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Saffron (pronounced /ˈsæfrən/ or /ˈsæfrɒn/)^[1] is a spice derived from the flower of Crocus sativus, commonly known as the saffron crocus. Crocus is a genus in the family Iridaceae. Saffron crocus grows to 20–30 cm (8–12 in) and bears up to four flowers, each with three vivid crimson stigmas, which are the distal end of a carpel.^[2] The styles and stigmas are collected and dried to be used as a seasoning and colouring agent in cooking. Saffron, long among the world’s most costly spices by weight,^[3][4][5] is native to Greece orSouthwest Asia^[6][4] and was first cultivated in Greece.^[7] As a genetically monomorphic clone,^[8] it was slowly propagated throughout much of Eurasia and was later brought to parts of North Africa, North America, and Oceania. The saffron crocus, unknown in the wild, probably descends from Crocus cartwrightianus, which originated in Crete;^[8] C. thomasii and C. pallasii are other possible precursors.^[9][10] The saffron crocus is a triploid that is “self-incompatible” and male sterile; it undergoes aberrant meiosis and is hence incapable of independent sexual reproduction—all propagation is by vegetative multiplication via manual “divide-and-set” of a starter clone or by interspecific hybridisation.^[11][10] If C. sativus is a mutant form of C. cartwrightianus, then it may have emerged via plant breeding, which would have selected for elongated stigmas, in late Bronze Age Crete.^[12] Saffron’s taste and iodoform- or hay-like fragrance result from the chemicals picrocrocin and safranal.^[13][14] It also contains a carotenoid pigment, crocin, which imparts a richgolden-yellow hue to dishes and textiles. Its recorded history is attested in a 7th-century BC Assyrian botanical treatise compiled under Ashurbanipal,^[15] and it has been traded and used for over four millennia. Iran now accounts for approximately 90% of the world production of saffron.^[16] Saffron is obtained from dried style and stigma of reddish-orange flowers of a plant. Kesar or Saffron is the most expensive spice of world as stigmas of about 60, 000 hand collected flowers provide only half- kilograms of it. Saffron is used as coloring and flavoring ingredient in the preparation of various dishes. It is also used as traditional medicine for many diseases and in cosmetics. Saffron has a distinct aromatic odour and a bitter, pungent taste. Medicinally it is stimulant (stimulates levels of physiological or nervous activity), aphrodisiac, improves digestion and appetite. It increases blood flow in pelvic region on oral intake. Its over-doses is a narcotic poison. Saffron is always used in small doses. It is a popular remedy for promoting menstruation.

 

Saffron is the most expensive herb harvested from the stigma of the Crocus sativus flower. It is dark orange and thread like in appearance, with a spicy flavor, nice yellow to orange color and pungent odor.

•   The plant is grown in India, Spain, France, Italy, the Middle East, and the eastern Mediterranean region.
• Over 200,000 crocus stigmas must be harvested to produce one pound of saffron.
• Saffron is harvested by drying the orange stigma which are 3 of them in one Crocus sativus flower over fire.
• This volume makes the herb extremely expensive and quite often adulterated.
• Saffron is prescribed as a herbal remedy to stimulate the digestive system, ease colic and stomach discomfort, and minimize gas.
• It is also used as an emmenagogue, to stimulate and promote menstrual flow in women.
• Additional human studies have indicated that saffron has powerful antioxidant properties; that is, it helps to protect living tissues from free radicals and other harmful effects of oxidation.
• Two chemical components of saffron extract, crocetin and crocin, reportedly improved memory and learning skills. These properties indicate that saffron extract may be a useful treatment for neurodegenerative disorders and related memory impairment.
• In ancient India, robes were traditionally dyed a golden color from the crocin chemical dye that is found in saffron.
• In fact, after Buddha had died, the Buddhist priests made this golden saffron color their official color of their robes.
• Saffron was used by Greeks and Romans as a perfume on behalf of its pleasant aroma.
• Cleopatra used to use saffron as a type of cosmetic. And now a days it is used in face creams as a fairness cream.
• In the Middle Ages, one could be sentenced to the punishment of being buried alive if they tried to alter saffron by adding in other substances.
• Romans used to take baths infused with saffron.
• In order to cure hang-overs, Romans would sleep with expensive pillows that were stuffed with saffron.
• Saffron is extensively used in Indian Cuisine and Middle Eastern Cuisine.

---

Scientific classification

• Kingdom:Plantae
• Division:Magnoliophyta
• Class:Liliopsida
• Order:Asparagales
• Family:Iridaceae
• Genus:Crocus
• Species:C. sativus

Vernacular Names

SANSKRIT:Bhavarakta, Saurab, Mangalya, Kumkum ENGLISH:Saffron, Crocus PERSIAN:Zafrahn;Zipharana;GUJARATI:Keshar, Kesar KANNADA:Kunkuma, Kesari, MALAYALAM:Kunkuma Puvu MARATHI:Keshar PUNJABI:Kesar, Keshar TAMIL:Kungumapuvu TELUGU:Kunkuma Puvvu URDU:Zafran Parts Used:Dried stigmas and tops of the styles of Crocus sativus flowers. Habitat:Saffron is Cultivated in Kashmir, Kishtwar (Jammu) and in Nepal. Commercially, it is grown in Spain, France, Italy, Greece, Turkey, and China. Energetics:Pungent, bitter, Hot in potency

Plant description

Perennial tuber plant;Leaves radical,  linear,  dark green above,  pale green below,  enclosed in a membranous sheath;large Apurple or lilac colored flowers;Corolla in two segments, between which the long styles hang out;Stigmas three, large, nearly an inch long, rolled at the edges, bright orange bitter and warming taste.

Constituents of Saffron

Saffron contains three crystalline colouring matters ?-crocetin, ?-crocetin and ?-crocetin. It also contains essential oil a number of carotenoid pigments. The essential oil obtained from stigmas contains thirty-four or more components, viz. terpenes, terpene alcohols, and esters.

Medicinal Uses of Saffron

Saffron is used as condiment and colouring ingredient in several dishes. It is also used as a medicinal herb in fevers, enlargement of the liver, cough and asthma, anaemia, seminal debility rheumatism and neuralgia. Saffron is nervine tonic, sedative, antispasmodic expectorant, stomachic, diaphoretic and emmenagogue. In low doses Saffron stimulates gastric secretion and thus improves digestion. In large dose it increases flow of blood in pelvic region, stimulate uterine smooth muscles and can cause abortion.

1. Saffron oral use gives relief in respiratory ailments. In cough and cold a pinch of Saffron is taken with a glass of milk.
2. In painful urination and other urinary disorder the decoction of Saffron or infused tea should be taken.
3. In irritation in eyes, crushed saffron should be mixed with honey and this should be applied in eyes.
4. In looseness of bowels saffron is given children with ghee. It can also be given with half a teaspoon of lemon juice.
5. For pneumonia in kids, few threads of saffron are added to 10-15 ml juice of bitter gourd leaves and given twice a day.
6. Saffron is added to meals for regulating the menstrual cycle. It also gives relief in painful menstruation, PMS (premenstrual syndrome) and promotes fertility.
7. For sexual weakness, about 250 mg of saffron is taken with milk twice a day for one week.
8. Saffron improves digestion and appetite.
9. To get relief from dry cough one should drink one hot glass of milk added with turmeric, and few strands of saffron.
10. Saffron in paste form is applied topically for head-ache.
11. Its external application is also useful in sores, bruises and skin diseases. It is applied on face for improving complexion and treating hyper-pigmented spots.
12. It is also used for patchy loss of hair. For this purpose a paste of liquorice (mulethi) made by grinding the pieces in milk with a pinch of saffron is applied over the bald patches in the night before going to bed.
13. A famous Ayurvedic preparation containing Kesar or saffron is kumkumadi tailam. This medicated saffron/kumkum oil is applied on pimples marks, dark spots, dark circles, wrinkles etc.

The recommended doses of Saffron below one gram. Toxic dose is 1.5g–5 g.

Etymology

A degree of uncertainty surrounds the origin of the English word, “saffron” although it can be traced to have stemmed immediately from 12th-century Old French term safran, which comes from the Latin word safranum. Safranum comes from the Persian intercessor زعفران, or za’ferân. Old Persian is the first language in which the use of saffron in cooking is recorded, with references dating back thousands of years.

Species

Description

Köhler’s Medicinal Plants:

corolla	stamens
corm	stigma

The domesticated saffron crocus, Crocus sativus, is an autumn-flowering perennial plant unknown in the wild. Its progenitors are possibly the eastern Mediterranean autumn-flowering Crocus cartwrightianus,^[17][10] which is also known as “wild saffron”^[18] and originated in Greece.^[14] The saffron crocus probably resulted when C. cartwrightianus was subjected to extensive artificial selection by growers seeking longer stigmas. C. thomasii and C. pallasii are other possible sources.^[9][10] It is a sterile triploid form, which means that three homologous sets of chromosomes compose each specimen’s genetic complement; C. sativus bears eight chromosomal bodies per set, making for 24 in total.^[2] Being sterile, the purple flowers of C. sativus fail to produce viable seeds; reproduction hinges on human assistance: clusters of corms, underground, bulb-like, starch-storing organs, must be dug up, divided, and replanted. A corm survives for one season, producing via this vegetative division up to ten “cormlets” that can grow into new plants in the next season.^[17] The compact corms are small, brown globules that can measure as large as 5 cm (2.0 in) in diameter, have a flat base, and are shrouded in a dense mat of parallel fibres; this coat is referred to as the “corm tunic”. Corms also bear vertical fibres, thin and net-like, that grow up to 5 cm above the plant’s neck.^[2]

C. sativus.

The plant grows to a height of 20–30 cm (8–12 in), and sprouts 5–11 white and non-photosynthetic leaves known ascataphylls. These membrane-like structures cover and protect the crocus’s 5 to 11 true leaves as they bud and develop. The latter are thin, straight, and blade-like green foliage leaves, which are 1–3 mm in diameter, either expand after the flowers have opened (“hysteranthous”) or do so simultaneously with their blooming (“synanthous”).C. sativus cataphylls are suspected by some to manifest prior to blooming when the plant is irrigated relatively early in the growing season. Its floral axes, or flower-bearing structures, bear bracteoles, or specialised leaves that sprout from the flower stems; the latter are known as pedicels.^[2] After aestivating in spring, the plant sends up its true leaves, each up to 40 cm (16 in) in length. In autumn, purple buds appear. Only in October, after most other flowering plants have released their seeds, do its brilliantly hued flowers develop; they range from a light pastel shade of lilac to a darker and more striated mauve.^[19] The flowers possess a sweet, honey-like fragrance. Upon flowering, plants average less than 30 cm (12 in) in height.^[20] A three-pronged style emerges from each flower. Each prong terminates with a vivid crimson stigma 25–30 mm (0.98–1.18 in) in length.^[17]

Cultivation

Saffron bulbs for vegetative reproduction

Crocus sativus thrives in the Mediterranean maquis, an ecotype superficially resembling the North American chaparral, and similar climates where hot and dry summer breezes sweep semi-arid lands. It can nonetheless survive cold winters, tolerating frosts as low as −10 °C (14 °F) and short periods of snow cover.^[17][21] Irrigation is required if grown outside of moist environments such as Kashmir, where annual rainfall averages 1,000–1,500 mm (39–59 in); saffron-growing regions in Greece (500 mm or 20 in annually) and Spain (400 mm or 16 in) are far drier than the main cultivating Iranian regions. What makes this possible is the timing of the local wet seasons; generous spring rains and drier summers are optimal. Rain immediately preceding flowering boosts saffron yields; rainy or cold weather during flowering promotes disease and reduces yields. Persistently damp and hot conditions harm the crops,^[22] and rabbits, rats, and birds cause damage by digging up corms. Nematodes, leaf rusts, and corm rot pose other threats. Yet Bacillus subtilis inoculation may provide some benefit to growers by speeding corm growth and increasing stigma biomass yield.^[23]

Saffron harvesting, Torbat-e Heydarieh, Iran

The plants fare poorly in shady conditions; they grow best in full sunlight. Fields that slope towards the sunlight are optimal (i.e., south-sloping in the Northern Hemisphere). Planting is mostly done in June in the Northern Hemisphere, where corms are lodged 7–15 cm (2.8–5.9 in) deep; its roots, stems, and leaves can develop between October and February.^[2] Planting depth and corm spacing, in concert with climate, are critical factors in determining yields. Mother corms planted deeper yield higher-quality saffron, though form fewer flower buds and daughter corms. Italian growers optimise thread yield by planting 15 cm (5.9 in) deep and in rows 2–3 cm (0.79–1.18 in) apart; depths of 8–10 cm (3.1–3.9 in) optimise flower and corm production. Greek, Moroccan, and Spanish growers employ distinct depths and spacings that suit their locales. C. sativus prefers friable, loose, low-density, well-watered, and well-drained clay-calcareous soils with high organic content. Traditional raised beds promote good drainage. Soil organic content was historically boosted via application of some 20–30 tonnes of manure per hectare. Afterwards, and with no further manure application, corms were planted.^[24] After a period of dormancy through the summer, the corms send up their narrow leaves and begin to bud in early autumn. Only in mid-autumn do they flower. Harvests are by necessity a speedy affair: after blossoming at dawn, flowers quickly wilt as the day passes.^[25] All plants bloom within a window of one or two weeks.^[26]Roughly 150 flowers together yield 1 g (0.035 oz) of dry saffron threads; to produce 12 g (0.42 oz) of dried saffron (or 72 g (2.5 oz) moist and freshly harvested), 1 kg (2.2 lb) of flowers are needed; 1 lb (0.45 kg) yields 0.2 oz (5.7 g) of dried saffron. One freshly picked flower yields an average 30 mg (0.0011 oz) of fresh saffron or 7 mg (0.00025 oz) dried.^[24]

Spice

Chemistry

Structure of picrocrocin:^[27]

β–D-glucopyranose derivative

safranal moiety

  Picrocrocin is a monoterpene glycoside precursor of safranal. It is found in the spice saffron, which comes from the crocus flower.Picrocrocin has a bitter taste, and is the chemical most responsible for the taste of saffron. During the drying process, picrocrocin liberates the aglycone (HTCC, C₁₀H₁₆O₂) due to the action of the enzyme glucosidase. The aglycone is then transformed to safranal by dehydration. Picrocrocin is a degradation product of the carotenoidzeaxanthin. Caballero-Ortega H, Pereda-Miranda R, Abdullaev FI (2007). “HPLC quantification of major active components from 11 different saffron (Crocus sativus L.) sources”. Food Chemistry 100 (3): 1126–1131. doi:10.1016/j.foodchem.2005.11.020.

• Pfander, H.; Schurtenberger, H.; Biosynthesis of C20-carotenoids in Crocus sativus. Phytochemistry 1982, 21, 1039-1042. http://dx.doi.org/10.1016/S0031-9422(00)82412-7

Picrocrocin

CAS Registry Number: 138-55-6

CAS Name: (4R)-4-(b-D-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde

Additional Names: saffron-bitter

Molecular Formula: C16H26O7

Molecular Weight: 330.37

Percent Composition: C 58.17%, H 7.93%, O 33.90%

Literature References: From stigmas of Crocus sativus L., Iridaceae. Isoln: Kayser, Ber. 17, 2228 (1884). Structure: Kuhn, Winterstein, Ber. 67, 344 (1934). Exerts sex-determining influences in the plant organism: Kuhn, Angew. Chem. 53, 1 (1940). Its moieties are glucose and safranal, q.q.v. Abs config: Buchecker, Eugster, Helv. Chim. Acta 56, 1121 (1973). Synthesis: H. Mayer, J.-M. Santer, Helv. Chim. Acta 63, 1463 (1980).

Properties: Crystals, mp 154-156°. [a]D20 -58° (c = 0.6). Bitter taste. Alkali unstable. Sol in water, alcohol; slightly sol in chloroform, ether. Practically insol in petr ether, benzene.

Melting point: mp 154-156°

Optical Rotation: [a]D20 -58° (c = 0.6)

Picrocrocin

1H NMR spectrum	Predict NMR spectrum (requires installed Java plugin) Predict NMR spectrum (requires HTML5 compatible browser)
13C NMR spectrum	Predict NMR spectrum (requires HTML5 compatible browser)

Picrocrocin

Names
IUPAC names 4-(β-D-glucopyranosyloxy)- 2,6,6-trimethyl-1-cyclohexene- 1-carboxaldehyde
Identifiers
CAS number	138-55-6
ChemSpider	115678
InChI[show]
Jmol-3D images	Image (138-55-6)
PubChem	130796
SMILES[show]
Properties
Molecular formula	C16H26O7
Molar mass	330.37 g/mol
Density	1.31 g/mL
Melting point	154 to 156 °C (309 to 313 °F; 427 to 429 K)
Boiling point	520.4 °C (968.7 °F; 793.5 K)
Except where noted otherwise, data is given for materials in their standard state (at 25 °C (77 °F), 100 kPa)

The chemical structure of the main bioactive compounds from the dried stigmas of Crocus sativus L. – See more at: http://www.mdpi.com/2304-8158/3/3/403/htm#sthash.iOWsNDGb.dpuf

Saffron contains more than 150 volatile and aroma-yielding compounds. It also has many nonvolatile active components,^[28] many of which are carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes. However, saffron’s golden yellow-orange colour is primarily the result of α-crocin. This crocin is trans-crocetin di-(β-D-gentiobiosyl) ester; it bears the systematic (IUPAC) name 8,8-diapo-8,8-carotenoic acid. This means that the crocin underlying saffron’s aroma is a digentiobiose ester of the carotenoid crocetin.^[28] Crocins themselves are a series ofhydrophilic carotenoids that are either monoglycosyl or diglycosyl polyene esters of crocetin.^[28] Crocetin is a conjugated polyene dicarboxylic acidthat is hydrophobic, and thus oil-soluble. When crocetin is esterified with two water-soluble gentiobioses, which are sugars, a product results that is itself water-soluble. The resultant α-crocin is a carotenoid pigment that may comprise more than 10% of dry saffron’s mass. The two esterified gentiobioses make α-crocin ideal for colouring water-based and non-fatty foods such as rice dishes.^[7]

Esterification reaction betweencrocetin and gentiobiose. Components of α–crocin:

β–D-gentiobiose

crocetin

The bitter glucoside picrocrocin is responsible for saffron’s flavour. Picrocrocin (chemical formula:C 16H 26O 7; systematic name: 4-(β-D-glucopyranosyloxy)-2,6,6- trimethylcyclohex-1-ene-1-carboxaldehyde) is a union of an aldehyde sub-element known as safranal (systematic name: 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxaldehyde) and a carbohydrate. It has insecticidal and pesticidal properties, and may comprise up to 4% of dry saffron. Picrocrocin is a truncated version of the carotenoidzeaxanthin that is produced via oxidative cleavage, and is the glycoside of the terpene aldehyde safranal. The reddish-coloured zeaxanthin is, incidentally, one of the carotenoids naturally present within the retina of the human eye.^[29] When saffron is dried after its harvest, the heat, combined with enzymatic action, splits picrocrocin to yield D–glucose and a free safranal molecule.^[27] Safranal, a volatile oil, gives saffron much of its distinctive aroma.^[13][30] Safranal is less bitter than picrocrocin and may comprise up to 70% of dry saffron’s volatile fraction in some samples.^[29] A second element underlying saffron’s aroma is 2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one, which produces a scent described as saffron, dried hay-like.^[31] Chemists find this is the most powerful contributor to saffron’s fragrance, despite its presence in a lesser quantity than safranal.^[31] Dry saffron is highly sensitive to fluctuating pH levels, and rapidly breaks down chemically in the presence of light andoxidising agents. It must, therefore, be stored away in air-tight containers to minimise contact with atmospheric oxygen. Saffron is somewhat more resistant to heat.

Grades and ISO 3632 categories[edit]

Red threads and yellow styles.

Saffron is not all of the same quality and strength. Strength is related to several factors including the amount of style picked along with the red stigma. Age of the saffron is also a factor. More style included means the saffron is less strong gram for gram, because the colour and flavour are concentrated in the red stigmas. Saffron from Iran, Spain and Kashmir is classified into various grades according to the relative amounts of red stigma and yellow styles it contains. Grades of Iranian saffron are: “sargol” (red stigma tips only, strongest grade), “pushal” or “pushali” (red stigmas plus some yellow style, lower strength), “bunch” saffron (red stigmas plus large amount of yellow style, presented in a tiny bundle like a miniature wheatsheaf) and “konge” (yellow style only, claimed to have aroma but with very little, if any, colouring potential). Grades of Spanish saffron are “coupé” (the strongest grade, like Iranian sargol), “mancha” (like Iranian pushal), and in order of further decreasing strength “rio”, “standard” and “sierra” saffron. The word “mancha” in the Spanish classification can have two meanings: a general grade of saffron or a very high quality Spanish-grown saffron from a specific geographical origin. Real Spanish-grown La Mancha saffron has PDO protected status and this is displayed on the product packaging. Spanish growers fought hard for Protected Status because they felt that imports of Iranian saffron re-packaged in Spain and sold as “Spanish Mancha saffron” were undermining the genuine La Mancha brand. Countries producing less saffron do not have specialised words for different grades and may only produce one grade. Artisan producers in Europe and New Zealand have offset their higher labour charges for saffron harvesting by targeting quality, only offering extremely high grade saffron. In addition to descriptions based on how the saffron is picked, saffron may be categorised under the international standard ISO 3632 after laboratory measurement of crocin (responsible for saffron’s colour), picrocrocin (taste), and safranal (fragrance or aroma) content.^[32] However, often there is no clear grading information on the product packaging and little of the saffron readily available in UK is labelled with ISO category. This lack of information makes it hard for customers to make informed choices when comparing prices and buying saffron. Under ISO 3632, determination of non-stigma content (“floral waste content”) and other extraneous matter such as inorganic material (“ash“) are also key. Grading standards are set by the International Organization for Standardization, a federation of national standards bodies. ISO 3632 deals exclusively with saffron and establishes three categories: III (poorest quality), II, and I (finest quality). Formerly there was also category IV, which was below category III. Samples are assigned categories by gauging the spice’s crocin and picrocrocin content, revealed by measurements of specific spectrophotometric absorbance. Safranal is treated slightly differently and rather than there being threshold levels for each category, samples must give a reading of 20-50 for all categories. These data are measured through spectrophotometry reports at certified testing laboratories worldwide. Higher absorbances imply greater levels of crocin, picrocrocin and safranal, and thus a greater colouring potential and therefore strength per gram. The absorbance reading of crocin is known as the “colouring strength” of that saffron. Saffron’s colouring strength can range from lower than 80 (for all category IV saffron) up to 200 or greater (for category I). The world’s finest samples (the selected, most red-maroon, tips of stigmas picked from the finest flowers) receive colouring strengths in excess of 250, making such saffron over three times more powerful than category IV saffron. Market prices for saffron types follow directly from these ISO categories. Sargol and coupé saffron would typically fall into ISO 3632 category I. Pushal and mancha would probably be assigned to category II. On many saffron packaging labels, neither the ISO 3632 category nor the colouring strength (the measurement of crocin content) is displayed. However, many growers, traders, and consumers reject such lab test numbers. Some people prefer a more holistic method of sampling batches of threads for taste, aroma, pliability, and other traits in a fashion similar to that practised by experienced wine tasters.^[33] However, ISO 3632 grade and colouring strength information allow consumers to make instant comparisons between the quality of different saffron brands, without needing to purchase and sample the saffron. In particular, consumers can work out value for money based on price per unit of colouring strength rather than price per gram, given the wide possible range of colouring strengths that different kinds of saffron can have. Despite attempts at quality control and standardisation, an extensive history of saffron adulteration, particularly among the cheapest grades, continues into modern times. Adulteration was first documented in Europe’s Middle Ages, when those found selling adulterated saffron were executed under the Safranschou code.^[34] Typical methods include mixing in extraneous substances like beets, pomegranate fibres, red-dyed silk fibres, or the saffron crocus’s tasteless and odourless yellow stamens. Other methods included dousing saffron fibres with viscid substances like honey or vegetable oil to increase their weight. However, powdered saffron is more prone to adulteration, with turmeric, paprika, and other powders used as diluting fillers. Adulteration can also consist of selling mislabelled mixes of different saffron grades. Thus, in India, high-grade Kashmiri saffron is often sold and mixed with cheaper Iranian imports; these mixes are then marketed as pure Kashmiri saffron, a development that has cost Kashmiri growers much of their income.^[35][36]

Types

Saffron from different producer countries, picked and dried in different ways gives rise to different end qualities.

