 100-55, marketed by Rohm GmbH, thickness of the coated film and the release of drug can begin Darmstadt, Germany. Typical cellulose acetate phthalates in any part of the GI tract. Suitable membrane materials for have an acetyl content of 17-26% and a phthalate content of Such a membrane include ethylcellulose. from 30-40% with a viscosity of ca. 45-90 cp. Typical cellu 0412. In another embodiment, drug dosage forms are pro lose acetate trimellitates have an acetyl content of 17-26%, a vided that comprise a Sustained release device housing a trimellityl content from 25-35% with a viscosity of ca. 15-20 formulation of the invention. In this embodiment, the drug cS. An example of a cellulose acetate trimellitate is the mar containing formulation is uniformly mixed with a Sustained keted product CAT (Eastman Kodak Company, USA). release polymer. These Sustained release polymers are high Hydroxypropyl methylcellulose phthalates typically have a molecular weight water-soluble polymers, which when in molecular weight of from 20,000 to 130,000 daltons, a contact with water, Swell and create channels for water to hydroxypropyl content of from 5 to 10%, a methoxy content diffuse inside and dissolve the drug. As the polymers swell of from 18 to 24% and a phthalyl content from 21 to 35%. An and dissolve in water, more of drug is exposed to water for example of a cellulose acetate phthalate is the marketed prod dissolution. Such a system is generally referred to as Sus uct CAP (Eastman Kodak, Rochester N.Y., USA). Examples tained release matrix. Suitable materials for such a device of hydroxypropyl methylcellulose phthalates are the mar include hydropropyl methylcellulose, hydroxypropyl cellu keted products having a hydroxypropyl content of from lose, hydroxyethyl cellulose and methyl cellulose. 6-10%, a methoxy content of from 20-24%, a phthalyl con 0413. In another embodiment, drug dosage forms are pro tent of from 21-27%, a molecular weight of about 84,000 vided that comprise an enteric coated device housing a sus daltons, sold under the trademark HP50 and available from tained release formulation of the invention. In this embodi Shin-Etsu Chemical Co. Ltd., Tokyo, Japan, and having a ment, the drug containing product described above is coated hydroxypropyl content, a methoxyl content, and a phthalyl with an enteric polymer. Such a device would not release any content of 5-9%, 18-22% and 27-35%, respectively, and a drug in the stomach and when the device reaches the intestine, molecular weight of 78,000 daltons, known under the trade the enteric polymer is first dissolved and only then would the mark HP55 and available from the same supplier. drug release begin. The drug release would take place in a 0416) The therapeutic agents may be provided in coated or Sustained release fashion. uncoated capsules. The capsule material may be eitherhard or 0414 Enterically coated, osmotically activated devices soft, and as will be appreciated by those skilled in the art, can be manufactured using conventional materials, methods typically comprises a tasteless, easily administered and water and equipment. For example, osmotically activated devices soluble compound Such as gelatin, starch oracellulosic mate may be made by first encapsulating, in a pharmaceutically rial. The capsules are preferably sealed, such as with gelatin acceptable soft capsule, a liquid or semi-solid formulation of bands or other biologically amenable sealant material. See, the compounds of the invention as described previously. This for example, Remington: The Science and Practice of Phar interior capsule is then coated with a semipermeable mem macy, Nineteenth Edition (Easton, Pa.; Mack Publishing Co., brane composition (comprising, for example, cellulose 1995), which describes materials and methods for preparing acetate and polyethylene glycol 4000 in a suitable solvent encapsulated pharmaceuticals. Such as a methylene chloride-methanol admixture), for 0417. A product containing therapeutic compound(s) of example using an air Suspension machine, until a sufficiently the invention can be configured as a Suppository. The thera thick laminate is formed, e.g., around 0.05 mm. The semiper peutic compound(s) of the invention can be placed anywhere US 2014/021248.6 A1 Jul. 31, 2014 34 within or on the suppository to favorably affect the relative gels, aqueous Suspensions, aqueous liposomal dispersions, release of the therapeutic compound(s). The nature of the aqueous emulsions, aqueous microemulsions and combina release can be Zero order, first order, or sigmoidal, as desired. tions thereof. Non-aqueous preparations include, for 0418 Suppositories are solid dosage forms of medicine example, non-aqueous gels, non-aqueous Suspensions, non intended for administration via the rectum. Suppositories are aqueous liposomal dispersions, non-aqueous emulsions, non compounded so as to melt, soften, or dissolve in the body aqueous microemulsions and combinations thereof. The vari cavity (around 98.6 F) thereby releasing the medication ous forms of the nasal delivery systems can include a buffer to contained therein. Suppository bases should be stable, nonir maintain pH, a pharmaceutically acceptable thickening agent ritating, chemically inert, and physiologically inert. Many and a humectant. The pH of the buffer can be selected to commercially available Suppositories contain oily or fatty optimize the absorption of the therapeutic agent(s) across the base materials, such as cocoa butter, coconut oil, palm kernel nasal mucosa. oil, and palm oil, which often melt or deform at room tem 0423. With respect to the non-aqueous nasal formulations, perature necessitating cool storage or other storage limita suitable forms of buffering agents can be selected such that tions. U.S. Pat. No. 4,837.214 to Tanaka et al. describes a when the formulation is delivered into the nasal cavity of a suppository base comprised of 80 to 99 percent by weight of mammal, selected pH ranges are achieved therein upon con a lauric-type fathaving a hydroxyl value of 20 or smaller and tact with, e.g., a nasal mucosa. In the present invention, the pH containing glycerides of fatty acids having 8 to 18 carbon of the compositions should be maintained from about 2.0 to atoms combined with 1 to 20 percent by weight diglycerides about 6.0. It is desirable that the pH of the compositions is one offatty acids (which erucic acid is an example of). The shelf which does not cause significant irritation to the nasal mucosa life of these type of Suppositories is limited due to degrada of a recipient upon administration. An aerosol or spray device tion. Other Suppository bases contain alcohols, Surfactants, may be used in conjunction with the nasal delivery systems of and Such diluents which raise the melting temperature but the invention. also can lead to poor absorption of the medicine and side 0424 The viscosity of the compositions of the present effects due to irritation of the local mucous membranes (see invention can be maintained at a desired level using a phar for example, U.S. Pat. No. 6,099,853 to Hartelendy et al., U.S. maceutically acceptable thickening agent. Thickening agents Pat. No. 4,999,342 to Ahmad et al., and U.S. Pat. No. 4,765, that can be used in accordance with the present invention 978 to Abidi et al.). include methyl cellulose, Xanthan gum, carboxymethyl cel 0419. The base used in the pharmaceutical suppository lulose, hydroxypropyl cellulose, carbomer, polyvinyl alco composition of this invention includes, in general, oils and hol, alginates, acacia, chitosans and combinations thereof. fats comprising triglycerides as main components such as The concentration of the thickening agent will depend upon cacao butter, palm fat, palm kernel oil, coconut oil, fraction the agent selected and the viscosity desired. Such agents can ated coconut oil, lard and WITEPSOL(R), waxes such as lano also be used in a powder formulation discussed above. lin and reduced lanolin; hydrocarbons such as VASELINER, 0425 The compositions of the present invention can also squalene, squalane and liquid paraffin, long to medium chain include a humectant to reduce or prevent drying of the mucus fatty acids such as caprylic acid, lauric acid, Stearic acid and membrane and to prevent irritation thereof. Illustratively, oleic acid; higher alcohols such as lauryl alcohol, cetanol and Suitable humectants that can be used in the present invention Stearyl alcohol; fatty acid esters such as butyl Stearate and include Sorbitol, mineral oil, vegetable oil and glycerol; dilauryl malonate; medium to long chain carboxylic acid Soothing agents; membrane conditioners; Sweeteners; and esters of glycerin Such as triolein and tristearin; glycerin combinations thereof. The concentration of the humectant in Substituted carboxylic acid esters such as glycerin acetoac the present compositions will vary depending upon the agent etate; and polyethylene glycols and its derivatives, such as selected. macrogols and cetomacrogol. They may be used either singly 0426 One or more therapeutic agents may be incorporated or in combination of two or more. If desired, the composition into the nasal delivery system or any other delivery system of this invention may further include a Surface-active agent, a described herein. coloring agent, etc., which are ordinarily used in Supposito 0427. A composition formulated for topical administra 1S. tion may be liquid or semi-solid (including, for example, a 0420. The pharmaceutical compositions of this invention gel, lotion, emulsion, cream, ointment, spray or aerosol) or may be prepared by uniformly mixing predetermined may be provided in combination with a “finite' carrier, for amounts of the active ingredient, the absorption aid and example, a non-spreading material that retains its form, optionally the base, etc. in a stirrer or a grinding mill, at an including, for example, a patch, bioadhesive, dressing orban elevated temperature if required. The resulting composition dage. It may be acqueous or non-aqueous; it may be formu may be formed into a Suppository in unit dosage form by, for lated as a solution, emulsion, dispersion, a suspension or any example, casting the mixture in a mold, or by forming it into other mixture. a gelatin capsule using a capsule filling machine. 0428. Some modes of administration include topical 0421. The compositions according to the present inven application to the skin, eyes or mucosa. Thus, typical vehicles tion also can be administered as a nasal spray, nasal drop, are those Suitable for pharmaceutical or cosmetic application Suspension, gel, ointment, cream or powder. The administra to body surfaces. The compositions provided herein may be tion of a composition can also include using a nasal tampon or applied topically or locally to various areas in the body of a a nasal sponge containing or impregnated with a composition patient. As noted above, topical application is intended to of the present invention. refer to application to the tissue of an accessible body Surface, 0422 The nasal delivery systems that can be used with the Such as, for example, the skin (the outer integument or cov present invention can take various forms including aqueous ering) and the mucosa (the mucous-producing, secreting and/ preparations, non-aqueous preparations and combinations or containing Surfaces). Exemplary mucosal Surfaces include thereof. Aqueous preparations include, for example, aqueous the mucosal Surfaces of the eyes, mouth (such as the lips, US 2014/021248.6 A1 Jul. 31, 2014 tongue, gums, cheeks, Sublingual and roof of the mouth), having 9 to 22 carbon atoms. Suitable examples include, but larynx, esophagus, bronchial, nasal passages, vagina and rec are not limited to, pelargonic, lauric, myristic, palmitic, tum/anus; in Some embodiments, preferably the mouth, lar Stearic, isostearic, hydroxy Stearic, oleic, linoleic, ricinoleic, ynx, esophagus, vagina and rectum/anus; in other embodi arachidonic, behenic, and erucic acids. (i) Fatty alcohols hav ments, preferably the eyes, larynx, esophagus, bronchial, ing 10 to 22 carbon atoms, such as, but not limited to, lauryl, nasal passages, and vagina and rectum/anus. As noted above, myristyl, cetyl, hexadecyl, Stearyl, isostearyl, hydroxys local application herein refers to application to a discrete tearyl, oleyl, ricinoleyl, behenyl, erucyl, and 2-octyl dodecyl internal area of the body, Such as, for example, a joint, soft alcohols. () Fatty alcohol ethers, including, but not limited to tissue area (such as muscle, tendon, ligaments, intraocular or ethoxylated fatty alcohols of 10 to 20 carbonatoms, such as, other fleshy internal areas), or other internal area of the body. but are not limited to, the lauryl, cetyl, Stearyl, isostearyl, Thus, as used herein, local application refers to applications oleyl, and cholesterol alcohols having attached thereto from 1 to discrete areas of the body. to 50 ethylene oxide groups or 1 to 50 propylene oxide groups 0429. Also in certain embodiments, including embodi or mixtures thereof. (k) Ether-esters, such as fatty acid esters ments that involve aqueous vehicles, the compositions may of ethoxylated fatty alcohols. (1) Lanolin and derivatives, also contain a glycol, that is, a compound containing two or including, but not limited to, lanolin, lanolin oil, lanolin wax, more hydroxy groups. A glycol which is particularly pre lanolin alcohols, lanolin fatty acids, isopropyl lanolate, ferred for use in the compositions is propylene glycol. In ethoxylated lanolin, ethoxylated lanolin alcohols, ethoxy these embodiments, the glycol is preferably included in the lated cholesterol, propoxylated lanolin alcohols, acetylated compositions in a concentration of from greater than 0 to lanolin, acetylated lanolin alcohols, lanolin alcohols about 5 wt.%, based on the total weight of the composition. linoleate, lanolin alcohols ricinoleate, acetate of lanolin alco More preferably, the compositions contain from about 0.1 to hols ricinoleate, acetate of ethoxylated alcohol(S)-esters, less than about 5 wt.% of a glycol, with from about 0.5 to hydrogenolysis of lanolin, ethoxylated hydrogenated lanolin, about 2 wt.% being even more preferred. Still more prefer ethoxylated Sorbitollanolin, and liquid and semisolid lanolin ably, the compositions contain about 1 wt.% of a glycol. absorption bases. (m) polyhydric alcohols and polyether 0430 For local internal administration, such as intra-ar derivatives, including, but not limited to, propylene glycol, ticular administration, the compositions are preferably for dipropylene glycol, polypropylene glycol M.W. 2000 mulated as a solution or a Suspension in an aqueous-based 4000, polyoxyethylene polyoxypropylene glycols, polyox medium, Such as isotonically buffered saline or are combined ypropylene polyoxyethylene glycols, glycerol, ethoxylated with a biocompatible Support or bioadhesive intended for glycerol, propoxylated glycerol, Sorbitol, ethoxylated Sorbi internal administration. tol, hydroxypropyl sorbitol, polyethylene glycol M.W. 200 6000), methoxy polyethylene glycols 350, 550, 750, 2000, 0431 Lotions, which, for example, may be in the form of 5000, poly(ethylene oxide) homopolymers M.W. 100,000 a suspension, dispersion or emulsion, contain an effective 5,000,000, polyalkylene glycols and derivatives, hexylene concentration of one or more of the compounds. The effective glycol (2-methyl-2,4-pentanediol), 1,3-butylene glycol, 1.2, concentration is preferably to deliver an effective amount, typically at a concentration of between about 0.1-50%, by 6.-hexanetriol, ethohexadiol USP (2-ethyl-1,3-hexanediol), weight, or more of one or more of the compounds provided Cs-Cs vicinal glycol and polyoxypropylene derivatives of herein. The lotions also contain by weight from 1% to 50% of trimethylolpropane. (n) polyhydric alcohol esters, including, an emollient and the balance water, a suitable buffer, and but not limited to, ethylene glycol mono- and di-fatty acid other agents as described above. Any emollients known to esters, diethylene glycol mono- and di-fatty acid esters, poly those of skill in the art as suitable for application to human ethylene glycol M.W. 200-6000, mono- and di-fatty esters, skin may be used. These include, but are not limited to, the propylene glycol mono- and di-fatty acid esters, polypropy following: (a) Hydrocarbon oils and waxes, including min lene glycol 2000 monooleate, polypropylene glycol 2000 eral oil, petrolatum, paraffin, ceresin, oZokerite, microcrys monostearate, ethoxylated propylene glycol monostearate, talline wax, polyethylene, and perhydrosqualene. b) Silicone glyceryl mono- and di-fatty acid esters, polyglycerol poly oils, including dimethylpolysiloxanes, methyl phenylpolysi fatty acid esters, ethoxylated glyceryl monostearate, 1,3-bu loxanes, water-soluble and alcohol-soluble silicone-glycol tylene glycol monostearate, 1,3-butylene glycol distearate, copolymers. (c) Triglyceride fats and oils, including those polyoxyethylene polyol fatty acid ester, Sorbitan fatty acid derived from vegetable, animal and marine sources. esters, and polyoxyethylene Sorbitan fatty acid esters. (o) Examples include, but are not limited to, castor oil, safflower Wax esters, including, but not limited to, beeswax, sper oil, cotton seed oil, corn oil, olive oil, cod liver oil, almond oil, maceti, myristyl myristate, and Stearyl Stearate and beeswax avocado oil, palm oil, sesame oil, and soybean oil. (d) derivatives, including, but not limited to, polyoxyethylene Acetoglyceride esters, such as acetylated monoglycerides. (e) sorbitol beeswax, which are reaction products of beeswax Ethoxylated glycerides, such as ethoxylated glyceryl with ethoxylated sorbitol of varying ethylene oxide content monostearate. (f) Alkyl esters of fatty acids having 10 to 20 that form a mixture of ether-esters. (p) Vegetable waxes, carbon atoms. Methyl, isopropyl and butyl esters of fatty including, but not limited to, carnauba and candelilla waxes. acids are useful herein. Examples include, but are not limited (q) phospholipids, such as lecithin and derivatives. (r) Sterols, to, hexyl laurate, isohexyl laurate, isohexyl palmitate, isopro including, but not limited to, cholesterol and cholesterol fatty pyl palmitate, isopropyl myristate, decyl oleate, isodecylole acid esters. (S) Amides, such as fatty acid amides, ethoxylated ate, hexadecyl Stearate, decyl Stearate, isopropyl isostearate, fatty acid amides, and solid fatty acid alkanolamides. diisopropyladipate, diisohexyladipate, dihexyldecyladipate, 0432. The lotions further preferably contain, by weight, diisopropyl sebacate, lauryl lactate, myristyllactate, and cetyl from 1% to 10%, or preferably from 2% to 5%, of an emul lactate. (g) Alkenyl esters offatty acids having 10 to 20 carbon sifier. The emulsifiers can be nonionic, anionic or cationic. atoms. Examples thereof include, but are not limited to, oleyl Examples of satisfactory nonionic emulsifiers include, but are myristate, oleyl Stearate, and oleyl oleate. (h) Fatty acids not limited to, fatty alcohols having 10 to 20 carbon atoms, US 2014/021248.6 A1 Jul. 31, 2014 36 fatty alcohols having 10 to 20 carbonatoms condensed with 2 propellants are used as understood in the art in a quantity and to 20 moles of ethylene oxide or propylene oxide, alkylphe under a pressure Suitable to expel the contents of the con nols with 6 to 12 carbon atoms in the alkyl chain condensed tainer. with 2 to 20 moles of ethylene oxide, mono- and di-fatty acid 0437 Suitably prepared solutions and suspensions may esters of ethylene oxide, mono- and di-fatty acid esters of also be topically applied to the eyes and mucosa. Solutions, ethylene glycol where the fatty acid moiety contains from 10 particularly those intended for ophthalmic use, may be for to 20 carbon atoms, diethylene glycol, polyethylene glycols mulated as 0.01%-10% isotonic solutions, pH about 5-7, with of molecular weight 200 to 6000, propylene glycols of appropriate salts, and preferably containing one or more of molecular weight 200 to 3000, glycerol, sorbitol, sorbitan, the compounds herein at a concentration of about 0.1%, pref polyoxyethylene Sorbitol, polyoxyethylene Sorbitan and erably greater than 1%, up to 50% or more. Suitable oph hydrophilic wax esters. Suitable anionic emulsifiers include, thalmic solutions are known see, e.g., U.S. Pat. No. 5,116, but are not limited to, the fatty acid Soaps, e.g., Sodium, 868, which describes typical compositions of ophthalmic potassium and triethanolamine soaps, where the fatty acid irrigation Solutions and Solutions for topical application. moiety contains from 10 to 20 carbon atoms. Other suitable Such solutions, which have a pH adjusted to about 7.4, con anionic emulsifiers include, but are not limited to, the alkali tain, for example, 90-100 mM sodium chloride, 4-6 mM metal, ammonium or Substituted ammonium alkyl Sulfates, dibasic potassium phosphate, 4-6 mM dibasic sodium phos alkylarylsulfonates, and alkyl ethoxyether Sulfonates having phate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chlo 10 to 30 carbon atoms in the alkyl moiety. The alkyl ethoxy ride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium ether sulfonates contain from 1 to 50 ethylene oxide units. acetate, 10-20 mM D.L.-sodium, B-hydroxybutyrate and Among satisfactory cationic emulsifiers are quaternary 5-5.5 mM glucose. ammonium, morpholinium and pyridinium compounds. Cer 0438 Gel compositions can be formulated by simply tain of the emollients described in preceding paragraphs also admixing a Suitable thickening agent to the previously have emulsifying properties. When a lotion is formulated described solution or Suspension compositions. Examples of containing Such an emollient, an additional emulsifier is not Suitable thickening agents have been previously described needed, though it can be included in the composition. with respect to the lotions. 0433. The balance of the lotion is water or a C or C. 0439. The gelled compositions contain an effective alcohol, or a mixture of water and the alcohol. The lotions are amount of therapeutic agent(s) of the invention, typically at a formulated by simply admixing all of the components concentration of between about 0.1-50% by weight or more together. Preferably the compound, such as loperamide, is of one or more of the compounds provided herein.; from 5% dissolved, suspended or otherwise uniformly dispersed in the to 75%, preferably from 10% to 50%, of an organic solvent as mixture. previously described; from 0.5% to 20%, preferably from 1% 0434. Other conventional components of such lotions may to 10% of the thickening agent; the balance being water or be included. One Such additive is a thickening agentata level other aqueous or non-aqueous carrier, Such as, for example, from 1% to 10% by weight of the composition. Examples of an organic liquid, or a mixture of carriers. Suitable thickening agents include, but are not limited to: 0440 The formulations can be constructed and designed cross-linked carboxypolymethylene polymers, ethyl cellu to create steady state plasma levels. Steady State plasma con lose, polyethylene glycols, gum tragacanth, gum kharaya, centrations can be measured using HPLC techniques, as are Xanthan gums and bentonite, hydroxyethyl cellulose, and known to those of skill in the art. Steady state is achieved hydroxypropyl cellulose. when the rate of drug availability is equal to the rate of drug elimination from the circulation. In typical therapeutic set 0435 Creams can be formulated to contain a concentra tings, the therapeutic agent(s) of the invention will be admin tion effective to deliver an effective amount of therapeutic istered to patients either on a periodic dosing regimen or with agent(s) of the invention to the treated tissue, typically at a constant infusion regimen. The concentration of drug in the between about 0.1%, preferably at greater than 1% up to and plasma will tend to rise immediately after the onset of admin greater than 50%, preferably between about 3% and 50%, istration and will tend to fall over time as the drug is elimi more preferably between about 5% and 15% therapeutic nated from the circulation by means of distribution into cells agent(s) of the invention. The creams also contain from 5% to and tissues, by metabolism, or by excretion. Steady state will 50%, preferably from 10% to 25%, of an emollient and the be obtained when the mean drug concentration remains con remainder is water or other Suitable non-toxic carrier, such as stant over time. In the case of intermittent dosing, the pattern an isotonic buffer. The emollients, as described above for the of the drug concentration cycle is repeated identically in each lotions, can also be used in the cream compositions. The interval between doses with the mean concentration remain cream may also contain a Suitable emulsifier, as described ing constant. In the case of constant infusion, the mean drug above. The emulsifier is included in the compositionata level concentration will remain constant with very little oscillation. from 3% to 50%, preferably from 5% to 20%. The achievement of steady state is determined by means of 0436 These compositions that are formulated as solutions measuring the concentration of drug in plasma over at least or Suspensions may be applied to the skin, or, may be formu one cycle of dosing Such that one can verify that the cycle is lated as an aerosol or foam and applied to the skin as a being repeated identically from dose to dose. Typically, in an spray-on. The aerosol compositions typically contain, by intermittent dosing regimen, maintenance of steady state can weight, from 25% to 80%, preferably from 30% to 50%, of a be verified by determining drug concentrations at the con Suitable propellant. Examples of Such propellants are the secutive troughs of a cycle, just prior to administration of chlorinated, fluorinated and chlorofluorinated lower molecu another dose. In a constant infusion regimen where oscilla lar weight hydrocarbons. Nitrous oxide, carbon dioxide, tion in the concentration is low, steady state can be verified by butane, and propane are also used as propellant gases. These any two consecutive measurements of drug concentration. US 2014/021248.6 A1 Jul. 31, 2014 37

