High-purity large-scale preparation of stannsoporfin.

___I KNOW___

SECMWISU Oí ECONOMY

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Mexican Institute of Industrial Property

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PATENT TITLE NO. 345035

Owner (s): INFACARE PHARMACEUTICAL CORPORATION

Address: 600 West Germantown Pike, Suite 400, Plymouth Meeting, Pennsylvania, 19462,

USA

Name: LARGE-SCALE AND HIGH PURITY PREPARATION OF ESTANSOPORPHINE.

Classification: Int.CL8: A61K31 / 555; C07D487 / 22; C07D498 / 22; C07F7 / 00; C07F7 / 22

Inventor (s): GEORGE S. DRUMMOND; ROBERT CAROSELLI; KEITH A. COOKE; Daniel

LEVIN; DAVID G. ROE; CHRISTOPHER P. BOUCHER

REQUEST

Number: International filing date:

MX / a / 2011/007519 October 4, 2007

Divisional Patent Number: 290015

PRIORITY

Country: Date: Number:

US October 4, 2006 60 / 849,641

US - February 28, 2007 60 / 904,601

Validity: Twenty years

Expiration Date: October 4, 2027

The reference patent was granted b ased on articles 1^gold, 2nd fraction V, 6^gold fraction llt, and 58 of the Industrial Property Law.

In accordance with article 2® of the Industrial Property Law, this patent has a validity of twenty years, non-derogable, from the date of filing of the international application and will be subject to payment. SmMmMI to keep the rights in force. · '4

Whoever subscribes to this title does so based on the provisions of articles 8 “sections III and 7 bis 2 of the Industrial Property Law (Official Cfano de la Federación (D®f $ -27 / 06 / 19í &lt;1. Resumed to 06 / 02/1994 10/25/1996, 12/26/1967, 05/17/1999, 01/26/2004, 06/16/200 01/25/2 ") 6, 06/05 / 2009,06 / 01 / 2010; tt / 08H010, 06/26/2010, 01/27/2012 and 04/09/2012); Articles 1, 3 »fraction V Subsection a), 4^gold and 12th sections I and III of the Regulations of the Mexican Institute of Industrial Health (DOF 12/14/19, amended on 07/01/20 02, 07/15/2004 | 07/28/2064 and 09/07/2007 ); Articles 1, 3^or, 4^or, 5^or fraction V subsection a), 16 sections I and III and 30 of the Organic Statute of the Mexican Institute of Property lndutótel (DOF 12/27/1999, amended 10/10/2002, 07/29/2004, 08/04/2004 and 13 / C9 / 2007); 1st, 3rd and 5^or Subsection a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

Issue Date: January 16, 2017

THE DIVISIONAL DIRECTOR OF PATENTS

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NAHANNY CANAL REYES

Arenal No, 550 Floor 1, Col, Pueblo Santa María Tepepan. Xochimilco, C P. 16020.

Mexico City

Tea!. (55) 53 34 07 00

MX / 2017/4774

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LARGE-SCALE AND HIGH-plu? F7A hf PREPARATION

ESTANSOPORPHINE

CROSS REFERENCE TO RELATED REQUESTS

This application claims the priority benefit of United States Provisional Patent Application No. 60 / 849,641, filed on October 4, 2006, and United States Provisional Patent Application No. 60 / 904,601, filed February 28, 2007. · The contents of these applications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention pertains to methods for synthesizing stansoporfin (tin (IV) dichloride mesoporphyrin IX) in large quantities with high purity, and the compositions thus produced.

BACKGROUND

Stansoporfin, or stannous (IV) dichloride mesoporphyrin IX, is an inhibitor of the enzyme heme oxygenase. Stansoporfin has been proposed for therapeutic use in various diseases, such as infantile hyperbilirubinemia (US 4,657,902; US 4,668,670;

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MEXICAN INSTITUTE

WO 94/28906) and psoriasis (US 4,782,049). Because of this pharmaceutical utility, stansoporfin preparation methods are of great interest.

Infant hyperbilirubinemia (also known as infantile jaundice or neonatal hyperbilirubinemia) occurs in a newborn when the liver is unable to conjugate bilirubin so that it can be excreted at a rate commensurate with the formation of bilirubin. Bilirubin comes from the release of the heme group as part of the physiological conversion of fetal to adult hemoglobin at birth. The enzyme heme oxygenase oxidizes the heme group to biliverdin; the enzyme biliverdin reductase then reduces biliverdin to bilirubin. Bilirubin in high serum levels is a neurotoxic substance. In adult humans, the liver rapidly converts bilirubin to a conjugated excretable form. However, in newborn humans the liver is still developing and absorption and conjugation by the liver is not as efficient as in adults. Additionally, hemolysis can occur at a relatively higher rate than in adults. All of these factors can lead to excessive bilirubin in the infant. For some infants, high serum bilirubin levels can have detrimental physiological consequences. Bilirubin is yellow in color and infants with excess bilirubin suffer from jaundice, with a yellow coloration on their skin and in the sclera of their eyes.