The various saffron crocus cultivars give rise to thread types that are often regionally distributed and characteristically distinct. Varieties (not varieties in the botanical sense) from Spain, including the tradenames “Spanish Superior” and “Creme”, are generally mellower in colour, flavour, and aroma; they are graded by government-imposed standards. Italian varieties are slightly more potent than Spanish. The most intense varieties tend to be Iranian. Various “boutique” crops are available from New Zealand, France, Switzerland, England, the United States, and other countries—some of them organically grown. In the U.S., Pennsylvania Dutch saffron—known for its “earthy” notes—is marketed in small quantities.^[37][38] Consumers may regard certain cultivars as “premium” quality. The “Aquila” saffron, or zafferano dell’Aquila, is defined by high safranal and crocin content, distinctive thread shape, unusually pungent aroma, and intense colour; it is grown exclusively on eight hectares in the Navelli Valley of Italy’s Abruzzo region, near L’Aquila. It was first introduced to Italy by a Dominican monk from Inquisition-era Spain. But the biggest saffron cultivation in Italy is in San Gavino Monreale, Sardinia, where it is grown on 40 hectares, representing 60% of Italian production; it too has unusually high crocin, picrocrocin, and safranal content. Another is the “Mongra” or “Lacha” saffron of Kashmir (Crocus sativus ‘Cashmirianus’), which is among the most difficult for consumers to obtain. Repeated droughts, blights, and crop failures in the Indian-controlled areas of Kashmir combine with an Indian export ban to contribute to its prohibitive overseas prices. Kashmiri saffron is recognisable by its dark maroon-purple hue; it is among the world’s darkest, which hints at strong flavour, aroma, and colouring effect.

History

A detail from the “Saffron Gatherers” fresco of the “Xeste 3″ building. It is one of many depicting saffron; they were found at the Bronze Age settlement ofAkrotiri, on the Aegean island of Santorini.

The documented history of saffron cultivation spans more than three millennia.^[17] The wild precursor of domesticated saffron crocus wasCrocus cartwrightianus. Human cultivators bred wild specimens by selecting for unusually long stigmas; thus, a sterile mutant form of C. cartwrightianus, C. sativus, likely emerged in late Bronze Age Crete.^[12]

Eastern

Buddhist adepts pray in the Hundred Dragons Hall, Buddha Tooth Relic Temple and Museum, Singapore, wearing saffron-coloured robes.

Saffron was detailed in a 7th-century BC Assyrian botanical reference compiled under Ashurbanipal.^[15]Documentation of saffron’s use over the span of 4,000 years in the treatment of some 90 illnesses has been uncovered.^[39] Saffron-based pigments have indeed been found in 50,000 year-old depictions of prehistoric places in northwest Iran.^[40][41] The Sumerians later used wild-growing saffron in their remedies and magical potions.^[42] Saffron was an article of long-distance trade before the Minoan palace culture’s 2nd millennium BC peak. Ancient Persians cultivated Persian saffron (Crocus sativus ‘Hausknechtii’) in Derbena, Isfahan, and Khorasan by the 10th century BC. At such sites, saffron threads were woven into textiles,^[40] ritually offered to divinities, and used in dyes, perfumes, medicines, and body washes.^[43]Saffron threads would thus be scattered across beds and mixed into hot teas as a curative for bouts of melancholy. Non-Persians also feared the Persians’ usage of saffron as a drugging agent and aphrodisiac.^[44] During his Asian campaigns, Alexander the Great used Persian saffron in his infusions, rice, and baths as a curative for battle wounds. Alexander’s troops imitated the practice from the Persians and brought saffron-bathing to Greece.^[45] Conflicting theories explain saffron’s arrival in South Asia. Kashmiri and Chinese accounts date its arrival anywhere between 2500–900 years ago.^[46][47][48] Historians studying ancient Persian records date the arrival to sometime prior to 500 BC,^[7] attributing it to a Persian transplantation of saffron corms to stock new gardens and parks.^[49] Phoenicians then marketed Kashmiri saffron as a dye and a treatment for melancholy. Its use in foods and dyes subsequently spread throughout South Asia. Buddhist monks wear saffron-coloured robes; however, the robes are not dyed with costly saffron but turmeric, a less expensive dye, or jackfruit.^[50] Monks’ robes are dyed the same colour to show equality with each other, and turmeric or ochre were the cheapest, most readily available dyes. Gamboge is now used to dye the robes.^[51] Some historians believe that saffron came to China with Mongol invaders from Persia.^[52] Yet saffron is mentioned in ancient Chinese medical texts, including the forty-volume pharmacopoeia titled Shennong Bencaojing (神農本草經: “Shennong’s Great Herbal”, also known as Pen Ts’ao or Pun Tsao), a tome dating from 300–200 BC. Traditionally credited to the fabled Yan (“Fire”) Emperor (炎帝) Shennong, it discusses 252 phytochemical-based medical treatments for various disorders.^[53] Nevertheless, around the 3rd century AD, the Chinese were referring to saffron as having a Kashmiri provenance. According to Chinese herbalist Wan Zhen, “[t]he habitat of saffron is in Kashmir, where people grow it principally to offer it to the Buddha.” Wan also reflected on how it was used in his time: “The flower withers after a few days, and then the saffron is obtained. It is valued for its uniform yellow colour. It can be used to aromatise wine.”^[48]

Wider Near East, Western Europe and the USA

Preserved “safran”, Staatliches Museum für Naturkunde, Karlsruhe, Germany.

The Minoans portrayed saffron in their palace frescoes by 1600–1500 BC; they hint at its possible use as a therapeutic drug.^[39][54] Ancient Greek legends told of sea voyages to Cilicia, where adventurers sought what they believed were the world’s most valuable threads.^[21] Another legend tells of Crocus and Smilax, whereby Crocus is bewitched and transformed into the first saffron crocus.^[40] Ancient perfumers in Egypt, physicians inGaza, townspeople in Rhodes,^[55] and the Greek hetaerae courtesans used saffron in their scented waters, perfumes and potpourris, mascaras and ointments, divine offerings, and medical treatments.^[44] In late Hellenistic Egypt, Cleopatra used saffron in her baths so that lovemaking would be more pleasurable.^[56] Egyptian healers used saffron as a treatment for all varieties of gastrointestinal ailments.^[57] Saffron was also used as a fabric dye in such Levantine cities as Sidon and Tyre inLebanon.^[58] Aulus Cornelius Celsus prescribes saffron in medicines for wounds, cough, colic, and scabies, and in the mithridatium.^[59] Such was the Romans’ love of saffron that Roman colonists took it with them when they settled in southern Gaul, where it was extensively cultivated until Rome’s fall. Competing theories state that saffron only returned to France with 8th-century AD Moors or with the Avignon papacy in the 14th century AD.^[60] European saffron cultivation plummeted after the Roman Empire went into eclipse. As with France, the spread of Islamic civilisation may have helped reintroduce the crop to Spain and Italy.^[61] The 14th-century Black Death caused demand for saffron-based medicaments to peak, and Europe imported large quantities of threads via Venetian and Genoan ships from southern and Mediterranean lands such as Rhodes. The theft of one such shipment by noblemen sparked the fourteen-week-long Saffron War.^[62] The conflict and resulting fear of rampant saffron piracy spurred corm cultivation in Basel; it thereby grew prosperous.^[63] The crop then spread to Nuremberg, where endemic and insalubrious adulteration brought on the Safranschou code—whereby culprits were variously fined, imprisoned, and executed.^[64] Saffron cultivation was introduced into England in around 1350, the story being that corms were smuggled from the Levant in a special hollow compartment of a pilgrim’s staff .^[65]The crop seems to have been initially grown in monastic gardens for medicinal use, only being planted in the less kind conditions of open fields many decades later. Soil and climatic conditions meant that by the sixteenth century, saffron cultivation had centred on Eastern England. The Essex town of Saffron Walden, named for its new speciality crop, emerged as a prime saffron growing and trading centre. However, an important omission in a botanical book published in the 1790s meant that the true extent of saffron growing in the eastern counties has been long overlooked .^[66] North Norfolk (especially the area around Walsingham), southern Cambridgeshire and a small area of west Suffolk also produced saffron. Some was also grown in Gloucestershire and other “Westerlie Parts” according to one source. The evidence for this comes from several angles including titherecords, estate records and field names. In Norfolk, customs records show locally grown saffron was exported to the Low Countries .^[67] (The crop has recently been re-introduced to Norfolk and award-winning ISO 3632 category I saffron is grown at Burnham Norton. However, an influx of more exotic spices—chocolate, coffee, tea, and vanilla—from newly contacted Eastern and overseas countries caused European cultivation and usage of saffron to decline.^[68][69] The last grower in England appears to have been John Knott of Duxford in Cambridgeshire, who delivered his crop to London apothecaries until around 1818 .^[70] It would be nearly two centuries before saffron was commercially grown in England again. Only in southern France, Italy, and Spain did the clone significantly endure.^[71] Europeans introduced saffron to the Americas when immigrant members of the Schwenkfelder Church left Europe with a trunk containing its corms. Church members had grown it widely in Europe.^[37] By 1730, the Pennsylvania Dutch cultivated saffron throughout eastern Pennsylvania. Spanish colonies in the Caribbean bought large amounts of this new American saffron, and high demand ensured that saffron’s list price on the Philadelphia commodities exchange was equal to gold.^[72] Trade with the Caribbean later collapsed in the aftermath of the War of 1812, when many saffron-bearing merchant vessels were destroyed.^[73] Yet the Pennsylvania Dutch continued to grow lesser amounts of saffron for local trade and use in their cakes, noodles, and chicken or trout dishes.^[74] American saffron cultivation survives into modern times, mainly in Lancaster County, Pennsylvania.^[37]

Trade and use

Trade

“Ispanya saffron” at market in Turkey.

Sale of saffron and other spices in Iran

Almost all saffron grows in a belt from Spain in the west to India in the east. The other continents, except Antarctica, produce smaller amounts. Some 300 t (300,000 kg) of dried whole threads and powder are gleaned yearly,^[14] of which 50 t (50,000 kg) is top-grade “coupe” saffron.^[76] Iran answers for around 90–93% of global production and exports much of it.^[16] A few of Iran’s drier eastern and southeastern provinces, including Fars, Kerman, and those in the Khorasan region, glean the bulk of modern global production. In 2005, the second-ranked Greece produced 5.7 t (5,700 kg), while Morocco and Kashmir, tied for third rank, each produced 2.3 t (2,300 kg).^[16] In recent years, Afghan cultivation has risen. Azerbaijan, Morocco, and Italy are, in decreasing order, lesser producers. Prohibitively high labour costs and abundant Iranian imports mean that only select locales continue the tedious harvest in Austria, Germany, and Switzerland—among them the Swiss village of Mund, whose annual output is a few kilograms.^[14] Microscale production of saffron can be found in Tasmania,^[77] China, Egypt, England (the village of Burnham Norton^[78]) France, Israel, Mexico, New Zealand, Turkey (mainly around the town of Safranbolu), California, and Central Africa.^[4][28] To glean 1 lb (450 g) of dry saffron requires the harvest of 50,000–75,000 flowers; a kilogram requires 110,000–170,000 flowers.^[79][80] Forty hours of labour are needed to pick 150,000 flowers.^[81] Stigmas are dried quickly upon extraction and (preferably) sealed in airtight containers.^[82] Saffron prices at wholesale and retail rates range from US$500 to US$5,000 per pound, or US$1,100–11,000/kg, equivalent to £2,500/€3,500 per pound or £5,500/€7,500 per kilogram. In Western countries, the average retail price in 1974 was $1,000/£500/€700 per pound, or US$2,200/£1,100/€1,550 per kilogram.^[4] In February 2013, a retail bottle containing 0.06 ounces could be purchased for $16.26 or the equivalent of $4,336 per pound or as little as about $2,000/pound in larger quantities. A pound contains between 70,000 and 200,000 threads. Vivid crimson colouring, slight moistness, elasticity, and lack of broken-off thread debris are all traits of fresh saffron.

Use

Crushed saffron threads are soaked in hot—but not boiling—water for several minutes prior to use in cuisine. This helps release the beneficial components.

Saffron’s aroma is often described by connoisseurs as reminiscent of metallic honey with grassy or hay-like notes, while its taste has also been noted as hay-like and sweet. Saffron also contributes a luminous yellow-orange colouring to foods. Saffron is widely used in Indian, Persian, European, Arab, and Turkish cuisines. Confectioneries and liquors also often include saffron. Common saffron substitutes include safflower (Carthamus tinctorius, which is often sold as “Portuguese saffron” or “açafrão”), annatto, and turmeric (Curcuma longa). Saffron has also been used as a fabric dye, particularly in China and India, and in perfumery.^[83] It is used for religious purposes in India, and is widely used in cooking in many cuisines, ranging from the Milanese risotto of Italy to the bouillabaisse of France to the biryani with various meat accompaniments in South Asia. Saffron also has a long history of use in traditional medicine.^[84]

Biomedical research

There is some evidence to suggest that saffron may help alleviate the symptoms of major depressive disorder.^[85][86] Preclinical studies indicate that saffron could be a promising candidate for cancer chemoprevention studies.^[87] Early studies suggest that it may protect the eye from the direct effects of bright light, and from retinal stress in additional to slowing down macular degeneration and retinitis pigmentosa.^[88] (Most saffron-related research refers to the stigmas, but this is often not made explicit in research papers.) Some studies suggest that saffron may help relieve the symptoms of premenstrual syndrome.^[89][90]

Notes

Citations

References

Books

Journal articles

Miscellaneous

Other

External links

Contraindications, Interactions, and Side Effects (Saffron)

Saffron use in large dose is contraindicated in pregnancy. It may cause contraction of uterus and abortion. Severe side effects may result from ingesting 5 g saffron. No side-effect when used in proper doses.

CDK Inhibitor, MK 7965, DINACICLIB, SCH 727965

REVIEW…….http://www.mdpi.com/2072-6694/6/4/2224/htm

One of the most popular CDK inhibitor in clinical trials in the recent years was dinaciclib (MK-7965, SCH 727965) (Figure 3), the inhibitor of CDK1, CDK2, CDK5, and CDK9. A Phase I trial on the effect of dinaciclib in combination with aprepitant was performed in patients with advanced malignancies [44]. Aprepitant is used for the prevention of chemotherapy-induced nausea and vomiting, is known as an inhibitor and inducer of CYP3A4, which metabolizes dinaciclib.

Coadministration of dinaciclib with aprepitant resulted in no clinically significant effect on the pharmacokinetics and did not alter the safety profile of dinaciclib. The first Phase I clinical trial on dinaciclib as a single agent was performed on patients with advanced malignancies [68]. Forty-eight patients with various solid tumors were treated and 10 of them achieved prolonged stable disease for at least four treatment cycles. Adverse effects were mild, the most common being nausea, anemia, decreased appetite and fatigue.

A phase II multi-center study of dinaciclib for relapsed and/or refractory AML was performed on 20 patients [69]. Temporary decrease in peripheral blood and/or bone marrow blasts was observed in 60% of patients. Four of 13 (31%) patients with circulating blasts had >50% decrease and 6 (46%) >80% decrease in the absolute blast count within 1–8 days of the first dinaciclib dose. Toxicities included diarrhea, fatigue, transaminitis, and manifestations of tumor lysis syndrome, with one patient who deceased of acute renal failure. Another Phase II study was performed of dinaciclib versus erlotinib in patients with non-small cell lung cancer [70].

Unfortunately, it was found that dinaciclib was not successful as monotherapy in non-small cell lung cancer. Most common toxicities included neutropenia, leukopenia, vomiting, and diarrhea. Yet another Phase II study was performed on dinaciclib versus capecitabine in patients with advanced breast cancer [71]. Dinaciclib treatment demonstrated antitumor activity in two of seven patients with ER-positive and ERBB 2-negative metastatic breast cancer, however efficacy was not superior to capecitabine (p = 0.991).

Toxicities included neutropenia, leukopenia, increase in aspartate aminotransferase, and febrile neutropenia. Phase I nonrandomized dose-escalation trial was performed, where patients with relapsed or refractory chronic lymphocytic leukemia were treated with dinaciclib and rituximab [72]. Four out of six patients achieved stable disease, and one patient achieved complete response. Drug-related adverse events were mostly hematological, digestive and metabolic and no dose-limiting toxicities were observed. Dinaciclib was also moved into Phase III development for refractory chronic lymphocytic leukemia [73]. Phase I/II clinical trial Dinaciclib in patients with relapsed multiple myeloma showed promise as single agent [74]. The overall confirmed response rate was 3 of 27 (11%). Adverse effects included leukopenia, thrombocytopenia, gastrointestinal symptoms, alopecia, and fatigue. –

FOR REF See more at: http://www.mdpi.com/2072-6694/6/4/2224/htm#sthash.amBuLwq1.dpuf

Dinaciclib (SCH-727965) is an experimental drug that inhibits cyclin-dependent kinases (CDKs.^[1] It is being evaluated in clinical trials for various cancer indications.^[2]

Mechanisms of action

• Cyclin-dependent kinase inhibitor dinaciclib interacts with the acetyl-lysine recognition site of bromodomains.^[3]
• Dinaciclib (SCH727665) inhibits the unfolded protein response (UPR) through a CDK1 and CDK5-dependent mechanism.^[4]

Anti-tumoral action

• In melanoma
  • The anti-melanoma activity of dinaciclib is dependent on p53 signaling.^[5]

• In chronic lymphocytic leukemia (CLL)
  • Dinaciclib promotes apoptosis and abrogates microenvironmental cytokine protection in chronic lymphocytic leukemia cells.^[6]

• In pancreatic cancer
  • Dinaciclib inhibits pancreatic cancer growth and progression in murine xenograft models.^[7]

• In osteosarcoma
  • Dinacliclib induces the apoptosis of osteosarcoma cells.^[8]
  • Apoptosis of osteosarcoma cultures can be induced by the combination of the cyclin-dependent kinase inhibitor SCH727965 and a heat shock protein 90 inhibitor.^[9]

Systematic (IUPAC) name
(S)-3-(((3-Ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)pyridine 1-oxide
Clinical data
Legal status	Investigational
Identifiers
CAS number	779353-01-4
ATC code	?
PubChem	CID 46926350
ChemSpider	25027387
ChEMBL	CHEMBL2103840
Synonyms	SCH-727965
Chemical data
Formula	C21H28N6O2

Clinical trials

• Phase 1^[10]
• Phase 2
  • Advanced breast cancer^[11]
  • Non-small cell lung cancer (NSCLC)^[12]

/////////////////////////////////////////////////

http://www.google.com.tr/patents/US8076479

One example of these inhibitors is the compound of Formula II.

The synthesis of the compound of Formula II is described in the ‘878 publication according to Scheme II:
Scheme II:

Step 1—Amidization to Form Substituted Pyrazole

http://www.google.com.tr/patents/US8076479

Step 2—Formation and Dehalogenation of pyrazolo[1,5a]pyrimidine

Step 3—Amination (Two Separate, Sequential Reactions)

As described in the ‘878 publication, Synthetic Scheme II leading to the compound of Formula II has several disadvantages from the standpoint of commercial scale synthesis. In step 1, the starting material (compound “C”) used in the formation of compound “D” is a sticky, viscous oil which is difficult to process (weigh, transfer, and blend). Moreover, step 1, as described in the ‘878 publication, requires isolation and chromatographic purification of compounds C and D prior to carrying out each subsequent derivatization reaction. In addition, as described in the ‘878 publication, the reaction of compound C with malonate diester is carried out using the diester as a solvent. After isolation and purification of the resultant malonate adduct, compound D, ring closure to form diketone compound E is carried out in methanol. In accordance with the procedure described in the ‘878 publication, compound E is isolated and dried, then converted to the corresponding dichloride in N,N-dimethyl aniline by treatment with phosphorous oxychloride (POCl₃). The dichloride thus formed was isolated and purified by chromatography prior to the sequential amination reactions. Additionally, the compounds of Formula G and of Formula II require chromatography purification and isolations, as described in the ‘878 publication.

As further described in the ‘878 publication, each of the amination reactions were run separately with isolation and chromatographic purification between amination reactions. Accordingly, the ‘878 publication describes the preparation of the compound of Formula II utilizing a scheme consisting of five separate reaction steps with intervening isolation and purification of the products, each sequential step being carried out in a different solvent system. The overall yield of the compound of Formula II reported for this synthesis, based on starting compound C (Scheme II) is about 20%.