0441. Accordingly, the invention herein is suited for ease treated or lessened by a compound of this invention is a chronic, acute, symptomatic, therapeutic, or prophylactic myocardial contractility disease or disorder oran acute coro treatment of human or animal diseases or disorders compris nary syndrome. ing maladies of malignant or benign growth; disorders of 0446. In addition to the monotherapies described above, a metabolism; exaggerated inflammation and allergic compound of the invention is also therapeutic in combination responses; cardiovascular diseases; and complications asso with existing therapies directed to hypertension and tissue ciated with transplantation. Additionally, in an embodiment, Swelling. In this context, a combination is defined as a fixed the compounds of this invention act as potent and selective proportion of the compound of the invention and another anti-pain or tension agents. In a further embodiment, the non-imidazoline type compound or compounds to be admin compounds of this invention act as potent and selective istered to the patient simultaneously, as in a kit, or at separate inhibitors of neurogenic spasmodic muscle contractions. and distinct, or predetermined time periods or time intervals. 0442. In an embodiment, the compounds and composi The inhibitor compound or compounds need not be restricted tions of the invention are provided for use in treating or to Small molecular compounds such as those of this invention. reducing the severity of organ transplantation rejection. The non-Imidazoline inhibitor compound may be a biologic 0443) A compound of the invention may also be therapeu Such as an antibody, receptor, binding protein, lipid, Sugar or tic for diseases of inflammation and allergy as manifested in the like. Furthermore, the other component or components of cells required to mount an inflammatory response, Such as the combination may also represent energy in the form of neutrophils, macrophage, mast cells, T-cells, B-cells, plasma radiation, or sources from the full range of the electromag cells, dendritic cells and eosinophils. The pain and tension netic spectrum Such heat, Sound, X-ray or the like. Sources of associated with inflammatory diseases or conditions which irradiation, which may be externally or internally applied, may be treatable by the invention include, but are not limited include cobalt, gold, tritium, and radioisotopes capable of to, autoimmune diseases and common arthritis types, includ Supplying effective translational energy for killing pain caus ing rheumatoid arthritis, osteoarthritis, ankyolsing spondyli ing malignant tumors and tumorivasculary tissues. tis, psoriatic arthritis; psoriasis, systemic lupus erythemato 0447. By analogy, the compounds of this invention maybe Sus, glomerulonephritis, Scleroderma, general renal failure, combined with existing non-Isometheptene effective anti inflammatory bowel disease, ulcerative colitis, Crohn's dis pain and anti-cramping therapies for patients suffering from ease, pancreatitis, multiple Sclerosis; inflammation due to metabolic diseases, inflammatory and allergic disorders, ath hyper-responsiveness to cytokine production, chronic erosclerosis, cardiovascular disease, as described above for obstructive pulmonary, airway or lung disease (COPD, monotherapeutic uses. COAD or COLD), acute respiratory distress syndrome 0448. Accordingly, the invention also provides com (ARDS) and occupation-related diseases such as aluminosis, pounds and pharmaceutical compositions thereof, which are anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silico useful as pain inhibitors, as well as methods for using Such sis, tabacosis and byssinosis. Additionally, a compound of the compounds to treat, ameliorate, reduce, eliminate, or prevent present invention encompasses treatment of parasite-related a painful condition, disease, or pathology, associated with diseases involving hypereosinophilia. A compound of the neurovascular spasms. In some embodiments, the invention invention is also therapeutic for diseases and conditions provides methods for using the compounds of the invention to related to immediate-type hypersensitivity, also referred to as treat, ameliorate, reduce the severity of, or prevent diseases or allergic responses, conditions and diseases. These diseases disorders, e.g., inflammatory diseases, allergic diseases, etc. and conditions include, but are not limited to, asthma (extrin 0449. A biological sample refers to an in vitro or ex vivo sic or intrinsic), asthma related sequelae including Small and sample and includes, without limitation, cell cultures or large airway hyperactivity, bronanaphylaxis, aspirin-induced extracts thereof; cell, tissue or organ samples, or extracts asthma, allergic airway inflammation, urticaria, Steven thereof biopsied material obtained from a subject, i.e., an Johnson syndrome, atopic dermatitis, bolus pemphigoid and animal or mammalian Subject, including humans, or extracts the like. A compound of the invention is therapeutic for dis thereof, and blood, plasma, serum, urine, saliva, feces, semen, eases involving neutrophils, macrophages, mast cells, T-cells, tears, body cavity lavage material, or other body fluids or B-cells, plasma-cells, basophiles, eosinophiles and mast extracts thereof. cells. 0450. In another embodiment, one or more of the neu 0444. A compound of the present invention may also be rovascular spasm ameliorating compounds of the present used in treatment or therapy for discomfort and pain due to invention, orpharmaceutically acceptable compositions con inflammation associated with metabolic diseases, such as taining Such compounds, are employed for coating or lining diabetes and obesity. an implantable medical device, e.g., Stents, catheters, grafts, 0445 Cardiovascular diseases, acute heart failure, vascular grafts, prostheses and artificial valves. As will be enlargement of the heart, and atherosclerosis are also diseases appreciated by one having skill in the art, vascular grafts have that are suitable for ameliorating treatment ortherapy using a been used to overcome restenosis, or a re-narrowing of a compound of the invention described herein. Nonlimiting vessel wall following injury or Surgery. In some patients, the examples of cardiovascular diseases also include pulmonary implantation of a stent or another type of implantable device hypertension, deep venous thrombosis, stroke, myocardial may be associated with a risk of clot formation (embolism) or infarction, myocardial contractility diseases or disorders, platelet activation. To overcome or mitigate this risk, the stent ischemia, thromboembolism, pulmonary embolism, acute or device can be coated (pre-coated) with a compound of the arterialischemia, peripheral thrombotic occlusions, coronary invention or a pharmaceutically acceptable composition artery disease and acute coronary syndrome (ACS). In an thereof. In accordance with the invention, Such coating (or embodiment, the cardiovascular disease treated or lessened pre-coating) may reduce or prevent inflammation reactions or by a compound of this invention is artherosclerosis. In an undesirable cell proliferation with concomitant pain and embodiment, the neurovascular effect on cardiovascular dis muscle pain following implantation. US 2014/021248.6 A1 Jul. 31, 2014