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Infants who have elevated levels of on · -guo rο-cte-triTlTrubιná are at risk of developing querníctero, a rare but potentially devastating neurological condition which can result in severe lifelong disabilities and complications such as athetosis, hearing loss , vision problems, and dental problems (See Centers for Disease Control and Prevention World-Wide-Web.cdc.gov/ncbddd/dd/kernicterus.htm.) accordingly, infants should be carefully monitored after birth, therapeutic intervention will be initiated if the infant's bilirubin level is excessive. The American Academy of Pediatrics has published a Clinical Practice Guideline for evaluating newborns for hyperbilirubinemia and treating newborns who are at risk; see Pediatrics 1 14: 297-316 (2004). As health care costs have risen in the United States, apparently healthy newborns and their mothers are discharged quickly, sometimes within 24 to 48 hours after birth. However, it is considered that this practice may have contributed to an increase in cases of querníctero, which has been virtually eliminated from developed countries; see Hansen TWR, Acta Paediatr. 89: 1 1551157 (2000)). Because prompt discharge of patients can delay the detection of jaundice and hyperbilirubinemia in infants, means of promptly treating hyperbilirubinemia are desirable. The newborn's unique medical status

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Born also requires as safe as possible means of treatment, since the side effects that are tolerable in adults may be completely unacceptable in neonates.

Currently approved and commonly used treatments for hyperbilirubinemia include phototherapy and exchange transfusion. Phototherapy involves radiation of the involves irradiating the newborn with light in the range of 430 to 490 nm (blue light). Light converts bilirubin to lumirubin and phorobilirubin, which are more easily excreted by the infant and therefore, results in decreased bilirubin levels.

Stansoporfin (stannous (IV) dichloride mesoporphyrin IX) has been shown to be of therapeutic value in the treatment of hyperbilirubinemia; see Valaes et al., Pediatrics 93: 1-11 (1994) and Kappas et al., Pediatrics, 95: 468-474 (1995). Other indications in which stansoporfin can be used are described in US 4,692,440 (to increase the rate of excretion of the heme group), WO 89/02269 (to counteract the toxicity of cancer therapy), US 4,782,049 (to treat psoriasis) and other publications.

United States Patent No. 6,818,763, United States Patent Application Publication 2004/0210048, and United States Patent Application No. 11 / 096,359 describe methods for synthesizing stansoporfin. However, it is desirable

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INSTITUTO MEXICANO JA

OF THE FROHEDAD XTL * -XXi _: . INDUSTRIAL is still developing methods to produce stansoporfin with higher purity, due to the therapeutic advantages of using as pure a substance as possible and also due to the strict requirements of regulatory agencies.

The present application describes methods for synthesizing stansoporfin at a level and purity not achieved heretofore, as well as large-scale preparations of pure stansoporfin. The present application also describes a new method of inserting tin and other metals into porphyrin rings. This new method can significantly decrease the time required for stansoporfin synthesis.

DESCRIPTION OF THE INVENTION

The present invention encompasses, in certain respects, high purity stansoporfin in a large scale (volume) amount, and methods for making such stansoporfin compositions. The invention also encompasses other synthetic methods and chemical compositions as described herein.

In one embodiment, the invention encompasses a composition of matter comprising high purity stansoporfin in a large scale amount (or volume). In another embodiment, the invention comprises a material composition comprising high-purity stansoporfin in a large-scale quantity.

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(volume) when produced in an indlvkluol batch, that is, a quantity of high purity stansoporfin on a large scale (or volume) of individual batch. The high purity stansoporfin can be about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or at least about 99.5% pure. minus about 99.8%. The amount of any impurity in the high purity stansoporfin can be less than about 0.1%, less than about 0.09%, less than about 0.08%, or about 0.07% or less; in another embodiment, any individual impurity is any individual impurity related to the product. The large-scale amount (volume) of stansoporfin can be at least about 10 grams, at least about 25 grams, at least about 50 grams, at least about 100 grams, at least about 200 grams, at least about 200 grams. minus about 500 grams, at least about 1.0 kg, at least about 2.0 kg, or at least about 5.0 kg. In one embodiment, the high purity stansoporfin as described above is variously produced in a single batch.

In an alternative embodiment, the large-scale quantity of stansoporfin has a purity of at least about 97%, of at least about 98%, of at least about 98.5%, of at least about 99%, of

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at least approximately approximately 99.5%, of at least «W * M ** M *« *******ⁿ***^{Min, l}*^&lt;&gt; * FR ^ teM &lt;.W * .. 'V.' ^&gt; «

99.8%, and has no impurity present in an amount greater than about 0.2%, and more preferably has no impurity present in an amount greater than about 0.15%, and even more preferably has no impurity present in a greater amount to about 0.12%. In one embodiment, the high purity stansoporfin is produced in a single batch.

In further embodiments, the amount of palladium impurities present in the large-scale amount of high purity stansoporfin is less than about 20 ppm, less than about 15 ppm, less than about 10 ppm, or less than about 5 ppm. In one embodiment, the high purity stansoporfin is produced in a single batch.