Example 1Preparation of Diketone Compound E (Scheme VI) 3-Ethylpyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione

To a 250 ml, three-necked flask equipped with a thermometer, a reflux condenser and mechanical stirrer was charged 3-amino-4-ethylpyrazole oxalate (10 g, 50 mmole), dimethylmalonate (10 ml, 88 mmole), methyl alcohol (80 ml) and sodium methoxide (50 ml, 245 mmole, 25% in methyl alcohol). The batch was heated at reflux for 16 hours then cooled to room temperature. Celite (5 g) and water (60 ml) were added to the batch and agitated for 10 minutes. The batch was filtered to remove the solid residue. The filtrate was pH adjusted to pH˜3 with aqueous HCl (10 ml) to effect precipitation. The precipitate (compound “E”) was filtered and washed with water (40 ml). The wet cake was dried for 18 hours in vacuum oven maintained in the range of oven at 45° C. to 55° C., to give a solid product (84.3%, 7.5 g). C₈H₉N₃O₃, Mp: 200-205° C.; NMR in DMSO-d6: 1.05 (t, 3H), 2.23 (q, 2H), 3.26 (bs, 1H), 3.89 (bs, 1H), 7.61 (s, 1H), 11.50(bs, 1H).

Example 2Preparation of Dichloride Compound F (Scheme VI) 5,7-Dichloro-3-Ethylpyrazolo[1,5-a]pyrimidine

Into a 3-neck flask fitted with an inert gas inlet, a reflux condenser and a mechanical stirring apparatus and containing 83 liters of acetonitrile was placed 3-Ethylpyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (E) prepared as described in Step 1 (11.0 kg, 61.5 mole), N,N-dimethylaniline (8.0 L, 63 mole) and POCl₃ (7 kg, 430 mole). With stirring the mixture was brought to reflux and maintained under refluxing conditions for 15 hours. The reaction mixture was sampled periodically to monitor the amount of compound “E” present. After the conversion was complete, the solution was cooled to 15° C. Into the cooled reaction mixture was added water which had been cooled to a temperature of less than 20° C. The product is filtered and washed with 4 aliquots of acetonitrile-water (1:3) which had been cooled to a temperature of 20° C. followed by a wash with 10× water. The wet cake is dried in a vacuum oven maintained at 40° C. for at least 15 hours to yield the compound “F” (86.7%); ¹H NMR (CDCl₃): 1.32(t, 3H), 2.81 (q, 2H), 6.92 (s, 1H), 8.10 (s, 1H)

mp: 90-95° C.

Example 3Preparation of Compound G (Scheme VI) 5-Chloro-3-Ethyl-N-[(1-oxido-pyridinyl)methyl]pyrazolo-[1,5-a]pyrimidine-5.7(4H,6H)-dion-7-amine

Into a 3-liter, three-necked flask equipped with a thermometer, a reflux condenser and mechanical stirrer was charged an aliquot of the dichloride compound “F” prepared in Step 2 (150 g, 0.69 mole), potassium phosphate tribasic monohydrate (338.0 g, 1.47 mole), the dihydrochloride salt of N-oxide-pyridin-3-yl-methylamine, compound F1a (142.5 g, 0.72 mole), water (1500 ml) and acetonitrile (300 ml). The batch was heated at reflux for 6 hours. At the end of the refluxing period the batch was cooled to room temperature over 2 hours and then held at room temperature for 4 hours. The resulting precipitate was filtered and washed with water (600 ml). The wet cake was returned to the flask with water (1500 ml) and acetonitrile (300 ml), and heated to reflux. Reflux was maintained for 6 hours additional. At the end of the second reflux period the reaction mixture was cooled to room temperature over a 2 hour period and left to stand at room temperature for 4 hours. The resulting precipitate was filtered and washed with water (600 ml). The wet cake was dried in an air draft oven at 50° C. for 18 hours to give the first amine adduct “G” material (179 g, 84.9%). mp: 187-189C; NMR in CDCl3, 1.26(t, 3H), 2.73(q, 2H), 4.60(d, 2H), 5.87(s, 1H), 6.83(bs, 1H), 7.33(t, 1H), 7.70(d, 1H), 7.84(s, 1H), 8.58(d, 1H), 8.64(d, 1H).

Example 4

Preparation of the Compound of Formula II (Scheme VI) 1-[3-Ethyl-7-[(1-oxido-3-pyridinyl)methyl]amino]pyrazolo[1,5-a]pyrimidin-5-yl]-2(s)-piperidinemethanol

Into a three-neck flask fitted with a mechanical stirrer and a reflux condenser were placed the first amine adduct prepared in Step 3, compound “G”, (7 kg, 23 mole), amino-alcohol compound G1a (5.6 kg, 43.3 mole), sodium carbonate (3.5 kg, 33.0 mole), 110 ml of water and 1-methyl-2-pyrrolidinone (NMP) (11 L). The reaction mixture was heated to 150° C. for 4 days. After chromatography indicated that the reaction was complete (90-95% substrate consumed), the reaction mixture was cooled to room temperature and quenched by adding water. The mixture was then extracted with ethyl acetate. The batch was dried by distillation of the water azeotrope under atmospheric pressure and concentrated to about 28 L volume. THF was added and the solution was heated to reflux until all the solids dissolve. Ethyl acetate and trietylamine are added to the hot solution. The batch was cooled to ambient and then agitated with the temperature maintained in the range of from 20° C. to 25° C. for 12 hours. The solids were collected by filtration, washed first with ethyl acetate then water, and dried in the filter under vacuum for 24 hours with the temperature maintained at from 40° C. to 50° C., yielding 4.9 kg, 51.3% of the compound of Formula II.

DSC, 168.6° C.; Specific Rotation (10 mg/ml in MeOH, 20° C.), −117.8 °;

¹HNMR (400 MHz, DMSO): 8.31 ppm (1H, s), 8.11-8.13 ppm (1H, td, J=5.7 Hz, J=1.4 Hz), 7.97 ppm (1H, t, J=6.7 Hz), 7.68 ppm (1H, s), 7.41 ppm (1H, s), 7.37-7.43 ppm (1H, dd), 5.55 ppm (1H, s), 4.85 ppm (1H, t, J=5.4 Hz), 4.49-4.59 ppm (3H, m), 4.24-4.28 ppm (1H, broad), 3.27-3.46 ppm (2H, m), 2.76-2.83 ppm (1H, t, J=13.0 Hz), 2.45-2.50 ppm (2H, q, J=7.5 Hz), 1.72-1.79 (1H, m), 1.54-1.68 ppm (6H, m), 1.30-1.34 ppm (1H, m), 1.16 ppm (3H, t, J=7.5 Hz)

References

External links

• dinaciclib at the US National Library of Medicine Medical Subject Headings (MeSH)

DINACICLIB

Patent                                                         Submitted                                                                Granted

Process and intermediates for the synthesis of (3-alkyl-5-piperidin-1-yl-3,3a-dihydro-pyrazolo[1,5-a]pyrimidin-7-yl)-amino derivatives and intermediates [US8076479]2008-03-06   GRANT2011-12-13

Process for resolving chiral piperidine alcohol and process for synthesis of pyrazolo[1,5-a] pyrimidine derivatives using same [US7786306]2008-02-28   GRANT2010-08-31

Sequential Administration of Chemotherapeutic Agents for Treatment of Cancer [US2011129456]2011-06-02

TARGETING CDK4 AND CDK6 IN CANCER THERAPY [US2011009353]2011-01-13

Pyrazolopyrimidines as cyclin dependent kinase inhibitors [US2007225270]2007-09-27

PYRAZOLO[1,5-a]PYRIMIDINES [US2007275963]2007-11-29

Novel pyrazolopyrimidines as cyclin dependent kinase inhibitors [US2007281951]2007-12-06

Novel pyrazolopyrimidines as cyclin dependent kinase inhibitors [US2008050384]2008-02-28

Novel pyrazolopyrimidines as cyclin dependent kinase inhibitors [US2007054925]2007-03-08

FLUDABARINE

Systematic (IUPAC) name
[(2R,3R,4S,5R)-5-(6-amino-2-fluoro-purin-9-yl)- 3,4-dihydroxy-oxolan-2-yl]methoxyphosphonic acid
Clinical data
Trade names	Fludara
AHFS/Drugs.com	monograph
MedlinePlus	a692003
Pregnancy category	D
Legal status	AU: Prescription Only (S4) UK: POM
Routes	Intravenous, oral
Pharmacokinetic data
Bioavailability	55%
Protein binding	19 to 29%
Half-life	20 hours
Excretion	Renal
Identifiers
CAS number	75607-67-9
ATC code	L01BB05
PubChem	CID 657237
DrugBank	DB01073
ChemSpider	571392
UNII	P2K93U8740
KEGG	D01907
ChEBI	CHEBI:63599
ChEMBL	CHEMBL1568
Chemical data
Formula	C10H13FN5O7P
Molecular mass	365.212 g/mol

……………….

Fludarabine or fludarabine phosphate (Fludara) is a chemotherapy drug used in the treatment of hematological malignancies(cancers of blood cells such as leukemias and lymphomas). It is a purine analog, which interferes with DNA synthesis.

Indications

Fludarabine is highly effective in the treatment of chronic lymphocytic leukemia, producing higher response rates than alkylating agents such as chlorambucil alone.^[1] Fludarabine is used in various combinations with cyclophosphamide, mitoxantrone,dexamethasone and rituximab in the treatment of indolent non-Hodgkins lymphomas. As part of the FLAG regimen, fludarabine is used together with cytarabine and granulocyte colony-stimulating factor in the treatment of acute myeloid leukaemia. Because of its immunosuppressive effects, fludarabine is also used in some conditioning regimens prior to allogeneic stem cell transplant.

Pharmacology

Fludarabine is a purine analog, and can be given both orally and intravenously. Fludarabine inhibits DNA synthesis by interfering withribonucleotide reductase and DNA polymerase. It is active against both dividing and resting cells. Being phosphorylated, fludarabine is ionized at physiologic pH and is effectually trapped in blood. This provides some level of specificity for blood cells, both cancerous and healthy.

Side effects

Fludarabine is associated with profound lymphopenia, and as a consequence, increases the risk of opportunistic infectionssignificantly. Patients who have been treated with fludarabine will usually be asked to take co-trimoxazole or to use monthly nebulised pentamidine to prevent Pneumocystis jiroveci pneumonia. The profound lymphopenia caused by fludarabine renders patients susceptible to transfusion-associated graft versus host disease, an oftentimes fatal complication of blood transfusion. For this reason, all patients who have ever received fludarabine should only be given irradiated blood components.

Fludarabine causes anemia, thrombocytopenia and neutropenia, requiring regular blood count monitoring. Some patients require blood and platelet transfusion, or G-CSF injections to boost neutrophil counts.

Fludarabine is associated with the development of severe autoimmune hemolytic anemia in a proportion of patients.^[2]

Difficulties are often encountered when harvesting peripheral blood stem cells from patients previously treated with fludarabine.^[3]

History

Fludarabine was produced by John Montgomery and Kathleen Hewson of the Southern Research Institute in 1968.^[4] Their previous work involved 2-fluoroadenosine, which was unsafe for use in humans; the change to this arabinose analogue was inspired by the success of vidarabine.^[4]

• Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine) (II) is a purine nucleoside antimetabolite resistant to adenosine deaminase, employed for the treatment of leukemia.
• Fludarabine is usually administered as a pro-drug, fludarabine phosphate, which is also the natural metabolite. Fludarabine was firstly synthesised by Montgomery (US 4,188,378 and US 4,210,745) starting from 2-aminoadenine. The method comprised acetylation of 2-aminoadenine, reaction with a benzyl-protected chlorosugar, deacetylation of the amino groups, diazotization and fluorination of the 2-amino group followed by deprotection of the sugar residue.
• Fludarabine phosphate can be obtained according to conventional phosphorylation methods, typically by treatment with trimethylphosphate and phosphoryl chloride. Recently, a method for preparing highly pure fludarabine, fludarabine phosphate and salts thereof has been disclosed by Tilstam et al. (US 6,46,322).
• Enzymatic synthesis has been regarded as a valid alternative to conventional methods for the synthesis of nucleosides and nucleotides derivatives. EP 0 867 516 discloses a method for the preparation of sugar nucleotides from sugar 1-phosphates and nucleosides monophosphates by use of yeast cells having nucleoside diphosphate-sugar pyrophosphorylase activity. EP 0721 511 B1 discloses the synthesis of vidarabine phosphate and fludarabine phosphate by reacting an arabinonucleotide with an arylphosphate in the presence of a microorganism able to catalyse the phosphorylation of nucleosides. This method is particularly convenient in that it does not require purified enzymes, but it does not allow to synthesise vidarabine and fludarabine.

………………………………………………………………

paper

Simple Modification To Obtain High Quality Fludarabine

http://pubs.acs.org/doi/abs/10.1021/op3000509

A simple and improved debenzylation process is described to obtain fludarabine in greater than 99.8% purity and 90–95% yield.

……………………………………………………………………….

Patents

http://www.google.com/patents/EP1464708A1?cl=en

• The present invention relates to a process for the preparation of fludarabine phosphate (I) illustrated in the scheme and comprising the following steps:

• a) reaction of 2-fluoroadenine with 9-β-D-arabinofuranosyl-uracil in the presence of Enterobacter aerogenes to give crude fludarabine (II);
• b) treatment of crude fludarabine with acetic anhydride to 2′,3′,5′-tri-O-acetyl-9-β-D-arabinofuranosyl-2-fluoroadenine (III);
• c) hydrolysis and recrystallisation of intermediate (III) to give pure fludarabine;
• d) phosphorylation of fludarabine to give fludarabine phosphate (I).
• Step a) is carried out in a 0.03 – 0.05 M KH₂PO₄ solution, heated to a temperature comprised between 50 and 70°C, preferably to 60°C, adjusted to pH 7 with KOH pellets and added with 2-fluoroadenine, Ara-U and EBA. The concentration of 2-fluoroadenine in the solution ranges from 0.02 to 0.03 M, while 9-β-D-arabinofuranosyl-uracil is used in a strong excess; preferably, the molar ratio between 9-β-D-arabinofuranosyl-uracil and 2-fluoroadenine ranges from 5:1 to 7:1, more preferably from 5.5:1 to 6.5:1. 2 – 2.5 1 of cell culture per 1 of KH₂PO₄ solution are used. The mixture is stirred at 60°C, adjusting the pH to 7 with a 25% KOH solution and the reaction is monitored by HPLC. Once the reaction is complete (about 24-26 hours), the cell material is separated by conventional dialysis and the permeated solutions are recovered and kept cool overnight. Crystallised fludarabine contains 10% 9-β-D-arabinofuranosyl adenine, which can be conveniently removed by means of steps b) and c).
• In step b) crude fludarabine from step a) is dissolved in 9-11 volumes of acetic anhydride, preferably 10 volumes and reacted at 90 – 100°C under stirring, until completion of the reaction (about 10 – 12 h). Acetic anhydride is co-evaporated with acetone and the product is suspended in water.
• The hydrolysis of step c) is carried out with methanol and ammonium hydroxide. Typically, compound (III) from step b) is suspended in 9-11 volumes of methanol and 2.5 – 3.5 volumes of 25% NH₄OH and stirred at room temperature until complete hydrolysis (about 20 hours; the completion of the reaction can be promoted by mildly warming up the mixture to 30-32°C). Fludarabine precipitates by cooling the mixture to 10°C and is further hot-crystallised with water, preferably with 50 – 70 ml of water per gram of fludarabine or with a water/ethanol mixture (1/1 v/v) using 30 – 40 ml of mixture per gram of fludarabine. Fludarabine is recovered as the monohydrate and has a HPLC purity higher than 99%.
• Even though the conversion of fludarabine into fludarabine phosphate (step d) can be carried out according to any conventional technique, for example as disclosed in US 4,357,324, we have found that an accurate control of the reaction and crystallisation temperature allows to minimise product decomposition and significantly improves the yield. According to a preferred embodiment of the invention, the reaction between phosphorus oxychloride, triethylphosphate and fludarabine is carried out at -10°C, and fludarabine phosphate is precipitated from water at 0°C.
• In summary, the present invention allows to obtain the following advantages: fludarabine is prepared by enzymatic synthesis without the use of pure enzymes and is therefore particularly suitable for industrial scale; fludarabine is easily recovered and purified from 9-β-D-arabinofuranosyl adenine by acetylation without the need of chromatographic purification, since the triacetyl-derivative precipitates from water with high purity and yield; fludarabine phosphate can be obtained in high yield by controlling the reaction and crystallisation temperature in the phosphorylation step.
• The following examples illustrate the invention in more detail.

EXAMPLES

Example 1 – Crude 9-β-D-arabinofuranosyl-2-fluoroadenine (II)

• A solution of KH₂PO₄ (123 g, 0,9 moles) in water (13 l) was heated to 60°C under stirring and the pH adjusted to 7 with KOH pellets (130 g, 2.32 moles), then added with Ara-U (1451 g, 5.94 moles), 2-fluoroadenine (150 g, 0.98 moles) and EBA (ATCC® n° 13048) cell culture (30 l).
• The mixture was stirred at 60°C for 24-26 hours, adjusting the pH to 7 with a 25% KOH solution and monitoring the reaction by HPLC.
• After 24-26 hours the cell material was separated by dialysis at 50°-55°C, diluting the mixture with water. The permeated yellow clear solutions were collected, pooled (50 l) and left to stand at 0°-5°C overnight. The resulting crystalline precipitate was filtered and washed with cold water (2 l).
• The product was dried at 45°C under vacuum for 16 hours to give 110 g of the crude compound (II) which was shown by HPLC to be a mixture of (I) (90%) and 9-β-D-arabinofuranosyl adenine (10%).

Example 2

Pure 9-β-D-arabinofuranosyl-2-fluoroadenine (II)

• 9-β-D-arabinofuranosyl-2-fluoroadenine (II) (30 g, 0,095 moles) was suspended in acetic anhydride (300 ml) and heated to 95°C under stirring.
• After 7 hours a clear solution was obtained and left to react at 95°C for further 2-3 hours until the acetylation was completed.
• The resulting yellow solution was then concentrated under vacuum at 45°C and the residue was co-evaporated with acetone (2 x 50 ml) and suspended in water (600 ml). The water suspension was cooled to room temperature and left under stirring for 1 hour.
• The product was collected by filtration and washed with water (2 x 100 ml) to give 34 g of wet 2′,3′,5′-tri-O-acetyl-9-β-D-arabinofuranosyl-2-fluoroadenine (III).
• Wet compound (III) was suspended in methanol (300 ml) and added with 25% NH₄OH (100 ml). The mixture was left to stand at room temperature overnight and after 19 hours was warmed to 30°-32°C for 3 hours, until no starting material was detected by HPLC.
• The suspension was cooled to 10°C for 1 hour, then the product was collected by filtration and washed with a methanol-water mixture (2 x 25 ml, 3:1 v/v). The product was dried under vacuum at 45°C overnight to give 17.5 g of fludarabine (II) (98.4% HPLC purity).

Method A

• Re-crystallisation of compound (II) (17.5 g, 0.061 moles) was also carried out by suspending the product in water (875 ml) and heating to 95°C until a clear solution was obtained. The solution was allowed to cool spontaneously to room temperature and the crystalline product was filtered, washed with cold water (2 x 50 ml) and dried under vacuum at 45°C overnight, to give 15.5 g of pure fludarabine (II) as the monohydrate (99.3% HPLC purity).
• The monohydrate was further dried under vacuum at 90°C for 24 hours to give pure anhydrous fludarabine (II).

Method B

• Fludarabine (II) (35 g, 0.123 moles) was also re-crystallized by suspending the product in a water/ethanol mixture (1/1, v/v) (1050 ml) and heating to 80°C until a clear solution was obtained. The solution was allowed to cool spontaneously to room temperature and the crystalline product was filtered, washed with a water/ethanol mixture (2 x 50 ml) and dried under vacuum at 45°C overnight, to give 32 g of pure fludarabine (II) as the monohydrate ( 99% HPLC purity ).
• The monohydrate was further dried under vacuum at 90°C for 24 hours to give pure anhydrous fludarabine (II).

Example 3 – 9-β-D-arabinofuranosyl-2-fluoroadenine-5′-phosphate (I)Method A

• Phosphorous oxychloride (5 g, 3 ml, 0.033 moles) was added to cold (0°C, ice-bath) triethylphosphate (50 ml) and the solution was kept at 0°C for 1 hour, thereafter added with anhydrous fludarabine (II) (5 g, 0.018 moles) under stirring.
• After about 3 hours, the reaction mixture became homogeneous and turned light-yellow and was kept at 0°C overnight. Once the phosphorylation was completed (about 23 hours) the mixture was added with water (10 ml) and the solution was stirred for 3 hours at 0°C. The mixture was then poured into cold (0°C) methylene chloride (400 ml) and kept at 0°C under stirring until a clear methylene chloride phase was obtained (at least 1 hours).
• The methylene chloride phase was removed by decantation and the residual yellowish oil was dissolved in warm (50°C) water (30 ml). The solution was allowed to cool spontaneously to room temperature overnight and the resulting crystalline product was collected by filtration and washed with water (10 ml) and ethanol (2 x 10 ml).
• The product was dried at room temperature under vacuum for 24 hours to give 4 g of compound (I).
• Compound (I) was re-crystallised as follows: compound (I) (4 g) was dissolved in 60 ml of preheated deionized water (73°-75°C) and the solution was stirred and rapidly cooled to 50°C to minimize product decomposition. The solution was then allowed to cool spontaneously to room temperature: the precipitation started at 40°C. The resulting precipitate was collected by filtration and washed with water (10 ml) and ethanol (2 × 10 ml). The product was dried at room temperature under vacuum for 24 hours to give 2.5 g of compound (I).