0451 General description of suitable coatings and coated years) treatment. Thus, migraine headache is considered a implantable devices may be found in U.S. Pat. Nos. 6,099, chronic condition that requires multiple acute treatments. 562; 5,886,026 and 5,304,121. The coatings are typically 0457 Metabolism: biocompatible polymeric materials such as a hydrogel poly 0458 Based on the published results of the completed mer, polymethyldisiloxane, polycaprolactone, polyethylene nonclinical pharmacokinetic and drug metabolism studies on glycol, polylactic acid, ethylene vinyl acetate and mixtures or Isometheptene, the following is an overview of the key find combinations thereof. Optionally, the coatings may be cov ings: ered with a suitable topcoat of a material such as fluorosili 0459 1. Isometheptene is metabolized to 6-amino-2-me cone, polysaccharides, polyethylene glycol, phospholipid, or thyl-heptan-2-ol (heptaminol) in rats and humans and this a combination thereof, to impart controlled release character istics for the coated compounds or compositions. An implant metabolite is excreted in the urine. able medical device coated or lined with a compound or 0460 2. Other human metabolites of Isometheptene composition according to the present invention is a further include N-desmethyl-Isometheptene, which metabolite embodiment embraced by the invention. Additionally, the undergoes acid hydrolysis to form 2-methyl-6-methylamino compounds may be coated on an implantable medical device 2-hepten-1-ol. through the use of beads or particles or through co-formula 0461) 3. Based on results in rats, the metabolism of tion with a polymer or other molecule to provide a drug depot, Isometheptene and urinary excretion of formed metabolites which allows the compound (drug) to be released over a that may be stereospecific. longer time period relative to the administration of an aque 0462 Isometheptene is usually administered as pharma ous formulation of the compound (drug). ceutically acceptable salt such as Isometheptene mucate, 0452. The combination drug product (CDP) preparations bitartrate, or hydrochloride. for the treatment of migraine may include injectable solutions 0463) Isometheptene (or Isometheptene mucate salt) is a for epidermal, parenteral, peroral, or intravenous administra sympathomimetic amine known to cause to constrict blood tion, or crystalline powders encapsulated in capsules or com vessels of both arteries and veins. pressed in tablets for oral or Sublingual administration. 0464 Drug Description 0453 Thus, Isometheptene may be one of the active ingre 0465. Each red capsule contains Isometheptene Mucate dients in a combination drug product (CDP) containing USP, 65mg, Dichloralphenazone USP 100 mg, and Acetami Isometheptene, caffeine, and acetaminophen. The proposed nophen USP 325 mg. clinical indication is migraine and 2 formulations (a tablet 0466 Isometheptene Mucate is a white crystalline powder and a capsule formulation) of the CDP will be evaluated. having a characteristic aromatic odor and bitter taste. It is an 0454. The CDP tablet formulation is to be same as Prodrin, unsaturated aliphatic amine with sympathomimetic proper which contains 135 mg of Isometheptene, 20 mg of caffeine, ties. and 500 mg of acetaminophen (about 1.93 mg/kg of 0467 Dichloralphenazone is a white, microcrystalline Isometheptene, 0.29 mg/kg of caffeine, and 7.14 mg/kg of powder, with slight odor and tastes saline at first, becoming acetaminophen for a 70 kg human). acrid. It is a mild sedative. 0455 The CDP capsule formulation is to be same as 0468 Acetaminophen, a non-Salicylate, occurs as a white, MigraLam or MigraTen, both of which contains 65 mg of odorless, crystalline powder, possessing a slightly bittertaste. Isometheptene, 100 mg of caffeine, and 325 mg of acetami 0469 Midrin (acetaminophen, Isometheptene and dichlo nophen (about 0.92 mg/kg of Isometheptene, 1.43 mg/kg of ralphenazone) capsules contain FD&C Yellow No. 6 as a caffeine, and 4.64 mg/kg of acetaminophen for a 70 kg color additive. human). 0470 Since CDPs containing, e.g., Isometheptene, caf 0456. According to information on the Internet (WebMD feine, and acetaminophen have been used for years (i.e., have Professional), the recommended oral dosing regimen for a Substantial clinical experience) for the treatment of migraine, CDP containing Isometheptene (racemic compound), the proposed use of the Isometheptene R-enantiomer (isomer acetaminophen, and dichloralphenaZone for the symptomatic 2), taken alone or in combination compositions, is not relief of migraine headache in adults is 2 capsules (each believed to have toxic or other undesirable side effects upon containing 65 mg of Isometheptene mucate, 325 mg acetami US nophen, and 100 mg of dichloralphenazone) initially, fol 0471) Isometheptene R-enantiomeras an active ingredient lowed by 1 capsule every hour until the headache is relieved. in a CDP with caffeine and acetaminophen as the other active The dosage should not exceed 5 capsules in 12 hours. Based ingredients is to be further evaluated for the treatment of on information obtainable on the Internet, a similar dosing migraine. The proposed clinical route and frequency of dos regimen is recommended for CDPs containing Isomethep ing for the CDP are oral and as needed (dosing no more than tene (racemic), caffeine, and acetaminophen. While no clini once an hour with no more than doses during a 12-hour cal trials on CDPs containing Isometheptene U.S.P. in chil period). To support the clinical development of Isomethep dren from 8 to 12 years of age have been reported in the tene as a CDP, the literature search identified 4 papers that published literature, a publication (Solomon, 1995) indicated provided some information on pharmacokinetics of the recommended dosing regimen for this age group is 1 Isometheptene alone but none for combinations with other capsule initially, followed by 1 capsule every hour to a limit of agents. No pharmacokinetic papers were found on the CDPs 3 capsules per day. The above dosage regimens for a CDP to be evaluated during the proposed clinical trials on containing Isometheptene, caffeine, and acetaminophen are Isometheptene to be administered orally as one of the active for the acute management of migraine. In humans, migraine agents in the CDP. headaches are recurring events that may require frequent 0472. Alternatively, the treatment with S-Isometheptene (more than once a month) acute treatment and chronic (over follows similar procedures. US 2014/021248.6 A1 Jul. 31, 2014 39