In another embodiment, the invention encompasses a large-scale, high-purity stansoporfin manufacturing method, comprising the steps of: a) exposing a metallic hydrogenation catalyst to a hydrogen atmosphere to form pre-hydrogenated catalyst; and b) contacting hemin with the pre-hydrogenated catalyst and keeping the hemin and catalyst under one or more combinations of temperature, hydrogen pressure, and sufficient time to remove iron from hemin and reduce vinyl groups from hemin to ethyl groups, forming mesoporphyrin IX. In another embodiment, the invention encompasses a method for making high-purity stansoporfin on a large scale,

IMPI Mexican Institute OF INDUSTRIAL PROPERTY, which comprises the steps of: a) exposing a metallic hydrogenation catalyst to a hydrogen atmosphere to form a pre-hydrogenated catalyst; b) contacting the hemin with the pre-hydrogenated catalyst and keeping the hemin and the catalyst under one or more combinations of temperature, hydrogen pressure, and sufficient time to remove the iron from the hemin and reduce the vinyl groups from the hemin to ethyl groups, thereby forming mesoporphyrin IX; and c) reacting mesoporphyrin IX with a tin (II) salt to form stansoporfin using a controlled rate of oxidation. In one embodiment, the metallic hydrogenation catalyst comprises palladium, palladium on carbon, platinum, platinum on carbon, nickel, or nickel aluminum catalyst. In another embodiment, the metal hydrogenation catalyst is palladium. In another embodiment, the metal hydrogenation catalyst is palladium on carbon. The can method produces a large scale quantity of high purity stansoporfin in a single batch.

In another embodiment, the invention encompasses a method for making high-purity stansoporfin on a large scale, comprising the steps of: a) exposing a metal hydrogenation catalyst to a hydrogen atmosphere to form pre-hydrogenated catalyst; b) contacting the pre-hydrogenated catalyst and keeping the hemin and the catalyst under one or more combinations of temperature, hydrogen pressure, and sufficient time to move the iron from the hemin and reduce the vinyl groups from the »* - ».uartrWMW»

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hemin to ethyl groups, thus forming mesoporphyrin IX; and c) reacting mesoporphyrin IX with tin (II) oxide to form stansoporfin. In one embodiment, the metal hydrogenation catalyst comprises palladium, palladium on carbon, platinum, platinum on carbon, nickel, or nickel aluminum catalyst. In another embodiment, the metal hydrogenation catalyst is palladium. In another embodiment, the metal hydrogenation catalyst is palladium on carbon. The can method produces a large-scale quantity of high purity stansoporfin in a single batch.

In another embodiment, the invention encompasses a method for making mesoporphyrin IX, comprising the steps of: a) exposing a palladium-on-carbon catalyst to a hydrogen atmosphere to form pre-hydrogenated palladium catalyst; and b) contacting hemin with the pre-hydrogenated catalyst and keeping the hemin and catalyst under one or more combinations of temperature, hydrogen pressure, and sufficient time to remove the iron from the hemin and reduce the vinyl groups from the hemin to ethyl groups, thus forming mesoporphyrin IX. In further embodiments, step b) is carried out at about 80 to 100 ° C, preferably at about 85 to 90 ° C, with hydrogen pressure at about 50 to 70 psi, preferably at about 55 to 60 psi, for about 1 to 3 hours, preferably about 1 to 1.5 hours; then at about 40 to 60 ° C

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preferably about 45 to 50 ° C, hydrogen pressure at about 50 to 70 psi, preferably about 55 to 60 psi, for about 18 to 48 hours, preferably about 24 hours.

In another embodiment, the invention encompasses an easily filterable mesoporphyrin IX dichloride preparation, wherein at least about 10 grams can be filtered in less than about 90 minutes, less than about 35 minutes, less than about 45 minutes, less than about 60 minutes, less than about 25 minutes, or less than about 10 minutes, starting from a solution in which the amount of solvent is present in a ratio of at least about 50-to-1 by weight to the amount of mesoporphyrin dichloride IX. In another embodiment, the invention encompasses an easily filterable mesoporphyrin IX dichloride preparation, wherein at least about 1000 grams can be filtered in less than about 1 day, less than about 12 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, or less than about 2 hours, from a solution where the amount of solvent is present in a ratio of at least about 50-to-1 by weight to the amount of mesoporphyrin dichloride IX. In one embodiment, the solvent is a mixture of water, hydrochloric acid, and

IMPI

MEXICAN USTITUTE. OF THE PROPERTY . INDUSTRIAL formic acid; hydrochloric acid can be about 31% hydrochloric acid before mixing.

In another embodiment, the invention encompasses a method for making an easily filterable mesoporphyrin IX dichloride preparation, comprising the step of adding an aqueous solution of hydrochloric acid to a formate solution of mesoporphyrin IX in formic acid. In one embodiment, the hydrochloric acid concentration in the aqueous solution is about 0.5 to 2.0 N. In another embodiment, the hydrochloric acid concentration in the aqueous solution is approximately 0.75 to 1.25 N. In another embodiment, the hydrochloric acid concentration in the aqueous solution is approximately 1.0 N.