Method B

• Phosphorous oxychloride (5 g, 3 ml, 0.033 mol) was added to cold (-10°C) triethylphosphate (50 ml) and the solution was kept at -10°C for 1 hour, thereafter anhydrous fludarabine (II) (5 g, 0,018 mol) was added with stirring at -10°C.
• After about 6 hours the reaction mixture turned light-yellow and became homogeneous. The mixture was kept at -10°C overnight and after 23 hours the phosphorylation was completed. After addition of 40 ml of cold water (2°C) the solution was stirred for 1 hour at 0°C and extracted with cold (0°C) methylene chloride (100 ml and two 50-ml portions).
• The aqueous solution was kept under vacuum at room temperature for 1 hour and allowed to stand at 0°C for 24 hours. The resulting crystalline product (I) was collected by filtration and washed with ethanol (2 x 20 ml).
• The product was dried at 40°C under vacuum for 24 hours (Yield: 5 g).
• A final crystallization was carried out as follows. Compound (I) (5 g) was dissolved in 75 ml of preheated deionized water (73°-75°C) and the solution was stirred and rapidly cooled to 50°C to minimize decomposition. The solution was then allowed to cool spontaneously to room temperature (the precipitation started at 40°C). The resulting precipitate was collected by filtration and washed with water (10 ml ) and ethanol (2 x 10 ml). The product was dried at 40°C under vacuum for 24 hours (Yield: 4 g).

……………………………………………….

http://www.google.com/patents/US20100290990

References

External links

• Fludara webpage

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO …..FOR BLOG HOME CLICK HERE

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SDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEV SPELVCSMLHLCSG [SEQ ID NO: 2].

BXQ-350

Cincinnati Children’s Hospital  ……..innovator

Bexion Pharmaceuticals……….under license

In February 2015, the US FDA granted saposin C Orphan designation for the treatment of glioblastoma multiforme

SAPOCIN C

Recombinant human Saposin C (SapC) bound to a liposomal formulation of the dioleoylphosphatidylserine

Bexion’s Saposin C – the active ingredient in the brain tumour therapy BXQ-350 – has been awarded Orphan Drug status by US regulators.

Read more at: http://www.pharmatimes.com/Article/15-02-17/US_Orphan_status_for_Bexion_s_brain_tumour_drug.aspx#ixzz3S3zXdHlO

Bexion Pharmaceuticals, under license from the Cincinnati Children’s Hospital, is investigating a human saposin C (SapC)/liposomal dioleoylphosphatidylserine (DOPS) conjugate, SapC-DOPS (BXQ-350), a nanovesicle-formulated pro-apoptotic sphingomyelinase activating molecular imaging agent and anticancer agent, for the potential diagnosis and treatment of cancer , . In October 2013, Bexion was planning a phase I first-in-human trial for the therapy of glioblastoma multiforme

Bexion Pharmaceuticals LLC announced today that the U.S. Food and Drug Administration (FDA) has granted the company Orphan Drug designation for Saposin C, active ingredient in its proprietary drug BXQ-350 for the potential treatment of glioblastoma multiforme.

The FDA’s Office of Orphan Drug Products Development reviews applications for Orphan Drug status to support development of medicines for underserved patient populations, or rare disorders that affect fewer than 200,000 people in the United States. The successful application submitted by Bexion and the FDA granting of Orphan Drug status entitles the company to a seven-year period of marketing exclusivity in the United States for BXQ-350, if it is approved by the FDA for the treatment of glioblastoma multiforme. Orphan Drug status also enables the company to apply for research grant funding for Phase I and II Clinical Trials, tax credits for certain research expenses, and a waiver from the FDA’s application user fee, as well as additional support from FDA and a potentially faster regulatory process.

Bexion was previously awarded a prestigious Phase II Bridge Award (Small Business Innovation Research Grant; SBIR) from the National Cancer Institute (NCI) to support the manufacture and clinical testing of BXQ-350.

“Orphan Drug status for BXQ-350 is an important milestone in the development of this new treatment modality,” stated Dr. Ray Takigiku, founder and CEO of Bexion. “Few treatment options are available for patients suffering from glioblastoma multiforme and this designation recognizes the unmet need that exists with this disease, as well as the unique attributes of BXQ-350. In addition, orphan designation allows Bexion to benefit from important financial, regulatory and commercial considerations and we have seen recently that products with orphan designation have become sought after assets.”

About Orphan Drug Designation
Orphan Drug designation is a status assigned to a medicine intended for use in rare diseases. In the U.S., the Orphan Drug Designation program confers Orphan Drug status to successful applicants for medicines intended for the safe and effective treatment, diagnosis or prevention of rare diseases or disorders that affect fewer than 200,000 people in the U.S. or that are not expected to recover the costs of developing and marketing a treatment.¹

The approval of an orphan designation request does not alter the standard regulatory requirements and process for obtaining marketing approval for investigational use. Sponsors must establish safety and efficacy of a compound in the treatment of a disease through adequate and well-controlled studies. However, the FDA review process may be speedier for Orphan Drugs than those which do not receive Orphan Drug designation.

About BXQ-350
In pre-clinical studies, Bexion’s first-in-class biologic, BXQ-350 has shown promising results in selectively inducing cell death in the laboratory. BXQ-350 is a proprietary nanovesicle formulation of Saposin C (sphingolipid activator protein C, or SapC) and the phospholipid dioleoylphosphatidylserine (DOPS).

About Bexion Pharmaceuticals
Bexion Pharmaceuticals is a privately held biotech company focused on the development and commercialization of innovative cures for cancer.  Initial products are based on a proprietary platform technology licensed from Cincinnati Children’s Hospital Medical Center.  The technology has demonstrated potential for development as a therapeutic, diagnostic and surgical imaging reagent, and as a carrier for other pharmaceutical agents, such as oligonucleotides.  For more information, visit www.bexionpharma.com or contact Margaret van Gilse atmvangilse@bexionpharma.com.

¹ U.S. Food and Drug Administration web site. “Regulatory Information: Orphan Drug Act.”http://www.fda.gov/regulatoryinformation/legislation/federalfooddrugandcosmeticactfdcact/significantamendmentstothefdcact/orphandrugact/default.htm.

Margaret van Gilse859-757-1652mvangilse@bexionpharma.com

SOURCE Bexion Pharmaceuticals LLC

Glioblastoma is the most common primary CNS malignant neoplasm in adults, and accounts for nearly 75% of the cases. Although there has been steady progress in their treatment due to improvements in neuro-imaging, microsurgery, and radiation, glioblastomas remain incurable. The average life expectancy is less than one year from diagnosis, and the five-year survival rate following aggressive therapy, including gross tumor resection, is less than 10%. Glioblastomas cause death due to rapid, aggressive, and infiltrative growth in the brain. The infiltrative growth pattern is responsible for the un-resectable nature of these tumors. Glioblastomas are also relatively resistant to radiation and chemotherapy, and therefore post-treatment recurrence rates are high. In addition, the immune response to the neoplastic cells is mainly ineffective in completely eradicating residual neoplastic cells following resection and radiation therapy.

One problem in treating glioblastoma is the tumor’s protection behind the blood-brain tumor barrier (BBTB). A significant obstacle in the development of therapeutics for glioblastoma is the inability of systemic therapies to efficiently cross the BBTB. Saposin C (SapC) is a sphingolipid- activating protein that functions to catabolize glycosphingolipids. SapC-DOPS forms stable nanovesicles which can efficiently cross the blood-brain tumor barrier and fuse with GBM cells inducing cell death.

Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus, which was discovered first for its properties as an antifungal agent. Streptomyces hygroscopicus has also been implicated as a cancer agent.

There remains a need in the art for new therapeutics for the treatment of glioblastoma.

…………………………………………………………………..

https://www.google.com/patents/US20040229799?cl=en22

Example 1Purification of Recombinant Saposin C

[0106] Recombinant saposin C was overexpressed in E. coli cells by using the isopropyl-1-thio-β-D-galactopyranoside inducing pET system (Qi et al. (1994) J. Biol. Chem. 269:16746-16753, herein incorporated by reference in its entirety). Expressed polypeptides with a His-tag were eluted from nickel columns. After dialysis, the polypeptides were further purified by HPLC chromatography as follows. A C4 reverse phase column was equilibrated with 0.1% trifluoroacetic acid (TFA) for 10 minutes. The proteins were eluted in a linear (0-100%) gradient of 0.1% TFA in acetonitrile over 60 minutes. The major protein peak was collected and lyophilized. Protein concentration was determined as previously described (Qi et al. (1994) J. Biol. Chem. 269:16746-16753).

Example 2Bath Sonication of Sanosin C and Dioleoylphosphatidylserine

[0107] Dioleoylphosphatidylserine (DOPS) was obtained from Avanti Polar Lipids (Alabaster AL). Twenty to thirty imoles of DOPS in chloroform were dried under N₂ and vacuum to lipid films. Five to ten μmoles saposin C polypeptide was added to the dried films and suspended in 50 μl McIlvanine buffer (pH 4.7). The suspension was then brought to a 1 ml volume with either cell culture medium or phosphate buffered saline (PBS) (Ausubel et al. (2002) Current Protocols in Molecular Biology. John Wiley & Sons, New York, New York, herein incorporated by reference). The mixture was sonicated in a bath sonicator for approximately 20 minutes. Ice was added as needed to prevent overheating the samples.

………………………………………………………………

http://www.google.com/patents/WO2014078522A1?cl=en

The SapC-DOPS composition comprises a phospholipid, an isolated saposin C-related polypeptide, wherein the polypeptide comprises an amino acid sequence at least 75% identical to the entire length of SEQ ID NO: 2, and a pharmaceutically acceptable carrier, wherein the phospholipid forms a nano vesicle incorporating the polypeptide. In certain embodiments, the polypeptide comprises an amino acid sequence at least 85% identical to the entire length of SEQ ID NO: 2. In certain embodiments, the polypeptide comprises an amino acid sequence at least 95% identical to the entire length of SEQ ID NO: 2. In certain embodiments, the polypeptide comprises an amino acid sequence at least 99% identical to the entire length of SEQ ID NO: 2.

The Sequence Listing, filed electronically and identified as SEQ_LIST_OSIF-2013- 102.txt, was created on November 12, 2013, is 5,548 in size, and is hereby incorporated by reference.

[0004] SEQ ID NO: 1

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*8 a 210 2iS

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:?e

<H ¾^■ yts ca« ¾»* **u v ΆΧ» s?s ass ¾«¾

:»o

L st S«x ri» r s

SEQ ID NO: 2

BEXION PHARMA

1. 1. Russell Street, Covington, KY 41011, United States

      $2.9 Million Grant Awarded to Covington-Based Bexion for Next Step in Cancer Fight

      921 Spring Street Covington, Kentucky 41016 United States

      112 East 4th Street, Covington, KY 41011.

      1182 Riverhouse Way Covington KY : 427657

Female Libido Enhancer  – Bremelanotide

Bremelanotide is a compound that is currently under investigation for its potential uses in managing reperfusion injury, female sexual dysfunction or hemorrhagic shock. The chemical may also see success in managing modulate inflammation or limiting the effects of ischemia.

N-Acetyl-L-norleucyl-L-alpha-aspartyl-L-histidyl-D-phenylalanyl-L-arginyl-L-tryptophyl-L-lysine (2-7)-lactam

Bremelanotide,  PT 141, CAS NO.: 189691-06-3

Bremelanotide ⁱ/ˌbrɛmɨˈlænətaɪd/ (formerly PT-141) is a compound under drug development by Palatin Technologies as a treatment for female sexual dysfunction, hemorrhagic shock and reperfusion injury. It functions by activating the melanocortin receptors MC1R and MC4R, to modulate inflammation and limiting ischemia.^[2] It was originally tested for intranasal administration in treating female sexual dysfunction but this application was temporarily discontinued in 2008 after concerns were raised over adverse side effects of increased blood pressure. As of December 2014, Palatin is conducting a human Phase 3 study^[3] using a subcutaneous drug delivery system that appears to have little effect on blood pressure.

Palatin, in collaboration with European licensee Gedeon Richter, is developing an sc formulation of the synthetic peptide bremelanotide (PT-141; BMT), a melanocortin MCR-4 agonist and a synthetically modified analog of PT-14, also analogous to alpha-melanocyte-stimulating hormone (alpha-MSH), for the potential treatment of female sexual dysfunction (FSD) including hypoactive sexual desire disorder (HSDD)

The Bremelanotide or PT-141 is a mean that explains the revolution caused by the medical world in a silent but attractive manner in the human health related study. Bremelanotide is the latest arrival from the company called Palatin Technologies which forms the basic treatment for the hemorrhagic shock and reperfusion injury.( In short about the company, the Palatin Technologies is the owner of this research and is located in New Jersey. Hence this medicine is a Jersey based Product. And regarding the product under research, is waiting for the approval from the Food and Drug Association. Once this is done, the company has targeted to reach those customers, whom the Viagra has approached. This has the effect of helping the male patients suffering with an erectile dysfunction syndrome. Also if it gets the approval as a treatment measure for the female sexual dysfunction, then this medicine is expected to bring a relief to the post-menopausal and also supports or provides their sexual happiness and also they are checking regarding thehyposexual desire disorder. This is expected to be a blockbuster, if released. So this medicine is waiting for a confirmation as well as an approval.

In February 2015, a randomized, double-blind, placebo-controlled, open-label extension, phase III trial (NCT02338960; BMT-302, Reconnect Study) was initiated in the US in premenopausal women (expected n = 550) with hypoactive sexual desire disorder to evaluate the efficacy and safety of bremelanotide. At that time, the trial was expected to complete in July 2017

Study – Potential Use Erectile Dysfunction

One study has explored the potential use of bremelanotide as a replacement for natural peptide melanocyte stimulating hormones for the sake of treating erectile dysfunction.

• The goal of this study was to determine if the effects of bremelanotide stimulating sexual desire that was shown in male rats could be replicated in the brains of female rats. To do this, hormone primed female rats in a control group and a test group that were treated with bremelanotide and known to have consummatory sexual disorders was introduced to a group of male rats and the reactions were measured.

• Heart racing, hops and darts, pacing and customary sexual behaviors were assessed while the brain was stimulated. The stimulation of specific molecular markers within the brain was examined to determine arousal in the female subjects.

• Results indicated that the females saw an increase in sexual behavior when bremelanotide was applied to the limbic and hypothalamic regions of their brains. It is suggested that this was because the chemical that stimulated the mPOA terminals, leading to activated dopamine in the brain.

Additional study is necessary to determine the extent of the effects bremelanotide has on the brain and natural stimulating chemicals.

Bremelanotide and Ongoing Research

This is an advanced research involved even now. This functions by activating the Melanocortin, which is a group of peptide hormones which includes the adrenocorticotropic hormone and also the different forms of the melanocyte stimulating hormones. These melanocortins are produced or prepared from the proopiomelanocortin in the pituitary glands. The melanocortin releases or exert their effects by making a bind with the melanocortin and thereby activating it).The Bremelanotide functions by activating the melanocortin receptors and thereby makes a modulation in the inflammation. This is actually produced for making use in treating the sexual dysfunction. Due to certain reasons; the process of researching was kept under hold in recently, since it created some adverse side effects of increased blood pressure. In the chemistry of the preparation of the bremelanotide, the Peptide Melanaton II forms the basic compound. This compound is tested using a sunless tanning agent.

The actual information about the peptide melanaton has the effect of making sexual arousal and speed as well as sudden erections and some other side effects. However, there are several other measures taken to test the property of the same under several other health situations to make a detailed study about the chemical compound structure to make a change in the combination of the chemical structure. This medicine has made a revolution in the field of science of the human structure. When made a deep verification of the compound structure of the chemical study showed the following information. The structural design has an appearance of white colored powder like material, which has an accurate purity of nearly 98%. The actual molecular weight of the compound formed is around 1025.2. This compound has the collective share of Amino acids in the composition, peptide and acetate contents also.

The study of the compound structure PT-141 has an enhanced support of making a recombination that produces a different profile of the same medicine but in a different standard with different properties that may support the human requirement.

Bremelanotide PT-141 is known for its aphrodisiac properties

Development

Bremelanotide was developed from the peptide hormone Melanotan II which underwent testing as a sunless tanningagent. In initial testing, Melanotan II did induce tanning but additionally caused sexual arousal and spontaneous erections as unexpected side effects in nine out of the ten original male volunteer test subjects.^[4]

In studies, bremelanotide was shown to induce lordosis in an animal model^[5] and was also effective in treating sexual dysfunction in both men (erectile dysfunction or impotence) and women (sexual arousal disorder). Unlike Viagra and other related medications, it does not act upon the vascular system, but directly increases sexual desire via the nervous system.^[6]

A Phase III clinical trial was scheduled to begin in the first half of 2007, but was delayed until August 2007. On August 30, Palatin announced that the U.S. Food and Drug Administration had expressed serious concerns regarding therisk/benefit ratio of bremelanotide with regards to the side effect of increased blood pressure. The FDA stated that it would consider alternate uses for bremelanotide, including as a treatment for individuals who do not respond to more established ED treatments. However, On May 13, 2008, Palatin Technologies announced it had “discontinued development of Bremelanotide for the treatment of male and female sexual dysfunction” while concurrently announcing plans to develop it as a treatment for hemorrhagic shock instead.^[7] The company additionally announced intentions to focus its attention on another compound, PL-6983, that causes lower blood pressure in animal models.^[8]Palatin has since re-initiated Bremelanotide studies for ED and FSD using a subcutaneous delivery method. On August 12, 2009, the company announced that in a double-blind study of 54 volunteers bremelanotide failed to evoke the hypertensive side effects seen with the nasal delivery system used in prior studies, concluding that “variability of uptake” inherent in intranasal administration of the drug resulted in “increases in blood pressure and gastrointestinal events…primarily related to high plasma levels in [only] a subset of patients” and that subcutaneous administration of the drug circumvented the potential for this side effect.^[8] Palatin has completed a human Phase 2B study utilizing subcutaneous administration and reported positive results.^[9]

Structure

Bremelanotide is a cyclic hepta-peptide lactam analog of alpha-melanocyte-stimulating hormone (alpha-MSH) that activates the melanocortin receptors MC3-R and MC4-R in thecentral nervous system. It has the amino acid sequence Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-OH or cyclo-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰]alpha-MSH-(4-10). It is a metabolite of Melanotan II that lacks the C-terminal amide function.

Bremelanotide

Systematic (IUPAC) name
(3S,6S,9R,12S,15S,23S)-15-[(N-acetyl-L-norleucyl)amino]-9-benzyl-6-{3-[(diaminomethylidene)amino]propyl}-12-(1H-imidazol-5-ylmethyl)-3-(1H-indol-3-ylmethyl)-2,5,8,11,14,17-hexaoxo-1,4,7,10,13,18-hexaa zacyclotricosane-23-carboxylic acid
Clinical data
Legal status	US: Unscheduled
Pharmacokinetic data
Half-life	120 minutes[1]
Identifiers
CAS number	189691-06-3
ATC code	None
PubChem	CID 9941379
ChemSpider	8116997
UNII	6Y24O4F92S
KEGG	D06569
ChEMBL	CHEMBL2070241
Chemical data
Formula	C50H68N14O10
Molecular mass	1025.2 g/mol

Sexual dysfunction, including both penile erectile dysfunction or impotence and female sexual dysfunction, are common medical problems. Significant effort has been devoted over the last twenty or more years to develop methods, devices and compounds for treatment of sexual dysfunction. While more effort has been undertaken for treatment of penile erectile dysfunction, female sexual dysfunction is also an area to which significant research and effort has been devoted.

At present, one commonly used orally administered drug for treatment of sexual dysfunction in the male is Viagra®, a brand of sildenafil, which is a phosphodiesterase 5 inhibitor, increasing the persistence of cyclic guanosine monophosphate and thereby enhancing erectile response. There are several other medical treatment alternatives currently available depending on the nature and cause of the impotence problem. Some men have abnormally low levels of the male hormone testosterone, and treatment with testosterone injections or pills may be beneficial. However, comparatively few impotent men have low testosterone levels. For many forms of erectile dysfunction, treatment may be undertaken with drugs injected directly into the penis, including drugs such as papaverin, prostaglandin E₁, phenoxybenzamine or phentolamine. These all work primarily by dilating the arterial blood vessels and decreasing the venous drainage. Urethral inserts, such as with suppositories containing prostaglandin, may also be employed. In addition, a variety of mechanical aids are employed, including constriction devices and penile implants.

A variety of treatments have also been explored for female sexual dysfunction, including use of sildenafil, although the Food and Drug Administration has not specifically approved such use. Testosterone propionate has also been employed to increase or augment female libido.

Melanocortin receptor-specific compounds have been explored for use of treatment of sexual dysfunction. In one report, a cyclic α-melanocyte-stimulating hormone (“α-MSH”) analog, called Melanotan-II, was evaluated for erectogenic properties for treatment of men with psychogenic erectile dysfunction. Wessells H. et al., J Urology 160:389-393 (1998); see also U.S. Pat. No. 5,576,290, issued Nov. 19, 1996 to M. E. Hadley, entitled Compositions and Methods for the Diagnosis and Treatment of Psychogenic Erectile Dysfunction and U.S. Pat. No. 6,051,555, issued Apr. 18, 2000, also to M. E. Hadley, entitled Stimulating Sexual Response in Females. The peptides used in U.S. Pat. Nos. 5,576,290 and 6,051,555 are also described in U.S. Pat. No. 5,674,839, issued Oct. 7, 1997, to V. J. Hruby, M. E. Hadley and F. Al-Obeidi, entitled Cyclic Analogs of Alpha-MSH Fragments, and in U.S. Pat. No. 5,714,576, issued Feb. 3, 1998, to V. J. Hruby, M. E. Hadley and F. Al-Obeidi, entitled Linear Analogs of Alpha-MSH Fragments. Melanotan-II is a peptide of the following formula:

Additional related peptides are disclosed in U.S. Pat. Nos. 5,576,290, 5,674,839, 5,714,576 and 6,051,555. These peptides are described as being useful for both the diagnosis and treatment of psychogenic sexual dysfunction in males and females. These peptides are related to the structure of melanocortins.

In use of Melanotan-II, significant erectile responses were observed, with 8 of 10 treated men developing clinically apparent erections, and with a mean duration of tip rigidity greater than 80% for 38 minutes with Melanotan-II compared to 3.0 minutes with a placebo (p=0.0045). The drug was administered by subcutaneous abdominal wall injection, at doses ranging from 0.025 to 0.157 mg/kg body weight. Transient side effects were observed, including nausea, stretching and yawning, and decreased appetite.