0473 Safety of the Isometheptene R-Enantiomer Example 3 0474 The pharmacology, pharmacokinetics, and toxicol ogy of Isometheptene enantiomer alone or as a CDP contain FDC ing, e.g., Isometheptene R-enantiomer, caffeine, and acetami 0483 nophen are important aspects for characterizing the efficacy of the inventive compound. R-Isometheptene enantiomer mucate: 50 mg 0475 No published papers were identified that provided Acetaminophen 325 mg, (or information on the safety pharmacology profiles of racemic Ibuprofen 200 mg) Isometheptene alone or as an active ingredient in a CDP. Dichloralphenazone (DCP) 100 mg 0476 Treatment with R-Isometheptene includes a daily oral dose or twice daily ranging from about 10 mg to about 50 mg, taken alone, or combined with other active ingredients Example 4 Such as a mild sedative, and a decongestant or proton pump inhibitor, such as Ibuprofen or Celebrex, at a dosage of about FDC 100 mg to about 900 mg. 0484 0477. One published report (Valdivia 2004) investigated the cardiovascular responses induced by Isometheptene (IV R-Isometheptene enantiomer mucate 32.5 mg at consecutive doses of 0.03, 0.1.0.3, 1, and 3 mg/kg with 5 to Dichloralphenazone 100 mg, and 15 minutes between doses) in male Wistar rats. Isomethep Acetaminophen 325 mg, (or tene alone produced dose-dependent increases in heart rate Ibuprofen 200, 400 or 800 mg) and diastolic blood pressure. Using co-administration of vari ous receptor antagonists, the Isometheptene-induced tachy cardic responses in rats apparently involved only an indirect 0485 The CDP tablet formulation is to contain 135 mg of (tyramine-like action) mechanism mediated by C.-adrenocep (R)-Isometheptene enantiomer, 20 mg of caffeine, and 500 tors and the corresponding vasopressor responses were medi mg of acetaminophen, (or Ibuprofen 800 mg). The CDP cap ated by a predominantly indirect (tyramine-like action) as Sule formulation is to contain 65 mg of Isometheptene isomer well as a minor direct (C.-adrenoceptors) sympathomimetic ((R)-enantiomer), 100 mg of caffeine, and preferably 325 mg mechanism. ofacetaminophen (or Ibuprofen, 200 mg, 400 mg. or 800mg). 0486 Injectable CDP solution may contain 50 mg 0478. These findings suggest that Isometheptene (R)- Isometheptene isomer 2 ((R)-enantiomer), combined with 30 enantiomer treatment, administered orally either alone or in a mg caffeine, and 125 mg of acetaminophen in 0.50 ml phos CDP. may have a lower potential adverse effects to the car diovascular system. phate buffered water or saline. 0479. In view of the ambiguous physiological effects of APPENDIX I tests involving the racemic Isometheptene compound or com position thereof, it is believed that the effect of its single Assays of Compounds Affinity with Imidazoline I1 active (R)-Isometheptene enantiomer (isomer 2) will be more Receptor specific with fewer side effects. Therefore, treatment of patients Suffering from migraine may be studied by applying 0487. The following experiment shows a reference drug increasing doses of Isometheptene (R)-enantiomer in order to binding assay of Imidazoline receptor, see FIG. 24: determine the efficacy of the composition in a pharmacologi cal profile of the induced cardiovascular responses and side Assay Characteristics: effects on heart rates. 0488 K (binding affinity): 10 nM 0480. For example, Isometheptene (R)-enantiomer as an 0489 B (receptor number): 50 fmol/mg protein active ingredient of a CDP with caffeine and acetaminophen as the other active ingredients is due to be evaluated for the Materials and Methods: treatment of migraine. 0490 Receptor Source: PC12 cell membranes Example 1 0491 Radioligand: "I-Iodoclonidine (1100 Ci/mmol) 0492 Final ligand concentration—2.0 nM 0493. Non-specific Determinant: Iodoclonidine 10.0 Variable Dosage uM 0481 Isometheptene mucate (racemic mixture): 50 mg. 0494 Reference Compound: Iodoclonidine 100 mg, 150 mg, 200 mg, 500 mg. 0495 Positive Control: Iodoclonidine 0496 Incubation Conditions: Reactions were carried out in 50 mMTRIS-HCl (pH 7.4) containing 5 mM EDTA, 5 mM Example 2 EGTA, 5 mM MgCl2 and 30 uM norepinephrine at room temperature for 60 minutes. The reaction was terminated by Variable Dosage rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to 0482 Isometheptene R-enantiomer mucate: 15 mg. 0 mg. control values in order to ascertain any interactions of test 50 mg, 100 mg, 150 mg, 200 mg or 500 mg. compound with the Imidazoline binding site. US 2014/021248.6 A1 Jul. 31, 2014 40