In another embodiment, the invention encompasses a method of inserting tin into mesoporphyrin IX, which comprises reacting mesoporphyrin IX with a tin salt in the absence of a proton scavenger.

In another embodiment, the invention encompasses a method of inserting tin into mesoporphyrin IX, which comprises reacting mesoporphyrin IX with a tin salt at a controlled rate of oxidation. In one embodiment, mesoporphyrin IX is reacted with a tin salt in a reaction vessel having a headspace, and the rate of oxidation is controlled by introducing an oxygen-containing gas into the headspace of the vessel. reaction. In another embodiment, I gas containing gas introduced into the upper space of the

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INSTITUTE MSXICANO DE LA PAOWBDAD • ·. Industrial - reaction vessel is about 3 to 22% oxygen in an inert gas, such as nitrogen. Otherwise, the oxygen-containing gas introduced into the headspace of the reaction vessel is air. In another embodiment, the oxygen-containing gas introduced into the headspace of the reaction vessel is about 4 to 15% oxygen in an inert gas, such as nitrogen. In another embodiment, the oxygen-containing gas introduced into the headspace of the reaction vessel is about 5 to 10% oxygen in an inert gas, such as nitrogen. In another embodiment, the oxygen-containing gas introduced into the headspace of the reaction vessel is about 6% oxygen in an inert gas, such as nitrogen. In another embodiment, the oxygen-containing gas introduced into the headspace of the reaction vessel is approximately 6% oxygen in nitrogen.

In any of the above embodiments, the large-scale quantity of high purity stansoporfin can be produced in a single batch.

In any of the above embodiments, the reagents, intermediates, and / or products can undergo additional purification caps. In certain embodiments, further purification comprises treating the reagent, intermediate, or product with diatomaceous earth and / or activated carbon. In one embodiment, treating the reagent, intermediate, or product with diatomaceous earth and / or activated carbon comprises dissolving or

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suspending the reagent, intermediate, and / or product in a solvent, adding diatomaceous earth and / or activated carbon, filtering the diatomaceous earth and / or activated carbon, and recovering the reagent, intermediate or product from the filtrate. In certain embodiments, further purification comprises triturating the reagent, intermediate, or product with hot acid, such as about 0.1 to 6N HCl in water, preferably about 3N HCl in water. In certain embodiments, one, two, or three of the diatomaceous earth treatment steps, activated carbon treatment, and hot acid grinding are performed sequentially, in any order, and can be repeated as desired.

In another embodiment, the invention encompasses a method of inserting a metal into a porphyrin compound or a salt thereof using a metal oxide. In another embodiment, the metal cation of the metal oxide is in an intermediate oxidation state. In another embodiment, the porphyrin compound is a mesoporphyrin or salt thereof, or a protoporphyrin or salt thereof, or a hematoporphyrin or salt thereof, or a deuteroporphyrin or salt thereof. In another embodiment, the porphyrin compound is mesoporphyrin IX or a salt thereof. In another embodiment, the porphyrin compound is mesoporphyrin IX dichloride. In another embodiment, the resulting product is a metallized porphyrin or a salt thereof. In another embodiment, the resulting product is a metallized mesoporphyrin or a salt thereof or a metallized protoporphyrin or a salt thereof or a metallized hematoporphyrin or

IMPI INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL a salt thereof or a metallized deuteroporphyrin and a-salt thereof. In other embodiments, the metal oxide is selected from tin oxide, zinc oxide, copper oxide, cadmium oxide, cobalt oxide, chromium oxide, iron oxide, aluminum oxide, titanium oxide, oxide of nickel, manganese oxide, silver oxide, gold oxide, vanadium oxide, platinum oxide, antimony oxide, or arsenic oxide. In other modalities, the metal oxide is selected from tin (II) oxide, zinc (II) oxide, copper (I) oxide, copper (II) oxide, cadmium (II) oxide, cobalt oxide (II), Cobalt (III) oxide, Cobalt (IV) oxide, Chromium oxide Co₃OR₄, (II), Chromium (III) oxide, Chromium (IV) oxide, Chromium (V) oxide, Chromium (VI) oxide, Iron (II) oxide, (III), Fe oxide₃OR₄, aluminum (III), titanium (II) oxide, titanium (III) oxide, titanium (IV) oxide, nickel (II) oxide, manganese (II) oxide, manganese (III) oxide, manganese (IV), manganese (Vil) oxide, silver (I) oxide, silver (II) oxide, gold (I) oxide, gold (III) oxide, vanadium (II) oxide, vanadium oxide (III), vanadium (IV) oxide, vanadium (V) oxide, platinum (II) oxide, platinum (IV) oxide, antimony (III) oxide, antimony (IV) oxide, antimony (V) oxide, arsenic (III) oxide, or arsenic (V) oxide. In other embodiments, the resulting product is a tin porphyrin, zinc porphyrin, copper porphyrin, cadmium porphyrin, cobalt porphyrin, chromium porphyrin, iron porphyrin, aluminum porphyrin, titanium porphyrin, nickel porphyrin I, mangan so porphyrin, silver porphyrin, gold porphyrin,