The minimum peptide fragment of native α-MSH needed for erectile response is the central tetrapeptide sequence, His⁶-Phe⁷-Arg⁸-Trp⁹ (SEQ ID NO:1). In general, all melanocortin peptides share the same active core sequence, His-Phe-Arg-Trp (SEQ ID NO:1), including melanotropin neuropeptides and adrenocorticotropin. Five distinct melanocortin receptor subtypes have been identified, called MC1-R through MC5-R, and of these MC3-R and MC4-R are believed to be expressed in the human brain. MC3-R has the highest expression in the arcuate nucleus of the hypothalamus, while MC4-R is more widely expressed in the thalamus, hypothalamus and hippocampus. A central nervous system mechanism for melanocortins in the induction of penile erection has been suggested by experiments demonstrating penile erection resulting from central intracerebroventricular administration of melanocortins in rats. While the mechanism of His-Phe-Arg-Trp (SEQ ID NO:1) induction of erectile response has not been fully elucidated, it has been hypothesized that it involves the central nervous system, and probably binding to MC3-R and/or MC4-R.

Other peptides and constructs have been proposed which are ligands that alter or regulate the activity of one or more melanocortin receptors. For example, International Patent Application No. PCT/US99/09216, entitled Isoquinoline Compound Melanocortin Receptor Ligands and Methods of Using Same, discloses two compounds that induce penile erections in rats. However, these compounds were administered by injection at doses of 1.8 mg/kg and 3.6 mg/kg, respectively, and at least one compound resulted in observable side effects, including yawning and stretching. Other melanocortin receptor-specific compounds with claimed application for treatment of sexual dysfunction are disclosed in International Patent Application No. PCT/US99/13252, entitled Spiropiperidine Derivatives as Melanocortin Receptor Agonists.

Both cyclic and linear α-MSH peptides have been studied; however, the peptides heretofore evaluated have had an amide or —NH₂ group at the carboxyl terminus. See, for example, Wessells H. et al., J Urology, cited above; Haskell-Luevano C. et al., J Med Chem 40:2133-39 (1997); Schiöth H. B. et al., Brit J Pharmacol 124:75-82 (1998); Schiöth H. B. et al., Eur J Pharmacol 349:359-66 (1998); Hadley M. E. et al., Pigment Cell Res 9:213-34 (1996); Bednarek M. A. et al., Peptides20:401-09 (1999); U.S. Pat. Nos. 6,054,556, 6,051,555 and 5,576,290; and, International Patent Applications PCT/US99/04111 and PCT/US98/03298. While significant research has been conducted in an effort to determine the optimal structure of α-MSH peptides, including a variety of structure-function, agonist-antagonist, molecular modeling and pharmacophore studies, such studies have relied upon peptides with an art conventional —NH₂ group at the carboxyl terminus. Further, it has long been believed that biologically active neuropeptides, including α-MSH peptides, are amidated, with an —NH₂ group at the carboxyl terminus, and that such amidation is required both for biological activity and stability. See, for example, Metabolism of Brain Peptides, Ed. G. O’Cuinn, CRC Press, New York, 1995, pp. 1-9 and 99-101.

…………………………………………….

Bioorganic and Medicinal Chemistry Letters, 2005 ,  vol. 15,  4  pg. 1065 – 1068

http://www.sciencedirect.com/science/article/pii/S0960894X04014842

……………………………………………….

US6794489

In a preferred embodiment, the invention provides the peptide

Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH  Compound 1

The peptide of Compound 1 has a formula of C₅₀H₆₈N₁₄O₁₀, and a net molecular weight of 1025.18. This peptide may be synthesized by solid-phase means and purified to greater than 96% purity by HPLC, yielding a white powder that is a clear, colorless solution in water. The structure of Compound 1 is:

In general, the peptide compounds of this invention may be synthesized by solid-phase synthesis and purified according to methods known in the art. Any of a number of well-known procedures utilizing a variety of resins and reagents may be used to prepare the compounds of this invention.

The peptides of this invention may be in the form of any pharmaceutically acceptable salt. Acid addition salts of the compounds of this invention are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. The acetate salt form is especially useful. Where the compounds of this invention include an acidic moiety, suitable pharmaceutically acceptable salts may include alkali metal salts, such as sodium or potassium salts, or alkaline earth metal salts, such as calcium or magnesium salts.

The invention provides a pharmaceutical composition that includes a peptide of this invention and a pharmaceutically acceptable carrier. The carrier may be a liquid formulation, and is preferably a buffered, isotonic, aqueous solution. Pharmaceutically acceptable carriers also include excipients, such as diluents, carriers and the like, and additives, such as stabilizing agents, preservatives, solubilizing agents, buffers and the like, as hereafter described.

EXAMPLE 1

Peptide Synthesis

The peptide Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH was synthesized by standard solid phase peptide synthesis methods, and is a cyclic heptapeptide melanocortin peptide analog with a free acid at the carboxyl terminus and an acetylated amino group at the amino terminus, with the structure:

The peptide has a net molecular weight of 1025.18, and is supplied in an acetate salt form. The peptide is a white, odorless amorphous hygroscopic powder, soluble in 0.9% saline, composed of C₅₀H₆₈N₁₄O₁₀. For synthesis, an Fmoc-Lys(R³)-p-alkoxybenzyl alcohol resin was transferred to a solid phase peptide synthesizer reactor with a mechanical stirrer. The R³group, such as 1-(1′-adamantyl)-1-methyl-ethoxycarbonyl (Adpoc), allyloxycarbonyl (Aloc) or 4-methyltrityl (Mtt), was removed and the next Fmoc-protected amino acid (Fmoc-Trp(Boc)-OH) was added to the resin through standard coupling procedures. The Fmoc protective group was removed and the remaining amino acids added individually in the correct sequence, by repeating coupling and deprotection procedures until the amino acid sequence was completed. After completion of coupling with the last Fmoc-amino acid derivative, Fmoc-Nle-OH, and cleavage of the Fmoc protective group, the exposed terminal amino group was acetylated with acetic anhydride and pyridine in N,N-dimethylformamide (DMF). The peptide-resin was dried and the Lys and Asp protective groups cleaved. The Lys and Asp deprotected peptide resin was suspended in a suitable solvent, such as DMF, dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP), a suitable cyclic coupling reagent, such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCCl/HOBt) was added, and coupling initiated by use of a suitable base, such as N,N-diispropylethylamine (DIPEA), sym-collidine or N-methylmorpholine (NMM). After cyclization, the peptide-resin was washed and the peptide cleaved from the resin and any remaining protective groups using trifluoroacetic acid (TFA) in the presence of water and 1,2-ethanedithiol (EDT). The final product was precipitated by adding cold ether and collected by filtration. Final purification was by reversed phase HPLC using a C₁₈ column. The purified peptide was converted to acetate salt by passage through an ion-exchange column.

…………………………………………..

WO2014071339

Compounds of the Invention.

in a preferred embodiment of the present invention, fie rneianocortin receptor agonist is;

Ac-Nie”Cyc/o{-Asp-His–D–Phe-Arg–Trp»Lys)–OH (bremeianotide)

The peptide of bremeianotide has a formula of C_saH_esN< C½, and a net mofecufar weight of 1025.18, This peptide may be synthesized by conventional means, including either solid-phase or Squid-phase techniques, and purified to greater than 99% purity by HPLC, yielding a white powder that is a clear, colorless solution in water. The structure of bremeianotide is:

in one embodiment of the invention, bremeianotide is synthesized by solid-phase synthesis and purified according to methods known in the art. Any of a number of ^‘well-known procedures utilizing a variety of resins and reagents may be used to prepare bremeianotide.

Bremeianotide may be in the form of any pharmaceutically acceptable salt. Acid addition salts of the compounds of this invention are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacefie, maieic, citric, tartaric, oxalic, succinic or methanesu!fonic acid. The acetate salt form is especially useful.

in a preferred embodiment, bremelanotide is an acetate salt form, and is formulated in a buffered aqueous solution including giycerin, and prepackaged in a syringe and auto-injector device. In alternative embodiments, bremelanotide is any pharmaceutically acceptable salt form, and is formulated in any pharmaceutically acceptable aqueous solution, the aqueous solution optionally including one or more salts, such as sodium chloride, one or more acids, such as citric acid, and one or more additional ingredients, including cellulose or derivatives thereof, saccharides o

polysaccharides such as dextrose, and any of a wide variety of surfactants, chelating agents and preservatives.

………………………………………….

US20050222014

In yet another embodiment of the present invention, the melanocortin receptor agonist is:
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH PT-141

The peptide of PT-141 has a formula of C₅₀H₆₈N₁₄O₁₀, and a net molecular weight of 1025.18. This peptide may be synthesized by conventional means, including either solid-phase or liquid-phase techniques, and purified to greater than 99% purity by HPLC, yielding a white powder that is a clear, colorless solution in water. The structure of PT-141 is:

In one embodiment of the invention, PT-141 is synthesized by solid-phase synthesis and purified according to methods known in the art. Any of a number of well-known procedures utilizing a variety of resins and reagents may be used to prepare PT-141.

PT-141 may be in the form of any pharmaceutically acceptable salt. Acid addition salts of the compounds of this invention are prepared in a suitable solvent from the peptide and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, citric, tartaric, oxalic, succinic or methanesulfonic acid. The acetate salt form is especially useful. Where the compounds of this invention include an acidic moiety, suitable pharmaceutically acceptable salts may include alkali metal salts, such as sodium or potassium salts, or alkaline earth metal salts, such as calcium or magnesium salts.

In a preferred embodiment, PT-141 is an acetate salt form, and is formulated in a buffered aqueous solution including glycerin, prepackaged in a metered unit dose intranasal delivery device. In alternative embodiments, PT-141 is any pharmaceutically acceptable salt form, and is formulated in any pharmaceutically acceptable aqueous solution, the aqueous solution optionally including one or more salts, such as sodium chloride, one or more acids, such as citric acid, and one or more additional ingredients, including cellulose or derivatives thereof, saccharides or polysaccharides such as dextrose, and any of a wide variety of surfactants, chelating agents and preservatives. In one preferred embodiment, PT-141 is administered to patients in volumes of 100 μL, with the quantity of PT-141 delivered determined by the concentration thereof. As described hereafter, in one preferred embodiment a metered unit dose contains 7.5 mg of PT-141.

While certain embodiments of the present invention are described primarily in the context of PT-141, it is to be understood that other melanocortin receptor agonists may be employed. For example, the metallopeptide melanocortin receptor agonists disclosed in WO 02/064091, filed on Feb. 13, 2001, and U.S. Ser. No. 10/640,755, filed on Aug. 13, 2003, both entitled Melanocortin Metallopeptides for Treatment of Sexual Dysfunction; and WO 01/13112, filed on Jun. 14, 2000, entitled Melanocortin Metallopeptide Constructs, Combinatorial Libraries and Applications, may be employed. In addition, the peptidomimetic melanocortin receptor agonists disclosed in U.S. Ser. No. 10/776,419, filed on Feb. 10, 2004, entitled Peptidomimetics of Biologically Active Metallopeptides; the pyrrolidine melanocortin receptor agonists disclosed in U.S. Ser. No. 10/766,657, filed on Feb. 10, 2004, entitled Pyrrolidine Melanocortin-Specific Compounds; and the bicyclic melanocortin receptor agonists disclosed in PCT/US04/01505, filed on Jan. 20, 2004, entitled Bicyclic Melanocortin-Specific Compounds, may also be employed. Also particular preferred are the piperazine melanocortin agonists disclosed in PCT/US04/01462, filed on Jan. 20, 2004 and U.S. Ser. No. 10/762,079, filed on Jan. 20, 2004, both entitled piperazine Melanocortin-Specific Compounds; the melanocortin agonists disclosed in WO 03/006620, filed on Jul. 11, 2002, entitled Linear and Cyclic Melanocortin Receptor-Specific Peptides; WO 04/005324, filed on Jul. 9, 2003, entitled Peptide Compositions for Treatment of Sexual Dysfunction; PCT/US00/18217, filed on Jun. 29, 2000 and U.S. Ser. No. 10/040,547, filed on Jan. 4, 2002, entitled Compositions and Methods for Treatment of Sexual Dysfunction; and U.S. Ser. No. 10/638,071, filed on Aug. 8, 2003, entitled Cyclic Peptide Compositions and Methods for Treatment of Sexual Dysfunction. The entire disclosure of each of the foregoing are incorporated here by reference. It is to be understood that the foregoing listing of patent applications disclosing melanocortin receptor agonists is intended to only be exemplary, and that other melanocortin receptor agonists, whether heretofore known or hereafter developed, may similarly be used in the practice of this invention.

…………………….

NMR prediction

External links

• Palatin Technologies The company that has developed bremelanotide.
• US 6,794,489 bremelanotide (PT-141) patent (Appl. No.:040547)
• US 6,579,968 bremelanotide (PT-141) patent (Appl. No.:066501)
• Bremelanotide Edguider Forum

Vinita Gupta, 43, Group President and CEO, Lupin Pharmaceuticals and Director, Lupin

India-based drugmaker Lupin has signed an agreement with Polish biopharmaceutical firm Celon Pharma to develop a fluticasone / salmeterol dry powder inhaler (DPI).

Under the deal, Lupin will take the responsibility for commercialisation of the product, which is a generic version of GlaxoSmithKline’s (GSK) Advair Diskus.

Lupin CEO Vinita Gupta said: “We are very pleased to partner with Celon given their experience in the development and manufacturing of fluticasone/salmeterol DPI in Europe…………..http://www.pharmaceutical-technology.com/news/newslupin-celon-pharma-partner-generic-version-gsks-advair-diskus-4514718?WT.mc_id=DN_News

Vinita Gupta, 43, Group President and CEO, Lupin Pharmaceuticals and Director, Lupin, is based in the United States, but has been in India a lot in the past one year.

Vinita Gupta

With an expanding role in Lupin’s universe, Vinita has been spending more time outside the US, at times taking her six-year-old son, Krish with her. “He is getting exposure at a much younger age,” she says. Gupta herself was exposed to business at the age of 11 by her father Desh Bandhu Gupta, Lupin’s founder and Chairman.

“We almost had a family board at home, discussing work,” she says. Currently work goes well indeed, with Gupta taking new initiatives in India and also making the business more global. “I am focusing on drivers for growth in our business for the next five years,” she says.

Gupta is married to US-based businessman Brij Sharma.

DB Gupta (centre) Chairman, Vinita Gupta (right) CEO and Nilesh Gupta

SILODOSIN

Alpha 1A adrenoceptor antagonist

Prostate hyperplasia

Kissei Pharmaceutical Co Ltd  INOVATOR

CAS 160970-54-7

2,3-Dihydro-1-(3-hydroxypropyl)-5-[(2R)-2-[[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl]amino]propyl]-1H-indole-7-carboxamide

160970-64-9 (racemate)
169107-04-4 (diHBr)

In February 2008, the FDA accepted for review an NDA for silodosin for the treatment of dysuria associated with BPH . In October 2008, the FDA approved the drug . In April 2009, Actavis launched silodosin for the treatment of the signs and symptoms of BPH .

SILODOSIN

1-(3-hydroxypropyl)-5-[(2R)-2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino]propyl]-2,3-dihydroindole-7-carboxamide

Kissei Pharmaceutical, Daiichi Sankyo (formerly Daiichi Seiyaku), Actavis (formerly Watson) and Recordati have developed and launched silodosin (Urief; Trupass; Rapaflo; Thrupas; Silodyx; Urorec; KMD-3213; Youlifu), an oral alpha 1A adrenoceptor antagonist selective for prostatic receptors . The product is comarketed in Europe by several licensees. The drug is indicated for the treatment of the signs and symptoms of benign prostatic hyperplasia (BPH).

Silodosin, a highly selective alpha1A-adrenoceptor antagonist, was launched in May 2006 in Japan for the oral treatment of urinary disturbance associated with benign prostatic hyperplasia (BPH). The product was launched in the U.S. for the treatment of signs and symptoms of benign prostatic hyperplasia in 2009. In 2009, a positive opinion was received in the E.U. for this indication and final approval was obtained in 2010. Launch in the E.U. took place the same year.

In May 2006, silodosin was launched as a capsule formulation in Japan. Actavis launched the drug in the US in April 2009. In June 2010, EU launched began, initially with Germany ; in November 2010, the drug was launched in France; by December 2010, the drug was launched in Spain.

In 2001, Kissei established an agreement with Daiichi Pharmaceutical to codevelop and comarket silodosin. An oral, once-daily formulation of silodosin filed in the U.S. by Watson (now Actavis) was approved in 2008. Watson (now Actavis) obtained exclusive rights in 2004 to develop and market the drug in the U.S.

PRODUCT Was developed and launched byKissei Pharmaceutical, Daiichi Sankyo, Actavis and Recordati. Family members of the product case EP0600675 have SPC protection in most EU states until 2018; while its Orange Book listed equivalent, US5387603, expire in the US in 2018 with US156 extension.

Silodosin (trade names Rapaflo (USA), Silodyx (Europe and South Africa), Rapilif (India), Silodal (India), Urief (Japan), Urorec (Russia)) is a medication for the symptomatic treatment of benign prostatic hyperplasia. It acts as an α₁-adrenoceptor antagonist with high uroselectivity (selectivity for the prostate).

Silodosin

Systematic (IUPAC) name
1-(3-hydroxypropyl)-5-[(2R)-({2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl}amino)propyl]indoline-7-carboxamide
Clinical data
Pregnancy category	US: B Not approved for use in women
Legal status	℞ Prescription only
Routes	Oral
Pharmacokinetic data
Bioavailability	32%
Protein binding	97%
Metabolism	Hepatic glucuronidation (UGT2B7-mediated); also minor CYP3A4 involvement
Half-life	13±8 hours
Excretion	Renal and fecal
Identifiers
CAS number	160970-54-7
ATC code	G04CA04
PubChem	CID 5312125
IUPHAR ligand	493
ChemSpider	4471557
UNII	CUZ39LUY82
ChEMBL	CHEMBL24778
Synonyms	KAD-3213, KMD-3213
Chemical data
Formula	C25H32F3N3O4
Molecular mass	495.534 g/mol

History

Silodosin received its first marketing approval in Japan in May 2006 under the tradename Urief, which is jointly marketed by Kissei Pharmaceutical Co., Ltd. and Daiichi Sankyo Pharmaceutical Co., Ltd.

Kissei licensed the US, Canadian, and Mexican rights for silodosin to Watson Pharmaceuticals, Inc. in 2004.

On February 12, 2008, Watson announced that the New Drug Application submitted to the United States Food and Drug Administration for silodosin has been accepted for filing. FDA approved this drug on October 9, 2008.^[1] Silodosin is marketed under the trade names Rapaflo in the US and Silodyx in Europe.^[2] and Rapilif in India (Ipca Urosciences)

Pharmacology

Since silodosin has high affinity for the α_1A adrenergic receptor, it causes practically no orthostatic hypotension (in contrast to other α₁ blockers). On the other side, the high selectivity seems to cause more problems with ejaculation.^[3]

As α_1A adrenoceptor antagonists are being investigated as a means to male birth control due to their ability to inhibit ejaculation but not orgasm, a trial with 15 male volunteers was conducted. While silodosin was completely efficacious in preventing the release of semen in all subjects, 12 out of the 15 patients reported mild discomfort upon orgasm. The men also reported the psychosexual side effect of being strongly dissatisfied by their lack of ejaculation.^[4]

///////////////////////////////

CN 103848772

http://www.google.com/patents/CN103848772A?cl=en

silodosin (Silodosin) is 〃 2 Japanese orange Johnson invented – receptor antagonist, for the treatment of benign prostatic hyperplasia or hypertrophy, and other related symptoms. Clinical trials showed that 25% of patients with benign prostatic hyperplasia need for drugs or surgery. Although prostatectomy is better, the mortality rate is not high, but patients bring varying degrees of damage. So look for an effective and safe non-surgical treatment, not only can control the further development of the disease, while relieving the symptoms of the patient.

  benign prostatic hyperplasia in older male patients have a higher prevalence, and clinical alternative drugs rarely, so the development of a benign prostatic hyperplasia drug treatment, not only has good social benefits, but also to bring good economic benefits. The study confirmed that silodosin is the treatment of benign prostatic hyperplasia in an important class of drugs.

Currently, the research reported in the published literature on the preparation of compounds of silodosin, are:

Early 1995, Kitazawa M et al patent US5387603, the reporter silodosin total synthesis method, but the method reaction step is long, the yield is not too high, not suitable for our industrial production.

  In 2009, 翟富民 et al patent CN102115455A, which reported a method for preparing Sailuoduoxin key intermediates. The appropriate method for improving existing methods, although shorter than the previous method step in the step, but low synthesis yield of the process, we can not meet the needs of industrial production.

  In summary, the compounds prepared silodosin more synthetic methods are constantly improved, but there are still a lot of flaws. Therefore, there is need for further research on the preparation of compounds of silodosin to get simple process, product yield, product easy separation of the new preparation. SUMMARY

  The present invention is to overcome the above problems of the prior art, there is provided a method for preparing important intermediates silodosin, the present invention is simple process, high yield, easy separation of the product, the method suitable for industrial production .

To achieve the above technical object, to achieve the above technical result, the present invention is realized by the following technical scheme:

One kind of silodosin preparation of important intermediate, comprising the steps of:

Step I) in a flask, 282g of raw materials 1-acetyl-5- (2-bromo-propyl) indoline, 222g phthalimide potassium salt and 700mL DMF, was heated at 110 ° C for 2h; After completion of the reaction, to which was added the right amount of water to wash away the excess solvent DMF and salt extraction desolventizing after EA, was 296g crude;

Step 2) In a flask was added 296g crude product obtained in step I, dissolved 800mL ethanol, was added 165mL of hydrazine hydrate, 50 ° C is heated to precipitate a white solid; After completion of the reaction, cooling suction filtered, the filter cake washed with ethanol, and then the mother liquor removing solvent under reduced pressure; After dissolving EA, washed with water to wash away the excess hydrazine, and finally the organic phase the solvent was removed to give 165g intermediate, i.e. 1-acetyl-5- (2-aminopropyl) indoline;

Step 3) In the three-necked flask, 165g of Intermediate 1-acetyl-5- (2-aminopropyl) indoline, dissolved 600mL methanol, stirred at room temperature, and thereto was slowly added dropwise bromine; the addition was complete After stirring at room temperature 5-6h; After completion of the reaction, slowly poured into saturated NaHSO3, and wash away excess bromine; extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate; After filtration, the solvent was removed in vacuo to give the crude product recrystallized from toluene to give 177g pure product;

Step 4) In a flask was added 177g of pure product obtained in Step 3 and 65g CuCN, after use 700mL DMF, was heated at 110 ° C for 3 to 5h; After completion of the reaction, the amount of water was added thereto, washing off excess solvent DMF and salt, EA desolventizing crude extract, after recrystallization 121g pure 1-acetyl-5- (2-bromo-propyl) -7-cyano-indoline that silodosin important intermediates;

Step (1), (2), (3), (4) synthesis reaction is:

Further, the step I) to step 4) by TLC plate tracking point detection reaction.