Literature Reference idazoxan in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 0.5 mM EDTA 0497 Steffen, G., Dendorfer, A., and Dominiak, P. Imida and 3 uMyohimbine. Zoline binding sites on PC12 cells and bovine chromaffin cells. Ann. N.Y. Acad. Sci. 763: 157-162 (1995), with modi 0510 Nonspecific binding is determined in the presence fications. of 10 uM cirazoline. 0511. Following incubation, the samples are filtered rap 0498 Piletz, J. E., Zhu, H. E., and Chikkala, D. N. Com idly under vacuum through glass fiber filters (Filtermat B, parison of Ligand Binding Affinities at Human I-Imidazo Wallac) presoaked with 0.3% PEI and rinsed several times line Binding Sites and the High Affinity State of Alpha-2 with an ice-cold buffer containing 50 mM Tris-HCl and 150 Adrenergic Subtypes. JPET: 279: 694-7002 (1996). mM NaCl using a 48-sample cell harvester (Mach II, 0499 Evaluation of the Affinity of Compounds for the Tomtec). The filters are dried then counted for radioactivity in Peripheral Imidazoline Receptor in Bovine Adrenal Medulla a scintillation counter (Betaplate 1204, Wallac) using a solid Glands Determined in a Radioligand Binding Assay. scintillator (Meltilex B/HS, Wallac). 0500 Experimental Protocol 0512. The results are expressed as a percent inhibition of 0501 Membrane homogenates of adrenal medulla glands the control radioligand specific binding. The standard refer (200g protein) are incubated for 60 minat 22°C. with 15 nM ence compound is idazoxan, which is tested in each experi Hclonidine in the absence or presence of the test com ment at several concentrations to obtain a competition curve pound in a buffer containing 137 mM NaCl, 2.68 mM KCl, from which its ICso is calculated. 0.5 mM MgCl, 8.1 mM NaHPO, 1.47 mM. KHPO, 0.5 0513 Bibliographic reference: BROWN, C. M., MAC mM EGTA, 0.5 mM EDTA, 0.5% ascorbic acid and 0.1% KINNON, A. C., McGRATH, J. C., SPEDDING, M. and BSA (pH 7.4). It also contains 10 uMRX821002 to block the KILPATRICKA. T. (1990), C-adrenoceptor subtypes and C2-adrenergic receptors. imidazoline-like binding sites in the rat brain, Brit. J. Phar 0502. Nonspecific binding is determined in the presence macol, 99: 803. of 10 uMrilmenidine. 0503) Following incubation, the samples are filtered rap idly under vacuum through glass fiber filters (GF/B, Packard) General information presoaked with 0.3% PEI and rinsed several times with ice Assay volume and format: 1 ml in 48-well plate Compound addition: 100x solution in solvent cold 50 mM Tris-HCl using a 96-sample cell harvester (Uni Maximum tolerable DMSO concentration: 1% filter, Packard). The filters are dried then counted for radio Expresscreen compatible: Yesbx No O activity in a Scintillation counter (Topcount, Packard) using a HTS compatible: Yes No x scintillation cocktail (Microscint 0, Packard). 0504 The results are expressed as a percent inhibition of 0514 TAAR1 cAMP Assay the control radioligand specific binding. 0515. The cAMP Hunter cell line expressing human 0505. The standard reference compound is rillmenidine, TAAR1 was expanded from freezer Stocks according to stan which is tested in each experiment at several concentrations dard procedures. Cells were seeded in a total volume of 20 uL to obtain a competition curve from which its ICs is calcu into white walled, 384-well microplates and incubated at 37° lated. C. for the appropriate time prior to testing. cAMP modulation 0506 Bibliographic reference: MOLDERINGS, G. J., was determined using the DiscoveRx HitHunter cAMP XS+ MOURA, D., FINK, K., BONISCH, H. and GOTHERT, M. assay. For agonist determination, cells were incubated with (1993), Binding of 3H]clonidine to I1-imidazoline sites in sample to induce response. Media was aspirated from cells bovine adrenal medullary membranes, Naun.-Sch. Arch. and replaced with 15 uL 2:1 HBSS/10 mM Hepes:cAMP XS--Ab reagent. Intermediate dilution of sample stocks was Pharm., 348: 70. performed to generate 4x sample in assay buffer. 4.5uL of 4x sample was added to cells and incubated at 37° C. or room General information: temperature for 30 or 60 minutes. Final assay vehicle con Assay volume and format: 200 l in 96-well plate: centration was 1%. After appropriate compound incubation, Compound addition: 100x solution in solvent assay signal was generated through incubation with 20 Jull Maximum tolerable DMSO concentration: 1% Expresscreen compatible: Yes No D cAMP XS+ ED/CL lysis cocktail for one hour followed by HTS compatible: Yes Nox incubation with 20LL cAMPXS+EA reagent for three hours at room temperature. Microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for 0507 Evaluation of the Affinity of Compounds for the chemiluminescent signal detection. Compound activity was Central Imidazoline I2 Receptor in the Rat Cerebral Cortex analyzed using CBIS data analysis Suite (ChemInnovation, Determined in a Radioligand Binding Assay. CA). For Gs agonist mode assays, percentage activity was 0508 Experimental Protocol: calculated using the following formula: % Activity=100%x 0509 Membrane homogenates of cerebral cortex (1 mg (mean RLU of test sample-mean RLU of vehicle control)/ protein) are incubated for 30 min at 22°C. with 2 nM H (mean RLU of MAX control-mean RLU of vehicle control). US 2014/021248.6 A1 Jul. 31, 2014 41

X-ray Data of (R)-Isometheptene Crystal

------SHELXTL XLMP CRYSTAL STRUCTURE REFINEMENT - MULTI-CPU VERSION - + Copyright (c) Bruker AXS Inc. 1993-2013 Version 2013/2 + + xSOO 61a. started at 14:26:54 on 19-Apr-2013 + ------

Command line parameters : x s 006ia -a 50000 -b3000 -c624 -t 4

- a sets the approximate maximum number of atoms including hydrogens. -b sets the Inaximum number of full-matrix parameters (leave unchanged for CGLS). For example -be 000 allows refinement of 1000 anisotropic atoms C 3000 with BLOC 1. For a 32-bit version, -b times the square root of the number of threads should not exceed about 655 OO ... - C sets the reflection t buffer size. This depends on the CPU cache size but will rarely need changing. -t sets the number of threads, otherwise the multi-CPU version sets this equal to the number of available CPUs. For optimal performance on systems with hyperthreading, usually the hyperthreading should be switched off or -t used to halve the number of threads; e.g. -t 4 rather than -t8 for an Intel i7 processor.

Running 4 threads on 4 processors

TITL xs OO 61 in C2 CELL 1.5 478 36. 3410 7.88266 9.82344 9 OOOO 94. O983 9 OOOO US 2014/021248.6 A1 Jul. 31, 2014 42

ZERR 4 O. OOOT O. OOO12 O. OOO 8 O. OOO O. OO17 O. OOO LATT - 7 SYMM - X , Y , - Z. SFAC C H N O UNIT 96 92 8 32

W = 28 O 6.86 F (OOO) = 1080. O Mu. O. 71 IIIIl-1 Cell Wit. 1970. 56 Rho = 1. 166

TEMP - 173

ACTA H.TAB BOND BOND SH CONF WPDB

L. S. 6 FMAP 2 PLAN 1 O OMIT -2 143. 8 OMIT - 5 12 OMIT - 7 3 OMIT - 16 2 11

TWIN

WGHT O. O39 OO 1, 1288 OO BASF O. O5322 FVAR 3. 98836 O. 51966 PART O rem anion

US 2014/021248.6 A1 Jul. 31, 2014 45

O. O2744 O. OOO 98 O. OO141 O OOO67 AFIX 1.37 H7A 2 O. 828 867 O. 297 389 O. 707 347 11. OOOOO - 1.5 OOOO H7B 2 O. 7923.81 O. 199838 O. 649.884 11. OOOOO -1.5 OOOO HTC 2 O. 8265 62 O. 218425 O. 556896 11. OOOOO -1.5 OOOO AFIX O N1 3 O. 834, 990 0.039478. O.7O6639 11 OOOOO O. O3931 O.O.3017 = ... O 1935 0.00134 O. OO 44 4 - O OO1.93 AFIX 23 H1A 2 O. 828 464 - O. O52741 O. 64 O877 11. OOOOO - 12 OOOO H1B 2 O. 823 466 O. O. 12955 O. T 924. 49 11. OOOOO - 2 OOOO AFIX O C8 l 0.876082 O. O. 43222 O. 73.4758 ll. OOOOO O. O3982 O. O4 674 = .03289 -0.00464 O. OOO22 - O OO285 AFIX 13 H.8 2 O. 881746 O. 141568 O 797O68 11 OOOOO -1. 2 OOOO AFIX O C9 1. O. 895 O 90 O. O782OO O. 605151 11. OOOOO O. O. 4557 O. O65 64 = O5385 O. O. 1229 O. O1295 - O OO2O5 AFIX 1.37 H9A 2 O. 884 468 O. OO5389 0. 531 613 11. OOOOO -1, 5 OOOO H9B 2 O. 921 519 O. O5 4 34 O O. 62O819 ll. OOOOO - 1.5 OOOO H9C 2 O. 89 1565 O 1974 76 O. 5792 66 11. OOOOO -1.5 OOOO AFIX O C10 1. O. 889 482 - O. 14596 O. 811526 11 OOOOO O. O4 O77 O. O5 675 = ... O 4 O16 O. OO 107 O. OOO 61 O . OO232 AFIX 23 H1 OA 2 O. 874 505 - O - 129428 O. 890.996 11 OOOOO -1. 20 OOO H1 OB 2 O. 91531 - O. O954 42 O. 847279 11 OOOOO -1. 2 OOOO AFIX O US 2014/021248.6 A1 Jul. 31, 2014 46