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IMPI MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY vanadium porphyrin, platinum porphyrin, antimony porphyrin, arsenic porphyrin, or a salt thereof. In other embodiments, the resulting product is tin mesoporphyrin, zinc mesoporphyrin, copper mesoporphyrin, cadmium mesoporphyrin, cobalt mesoporphyrin, chromium mesoporphyrin, iron mesoporphyrin, aluminum mesoporphyrin, titanium mesoporphyrin, nickel mesoporphyrin, nickel mesoporphyrin manganese, silver mesoporphyrin, gold mesoporphyrin, vanadium mesoporphyrin, platinum mesoporphyrin, antimony mesoporphyrin, arsenic mesoporphyrin, or a salt thereof. In other embodiments, the resulting product is a tin IX mesoporphyrin, zinc mesoporphyrin IX, copper mesoporphyrin IX, cadmium mesoporphyrin IX, cobalt mesoporphyrin IX, chromium mesoporphyrin IX, iron mesoporphyrin IX, aluminum mesoporphyrin IX, mesoporphyrin titanium IX, nickel mesoporphyrin IX, manganese mesoporphyrin IX, silver mesoporphyrin IX, gold mesoporphyrin IX, vanadium mesoporphyrin IX, platinum mesoporphyrin IX, antimony mesoporphyrin IX, arsenic mesoporphyrin IX, or a salt thereof. In other embodiments, the resulting product is tin protoporphyrin, zinc protoporphyrin, copper protoporphyrin, cadmium protoporphyrin, cobalt protoporphyrin, chromium protoporphyrin, iron protoporphyrin, aluminum protoporphyrin, titanium protoporphyrin, protoporphyrin, nickel protoporphyrin manganese, silver protoporphyrin, gold protoporphyrin, vanadium protoporphyrin, gold protoporphyrin

IMPI

INSTITUTO MBXlCANQ PE LA INDUSTRIAL PROPERTY

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platinum, antimony protoporphyrin, arsenic protoporphyrin, or a

<td>get out of them</td><td>. In</td><td>other fashion</td><td>lities, the product res</td><td>resulting s</td>

<td colspan="2">a hematoporphyrin</td><td colspan="2">tin, hematoporphyrin</td><td>zinc,</td>

<td>hematoporphyrin</td><td>of</td><td>copper,</td><td>hematoporphyrin</td><td>cadmium,</td>

<td>hematoporphyrin</td><td>of</td><td>cobalt,</td><td>hematoporphyrin</td><td>chrome,</td>

<td>hematoporphyrin</td><td>of</td><td>iron,</td><td>hematoporphyrin</td><td>aluminum,</td>

<td>hematoporphyrin</td><td>of</td><td>titanium,</td><td>hematoporphyrin</td><td>nickel,</td>

<td>hematoporphyrin</td><td>of</td><td>manganese</td><td>i, hematoporphyrin d</td><td>e silver,</td>

<td>hematoporphyrin</td><td>gold,</td><td colspan="3">Vanadium Hematoporphyrin, Hematoporphyrin</td>

<td colspan="3">platinum, hematoporphyrin</td><td colspan="2">antimony, hematoporphyrin d</td>

<td>arsenic, or a salt</td><td colspan="2">from the same.</td><td colspan="2">In other modalities, the product</td>

<td>resulting is a</td><td colspan="4">tin deuteroporphyrin, deuteroporphyrin d</td>

<td colspan="2">zinc, deuteroporphyrin</td><td colspan="2">copper, deuteroporphyrin</td><td>cadmium,</td>

<td>deuteroporphyrin</td><td>of</td><td>cobalt,</td><td>deuteroporphyrin</td><td>chrome,</td>

<td>deuteroporphyrin</td><td>of</td><td>iron,</td><td>deuteroporphyrin</td><td>aluminum,</td>

<td>deuteroporphyrin</td><td>of</td><td>titanium,</td><td>deuteroporphyrin</td><td>nickel,</td>

<td>deuteroporphyrin</td><td>of</td><td>manganese</td><td>, deuteroporphyrin d</td><td>e silver,</td>

gold deuteroporphyrin, vanadium deuteroporphyrin, platinum deuteroporphyrin, antimony deuteroporphyrin, arsenic deuteroporphyrin or a salt thereof.