The beneficial effects of the present invention are:

Preparation silodosin important intermediate of the present invention, mention of the method is simple, high reaction yield, product easily separated, suitable for industrial production and so on.

Preparation Method  A silodosin important intermediate, comprising the following steps: Step I) in a flask, 282g of raw materials 1-acetyl-5- (2-bromo-propyl) indoline, 222g o phthalimide potassium and 700mL DMF, heated at 110 ° C for 2h; After completion of the reaction, to which was added the right amount of water to wash away the excess solvent DMF and salt extraction desolventizing after EA, was 296g crude;

Step 2) In a flask was added 296g crude product obtained in step I, dissolved 800mL ethanol, was added 165mL of hydrazine hydrate, 50 ° C is heated to precipitate a white solid; After completion of the reaction, cooling suction filtered, the filter cake washed with ethanol, and then the mother liquor removing solvent under reduced pressure; After dissolving EA, washed with water to wash away the excess hydrazine, and finally the organic phase the solvent was removed to give 165g intermediate, i.e. 1-acetyl-5- (2-aminopropyl) indoline;

Step 3) In the three-necked flask, 165g of Intermediate 1-acetyl-5- (2-aminopropyl) indoline, dissolved 600mL methanol, stirred at room temperature, and thereto was slowly added dropwise bromine; the addition was complete After stirring at room temperature 5-6h; After completion of the reaction, slowly poured into saturated NaHSO3, and wash away excess bromine; extracted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate; After filtration, the solvent was removed in vacuo to give the crude product recrystallized from toluene to give 177g pure product;

Step 4) In a flask was added 177g of pure product obtained in Step 3 and 65g CuCN, after use 700mL DMF, was heated at 110 ° C for 3 to 5h; After completion of the reaction, the amount of water was added thereto, washing off excess solvent DMF and salt, EA desolventizing crude extract, after recrystallization 121g pure 1-acetyl-5- (2-bromo-propyl) -7-cyano-indoline that silodosin important intermediates;

Step (1), (2), (3), (4) synthesis reaction is:

Further, the step I) to step 4) by TLC plate tracking point detection reaction.

…………………………………………………

WO2013056842

http://www.google.com/patents/WO2013056842A1?cl=en

Silodosin is commercially available under the tradenames RAPAFLO^® or

UROPvEC as a capsule formulation for oral use containing 4 mg or 8 mg of the drug. The capsules are to be taken orally once daily for the treatment of the signs and symptoms of benign prostatic hyperplasia. US 5,387,603 and EP 0 600 675 disclose silodosin as a therapeutic agent for the treatment for dysurea associated with benign prostatic hyperplasia. The molecular structure of silodosin (XXV) is shown below.

(XXV)

The synthesis of silodosin is relatively complex and requires a sequence of multiple steps. A key intermediate compound in the synthesis of silodosin is the optically active amine compound represented by the general formula R-Y:

1

wherein, R represents a protecting group and R represents a cyano (CN) or carbamoyl (CONH₂) group. The intermediate compound R-Y bears the asymmetric carbon atom that imparts the optical activity to silodosin. Therefore, it is important to obtain the compound R-Y with high optical purity, because according to the methods reported in the state of the art the optical purity of the compound R-Y determines the optical purity of the final product silodosin.

JP 2001-199956 discloses a process for the preparation of a compound of formula R-Y, wherein l-(3-benzoyloxypropyl)-7-cyano-5-(2-oxopropyl)-2,3- dihydroindole or the corresponding 7-carbamoyl derivative is reacted with an optically active amine, namely L-2-phenylglycinol or L-l-phenylethanamine, to afford an imine compound of formula III as depicted in the below scheme 1. Scheme l . JP 2001-199956

R¹ = COPh; R² = CN or CONH₂; R³ = H or OH a = 1. cat. deprotection

2. frational crystallization with L-tartaric acid

b = 1. chromatographic separation

2. cat. deprotection

The optically active imine III is subjected to catalytic hydrogenation using platinum(IV) oxide as a catalyst affording the diastereomers IV in a ratio of 3.8:1. The chiral auxiliary II is subsequently removed by catalytic hydrogenation using 10% palladium on carbon, i. e. under the typical conditions which lead to the cleavage and removal of benzylic protecting groups from nitrogen or oxygen atoms. The catalytic deprotection reaction affords the desired intermediate compound R-Y with an optical purity corresponding to the ratio of the diasteromers obtained in the previous step, i. e. the ratio of compound R-Y to S-Y is approximately 3.8: 1, which corresponds to an optical purity of approximately 58.3% enantiomeric excess (e.e.).

In order to increase the optical purity of the intermediate R-Y JP 2001-199956 suggests to conduct a fractional crystallization of the desired enantiomer with L-tartaric acid. After a series of fractional crystallizations the compound R-Y is obtained with an optical purity of 97.6% enantiomeric excess. Alternatively, the diastereomers of the compound of formula IV are separated using chromatographic techniques as column chromatography on silicagel. The pure diastereomer R-TV affords the desired enantiomer R-Y with an optical purity of 100% e.e. after removal of the chiral auxiliary II with hydrogen using 10% palladium on carbon as catalyst.

Another approach for the synthesis of the key intermediate compound R-Y is reported in JP 2002-265444. The route of synthesis disclosed in said document is depicted in the below scheme 2.

Scheme 2. JP 2002-265444

R¹ = CH₂Ph (Bn); R² = CN The process involves the reaction of an enantiomeric mixture of the compound of formula VI with (I S, 2R)-2-benzylaminocyclohexane methanol (VII) to obtain a diastereomeric mixture containing the salt VIII. After a series of crystallizations the diastereomer VIII was obtained with an optical purity of 92.8% diastereomeric excess (d.e.). Subsequently, the salt VIII was treated with an acidic aqueous solution to release the acid R-Vl from the salt. After extraction from the aqueous solution with ethyl acetate the acid R-Vl is converted into its amide IX. The compound IX is finally subjected to a Hofmann type rearrangement reaction to obtain the desired intermediate compound R-V.

WO 201 1/030356 discloses a process for the preparation of the intermediate compound R-V, which avoids the resolution of the enantiomers of specific intermediate compounds using chiral auxiliaries or optically active bases. The route of synthesis described in WO 201 1/030356 starts from L-alanine (X), which is a naturally occurring optically active amino acid. The process described in

WO 2011/030356 is depicted in the below scheme 3.

R¹ = trimethylsilyl (TMS), tert-butyl dimethylsilyl (TBDMS), allyl, benzyl, propargyl R² = CN or CONH₂ The amino acid is protected by the addition of ethyl chloroformate and subsequently activated by the addition of oxalyl chloride to afford i?-(N-ethoxycarbonyl)alanine as an acyl chloride (XI). Said acyl chloride is reacted with hydroxy protected l-(3- hydroxypropyl)-7-cyano-2,3-dihydroindole of formula XII in a Friedel-Crafts acylation reaction, which gives a compound of formula XIII. The oxo group in compound XIII is reduced to afford a compound of formula XIV that is subsequently subjected to a hydrolysis reaction to yield the key intermediate compound R-Y. It is an object of the present invention to provide a process for preparing silodosin or a pharmaceutically acceptable salt thereof, which process affords the drug with high optical purity and with better yield compared to the prior art processes. This object is solved by the subject matter as defined in the claims.

Scheme 5. Conversion of com ound V to silodosin

R = protecting group

R² = CN or CONH₂

X = leaving group

Example 11. Silodosin (XXV)

A. The compound XXIV (18.0 g) was dissolved in methanol (150 ml) and 5% aqueous sodium hydroxide solution (50 ml). The reaction mixture was stirred at room temperature for 2 h. The deprotected compound XXIV, i. e. a compound of formula XXIV with R = hydrogen and R = cyano, was extracted with toluene. Subsequently, a 10% lactic acid solution (25 ml) was added to the toluene phase in order to extract the product in the aqueous phase. The aqueous solution was separated and then basified. The deprotected product was finally extracted with ethyl acetate. Removal of the solvent gives the deprotected compound to XXIV (R¹ = H and R² = CN; 1 1.0 g) as an oily mass.

B. A mixture of compound XXIV (R¹ = H and R² – CN; 10.0 g), DMSO (80 ml) and 5N NaOH solution (9.0 ml) was stirred for 15 min. at room temperature. An aqueous H₂0₂ (30%) solution (1 1.0 ml) was added to the reaction mixture, which was stirred at room temperature for additional 2 h after completion of the addition. Water was added to the reaction mixture, the product was extracted with ethyl acetate, and the solvent was subsequently evaporated to afford 9.0 g crude silodosin.

Example 12. Silodosin (XXV)

10.0 g of crude silodosin (optical purity = 85.0% e.e.) was dissolved in ethyl acetate (120 ml) at 55°C. The resulting clear solution was gradually cooled to 25°C under stirring. The suspension was further cooled to 15°C and stirred for 2 hours. The precipitated solid was filtered and dried at 50°C under vacuum to obtain 7.2 g of XXV with an optical purity of 97.5% e.e.

Example 13. Silodosin (XXV)

10.0 g of crude silodosin (optical purity = 98.5% e.e.) was dissolved in ethyl acetate (120 ml) at 55°C. The resulting clear solution was gradually cooled to 25 °C under stirring. The suspension was further cooled to 15°C and stirred for 2 hours. The precipitated solid was filtered and dried at 50°C under vacuum to obtain 7.2 g of XXV with an optical purity of 99.9% e.e.

Example 14. Silodosin (XXV)

10.0 g of crude silodosin (optical purity = 90.0 %e.e.) was dissolved in ethyl acetate (120 ml) at 55°C. The resulting clear solution was gradually cooled to 25°C under stirring. The suspension was further cooled to 15°C and stirred for 2 hours. The precipitated solid was filtered and dried at 50°C under vacuum to obtain 7.2 g of XXV with an optical purity of 97.0% e.e.

Example 15. Silodosin (XXV)

10.0 g of crude silodosin (optical purity = 92.0% e.e.) was dissolved in isopropyl acetate (160 ml) at 55°C. The resulting clear solution was gradually cooled to 25°C under stirring. The suspension was further cooled to 15°C and stirred for 2 hours. The precipitated solid was filtered and dried at 50°C under vacuum to obtain 8.2 g of XXV with an optical purity of 98.0% e.e. Example 16. Silodosin (XXV)

10.0 g of crude silodosin (optical purity = 98.0% e.e.) was dissolved in isopropyl acetate (160 ml) at 55°C. The resulting clear solution was gradually cooled to 25°C under stirring. The suspension was further cooled to 15°C and stirred for 2 hours. The precipitated solid was filtered and dried at 50°C under vacuum to obtain 8.0 g of XXV with an optical purity of 99.5% e.e.

………………………………

EP2475634

http://www.google.com/patents/EP2475634A2?cl=en

Scheme- 1.

Scheme-2.

Scheme-3.

Scheme-4.

Scheme-5.

Example-14

Preparation of Preparation of l-(3-Hydroxy-propyl)-5-(2(R)-{2-[2-(2, 2, 2-trifluoro- ethoxy)-phenoxy]-ethyIamino}-propyl)-2,3-dihydro-lH-indol-7-carboxylic acid amide (I)(Silodosin)

To a solution of Benzoic acid 3-[5(R)-(2-amino-propyl)-7-cyano-2, 3-dihydro-indol-l- yl]-propyl ester (XV) (3.5 g, 10 mmole) in Dimethyl sulphoxide (60 ml), charged Hydrogen peroxide (10% w/w) (11 ml). Then added 5 N sodium hydroxide solution (12.3 ml) and reaction mass was stirred for 2 hours. After completion of reaction water was added and extracted the product in ethyl acetate. Organic layer was washed with brine and dried over sodium sulphate. The solvent was evaporated below 40°C under reduced pressure and added methanol (25 ml). To this solution charged glacial acetic acid (0.25 g, 4mmole) and [2-(2, 2, 2-Trifluoro-ethoxy)-phenoxy]-acetaldehyde (VIII) (3 g, 0.0125 mole). Reaction mixture was stirred at 25-30°C for 1 hour. Then reacted with sodium cyanoborohydride (0.15 g, 2.8 mmoles) and heated at 40-45°C for 2 hours. After the completion of reaction solvent was distilled off below 40°C under reduced pressure and added water to the residue. Reaction mass was then acidified with aqueous mineral acid. The aqueous layer was then basified and product was extracted in ethyl acetate. Organic layer was washed with water and dried over sodium sulphate. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel using a mixture of ethyl acetate and hexane (5/95) as eluent to give 0.8g of (I) as yellow solid. Purity (by HPLC) = 98%

Example 15

Preparation of l-(3-hydroxypropyl)-5-[(2R)-({2-[2-(2, 2, 2-trifIuoroethoxy) phenoxy]-ethyl} amino) propyl]-2, 3-dihydro-lH-indole-7-carbonitriIe (XVII) A mixture of 3-[7-Cyano-5 (R)-[-2-{2-[2-(2,2,2-trifluoroethoxy)-phenoxy] ethyl} amino) propyl]-2,3-dihydro-lH-indol-l-yl}propyl benzoate (XVI) (6.0 g , 0.010 mole), methanol (30 ml) and aqueous solution of Sodium hydroxide ( 1.6 g in 8 ml of water) was stirred at ambient temperature for 6 hours. To the reaction mixture water (90ml) was added and product was extracted with ethyl acetate (90 ml). The organic layer was washed with saturated sodium bicarbonate solution followed by brine wash and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 3.85 g of (XVII). Example 16

Preparation of l-(3-Hydroxy-propyl)-5(R)-(2-{2-[2-(2, 2, 2-trifluoro-ethoxy)- phenoxy]-ethylamino}-propyl)-2, 3-dihydro-lH-indol-7-carboxylic acid amide (I) (Silodosin)

To a solution of l-(3-hydroxypropyl)-5(R)-[2-({2-[2-(2,2,2-trifluoroethoxy)phenoxy]- ethyl}amino)propyl]-2,3-dihydro-lH-indole-7-carbonitrile (XVII) (6.0 g , 0.013 mole) in dimethylsulfoxide (75 ml) was added 5 N sodium hydroxide solution (4.5 ml). To this reaction mixture, 30 % hydrogen peroxide (2.63 ml) was added slowly below 25°C. Reaction mixture was stirred at ambient temperature for 6 hours. Aqueous solution of sodium sulfite (2.1 in 150 ml water) was added to the reaction mixture. The reaction mixture was extracted with ethyl acetate. The combined ethyl acetate layer was extracted 2N hydrochloric acid. The aqueous layer was neutralized with sodium bicarbonate and extracted the product in ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution followed by brine wash and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was cooled to 5°C and filtered to get 4.51 g of (I) as solid.

…………………………………………………

WO2012147019

http://www.google.com/patents/WO2012147019A1?cl=en

The present invention provides a process for the preparation of Silodosin of formula (I). More particularly, the present invention provides the process for preparation of tartrate salt of 3-[7-cyano-5[(2R)-2-({2-[2-(2,2,2- trifluoroethoxy)phenoxy ] ethyl } amino)propyl] -2, 3 -dihydro- 1 H-indol- 1 -y 1 } propyl benzoate of formula (IV), which is a precursor in the preparation of Silodosin.

Background of the Invention:

A compound of 3-[7-cyano-5[(2R)-2-({2-[2-(2,2,2-trifluoroethoxy) phenoxy] ethyl}amino)propyl]-2,3-dihydro-lH-indol-l-yl}propyl benzoate (IV) is a key intermediate for preparation of Silodosin. The chemical name of Silodosin is l-(3- hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl} amino) propyl]-2,3-dihydro-lH-indole-7-carboxamide and structurally represented as

(IV)

(I)

U.S.Pat. No. 5,387,603 discloses Silodosin as therapeutic agents for the treatment of dysuria, urinary disturbance associated with benign prostatic hyperplasia.

U.S.Pat. No. 6,310,086 discloses a process for preparing a Silodosin analogue compound from reaction of (R)-3-{5-(2-aminopropyl)-7-cyano-2,3- dihydro- 1 H-indol- 1 -yl jpropylbenzoate with 2-(2-Ethoxyphenoxy)ethyl methane sulfonate and finally isolated as residue and purified by column chromatography on silicagel. The said literature process has certain drawbacks like use of column chromatography.

U.S.Pat. No. 7,834,193 (IN 3178/DELNP/2007) discloses the process for preparation of monooxalate salt of 3-{7-cyano-5-[(2R)-2-({2-[2-(2,2,2- trifluoroethoxy)phenoxy ] ethyl } amino)propyl] -2, 3 -dihydro- 1 H-indol- 1 -y 1 } propyl benzoate (IV). This patent specifically discloses the preparation of monooxalate salt of formula (IV) helps to remove N,N-dialkyl impurity to certain extend. CN 101993405 A discloses the reaction of (R)-5-(2-aminopropyl)-l-(3-(4- fluorobenzoyloxy)propyl)-7-cyanoindoline with 2-(2-(2,2,2-trifluoroethoxy) phenoxy)ethyl methane sulfonate followed by oxalic acid salt preparation.

The main drawback in the prior art process, the formation of N,N-dialkyl impurity compound of formula (VI), as disclosed in detailed description, in the preparation of Silodosin, during condensation of compound of formula (II) with compound of formula (III), the impurity which is not removable by crystallization method or precipitation technique and column chromatography purification is not suitable for commercial purpose. So considering the commercial importance of Silodosin, the present invention focus on the preparation of pure Silodosin, and surprisingly found that the isolation of formula (IV) as tartrate salt helps to prepare Silodosin having less than 0.2 % of N,N dialkyl impurity and with good yield. None of the prior arts teaches or motivates isolation of tartaric acid addition salt of formula (IV). The preparation of Silodosin from tartrate salt of 3-{7-cyano-5-[(2R)- 2-({2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl}amino)propyl]-2,3-dihydro-lH- indol-l-yl} propyl benzoate (IV) or its freebase of the present invention has purity of greater than 99.6 %.

Example 3

Preparation of l-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2-trifluoroethoxy) phenoxy] ethyl-} amino) propyl]-2,3-dihydro-lH-indole-7-carboxamide (Silodosin)

Method A: The compound of l-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2- trifluoroethoxy)phenoxy] ethyl- } amino)propyl] -2,3 -dihydro- 1 H-indole-7- carbonitrile of formula (V) in dimethylsulfoxide was treated with 48% hydrogen peroxide and 20% sodium hydroxide solution and stirred at room temperature till completion of reaction. After completion of reaction, reaction mass quenched with 5% sodium bisulphite solution and ethylacetate was added over it. The ethylacetate layer was separated and treated with 20 % aqueous hydrochloric acid. The aqueous layer separated, neutralized with sodium bicarbonate solution and extracted with ethylacetate. The separated organic layer was washed with 10% sodium bicarbonate solution, brine solution and dried under vacuum. The organic layer distilled upto residue under vacuum at 50-55°C. The obtained residue was crystallized in ethylacetate.

Method B: To the tartrate salt of 3-[7-cyano-5[(2R)-2-({2-[2-(2,2,2- trifluoroethoxy) phenoxy] ethyl}amino)propyl]-2,3-dihydro-lH-indol-l-yl}propyl benzoate (IV) (100 grams) in methanol, aqueous potassium hydroxide solution (38.38 grams) was added and stirred at room temperature till reaction completion. After completion of reaction, DM water and dichloromethane was added over it under stirring. Organic layer separated, washed with brine solution distilled under vacuum upto less than 1 volume. To the solution, dimethyl sulphoxide, 20% sodium hydroxide and hydrogen peroxide was added and stirred till completion of reaction. After completion of reaction, water containing sodium bisulfite was added to the reaction mass. The pH of the reaction mixture adjusted to about 8.5 using 10% sodium hydroxide and extracted in dichloromethane twice, washed with water, dried and concentrated upto 1-2 volume under vacuum. To the obtained solution, toluene was added over it at room temperature under stirring. The reaction mixture maintained for complete solid formation, filtered and dried under vacuum. Yield 58 grams. Example 4

Purification of Silodosin:

Method A: To the mixture of toluene and acetonitrile solvent, Silodosin was added over it and heated to 50° – 55 °C for complete dissolution. The reaction mass gradually cooled to room temperature and maintained for completion of solid formation. The obtained solid is filtered, washed with toluene and dried under vacuum. Method B: To the mixture of ethyl acetate and toluene solvent, Silodosin was added over it and heated to 60° – 65 °C for complete dissolution. The reaction mass gradually cooled to room temperature and maintained for completion of solid formation. The obtained solid is filtered, washed with toluene and dried under vacuum.

//////////////////////////////////////////

CN101993407

http://www.google.com/patents/CN101993407B?cl=en

silodosin for selective inhibition of urethral smooth muscle contraction and reduce the pressure within the urethra, but no significant impact on blood pressure, for the treatment of benign prostatic hyperplasia. At present, the method of synthesis Silodosin many reports, but the lack of high yield method for industrial production.

  JP200199956 reported that benzoic acid as a starting material, 1_ (3_ benzoyloxy-propyl) indoline hydrochloride (structural formula (1), R is a hydrogen atom) in 60% yield, then through the multi-step reaction was further prepared silodosin intermediate 1- (3-benzoyloxy-propyl) -5- (2-nitro-propyl) -7-cyano-indoline (structural formula VIII ), the total yield is low, and only 20 percent. Compound (VIII) with potassium carbonate, the reaction of hydrogen peroxide to yield compound (IX), impurities, and purified by column chromatography to be not suitable for industrial production. Compound (IX) under catalysis of molybdenum oxide, and L- (S) – benzyl glycyl alcohol asymmetric reactions, protecting groups may be due to steric hindrance is small, low chiral induction, is 3.8: I.

…………………………………………………..

WO 2015015512

see

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=E9E91192EB93FE4A861ABF346BF6AD06.wapp1nB?docId=WO2015015512&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Silodosin is (I) (formula 1, claim 1, page 31).