C11 l O 887981 - O - 27 955 6 O. 729 606 11. OOOOO O. O5079 O. O5548 = O. O 4590 -0. OO371 O. OO 106 O OO 148 AFIX 23 H1 1A 2 O. 862 621 - O - 29 7923 O 688522 11. OOOOO 20000 H11B 2 O. 9047 65 - 0.27 1591 O. 654796 11. OOOOO 20000 AFIX O C2 l O. 8991.69 - O. 425 602 O. 820456 11. OOOOO O. O5925 O. O5858 = O. O 66.79 - 0. - O. O.1167 O OO 7O6 AFIX 43 H12 2 O. 88 O945 - O. 4 6 5923 0.877 147 11 OOOOO 2 OOOO AFIX O C13 l O. 93O887 - O. 505015 O. 831839 11. OOOOO O. O7218 O. O6479 = O. 12357 - O ... O 25.18 -O. O2976 O ... O 1849 C14 1. O. 9375 4 3 - O. 64 5845 O. 938 741 ll. OOOOO O. 13726 O. O 6373 - O. 1815 O - O O2967 - O. 10210 O AFIX 1.37 H14A 2 O. 955 775 - O. 607385 1. O1 0448 11 OOOOO 5 OOOO H14B 2 O. 94 6774 - O. 747519 O. 894.967 11. OOOOO 5 OOOO H14C 2 O. 91 4 329 -O. 672521. O. 979 O 41 11. OOOOO 5 OOOO AFIX O C15 1 O. 9 615 69 - O. 4662O 4 O. 74 4.866 11. OOOOO O. 15932 = O. 296.45 - O. O3 694 O. O4293 O ... O 3960 AFIX 1.37 H15A 2 O. 9698.59 - O - 571 O 80 O. 703075 11 OOOOO 5 OOOO H5B 2 O. 982 111 - O - 4 15748 O. 8 OO 97 O 11 OOOOO 5 OOOO H15C 2 O. 95.2996 - O - 38 61.59 O. 673 13 O 11 . OOOOO 5 OOOO AFIX O

EADP C16 C16" EADP N2 N2' US 2014/021248.6 A1 Jul. 31, 2014 47

SIMU O. O1. C16 > C24 '

PART - 1 SAME C7 > C15 C16 l O 817876 - O. 575 492 1. 145O28 21. OOOOO O. O5879 O. O3379 = O. O2 613 OO387 - O. OO3O1 - O OO3O. AFIX 1.37 H16A 2 O. 824783 - O. 577 631 1. O5O 419 21. OOOOO 5 OOOO H16B 2 O. T 90.934 - O. 577 668 1. 146O 63 21. OOOOO 5 OOOO H16C 2 O. 828322 - O. 674 8 OO 1, 1937.96 21. OOOOO 5 OOOO AFIX O N2 3 O. 832.394 - O - 4 1859 O 1. 212875 21. OOOOO O. O 3675 0.03922 = O. O. 1831 ... 004 44 O. O. O551 - O OO 412 AFIX 2 3 H2A 2 O. 824. 418 - O. 32O2O8 55467 21. OOOOO 2 OOOO H2B 2 O. 821 O89 - O. 4 O 6530 1. 301,116 21. OOOOO 2 OOOO AFX O C17 1 O. 873 O14 - O. 41 1688 1.238798 21. OOOOO O. O. 4 O 98 O. O5984 = O. O3281 OO981 O. O. 1325 - O OO 784 AFIX 13 H7 2 O. 883981 - O. 467369 1. 15967 O 21. OOOOO ... 2 OOOO AFIX O C18 1. O. 88 5252 - O. 514 419 1. 367 O 56 21. OOOOO O. O537 6 O 1 O799 = O. O383 O -O. OO 215 O O1548 AFIX 1.37 H18A 2 O. 874314 - O. 4651.84 1. 44 6397 21. OOOOO 5 OOOO H18B 2 O. 912208 - O. 511356 1. 381 479 21. OOOOO 5 OOOO H18C 2 O. 877 O 41 - O. 63224 O 1. 355 O94 21. OOOOO 5 OOOO AEX O C19 1. O. 88 672 6 - O. 23 O538 1. 245134 21. OOOOO O. O5.783 O. O.8011 = US 2014/021248.6 A1 Jul. 31, 2014 48

O. O. 4 446 0. OO 148 O. OO847 - O ... O 1746 AFIX 23 H.9A 2 O. 881 834 - O. 1814.52 1. 334831 21. OOOOO -1. 20000 H19B 2 O 8727 64 -0. 16324 6 1. 173719 21.00000 -1. 20000 AFIX O C2O 1 O. 927 940 -O. 216222 1.225 018 21.00000 0.06125 O. O.9971 = O. O5744 - O. O1526 O. O11. 60 - O ... 02412 AFIX 23 H2OA 2 O. 941704 -O. 2991.18 1. 284 802 21. OOOOO -1. 20 000 H2OB 2 O. 932,335 - O. 243875 . 129221 21. OOOOO -1. 20000 AFIX O C21 1. O. 94 1852 -O, 04 0835 1. 257853 21. OOOOO O. O6898 O. O. 91.65 = O. O 6506 0. OOO85 0.00994 - O ... O 3485 AFIX 4.3 H21 2 O. 929.850 O. O. 486.53 1, 2O74O9 21.00000 -1. 20000 AFIX O C22 1. O. 968 782 O. OO 4998 1. 3483.89 21. OOOOO O. O5593 O. O94 69 = 0.08296 - O OO 832 O. O. 1894 - O ... O 1592 C23 1. O. 99.0770 -O. 116243 . 1. 445.192 21. OOOOO O. O9515 O. 11136 = O. 13723 ... O 1484 - O. O3882 - O OO 986 AFIX 1.37 H23A 2 O. 981 281 - O. 231853 1. 431,714 21. OOOOO - 1.5 OOOO H23B 2 O. 988239 -O. O81326 1. 539812 21. OOOOO -1.50 000 H23C 2 1. Oil 6854 -O. 113099 1. 425966 21. OOOOO -1.50 000 AFIX O C24 1. O. 978993 O. 888 66 1. 36,537 7 21. OOOOO O. O.7487 O. O9986 = O. 14217 - 0. O 1248 O. O156 - O O2779 AFIX 1.37 H24A 2 O. 9667 49 O. 25 4831 1. 290662 21. OOOOO - 5 OOOO H24B 2 1. OO 5789 O. 2014 O1 1. 363821 21. OOOOO -1. 50 000 US 2014/021248.6 A1 Jul. 31, 2014 49

H24C 2 O. 9711 O2 O. 23 O298 1. 452729 21. OOOOO 5 OOOO AFX O

PART - 2 SAME C7 > C15 C16" 1 O. 822474 - O 582 154 1. 157335 -21. OOOOO O5879 O. O3379 = O. O2 613 OO387 - O. OO3O1 - O ... O O3O1 AFIX 1.37 H16D 2 O. 833,363 - O - 6 O 9037 ... O 71487 -21 OOOOO 5 OOOO H16E 2 O. 795 514 - O. 583.338 1. 142778 -21 OOOOO 5 OOOO H16F 2 O. 83O331 - O. 666839 1. 226451 -2. OOOOO 5 OOOO AFIX O N2' 3 O. 834 865 - O. 412O62 1. 204537 -21. OOOOO ... O 3675 O. O3922 = ... O 1831 . OO 444 O. OO551 - O OO 412 AFIX 23 H2 1 2 O. 823993 - O. 3875 42 1. 29 1995 -21. OOOOO 2 OOOO H2' 2 2 O. 825 O77 - O. 327 OO 9 1. 1371.88 -21 OOOOO 2 OOOO AEIX O C17' 1 O - 8 75937 - O 390 4 22 1. 224743 -21. OOOOO ... O 3429 O. O6551 = ... O 4 O 99 ... O 1648 O. OO15 6 O. OOO 92 AFIX 13 H1.7" 2 O. 88 727 6 - O - 4 1552. 1. 1371.54 -21. OOOOO 2 OOOO AFIX O C18 1. 0.889488 - O - 52.2456 1. 333 484 -21. OOOOO ... O 4897 O. 11 O25 = ... O 44 85 ... O 3451 O. OOO59 O. OO325 AFIX 37 H18D 2 O. 877147 - O. 502778 1. 4175 67 -21 OOOOO 5 OOOO H18E 2 O. 916224 - O. 511358 1. 352396 -21 OOOOO 5 OOOO H18F 2 O. 883 681 - O. 6369 O1 1. 299397 -21 OOOOO 5 OOOO AFIX O US 2014/021248.6 A1 Jul. 31, 2014 50