In another embodiment, the invention encompasses a method of inserting tin into a porphyrin compound or a salt thereof using tin (II) oxide. In another embodiment, the porphyrin compound is a mesoporphyrin or salt thereof, or a protoporphyrin or salt thereof, or a hematoporphyrin or salt thereof. In other

IMPI

MEXICAN INSTITUTE OF LA. • ROFJEDAD

INDUSTRIAL

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modality, the porphyrin compound is mesop or.f.ix.in.a .. IX ... o. , i, or a. aal of it. In another embodiment, the porphyrin compound is mesoporphyrin IX dichloride. In another embodiment, the resulting product is a tin (IV) porphyrin or a salt thereof. In another embodiment, the resulting product is a tin (IV) mesoporphyrin or a salt thereof or tin (IV) protoporphyrin or a salt thereof or tin (IV) hematoporphyrin or a salt thereof. In another embodiment, the resulting product is tin (IV) mesoporphyrin IX or a salt thereof.

In another embodiment, the invention encompasses a method of inserting tin into a porphyrin compound or a salt thereof by providing a porphyrin compound or salt thereof, by providing tin (II) oxide, and contacting the oxide d tin (II) with the porphyrin compound or a salt thereof under acidic conditions, whereby the tin (II) oxide is inserted into the porphyrin ring to produce a porphyrin tin (IV) compound. In another embodiment, the tin (II) oxide is dissolved or suspended in acetic acid or formic acid, preferably acetic acid. In another embodiment, the compound porphyrin or a salt thereof is dissolved or suspended in formic acid or acetic acid, preferably formic acid. In another embodiment, the equivalent ratio nt of the total amount of tin (II) oxide used to the total amount of porphyrin compound or salt thereof used is from about two to six, preferably about four. In another modality, the solution or suspension of the computation

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Porphyrin or a salt thereof is added by-go-te-o to the 8θ4ιιχϊόα of tin (II) oxide. The drip addition can take place over a period of about three to nine hours, preferably longer than about six hours. The tin (II) oxide solution or suspension is maintained at a temperature of about 25-115 ° C, preferably about 50-75 ° C, most preferably about 60-65 ° C.

In another embodiment, the invention encompasses a method of inserting tin into a porphyrin compound or a salt thereof (such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX) by providing a mesoporphyrin compound or salt. thereof (such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX), providing stannous (II) oxide, and contacting the tin (II) oxide with the mesoporphyrin compound or salt thereof (such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX) under acidic conditions, whereby the tin oxide (II) is inserted into the porphyrin ring (such as a mesoporphyrin IX ring) to produce a compound a tin (IV) porphyrin compound. In another embodiment, the tin (II) oxide is dissolved or suspended in acetic acid or formic acid, preferably acetic acid. In another embodiment, the mesoporphyrin compound or salt thereof (such as mesoporphyrin IX or salt thereof, such as mesoporphyrin dichloride IX) is dissolved or suspended in formic acid or acetic acid, preferably formic acid. In other

IMPI

MEXICAN INSTITUTE

DE LA FROPIE.-mO INruSTRIAL modality, the equivalent ratio of the total amount of tin (II) oxide used to the total amount of the mesoporphyrin compound or salt thereof (such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX) used is about two to six, preferably up to about four. In another embodiment, the solution or suspension of the porphyrin compound or a salt thereof (such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX) is added dropwise to the tin oxide solution ( II). The dropwise addition can be carried out over a period of about three to nine hours, preferably greater than about six hours. The tin (II) oxide solution or suspension is maintained at a temperature of about 25-115 ° C, preferably about 50-75 ° C, more preferably about 60-65 ° C. After completion of the dropwise addition, the reaction mixture can be maintained at a temperature of about 60 to 65 ° C for about an additional 18 to 24 hours. The reaction mixture can be cooled and filtered after the additional reaction time.

In another embodiment, the invention encompasses a method for producing a tin (IV) porphyrin compound or salt thereof which comprises a) preparing a solution or suspension of an unmetallized porphyrin compound or a salt thereof; b) prepare a solution or suspension of stannous oxide (II), where the caps

<img file="MX345035B_D0018.tif" />

(a) and (b) can be presented in any order or simultaneously; and c) contacting the tin (II) oxide solution or suspension with the non-metallized porphyrin compound solution or suspension or salt thereof under suitable conditions to form the tin (IV) porphyrin compound or salt thereof. The tin (II) oxide solution or suspension and the non-metallized porphyrin compound solution or suspension or salt thereof can be prepared independently with formic acid or acetic acid; For example, the tin (II) oxide solution or suspension can be prepared with acetic acid, and the non-metallized porphyrin compound solution or suspension or salt thereof can be prepared with formic acid. The unmetallized porphyrin compound is selected from mesoporphyrins, protoporphyrins, hematoporphyrins, and salts thereof, such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin dichloride IX. The contacting step c) may comprise the addition of the solution or suspension of non-metallized porphyrin compound or salt thereof by means of dripping to the solution or suspension of tin (II) oxide under suitable conditions to form the porphyrin compound. tin (IV) or salt thereof. The dropwise addition can be completed in about 3 to 9 hours, such as in about 6 hours. The tin (II) oxide solution or suspension can be maintained at a temperature of about 60 to 65 ° C during the dropwise addition. After the completion of the addition of the iNsrrr · τ &lt;: ^ íxícano

Dk INDUSTRIAL PROPERTY solution or suspension of non-metallized porphyrin compound or salt thereof by means of dripping to the solution or suspension of tin (II) oxide, the reaction mixture can be kept at a temperature of approximately 60 to 65 ° C for approximately 18 to 24 additional hours. The reaction mixture can be cooled and filtered after the additional reaction time. In another embodiment, the method for producing a tin (IV) porphyrin compound or salt thereof is performed in the absence of a proton scavenger or proton sponge.