Process for the prepartion of pure polymorphic gamma form of silodosin – comprising dissolving any polymorphic form of silodosin in a solvent and seeding gamma form of silodosin.

Crude (I) (50 g) was dissolved in methanol, filtered and solvent was distilled under vacuum. The residue was dissolved in isopropanol at 50 degreeC, cooled and seed of (I) gamma form was added and further cooled and cyclohexane (500 mL) was added, solid was filtered, washed and dried to obtain pure polymorphic form gamma of (I) having a toluene content of 12 ppm (example 10, pages 29-30).

A process for the preparation of silodosin and/or its salt is claimed, comprising the reaction of 3-[5-((2R)-2-aminopropyl)-7-cyano-2,3-dihydro-1H-indol-1-yl]propyl benzoate(2R,3R)-monotartrate with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methanesulfonate to form a cyano benzyloxy intermediate, followed by hydrolysis to form a cyano hydroxy intermediate, which is then reacted with tartaric acid and hydrolyzed in the presence of an oxidizing agent to obtain the product. An alternate method of preparation of silodosin comprising the hydrolyses of tartrate salt of cyano hydroxy intermediate in the presence of an oxidizing agent, pure polymorphic form gamma of silodosin, and the cyano hydroxy intermediate are also claimed. Further processes for the prepartion of the pure polymorphic form gamma of silodosin are claimed, wherein the process involves the dissolution of of any polymorphic form of silodosin in a solvent by heating at 30-100 degree C, cooling before and after seeding with gamma form of silodosin, adding an antisolven, isolating the polymorph and optionally micronizing.

The present invention provides an improved and efficient process for the preparation of

It acts as an selective ai -adrenoceptor antagonist and is useful in the symptomatic treatment of benign prostatic hyperplasia (BPH). Chemically it is known as l-(3-hydroxypropyl)-5-[(2R)- ( { 2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino) propyl] indoline-7-carboxamide.

Silodosin and its pharmaceutically acceptable salts are first disclosed in US patent 5,387,603. Synthetic approach for the production of silodosin, is described in patent ‘603 can be represented as shown below in scheme 1.

l

Scheme 1

As represented in scheme 1, silodosin is prepared by the reaction of l-acetyl-5-(2r aminopropyl)indoline-7-carbonitrile with 2-[2-(2,2,2-trifiuoroethoxy)phenoxy] ethyl methanesulfonate in the presence of sodium bicarbonate in ethanol to give l-acetyl-5-[2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino]propyl]indoline-7-carbonitrile, which upon reaction with di-tert-butyldicarbonate in methylene chloride produces protected acetyl indoline carbonitrile compound. Further deacetylation with sodium hydroxide in ethanol followed by treatment with acetic acid provides protected indoline carbonitrile compound, which upon hydrolysis using dimethyl sulfoxide, 30% hydrogen peroxide, sodium hydroxide and acetic acid gives protected indoline carboxamide, which upon further reaction with 2-tert-butyldimethylsiloxy)ethyl-4-nitrobenzene sulfonate in the presence of cis-dicyclohexano-18 crown-6 and potassium carbonate in dioxane gives protected (tert-butyl-dimethylsiloxy) ethyl indoline carbonitrile. Further treatment with tetrabutylammonium fluoride in tetrahydrofuran produces N-boc protected hydroxy deprotected propyl indoline carbonitrile, which under goes facile deprotection of boc group upon treatment with trifluoroacetic acid, in methylene chloride to yield silodosin. The complete process is very complex, make use of pyrophoric reagents

which are very difficult to handle in large scale and have many extra steps involving protection and depfotection. Further in US patent ‘603, concrete detail of preparation and purification of silodosin have not been reported. Furthermore, isolated silodosin is characterized using IR, NMR and specific rotation but the patent is silent on product appearance and crystalline nature. There are several processes known for the preparation of silodosin and its intermediates viz; in JP 4634560; JP 4921646; JP-2006- 188470; WO2011/124704 and WO2011/101864. In most of the inventions, silodosin is prepared by following reaction as shown in scheme 2. Major disadvantages of these processes are the formation of N,N dialkyl impurity, and other impurities which forms during the condensation of 3-[5-((2/?)-2-aminopropyl)-7-cyano-2,3-dihydro-lH-indol-l-yl]propyl benzoate or its salts like monotartrate with 2-[2-(2,2,2-trifluoroethoxy)phenoxy] ethyl methanesulfonate. N,N dialkyl impurity forms in about 12-15% and may form due to reaction of one molecule of benzoate compound with two molecules of methanesulfonate compound. Removal of this impurity is not possible by simple purification

wherein R is benzoyl, benzyl, tetrahydropyranyl, 2-trimethylsilylethyl, dinitrophenyl, diphenyl methyl and the like

Scheme 2

US patent 7,834,193 discloses a process for preparation of silodosin with similar condensation of 3-[5-((2R)-2-arriinopropyl)-7-cyano-2,3-dihydro-lH-indol-l-yl]pfopyl benzoate or its salts like monotartrate with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methanesulfonate, but 3-{7-cyano-5-[(2R)-2-({2-(2,2,2-trifluoroethoxy)-phenoxy]ethyl}amino)propyl)-2,3-dihydro-lH-indol-l-yl)-propylbenzoate is purified by preparing monooxalate salt as shown below in

scheme 3. This patent specifically prepares monooxalate salt of 3- {7-cyano-5-[(2R)-2-({ 2- (2,2,2-trifluoroethoxy)-phenoxy]ethyl }amino)propyl)-2,3-dihydro-lH-indol-l-yl)-propyl benzoate to remove N,N÷dialkyl impurity, but impurity has not been removed completely, only a certain % of it, has been removed.

Scheme 3

In PCT publication WO2012/131710, preparation of silodosin is described wherein improved processes for preparation of 3-[5-((2R)-2-aminopropyl)-7-cyano-2,3-dihydro-lH-indol-l- yl]propyl benzoate have been disclosed which is then converted to silodosin by condensation with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methanesulfonate. In exemplified process, 3-[5- ((2R)-2-aminopropyl)-7-cyano-2,3-dihydro-lH-indol-l-yl]propyl benzoate is condensed with 2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethyl methanesulfonate and the resulting benzoate compound is hydrolyzed to give l-(3-hydroxy propyl)-5-[(2R)-2-({ 2-[2,2,2-trifluoroethoxy) phenoxy] ethyl }amino)propyr]-2,3-dihydro-lH-indol-7-carbonitrile.The carbonitrile compound is treated with oxalic acid to prepare its oxalate salt having purity greater than 99%, which is then hydrolyzed using a base to prepare free carbonitrile compound having purity greater than 99%, but this patent is silent about N, N- dialkyl impurity or its removal.

In PCT publication WO2012/147019, preparation of silodosin using 3-{ 7-cyano-5-[(2R)-2-({2- (2,2,2-trifluoroethoxy)-phenoxy]ethyl}amino)propyl)-2,3-dihydro-lH-indol-l-yl)-propyl benzoate tartrate salt has been described as shown below in scheme 4.

Scheme 4

One other PCT publication WO2012/147107 describes preparation of silodosin by preparing hydrochloride and acetic acid salts of l-(3-hydroxypropyl)-5-[(2R)-2-({2-[2,2,2-trifluoroethoxy) phenoxy] ethyl }amino)propyl]-2,3-dihydro-lH-indol-7-carbonitrile to remove N,N dialkyl impurity. It has been observed that in exemplified process, wherein hydroxy compound namely l-(3-hydroxy propyl)-5-[(2R)-2-({2-[2,2,2-trifluoroethoxy) phenoxy] ethyl }amino)propyl]-2,3-dihydro-lH-indol-7-carbonitrile is purified by preparing its acetate salt to, remove the impurities but still N, N-dialkyl impurity remains in an amount of 0.6%, which is difficult to remove in next stage or require extra purifications.

Beside to use highly pure silodosin, use of a pure polymorphic form of API is an essential requirement of drug formulation, these both aspects when address jointly, and obtained silodosin can be converted to pure polymorph then only a complete solution of prior art problems can be achieved. Apart from above mentioned process patents/publications which aimed to prepare the pure silodosin, there are exist some polymorph patents/publications which also aims to prepare pure polymorphic form of silodosin.

Polymorphism is considered as one of the- most important solid-state property of drug substance, since different polymorph have different physiochemical and biological properties and in pharmaceutical chemistry it is often desired to obtain one particular form that is biologically active and also offers ease of handling during formulation. The available literature references related to polymorph of silodosin are incorporated herein.

Japanese patent 3331048 (publication No.H07-330726), discloses a process for purification of silodosin wherein silodosin is dissolved in ethyl acetate, dried over anhydrous magnesium sulfate, solvent is distilled off and again dissolved in ethyl acetate at 70°C and crystallizes below room temperature. The resulting product is characterized by melting point, IR, NMR and specific rotation. Here also disclosure is silent about polymorphic form of product.

US patent publication US2006/0142374A1 (equivalent European patent EP1541554B 1) discloses polymorphic forms of silodosin including three crystalline polymorphic form of silodosin which are named as alpha (a), beta (β) and gamma (γ) and one amorphous form. These polymorphic forms have been characterized by X-ray powder diffraction pattern. In the patent publication, processes for the preparation of all these three crystalline forms have been disclosed. In. a given process, form alpha is prepared by dissolving crude silodosin in appropriate amount of ethyl acetate, ethyl formate, acetone, methyl ethyl ketone, acetonitrile, tetrahydrofuran or mixture of acetone and acetonitrile (1: 1), preferably ethyl acetate under heating, allowing to stand at room temperature to precipitate the crystal gradually. Similarly, form beta is prepared by dissolving crude silodosin in appropriate amount of methanol under heating, adding petroleum ether as a anti-solvent, crystal precipitation is ensured using vigorous stirring.

In a second process, to prepare the form beta, crude silodosin is dissolved in ethanol or 1-propanol and the reaction mass is cooled quickly. The crystalline form gamma is prepared by dissolving crude silodosin in appropriate amount of toluene or a mixture of acetonitrile and toluene (1:4) or ethyl acetate and toluene (1: 19), preferably in toluene, under heating, cooling to room temperature and allowing to precipitate gradually upon standing. In a second process to prepare form gamma, crude silodosin is dissolved in 2-propanol and the crystals are precipitated by adding an appropriate amount of toluene. In spite of disclosing three crystalline polymorphic forms, the patent publication prefers preparation and use of form alpha by highlighting the problems faced for preparation and use of other forms. It is disclosed that crystal form beta has manufacturing difficulties at industrial scale since precipitation occurs only when the nonpolar antisolvent is added to warm solution which leads to inconsistency in quality of crystals.

With the second process for preparation of form beta, desired level of yield and purity has not been achieved. Further, according to this publication, preparation of gamma form involves use of toluene which can not be removed completely from final product, because of its high boiling point and raises the problem of residual solvent. In the case of toluene, a class 2 solvent, its limits should not be more than 890 ppm. In the exemplified process, toluene content has not been disclosed, which clearly reflects that product was not suitable for pharmaceutical composition having problem of high residual content of toluene. Furthermore patent publication also states that all the three crystal forms donot have any difference in hygroscopicity and stabilities.

Thereafter, several patents/publications disclose preparation of polymorphic forms alpha and beta. For example a PCT publication WO2012/147107 discloses a process for preparation of beta form using isopropyl acetate and methyl isobutylketone. In another PCT publication WO2012/077138, preparation of alpha and beta forms are disclosed using various solvent , system. Similarly, in a Chinese patent CN102010359, crystalline form beta is prepared by dissolving the crude silodosin in alcoholic solvent by heating and the product is crystallized by cooling or by adding an antisolvent such as ketone or ether.

European patent EP2474529 discloses new polymorphic forms delta (δ) and eta (ε) of silodosin by using a solvent (tetrahydrofuran) and antisolvent (n-heptane, n-hexane, cyclohexane, tert butylmethyl ether).Further it discloses conversion of delta form to beta form by just heating the delta form at a particular temperature. The form delta can also be transformed into form eta by. slurrying in aqueous methanol. One new crystalline form designated as delta has also been disclosed in a Chinese patent publication CN102229558. An Indian patent application 478/MUM/2010, also discloses a new polymorphic form Zy-S which is prepared by using solvent such as esters, aromatic hydrocarbons, ketones, and alcohols.

All the above disclosures are silent about the preparation of gamma form of silodosin and only available disclosure reports that gamma form have problem of residual solvent, as impurity and is not suitable for pharmaceutical compositions.

Method C: l-(3-HydroxypropyI)-5-[(2R)-2-({2-[2,2,2-trifIuoroethoxy)phenoxy] ethyl} amino) propyl]-2,3-dihydro-lH-indol-7-carbonitrile tartrate (lOg) dissolved in dimethylsulfoxide (120 ml) and to this solution, was added 5 mol/L aqueous sodium hydroxide solution (15ml). To the reaction mixture, 30% hydrogen peroxide (5ml) was added and keeping the temperature below 25°C. The reaction mixture was stirred at 20-25°C, for 5 hours. To the reaction mixture, sodium sulfite (5g) dissolved in water (100ml) was added slowly. The reaction mixture was extracted with ethyl acetate (1x200ml) and ethyl acetate layer was concentrated under reduced pressure. The resulting product was dissolved in methanol and clear solution was filtered through micron filter paper of size 0.22 micron two times and filtrate was concentrated.The resulting compound was dissolved in toliiene (70ml) and isopropyl alcohol (7ml) at 50-55°C and the solution was cooled to 20-25°C, cyclohexane was added and stirred for further 4 hours, filtered and dried to give title compound having purity 99.86% and N,N-dialkyl impurity not detected by HPLC. Example 5: Preparation of pure Polymorphic Form Gamma (γ) of Silodosin

Silodosin (15g) having toluene content 1872 ppm, was micronized under air pressure. The micronized product was dried under vacuum at 55°C-60°C for 23.0 hours to afford pure polymorphic form gamma of silodosin having toluene content 460 ppm.

Example 6: Preparation of pure Polymorphic Form Gamma (γ) of Silodosin

Silodosin [having toluene content 1327 ppm] was micronized under air pressure. The micronized product was dried under vacuum at 55°C-60°C for 16 hours to afford pure polymorphic form gamma of silodosin having toluene content 350 ppm.

Example 7: Preparation of pure Polymorphic Form Gamma (γ) of Silodosin

Silodosin crude (3.0g) was dissolved in isopropanol (12ml) at 50°C and reaction mass was cooled to 35°C and seed of silodosin gamma form (O.lg) was added. Thereafter reaction mass was again cooled to 15-20°C and cyclohexane (30ml) was added to the reaction mass and stirred for further 0.5 hour. The resulting solid, thus obtained, was filtered, washed with cyclohexane and dried to afford pure polymorphic form gamma of silodosin having toluene content 34 ppm.

References

External links

• Watson Pharm., Inc.- Press Release
• Rapaflo Official Site

a1a-Adrenoceptor antagonist. Prepn: M. Kitazawa et al., EP 600675; eidem, US 5387603 (1994, 1995 both to Kissei).PRODUCT PATENT

Adrenoceptor binding study: K. Shibata et al., Mol. Pharmacol. 48, 250 (1995); and tissue selectivity: S. Murata et al., J. Urol. 164, 578 (2000).

Pharmacology: K. Akiyama et al., Pharmacology 64, 140 (2002).

Series of articles on pharmacology, pharmacokinetcs and toxicology: Yakugaku Zasshi 126, 187-263 (2006).

Review of development and therapeutic potential: F. Kamali, Curr. Opin. Cent. Peripher. Nerv. Syst. Invest. Drugs 1, 248-252 (1999)

Panobinostat

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

HDAC inhibitors, orphan drug

cas 404950-80-7 

2E)-N-hydroxy-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide

N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2E-2-propenamide (alternatively, N-hydroxy-3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-acrylamide)

Molecular Formula: C₂₁H₂₃N₃O₂   Molecular Weight: 349.42622

• Faridak
• LBH 589
• LBH589
• Panobinostat
• UNII-9647FM7Y3Z

A hydroxamic acid analog histone deacetylase inhibitor from Novartis.

NOVARTIS, innovator

Histone deacetylase inhibitors

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

FDA Approves Farydak (panobinostat) for Multiple Myeloma

February 23, 2015 — The U.S. Food and Drug Administration today approved Farydak (panobinostat) for the treatment of patients with multiple myeloma.

Multiple myeloma is a form of blood cancer that arises from plasma cells, a type of white blood cell, found in bone marrow. According to the National Cancer Institute, approximately 21,700 Americans are diagnosed with multiple myeloma and 10,710 die from the disease annually

read at

http://www.drugs.com/newdrugs/fda-approves-farydak-panobinostat-multiple-myeloma-4170.html?utm_source=ddc&utm_medium=email&utm_campaign=Today%27s+news+summary+-+February+23%2C+2015&utm_content=FDA+Approves+Farydak+%28panobinostat%29+for+Multiple+Myeloma

AND

FDA approves Farydak for treatment of multiple myeloma [press release].http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm435296.htmPublished February 23, 2015. Accessed february 23, 2015

FLAGS AND HITS

New drug approvals stats as on feb end 2015, 5.66 LAKH VIEWS ACROSS 202 COUNTRIES

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

syn……….http://newdrugapprovals.org/2014/01/23/panobinostat/

FDA approves Farydak for treatment of multiple myeloma [press release].http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm435296.htmPublished February 23, 2015. Accessed february 23, 2015

Atrasentan

A-147627, (+)-A-127722, ABT-627,173937-91-2,

Endothelin ET-A antagonist

Diabetic nephropathy; End stage renal disease; Renal disease

Atrasentan is an experimental drug that is being studied for the treatment of various types of cancer,^[1] including non-small cell lung cancer.^[2] It is also being investigated as a therapy for diabetic kidney disease.

Atrasentan failed a phase 3 trial for prostate cancer in patients unresponsive to hormone therapy.^[3] A second trial confirmed this finding.^[4]

It is an endothelin receptor antagonist selective for subtype A (ET_A). While other drugs of this type (sitaxentan, ambrisentan) exploit the vasoconstrictive properties of endothelin and are mainly used for the treatment of pulmonary arterial hypertension, atrasentan blocks endothelin induced cell proliferation.

In April 2014, de Zeeuw et al. showed that 0.5 mg and 1.25 mg of atrasentan reduced urinary albumin by 35 and 38% respectively with modest side effects. Patients also had decreased home blood pressures (but no change in office readings) decrease total cholesterol and LDL. Patients in the 1.25 mg dose group had increased weight gain which was presumably due to increased edema and had to withdraw from the study more than the placebo or 0.5 mg dose group.^[5] Reductions in proteinuria have been associated with beneficial patient outcomes in diabetic kidney disease with other interventions but is not an accepted end-point by the FDA.

The recently initiated SONAR trial^[6] will determine if atrasentan reduces kidney failure in diabetic kidney disease.

Useful for treating nephropathy and chronic kidney disease associated with Type II diabetes. For a prior filing see WO2015006219 , claiming the stable solid composition in the form of a tablet comprising atrasentan and an anti-oxidant. AbbVie (following its spin-out from Abbott), is developing atrasentan (phase III; February 2015) for treating chronic kidney disease, including diabetic nephropathy.

……………….

European Journal of Organic Chemistry

Enantioselective Synthesis of the Pyrrolidine Core of Endothelin Antagonist ABT-627 (Atrasentan) via 1,2-Oxazines

Year:2003
Volume:2003
Issue:18
page:3524-3533

………………….

http://www.google.com/patents/US20080132710

EXAMPLE 1

A mixture of bromoacetyl bromide (72.3 mL) in toluene (500 mL) at 0° C. was treated with dibutylamine (280 mL) in toluene (220 mL) while keeping the solution temperature below 10° C., stirred at 0° C. for 15 minutes, treated with 2.5% aqueous phosphoric acid (500 mL) and warmed to 25° C. The organic layer was isolated, washed with water (500 mL) and concentrated to provide the product as a solution in toluene.

EXAMPLE 25-((E)-2-nitroethenyl)-1,3-benzodioxole

3,4-methylenedioxybenzaldehyde (15.55 Kg) was treated sequentially with ammonium acetate (13.4 Kg,), acetic acid (45.2 Kg) and nitromethane (18.4 Kg), warmed to 70° C., stirred for 30 minutes, warmed to 80° C., stirred for 10 hours, cooled to 10° C. and filtered. The filtrant was washed with acetic acid (2×8 Kg) and water (2×90 Kg) and dried under a nitrogen stream then in under vacuum at 50° C. for 2 days.

EXAMPLE 3ethyl 3-(4-methoxyphenyl)-3-oxopropanoate

A mixture of potassium tert-amylate (50.8 Kg) in toluene (15.2 Kg) at 5° C. was treated with 4-methoxyacetophenone (6.755 Kg) and diethyl carbonate (6.4 Kg) in toluene over 1 hour while keeping the solution temperature below 10° C., warmed to 60° C. for 8 hours, cooled to 20° C. and treated with acetic acid (8 Kg) and water (90 Kg) over 30 minutes while keeping the solution temperature below 20° C. The organic layer was isolated, washed with 5% aqueous sodium bicarbonate (41 Kg) and concentrated at 50° C. to 14.65 Kg.

EXAMPLE 4ethyl 2-(4-methoxybenzoyl)-4-nitromethyl-3-(1,3-benzodioxol-5-yl)butyrate

A mixture of EXAMPLE 3 (7.5 Kg) in THF (56 Kg) was treated with EXAMPLE 3 (8.4 Kg), cooled to 17° C., treated with sodium ethoxide (6.4 g), stirred for 30 minutes, treated with more sodium ethoxide (6.4 g), stirred at 25° C. until HPLC shows less than 1 area % ketoester remaining and concentrated to 32.2 Kg.

EXAMPLE 5ethyl cis,cis-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrrolidine-3-carboxylate

Raney nickel (20 g), from which the water had been decanted, was treated sequentially with THF (20 mL), EXAMPLE 4 (40.82 g), and acetic acid (2.75 mL). The mixture was stirred under hydrogen (60 psi) until hydrogen uptake slowed, treated with trifluoroacetic acid, stirred under hydrogen (200 psi) until HPLC shows no residual imine and less than 2% nitrone and filtered with a methanol (100 mL) wash. The filtrate, which contained 13.3 g of EXAMPLE 5, was concentrated with THF (200 mL) addition to 100 mL, neutralized with 2N aqueous NaOH (50 mL), diluted with water (200 mL), and extracted with ethyl acetate (2×100 mL). The extract was used in the next step.