C19 1 O. 882397 - O. 205886 1.261806 -21. OOOOO ... 04804 O. O7212 = ... O 4329 - O ... O 1175 O. OO 994 - O O2 480 AFIX 2 3 H19C 2 O. 869 473 - O. 1814.79 1. 34.4983 -21 OOOOO 2 OOOO H19D 2 O. 870717 - O. 135389 1. 187312 -21. OOOOO 2 OOOO AFIX O C2O" 1. O. 9225 66 - O. 149044 1. 2874 O 9 -21. OOOOO ... O 5260 O. O. 94 41 = O5457 -O. O 1613 O. OO976 - O ... O 2394 AFIX 23 H2OC 2 O. 922 930 -0.027174 1. 31.1822 -21. OOOOO ... 2 OOOO H2OD 2 O. 933911 - 0.212512 1. 366 632 -21. OOOOO 2 OOOO AFIX O C21' 1 O. 945,346 -O. 17457 1. 17 OO 77 -21. OOOOO ... O 6350 O. 1 O75 6 = ... O 6153 - O. O 1127 O. O.1457 - O ... O 1850 AFIX 4.3 H2 1" 2 O. 93.9904 -O. 272 660 1. 116663 -21. OOOOO -1. 2OOOO AFIX O C22 1 O. 972 O93 - O. O78.333 1. 1299 69 -21. OOOOO ... O 6251 O. 1306O = ... O 6460 O. OO 888 -O. OOO25 - O O2 631 C23' 1 O. 991. 673 - O. 18703 1. OO 31.56 -21. OOOOO ... O 7.933 O. 1950 O = ... O 85.15 O. O2 434 O. OOO25 - O O37 O 9 AFIX 137 H23D 2 1. O17376 - O. 149 016 1. O29 O59 -21. OOOOO 5 OOOO H23E 2 O. 991 OO 5 -O. O.19171 O. 94.3252 -21 . OOOOO -1. 5 OOOO H23F 2 O. 979 327 - O - 21 4 088 0.95 4963 -21 OOOOO -1. 5 OOOO AFIX O C24 1 O. 98.2867 O. O.80901 1. 201647 -21 OOOOO 12470 O. 1267 O = O. O9922 0. O2 656 O. OO 427 - O ... O 31.89 AFIX 1.37 US 2014/021248.6 A1 Jul. 31, 2014 51

H24D 2 O. 96O708 O. 1399.62 1.227.738 -21 OOOOO - 1.5 OOOO H24E 2 O. 99 6238 O. 153625 1. 141054 -21 OOOOO -1.50 000 H24F 2 O. 998 797 O. O54 679 1.283 669 -21 OOOOO - 1.50 000 AFIX HKLF 4

Covalent radii and connectivity table for xs0061 in C2 O. 77 O 0.32O O. 7 OO O O. 660

C1 C2 . C2 C1 C3 C4 C4 C5 C5 C 6 C6 C5 O1 O2 O3 O4 O5 O6 O7 O8 C7 N C8 C1 O C9 C10 - C8 C11 C11 - C12 CO C12 - C13 C11 Cl3 - C12 C15 C14

US 2014/021248.6 A1 Jul. 31, 2014 53

Inconsistent equivalents etc.

h k l FOM 2 Sigma (For 2) N Esd of mean (For 2)

O - 8 O 37612. 37 356.11 2 25 O 6.95 3 - 1 O 129724 .34 664. 44 2 3581. 50 3 1. O 127969. O5 585. 70 2 38O2. 50 O 2 O 253 497.31 1019.99 2 10841. OO - 5 l 1 22 6222. 55 848. 73 2 5212. OO 37 - 5 2 232O. T 1 50. 47 2 318.39 -16 O 2 24.2097. 92 553. 34 9 3632. T 6 -8 O 2 19836O. 67 594.36 4 3814. 55 5 1 2 890 483.38 1884. 13 5 1128 O. 84 -1 - 9 3 6OO5 21 65. 54 4 399. 23 1 - 9 4 4688. 19 69. 62 2 5 65 54 -12 -8 6 306 0. 60 7 O. 89 2 415. 45 - 6 - 8 6 9264. O9 115. 16 2 698. 19 8 - 6 6 11183. O6 171. 69 4 867. 37 16 - 6 6 14121.38 183. 43 2 988. T 5 9 - 5 7 92.92. 60 157. 89 4 92.85 - 3 - 7 8 5566.82 104.30 2 597. 72 -14 - 6 8 10692. 13 161.21 2 1028. 60 17 - 5 8 261 O. 52 68.71 2 360. 35 19 - 5 8 307210 7 O. 44 2 4 O1. 68 -30 - 4 8 9274. 25 111. 27 2 623. 44 - 2 - 4 8 21511. 16 216. 40 5 1146. 35 O - 2 8 2.3586. T2 213. 84 5 115 O. 57 -26 2 8 775. 24 47.93 2 251, 68 -7 5 8 5725.96 135. 60 2 2553. 4 O - 6 6 8 5955. 66 126.86 2 65. 5 O O 6 8 10722. 69 179. 33 2 1 O21, 63 8 - 6 9 949. 98 34. 98 2 251. 73 - 3 -3 9 30.996. 34 25 O. OO 4 14 O3. 31 US 2014/021248.6 A1 Jul. 31, 2014 54

-21 -1 9 926O. 13 54.25 2 7485. 53 - 19 - 1 9 6100. T2 108.51 2 3369. 77 -24 O 9 1233.71 48.50 2 849.58 -30 2 9 729.83 36.95 2 248. 4 O -23 3 9 498.32 93. 33 2 21. 75.87 -3 3 9 31239. O6 230. 54 5 1239. 20 -10 4 9 519 O. 47 138. 84 2 88 O. 10 -3 5 9 11887. 99 182.78 2 1603. OO -21 -1 10 1736.59 48.23 2 1379. O1 - 19 - 1 1 0 6749. O7 18477 2 2977. 34 -22 O O. 5822.31 162. 13 2 1863. T 6 –20 0 10 8987.51. 216. 19 2 3429.31 - 18 O 1 O 2977. 96 139, 45 2 1 O58. T8 -16 O 1 O 4113. 75 97.81 2 3778. 91 - 19 1 10 5232.78 171, 84 2 1601.98 - 1 1 O 2745. 86 98.92 2 1678. T 6 - 18 2 10 25 65.30 52. 3O 2 2728. T 6 - 6 2 10 7 615, 25 36.94 2 8254. O7 18 2 10 613 O. 45 96.49 2 525. T 1. -17 3 10 959. A 6 50. 73 2 4 57.43 - 15 3 1 O 31.8. 21 32.13 2 25 4.96

* k etc. 'k k

66 Inconsistent equivalents

5499 Unique reflections, of which 0 suppressed

R (int) = 0. O287 R (sigma) = 0.0154 Friedel opposites not merged

Maximum memory for data reduction = 5898 / 65132

Number of data for d > 0.811A (CIF: max) and d > 0.833A (CIF: full) US 2014/021248.6 A1 Jul. 31, 2014 55

(ignoring systematic absences) : Unique reflections found (point group) 551.3 5076 Unique reflections possible (point group) 5514 5078 Uni cue reflections found (Laue group) 2969 2737 Unique reflections possible (Laue group) 297 O 2739 Unique Friedel pairs found 2544 2339 Unique Friedel pairs possible 2544 2339

Default effective X-H and X-D distances for T = -173. OC AFIX l = 2 3 4 4 (N 3 NJ 15 B 8 (O) 9 9 N 16 d (X-H) = 1. OO O. 99 O. 98 0.95 O. 88 O. 91 1. 12 O. 84 O. 95 O. 88 O. 95

Note that these distances are chosen to give the best fit to the X ray data and so avoid the introduction of systematic error. The true internuclear distances are longer and do not vary with temperature. The apparent Variation with temperature is caused by libration.

Least-squares cycle 1. Maximum vector length = 623 Memory required = 7228 / 61.2385

wR2 = 0. 1486 before cycle 1 for 5499 data and 4 O3 / 4 O3 parameters

Disagreeable restraints before cycle 1.

Observed Target Error Sigma Restraint

- O. O389 O. O1 OO SIMU U22 C21 C22' US 2014/021248.6 A1 Jul. 31, 2014 56

- O ... O 688 O. O2 OO SIMU U11 C22 C24 - O. O. 644 O. O2 OO SIMU U22 C22 C23" -O, O 622 O. O2 OO SIMU U11 C22 C24 '

Summary of restra ints applied in cycle 1.

ANTIBUMP DEX DANG SAME/SADI CHIV/Z, CHIV/NZ FLAT DELU RIGU SIMU ISOR SUMP

Number O O O 57 O O O O O 246 O O

rms sigma O. OOOO O. OOOO O. OOOO O. O321 O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O . O15 O O. OOOO O. OOOO

rms deviation O. OOOO O. OOOO O. OOOO O. 0290 O. OOOO O. OOOO O. 0000 O. OOOO O. OOOO O. O 18 O O. OOOO O. OOOO

GOOF at S = l. O54; Restrained Goo F = 1. O 6O for 3 O 4 restraints

Weight = 1 / si gma ^2 (For 2) + ( 0.1039 * P ) 2 + 1. 13 * P where P = ( Max ( Fo^2, O ) + 2 * For 2 ) / 3

N Value esd shift/esd parameter

1. 3. 98836 O. O1343 O. OOO OSF 2 O. 51966 O. OO 401 O OO1 FVAR 2 3 O. O5322 O. 27877 O. OOO BASF 1

Mean shift/es d = O. 001. Maximum = -0. OO5 for X C3 US 2014/021248.6 A1 Jul. 31, 2014 57

Max. shift = 0.000 A for H15B Max. dU = 0.000 for C1

Least-squares cycle 2 Maximum vector length = 623 Memory required = 7228 / 612385

wR2 = 0. 1486 before cycle 2 for 5.499 data and 403 / 4 O3 parameters

Disagreeable restraints before cycle 2

Observed Target Error Sigma Restraint

- O. O389 O. O1 OO SIMU U22 C21, C22' - O ... O 688 O. O2 OO SIMU U11 C22 C24 -0.0644 O. O2OO SIMU U22 C22" C23" -O ... O 622 O. O2 OO SIMU U11 C22 C24

Summary of restraints applied in cycle 2 :