Tin (IV) mesoporphyrin produced via any of the methods described above may undergo additional purification steps. In certain embodiments, further purification comprises treating tin (IV) mesoporphyrin with diatomaceous earth and / or activated carbon. In one embodiment, treating the tin (IV) mesoporphyrin with diatomaceous earth and / or activated carbon comprises dissolving or suspending the tin (IV) mesoporphyrin in a solvent, adding the diatomaceous earth and / or activated carbon, filtering the ti diatomaceous rra and / or activated carbon, and recover tin (IV) mesoporphyrin from the filtrate. In certain embodiments, further purification comprises triturating the tin (IV) mesoporphyrin with hot acid, such as about 0.1 to 6N HCl in water, preferably up to about 3N HCl in water, at a temperature of about 60 to 95 ° C. C, preferably at about 80 to 95 ° C, most notably at

<img file="MX345035B_D0019.tif" />

about 85 to 90 ° C. In certain embodiments, one, two, or all three steps of diatomaceous earth treatment, activated carbon treatment, and hot acid trituration are performed sequentially, in any order, and can be repeated as desired.

In another embodiment, the invention encompasses stansoporfin as produced through any of the processes described herein.

In another embodiment, the invention encompasses high-purity stansoporfin in a large-scale amount (or volume), wherein the high-purity stansoporfin is stable for at least about three months or at least about six months in storage. In another embodiment, the high purity stansoporfin in bulk is prepared as a single batch. In another embodiment, the invention encompasses high-purity stansoporfin in a large-scale amount (or volume), wherein the high-purity stansoporfin retains its high purity for at least about three months or at least about six months in storage. In another embodiment, the storage conditions are about 25 ° C and about 60% relative humidity. In another embodiment, the storage conditions are about 40 ° C and about 75% relative humidity. In another embodiment, the stansoporfin is stored in a polyethylene bag. In another embodiment, the stansoporfin is stored in a bag of

<img file="MX345035B_D0020.tif" />

IMPI i

INSTITUTO MEXICANO OH LA PXOUtOAO INDUSTRIAL polyethylene inside another polyethylene bag. In another way, the stansoporfin stored in a double bag is stored in a high-density polyethylene drum. In another embodiment, each polyethylene bag is approximately 4 mils thick (approximately 4/1000 of an inch, or approximately 0.1 millimeter).

In another embodiment, the invention encompasses a method for treating infantile hyperbilirubinemia, which comprises administering stansoporfin to a patient in need of such treatment, wherein the stansoporfin was produced in high purity and on a large scale. In another embodiment, the stansoporfin is produced as a single batch.

In another embodiment, the invention encompasses a method for preventing infantile hyperbilirubinemia, which comprises administering stansoporfin to a patient in need of said prevention, wherein the stansoporfin was produced in high purity and on a large scale. In another embodiment, the stansoporfin is produced as a single batch.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, stansoporfin is prepared in large quantity with high purity. In another embodiment of the present invention, stansoporfin is prepared in a large amount with high purity, where the large amount is

<img file="MX345035B_D0021.tif" />

<img file="MX345035B_D0022.tif" />

INSTITUTO MEXICANO t&gt; £ THE INDUSTRIAL NOUEOAD prepared as a single batch. Stansoporfin (tin (IV) dichloride mesoporphyrin IX; Chemical Abstráete Registry No. 106344-20-1) is also known by the trade name Stanate®, which is a registered trademark of InfaCare Pharmaceutical Corp., Plymouth Meeting, Pennsylvania. Stansoporfin has the following structure;

<img file="MX345035B_D0023.tif" />

which has the molecular formula C₃4H3₆Cl2N₄O4Sn and molecular weight 754.29.

By large quantity, large scale or volume s they represent at least about 10 grams. Other quantities for large-scale production of stansoporfin are at least about 25 grams, at least about 50 grams, at least about 100 grams, at least about 200 grams, at least about 500 grams, at least. about 1.0 kg, at least about 2.0 kg, or at least about 5.0 kg.

By "individual lot" s represents that the quantity of the specified product is synthesized at one time. A lot

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL individual is produced in a common way after a reaction (or series of reactions) carried out once (note that an individual preparation of the compound subjected as a whole to one or more reactions repeatedly such as purifications repeated, it is considered as a single batch). Thus a single batch excludes multiple preparations of a compound carried out at separate times, or in divided amounts, which are subsequently combined.