EXAMPLE 6ethyl trans,trans-2-(4-methoxyphenyl)-4-(1,3 -benzodioxol-5 -yl)pyrrolidine-3-carboxylate

Example 501E (38.1 g) was concentrated with ethanol (200 mL) addition to 100 mL, treated with sodium ethoxide (3.4 g), heated to 75° C., cooled to 25° C. when HPLC showed less than 3% of EXAMPLE 1E and concentrated. The concentrate was mixed with isopropyl acetate (400 mL), washed with water (2×150 mL) and extracted with 0.25 M phosphoric acid (2×400 mL). The extract was mixed with ethyl acetate (200 mL) and neutralized to pH 7 with sodium bicarbonate (21 g), and the organic layer was isolated.

EXAMPLE 7ethyl (2R,3R,4S)-(+)-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrrolidine-3-carboxylate, (S)-(+) mandelate

EXAMPLE 501F was concentrated with acetonitrile (100 mL) addition to 50 mL, treated with (S)-(+)-mandelic acid (2.06 g), stirred until a solution formed, stirred for 16 hours, cooled to 0° C., stirred for 5 hours and filtered. The filtrant was dried at 50° C. under a nitrogen stream for 1 day. The purity of the product was determined by chiral HPLC using Chiralpak AS with 95:5:0.05 hexane/ethanol/diethylamine, a flow rate of 1 mL/min. and UV detection at 227 nm. Retention times were 15.5 minutes for the (+)-enantiomer and 21.0 minutes for the (−)-enantiomer.

EXAMPLE 8(2R,3R,4S)-(+)-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonylmethyl)pyrrolidine-3-carboxylic acid

A mixture of EXAMPLE 7 (20 g) in ethyl acetate (150 mL) and 5% aqueous sodium bicarbonate was stirred at 25° C. until the salt dissolved and gas evolution stopped. The organic layer was isolated and concentrated. The concentrate was treated with acetonitrile (200 mL), concentrated to 100 mL, cooled to 10° C., treated with diisopropylethylamine (11.8 mL) and EXAMPLE 1 (10.5 g), stirred for 12 hours and concentrated. The concentrate was treated with ethanol (200 mL), concentrated to 100 mL, treated with 40% aqueous NaOH (20 mL), stirred at 60° C. for 4 hours, cooled, poured into water (400 mL), washed with hexanes (2×50 mL then 2×20 mL), treated with ethyl acetate (400 mL) and adjusted to pH 5 with concentrated HCl (12 mL). The organic layer was isolated and concentrated.

………………….

The Michael reaction between 3,4-(methylenedioxy)-beta-nitrostyrene (I) and ethyl (4-methoxybenzoyl)acetate (II) in the presence of DBU gave adduct (III) as a mixture of isomers. Hydrogenation of this nitro ketone over Raney-Ni afforded, after spontaneous cyclization of the resulting amino ketone, the pyrroline (IV). Further reduction of the imine with NaBH3CN yielded a mixture of three pyrrolidine isomers. The desired trans-trans isomer (VI) could not be separated from the cis-trans isomer by column chromatography. However, the pure cis-cis compound (V) was isomerized to (VI) with NaOEt in refluxing EtOH. The protection of the amine as the tert-butyl carbamate with Boc2O, and saponification of the ester function provided the racemic acid (VII). Resolution of (VII) was achieved by conversion to the mixed anhydride (VIII) with pivaloyl chloride, followed by condensation with the lithium salt of (S)-4-benzyl-2-oxazolidinone (IX), and chromatographic separation of the resulting diastereomeric imides. Alternatively, racemic (VII) could be resolved by crystallization of its salt with (R)-a-methylbenzylamine. Removal of the Boc group from the appropriate isomer (X) with HCl in dioxan, followed by alkylation with N,N-dibutylbromoacetamide (XI) in the presence of i-Pr2NEt furnished the pyrrolidinylacetamide (XII). Finally, hydrolysis of the imide with lithium hydroperoxide provided the target acid.

Cyclization of 5-(2-nitrovinyl)-1,3-benzodioxole (I) with ethyl 2-(4-methoxybenzoyl)acetate (II) by means of DBU in THF gives the 4-nitrobutyrate (III), which is reduced with H2 over Ni in ethanol to the corresponding amine, which undergoes immediate cyclization to give the pyrroline carboxylate (IV). Reduction of pyrroline (IV) with NaCNBH3 in THF affords the expected pyrrolidine as a mixture of the (trans,trans)-(V), (cis,cis)-(VI) and (cis,trans)-(VII) isomers. Using chromatography on silica gel, only the (cis,cis)-isomer (VI) is separated and completely isomerized to the (trans,trans)-isomer (V) by treatment with NaOEt in refluxing ethanol. Pure (trans,trans)-isomer (V) or the remaining mixture of (trans,trans)-(V) and (cis,trans)-(VII) is N-protected with Boc2O in dichloromethane to provide a mixture of carbamates. Then hydrolysis of the esters is performed with NaOH in ethanol/water at room temperature, and under these conditions only the (trans,trans)-isomer hydrolyzes, giving the racemic (trans,trans)-acid (VIII). Unreacted (cis,trans)-ester (VII) is easily removed by conventional methods. Condensation of the racemic acid (VIII) with the lithium salt of the chiral oxazolidinone (IX) by means of pivaloyl chloride yields the corresponding amide as a diastereomeric mixture of (X) and (XI) that are separated by chromatography. The desired isomer (XI) is deprotected with HCl in dioxane to afford the chiral pyrrolidine (XII), which is condensed with 2-bromo-N,N-dibutylacetamide (XIII) by means of diisopropylamine in acetonitrile to give the adduct (XIV). Finally, the chiral auxiliary of (XIV) is eliminated by means of LiOOH (LiOH + H2O2) in water.

References

1

• “Atrasentan”. NCI Dictionary of Cancer Terms. National Institute of Cancer.
• 2
• Chiappori, Alberto A.; Haura, Eric; Rodriguez, Francisco A.; Boulware, David; Kapoor, Rachna; Neuger, Anthony M.; Lush, Richard; Padilla, Barbara; Burton, Michelle; Williams, Charles; Simon, George; Antonia, Scott; Sullivan, Daniel M.; Bepler, Gerold (March 2008). “Phase I/II Study of Atrasentan, an Endothelin A Receptor Antagonist, in Combination with Paclitaxel and Carboplatin as First-Line Therapy in Advanced Non–Small Cell Lung Cancer”. Clinical Cancer Research 14 (5): 1464–9. doi:10.1158/1078-0432.CCR-07-1508. PMID 18316570.
• 3
• “Addition of experimental drug to standard chemotherapy for advanced prostate cancer shows no benefit in phase 3 clinical trial” (Press release). National Cancer Institute. April 21, 2011. Retrieved October 18, 2014.
• 4
• Quinn, David I; Tangen, Catherine M; Hussain, Maha; Lara, Primo N; Goldkorn, Amir; Moinpour, Carol M; Garzotto, Mark G; Mack, Philip C; Carducci, Michael A; Monk, J Paul; Twardowski, Przemyslaw W; Van Veldhuizen, Peter J; Agarwal, Neeraj; Higano, Celestia S; Vogelzang, Nicholas J; Thompson, Ian M (August 2013). “Docetaxel and atrasentan versus docetaxel and placebo for men with advanced castration-resistant prostate cancer (SWOG S0421): a randomised phase 3 trial”. The Lancet Oncology 14 (9): 893–900. doi:10.1016/S1470-2045(13)70294-8. PMID 23871417.
• 5
• de Zeeuw, Dick; Coll, Blai; Andress, Dennis; Brennan, John J.; Tang, Hui; Houser, Mark; Correa-Rotter, Ricardo; Kohan, Donald; Lambers Heerspink, Hiddo J.; Makino, Hirofumi; Perkovic, Vlado; Pritchett, Yili; Remuzzi, Giuseppe; Tobe, Sheldon W.; Toto, Robert; Viberti, Giancarlo; Parving, Hans-Henrik (May 2014). “The endothelin antagonist atrasentan lowers residual albuminuria in patients with type 2 diabetic nephropathy”. Journal of the American Society of Nephrology 25 (5): 1083–93. doi:10.1681/ASN.2013080830. PMID 24722445.
• 6

Clinical trial number NCT01858532 for “Study Of Diabetic Nephropathy With Atrasentan (SONAR)” at ClinicalTrials.gov

US-8962675, AbbVie Inc

Granted in February 2015, this patent claims novel crystalline anhydrous S-mandelate salt of atrasentan. Useful for treating nephropathy and chronic kidney disease associated with Type II diabetes.

Atrasentan

Systematic (IUPAC) name
(2R,3R,4S)-4-(1,3-Benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid
Clinical data
Legal status	?
Identifiers
CAS number	173937-91-2
ATC code	None
PubChem	CID 159594
ChemSpider	140321
UNII	V6D7VK2215
ChEMBL	CHEMBL9194
Chemical data
Formula	C29H38N2O6
Molecular mass	510.621 g/mol

READ MORE  ON SENTAN SERIES………..http://medcheminternational.blogspot.in/p/sentan-series.html

READ MORE ON SENTAN SERIES……http://medcheminternational.blogspot.in/p/sentan-series.html

Antagonists of Endothelin type A receptor ETA
Name	Structure
BQ-123
Bosentan
Atrasentan
Tezosentan
Sitaxsentan
Darusentan
Clazosentan
ZD-4054 (Zibotentan)
Ambrisentan
Tak-044
Avosentan

Sitasentan,TBC 11251

210421-64-0

N-(4-chloro-3-methyl-1,2-oxazol-5-yl)-2-[2-(6-methyl-1,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide

Sitaxentan sodium (TBC-11251) is a medication for the treatment of pulmonary arterial hypertension (PAH).^[1] It was marketed as Thelin by Encysive Pharmaceuticals until Pfizer purchased Encysive in February 2008. In 2010, Pfizer voluntarily removed sitaxentan from the market due to concerns about liver toxicity.^[2]

Sitaxentan belongs to a class of drugs known as endothelin receptor antagonists (ERAs). Patients with PAH have elevated levels of endothelin, a potent blood vessel constrictor, in their plasma and lung tissue. Sitaxentan blocks the binding of endothelin to its receptors, thereby negating endothelin’s deleterious effects.

Mechanism of action

Sitaxentan is a small molecule that blocks the action of endothelin (ET) on the endothelin-A (ET_A) receptor selectively (by a factor of 6000 compared to the ET_B).^[3] It is a sulfonamide class endothelin receptor antagonist (ERA) and is undergoing Food and Drug Administration (FDA) review for treating pulmonary hypertension. The rationale for benefit compared to bosentan, a nonselective ET blocker, is negligible inhibition of the beneficial effects of ET_B stimulation, such as nitric oxide production and clearance of ET from circulation. In clinical trials, the efficacy of sitaxentan has been much the same as bosentan, but the hepatotoxicity of sitaxentan outweighs its benefits. Dosing is once daily, as opposed to twice daily for bosentan.

Regulatory status

On December 10, 2010 Pfizer announced it would be withdrawing sitaxentan worldwide (both from marketing and from all clinical study use), citing that it is a cause of fatal liver damage.^[2]

Sitaxentan was approved for marketing in the European Union in 2006, in Canada in 2006^[4] and in Australia in 2007. By February 2008 it had been launched commercially in Germany, Austria, The Netherlands, the United Kingdom, Ireland, France, Spain and Italy.

In March 2006, the FDA recommended an approvable status to sitaxentan but said it would not yet approve the product. In July 2006, sitaxentan received a second approvable letter stating that efficacy outcome issues raised in the context of the STRIDE-2 study were still unresolved. In July 2007, Encysive commenced a formal dispute resolution process in a preliminary meeting with the FDA. In September 2007 the company announced that it was making preparations for another phase III clinical trial (intended to be named STRIDE-5) to overcome the FDA’s concerns.^[5] The takeover by Pfizer resulted in a reconfiguration and extension of these plans, to include combination therapy with sildenafil. The Sitaxentan Efficacy and Safety Trial With a Randomized Prospective Assessment of Adding Sildenafil (SR-PAAS) was an ongoing program of three clinical trials conducted in the United States (ClinicalTtrials.gov identifiers: NCT00795639, NCT00796666 and NCT00796510) with anticipated completion dates between June 2010 and January 2014.

N-(4-Chloro-3-methyl-5-isoxazolyl)-2-[2-(6-methyl-1,3-benzodioxol-5-yl)acetyl]-3-thiophenesulfonamide sodium salt, Sitaxsentan sodium salt, TBC-11251 sodium salt, Thelin

• CAS Number 210421-74-2
• Empirical Formula  C₁₈H₁₄ClN₂NaO₆S₂
• Molecular Weight 476.89

Adverse effects

Adverse effects observed with sitaxentan are class effects of endothelin receptor antagonists, and include :

• liver enzyme abnormalities (increased ALT and AST)
• headache
• edema
• constipation
• nasal congestion
• upper respiratory tract infection
• dizziness
• insomnia
• flushing

Because sitaxentan inhibits metabolism of warfarin, a decreased dose of warfarin is needed when co-administered with sitaxentan. This is because warfarin acts to prevent blood from clotting, and if it remains unmetabolized, it can continue to thin the blood.

http://www.google.com/patents/WO2007149568A2?cl=en

As used herein “sitaxsentan” refers to N-(4-chloro-3-methyl-5-isoxazolyl)-2-[2- methyl-4,5-(methylenedioxy)phenylacetyl]-thiophene-3-sulfonamide. Sitaxsentan is also known as TBCl 1251. Other chemical names for sitaxsentan include 4-chloro-3-methyl-5-(2- (2-(6-methylbenzo[d][l ,3]dioxol-5-yl)acetyl)-3-thienylsulfonamido)isoxazole and N-(4- chloro-3-methyl-5-isoxazolyl)-2-[3,4-(methylenedioxy)-6-methylphenylacetyl]-thiophene-3- sulfonamide.

The chemical name for sitaxsentan is N-(4-chloro-3-methyl-5-isoxazolyl)-2-[2- methyl-4,5-(methylenedioxy)phenylacetyl]-thiophene-3-sulfonamide, and its structural formula is as follows:

Sitaxsentan

Sitaxsentan is a potent endothelin receptor antagonist that has oral bioavailability in several species, a long duration of action, and high specificity for ETA receptors.

EXAMPLE 1

Preparation of 4-chloro-3-methyl-5-(2-(2-(6-methylbenzo[d] [l,3|dioxol-5-yl)aeetyl)-3- thienylsulfonamido)isoxazole, or N-(4-chloro-3-methyl-5-isoxazolyl)-2-[2-methy 1-4,5- (methylenedioxy)phenylacetyl]-thiophene-3-sulfonamide, or N-(4-chIoro-3-methyl-5- isoxazolyl)-2-[3,4-(methylenedioxy)-6-methylphenylacetyl]-thiophene-3-sulfonamide.

A. Preparation of (4-chIoro-3-methyl-5-(2-(2-(6-methylbenzo[d] [l,3]dioxol-5-yl)acetyl)- 3-thienylsuIfonamido)isoxazole 1. Preparation of 5-chloromethyI-6-methylbenzo[d][l,3]dioxole

To a mixture of methylene chloride (130 L), concentrated HCl (130 L), and tetrabuylammonium bromide (1.61 Kg) was added 5-methylbenzo[d][l,3]dioxole (10 Kg) followed by the slow addition of formaldehyde (14 L, 37 wt% in water). The mixture was stirred overnight. The organic layer was separated, dried with magnesium sulfate and concentrated to an oil. Hexane (180 L) was added and the mixture heated to boiling. The hot hexane solution was decanted from a heavy oily residue and evaporated to give almost pure 5-chloromethyl-6-methylbenzo[d][l,3]dioxole as a white solid. Recrystallization from hexane (50 L) gave 5-chloromethyl-6-methylbenzo[d][l,3]dioxole (80% recovery after recrystallization). 2. Formation of (4-chloro-3-methyl-5-(2-(2-(2-methyIbenzo[d][l,3]dioxol-5-yl) acetyl)-3-thienylsulfonamido)isoxazole

A portion of a solution of 5-chloromemyl-6-methylbenzo[d][l,3]di-oxole (16.8 g, 0.09 mol) in tetrahydrofuran (THF)(120 mL) was added to a well stirred slurry of magnesium powder, (3.3 g, 0.136 g-atom, Alfa, or Johnson-Mathey, -20 +100 mesh) in THF (120 mL) at room temperature. The resulting reaction admixture was warmed up to about 40-45⁰C for about 2-3 min, causing the reaction to start. Once the heating activated the magnesium, and the reaction began, the mixture was cooled and maintained at a temperature below about 8 ⁰C. The magnesium can be activated with dibromoethane in place of heat.

A flask containing the reaction mixture was cooled and the remaining solution of 5- chloromethlybenzo[d][l,3]dioxole added dropwise during 1.5 hours while maintaining an internal temperature below 8 ⁰C. Temperature control is important: if the Grignard is generated and kept below 8 ⁰C₅ Wurtz coupling is suppressed. Longer times at higher temperatures promote the Wurtz coupling pathway. Wurtz coupling can be avoided by using high quality Mg and by keeping the temperature of the Grignard below about 8 ⁰C and stirring vigorously. The reaction works fine at -20 ⁰C, so any temperature below 8 ⁰C is acceptable at which the Grignard will form. The color of the reaction mixture turns greenish.

The reaction mixture was stirred for an additional 5 min at 0 ⁰C, while N²-methoxy- N²-methyl-3-(4-chloro-3-methyl-5-isoazolylsulfamoyl)-2-thiophenecarboxamide (6.6 g, 0.018 mol) in anhydrous THF (90 mL) was charged into the addition funnel. The reaction mixture was degassed two times then the solution of N²-methoxy-N²-methyl-3-(4-chloro-3- methyl-5-isoxazolylsulfamoyl)-2-thiophenecarboxamide was added at 0 ⁰C over 5 min. TLC of the reaction mixture (Silica, 12% MeOHZCH₂Cl₂) taken immediately after the addition shows no N²-methoxy-N²-methyl-3-(4-chloro-3-methyl-5-isoxazolysulfamoyl)-2-thio- phenecarboxamide. The reaction mixture was transferred into a flask containing IN HCl (400 mL, 0.4 mol

HCl, ice-bath stirred), and the mixture stirred for 2 to 4 min, transferred into a separatory funnel and diluted with ethyl acetate (300 mL). The layers were separated after shaking. The water layer was extracted with additional ethyl acetate (150 mL) and the combined organics washed with half-brine. Following separation, THF was removed by drying the organic layer over sodium sulfate and concentrating under reduced pressure at about 39 ⁰C to obtain the title compound. EXAMPLE 2

1.0 g Sitaxentan was dissolved in 10 ml ethyl acetate and 5 ml hexanes were added. The formed suspension was heated until a clear solution was obtained. Upon cooling light yellow plates were formed. After filtration and drying under vacuum 515 mg of sitaxentan polymorph A was obtained as light yellow plates in very high purity.

EXAMPLE 3

Preparation of 4-chloro-3-methyl-5-(2-(2-(6-methyIbenzo[dJ [l,3]dioxol-5-yl)acetyl)-3- thienylsulfonamido)isoxazole, Sodium Salt

The crystalline sitaxsentan from Example 2 is dissolved in ethyl acetate and washed with saturated NaHCO₃ (5 x 10 mL). The solution is washed with brine, dried over Na₂SO₄ and concentrated in vacuo to obtain a solid residue. 10 mL OfCH₂Cl₂ is added and the mixture is stirred under nitrogen for 5 to 10 minutes. Ether (15 mL) is added and the mixture stirred for about 10 min. The product is isolated by filtration, washed with a mixture of CH₂Cl₂ /ether (1 :2) (10 mL) then with ether (10 mL) and dried under reduced pressure to obtain 4-Chloro-3-methyl-5-(2-(2-(6-methyIbenzo[d][l ,3]dioxol-5-yl)acetyl)-3- thienylsulfonamido)isoxazole, sodium salt.

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1HNMR(CDCl3):88.88(brs,1H),7.59(s,2H),6.72(s,1H),6.69(s,111),5.94(s,2H),4.22(s,2H),2.22(s,311),2.21(s,3H);

IR(KBrpellet):3455,3233,

3109,2899,1674,1632,1505,1487,1395,1373cm-1;

HRMS:[M+H]*455.0137

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see

Current Opinion in Investigational Drugs (PharmaPress Ltd.) (2001), 2(4), 531-536.

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Synthesis of Sitaxsentan sodium
Yingyong Huaxue (2007), 24, (11), 1310-1313. Publisher: (Kexue Chubanshe, ) CODEN:YIHUED ISSN:1000-0518.

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http://www.google.co.in/patents/WO2007106494A2?cl=en

Table 1: Sitaxsentan Sodium Lyophilized Formulation

References

• 2
• Citing liver damage, Pfizer withdraws Thelin, Associated Press, December 12, 2010
• 3
• Girgis, RE; Frost, AE; Hill, NS; Horn, EM; Langleben, D; McLaughlin, VV; Oudiz, RJ; Robbins, IM et al. (2007). “Selective endothelinA receptor antagonism with sitaxsentan for pulmonary arterial hypertension associated with connective tissue disease”. Annals of the rheumatic diseases 66 (11): 1467–72. doi:10.1136/ard.2007.069609. PMC 2111639. PMID 17472992.
• 4
• “UPDATE 1-Encysive gets Canadian approval for hypertension drug”. Reuters. 30 May 2007. Retrieved 2007-07-08.
• 5

External links

• http://www.drugs.com/nda/thelin_050714.html
• Mucke HAM: Sitaxsentan for the Oral Treatment of Pulmonary Arterial Hypertension: Benefits from Endothelin Receptor Subtype Selectivity? Clinical Medicine: Therapeutics 2009:1 111–121.
• ATS 2005. The International Conference of the American Thoracic Society. 20–25 May 2005. San Diego, CA.
• Robyn J. Barst, MD; Stuart Rich, MD, FCCP; Allison Widlitz, MS, PA; Evelyn M. Horn, MD; Vallerie McLaughlin, MD and Joyce McFarlin, RN : Clinical Efficacy of Sitaxsentan, an Endothelin-A Receptor Antagonist, in Patients With Pulmonary Arterial Hypertension Chest. 2002;121:1860-1868.
• American Heart Association. Primary or Unexplained Pulmonary Hypertension