ANTIBUMP DEIX DANG SAME/SADI CHIV/Z, CHIV/NZ FLAT DELU RIGU SIMU ISOR SUMP

Number O O O 57 O O O O O 246 O O

rms sigma O. OOOO O. OOOO O. OOOO O. O.321 O. OOOO O. OOOO O. 0000 O. OOOO O. OOOO O. O15 O O. OOOO O. OOOO

rms deviation O. OOOO O. OOOO O. OOOO O. O29 O O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O.8 O O. OOOO O. OOOO US 2014/021248.6 A1 Jul. 31, 2014 58

GOOF as S = 1. O54; Restrained Goo F = 1. O 60 for 3O4 restraints

Weight = 1 / C sigma^2 (For 2) + ( 0.1039 * P ) 2 + 1. 13 * P where P = ( Max ( For 2, O ) + 2 * For 2 ) / 3

N value esd shift/esd parameter

3. 98836 O. O1343 O. OOO OSF 2 O. 51966 O. OO4 O1 O. OOO FVAR 2 3 O. O5322 O. 27877 O. OOO BASF 1

Mean shift/es d = O. OOO Maximum = -0. OO2 for U12 O3

Max. shift = 0.000 A for H15A Max. dO = 0 . OOO for O4

Least-squares cycle 3 Maximum vector length = 623 Memory required = 7228 / 61.2385

wR2 = 0. 1486 before cycle 3 for 5 499 data and 403 A 4O3 parameters

Disagreeable restraints before cycle 3

Observed Target Error Sigma Restraint

- O. O389 O. O1 OO SIMU U22 C21, C22' - O, O 688 O. O2OO SMU U11 C22 C24 - O. O 64 4 O. O2OO SIMU U22 C22' C23' - O. O 622 O. O2OO SIMU U11 C22 C24 US 2014/021248.6 A1 Jul. 31, 2014 5 9

Summary of restraints applied in cycle 3

ANTIBUMP DEFIX DANG SAME/SADI CHIV/Z, CHIV/NZ FLAT DELU RIGU SIMU ISOR SUMP

Number O O O 57 O O O 246 O O

rms sigma O. OOOO O. OOOO O. OOOO O. O321 O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O15 O O. OOOO O. OOOO

rms deviation O. OOOO O. OOOO O. OOOO O. O29 O O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O18O O. OOOO O. OOOO

GOOF = S = 1. O54; Restrained Goo F = 1. O 60 for 3O4. restraints

Weight = 1 / sigma^2 (For 2) + ( 0.1039 * P ) 2 + 1. 13 * P where P = ( Max ( Fo^2, O ) + 2 * Fo? 2 ) / 3

N value esd shift/esd parameter

1 398836 O. O. 1343 O. OOO OSE 2 O 51966 O. OO4 O1 O. OOO FWAR 2 3 O. O5323 O. 27876 OOOO BASF

Mean shift/es d = O. OOO Maximum = -O. OO1 for x O8

Max. shift = O. OOO A for H16D Max. dO = O. OOO for C15

Least-squares cycle 4 Maximum vector length 623 Memory required = 7228 / 62385 US 2014/021248.6 A1 Jul. 31, 2014 60

wR2 = 0. 1486 before cycle 4 for 5499 data and 403 A 4 O3 parameters

Disagreeable restraints before cycle 4

Observed Target Error Sigma Restraint

- O. O389 OOOO SIMU U22 C21 C22' - O. O 688 O. O2 OO SIMU U11 C22 C24' - OO 64 4 O. O2 OO SIMU U22 C22' C23" - O. O622 O. O2 OO SIMU U11 C22 C24

Summary of restraints applied in cycle 4

ANTIBUMP DFIX DANG SAME/SADI CHIV/Z CHIV/NZ FLAT DELU RGU SIMU ISOR SUMP

Number O O O 57 O O O O O 246 O O

rms sigma O. OOOO O. OOOO. O. OOOO O. O32. O. OOOO O. OOOO O. OOOO 0.0000 O. OOOO O. O15 O O. OOOO O. OOOO

rms deviation O. OOOO O. OOOO O. OOOO O. O29O. O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O.80 OOOOO O. OOOO

GOOF = S = 1. O54; Restrained Goo F = 1. O 60 for 3O4 restraints

Weight = 1 / sigma ^2 (For 2) + ( 0.1039 * P ) 2 + 1. 13 k P. ) where P = ( Max ( Fo°2, O ) + 2 * FC^2 ) / 3 US 2014/021248.6 A1 Jul. 31, 2014 61

N value esd shift/esd parameter

1. 3. 98836 O. O1343 O. OOO OSF 2 O. 51966 O. OO 4O1 O. OOO FWAR 2 3 O. O5323 O. 27875 O. OOO BASF 1

Mean shift/es d = O. OOO Maximum = O. OOO for X C6

Max. shift = 0.000 A for H16F Max. CU = 0 . OOO for C23'

Least-squares cycle 5 Maximum vector length = 623 Memory required = 7228 / 612385

wR2 = O. 1486 before cycle 5 for 5 499 data and 403 / 4 O3 parameters

Disagreeable restraints before cycle .. 5

Observed Target Error Sigma Restraint

- O. O389 O. O1 OO SIMU U22 C21 C22' - O - O 688 O. O2 OO SIMU U11 C22 C24 ' - O. O 64 4 O. O2 OO SIMU U22 C22' C23" - O. O622 O. O2 OO SIMU U11 C22 C24 "

Summary of restraints applied in Cycle 5

ANTIBUMP DFIX DANG SAME/SADI CHIV/Z, CHIV/NZ FILAT DELU RIGU SIMU ISOR SUMP US 2014/021248.6 A1 Jul. 31, 2014 62

Number O O O 57 O O O O O 246 O O

rms sigma 0.0000 0.0000 O. OOOO O. O321 O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O150 OOOOO O. OOOO

rIts deviation O. OOOO O. OOOO O. OOOO O. 0290 O. OOOO O. OOOO O. OOOO 0.0000 0.0000 0.0180 0.0000 0.0000

Goo F = S = 1. O54; Restrained GooF = 1. O 6O for 3O4 restraints

Weight = 1 / sigma^2 (For 2) + ( O. 1039 * P ) 2 + 1. 13 * P where P = ( Max ( Fo^2, O ) + 2 * Fc 2 ) / 3

N Value esd shift/esd parameter

1. 3.98836 O. O. 1343 O. OOO OSE 2 O. 5966 O. OOA O1 O. OOO FVAR 2 3 O. O5323 O. 2 7875 O. OOO BASF

Mean shift/es d = O. OOO Maximum = O. OOO for x O8

Max. shift = 0 . OOO A for H16E Max. dU = 0 . OOO for C18"

Least-squares Cycle 6 Maximum vector length = 623 Memory required a 7228 612385

wR2 = 0. 1486 before cycle 6 for 5499 data and 4 O3 / 4 O3 parameters US 2014/021248.6 A1 Jul. 31, 2014 63

Disagreeable restraints before cycle 6

Observed Target Error Sigma Restraint

-O. O389 O. O. OO SIMU U22 C21 C22' -O ... O 688 O. O2 OO SMU U11 C22 C24 -O ... O 64 4 O. O2 OO SIMU U22 C22' C23" -O ... O 622 O. O2 OO SIMU U11, C22' C24 '

Summary of restraints applied in cycle 6

ANTIBUMP DFX DANG SAME/SADI CHIV/Z, CHIV/NZ FLAT DELU RIGU SIMU ISOR SUMP

Number O O O 57 O O O O O 246 O O

rms sigma O. OOOO O. OOOO O. OOOO O. O.321 O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O15 O O. OOOO O. OOOO

rms deviation O. OOOO O. OOOO O. OOOO O. O290 O. OOOO O. OOOO O. OOOO O. OOOO O. OOOO O. O18O O. OOOO O. OOOO

Goo F = S = 1. O54; Restrained Goo F = 1. O60 for 3O4 restraints

Weight = 1 / sigma ^2 (Fo^2) + ( O. 1039 * P ) 2 + 1. 13 * P where P = ( Max ( Fo^2, O ) + 2 * For 2 ) / 3

N value esd shift/esd parameter

1. 3. 98836 O. O. 1343 O. OOO OSF

US 2014/021248.6 A1 Jul. 31, 2014 73

O. 884 47 O. OO539 0. 531 61 1. OOOOO ... O 81.85 O. OOOOO OOOOO

O. 92 152 0.05434 O. 62082 1. OOOOO ... O 81.85 O. OOOOO . OOOOO

O. 891.57 O. 19748 O. 57927 1. OOOOO ... O 81.85 O. OOOOO OOOOO

C1 O O. 889 48 -O. 11460 O. 81153 1. OOOOO ... O 4 O77 O. O5675 O. OO 107 0.00061 O. O.O232 O ... 04 600 O. OO 617 O. OOOO 9 O. OOO 49 O. OOO33 O. OOOOO ... 00141 O. OO 185 O. OO .58 O. OO 154 O. OO 118 O. OO 136 O . OOOT1

H1 OA O. 874 50 - O. 1294.3 O - 891 OO 1. OOOOO O552O O. OOOOO OOOOO

HOB O. 91531 -O. O9544 O. 84728 1. OOOOO O552O O. OOOOO OOOOO

C1 O 88798 - O - 27956 O. 72961 1. OOOOO O. O5548 O. O 459 O -O. OO371 O. OO 106 O. OO 148 O ... O 5084 O. OO 619 O. OOOO 9 O. OOO 49 O. OOO 36 O. OOOOO OO1 66 O. O. O.194 O. OO 171 O. OO15 6 O. OO 132 O. OO15 O O . OOO 76

H1 1A O. 862 62 - O. 29792 O 68852 1. OOOOO ... O 61 OO O. OOOOO OOOOO

H11B O. 90 477 - O - 271.59 O. 654 8 O 1 OOOOO ... O 6100 O. OOOOO OOOOO

C12 O. 899.17 - O. 425 6.O O. 82O4 6 1. OOOOO O. O5925 O. O5858 O. O 6679 - O. OO3O8 - O. O. 1167 O. O.O 7 O 6 O ... O 6231