High purity represents a preparation that meets the following two criteria: 1) the overall purity level is about 97%; that is, the desired product (stansoporfin) accounts for at least 97% of the preparation; and 2) any individual product related impurities are present in an amount less than about 0.1% of the preparation. Purity is preferably measured by HPLC analysis. A product-related impurity is an impurity that requires characterization by means of the United States Food and Drug Administration standards; consequently the components of the drug product such as water are not considered as impurity.

By non-metallized porphyrin is meant a porphyrin that lacks a metal ion coordinated by one or more pyrrole nitrogens. A "metallized porphyrin" is a porphyrin having a metal ion coordinated by at least one nitrogenous pyrrole.

Through intermediate oxidation state, sr presents a

<img file="MX345035B_D0024.tif" />

IMPI INSTITUTO MHUCANC Oí LA PKOMIDAV INDUSTRIAL element, such as a metal, which is present in an oxidation state intermediate between its neutral state (no charge, or zero oxidation state) and its state with the plus the vada oxidation. By way of non-limiting example, iron commonly forms oxidation states of (0), (II), and (III); oxidation state (II) (ferrous state) is an intermediate oxidation state.

The purity of the preparation is important for the use of the compound as a pharmaceutical. The general level of purity can be at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or at least about 99.5%. a high purity preparation is also defined, as before, as a preparation with the further proviso that any individual impurities present are present in an amount less than about 0.1% of the preparation. (Note that the total amount of impurities may exceed 0.1% - for example, one impurity may be present at 0.08%, and another at 0.07%, totaling 0.15% - but when measured individually, no impurity is present at amounts equal to or exceeding about 0.1%.) In another embodiment, any individual impurities present are present in an amount less than about 0.09%. In another embodiment, any individual impurities present are present in an amount less than about 0.08% or less. In another embodiment, any individual impurities present are present in an amount less than about 0.07 - Water may be present in the preparation, even in significant amounts (at least about 1% to 5%), although not it is considered an impurity. Other residual solvents, such as acetone, formic acid, and acetic acid, are also not considered as impurities, especially if they present at or below the permissible levels described in the standards of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline — Impuriti s: Guideline for Residual Solvente, Q3C (R3), Step 4 version, November 2005 (World-Wide-Web.ich.org/LOB/media/MEDIA423.pdf).

In an alternate embodiment, stansoporfin has no impurity present in an amount greater than about 0.2%, and more preferably has no impurity present in an amount greater than about 0.15%, and even more preferably has no impurity present in a amount greater than about 0.12%.

The current synthesis produces stansoporfin that meets the two criteria listed above for high purity (overall purity of at least about 97%, without treating water and residual solvents as impurities and any impurities present are in an amount of about 0.1% or less). The second criterion, with respect to the level of individual impurities, is of interest due to regulatory requirements.

<img file="MX345035B_D0025.tif" />

The United States Food and Drug Administration - North America commonly requires detailed characterization of impurities at a level equal to 0.1%, as long as impurities are present at a level below 0.1% they do not need to be. characterized in detail unless they have unusually potent pharmacological or toxic effects at a level less than 0.1% (see Guidance for Industry publications: ANDAs: Impurities in Drug Substances, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (ODER), November 1999; available at World-Wide-Web-site-.fda.gov/cder/guidance/2452fnl.htm; and Guidance for Industry, Q3A Impurities in New Drug Substances, United States Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Biologies Evaluation and Research (CBER), February 2003 ICH, Revision 1, available at World-Wide-Web-site.fda.gov/cder/guidance/4164fnl.pdf). By meeting these threshold conditions established by the Food and Drug Administration, high-purity material has significant advantages over lower-purity material from a regulatory standpoint.

Another advantage of the invention as described in the pres nt is the expected reproducibility of the symptom, providing the ability to generate repeated batches of high purity stansoporfin in large quantity. Another advantage of the invention as sd writes in the pres nte s the capacity d I

<img file="MX345035B_D0026.tif" />

INSTITUTO MEXICANOBE LA FROVIEDAD INDUSTRIAL process and product to meet Good Manufacturing Practice (GMP) requirements, as defined by legal, regulatory or regulatory agency requirements in various countries (for example, the current Good Manufacturing Practice as specified in the United States Code of Federal Regulations, Title 21, Sections 210 and 211).

Another advantage of the invention as described herein is the production of high purity volume amounts of stansoporfin in a single batch, with concomitant advantages of increased homogeneity, low cost of synthesis, and relative ease of characterization.

Stansoporfin synthesis with high purity

Since porphyrins are light sensitive compounds, starting materials, intermediates, products and solutions or suspensions thereof will be protected from exposure to light and stored in a dark facility in containers that exclude light.

The synthesis of stansoporfin proceeds with hemin (iron (III) chloride protoporphyrin IX) as a starting material. The amounts required for large-scale synthesis are obtained from porcine red blood cells. DMF grade hemin is purchased from Harimex (Loenen, The Netherlands); The material is used without purification before use (the purity as supplied is

IMPI

INSTITUTO MEXICANO LA PROPERTY INDUSTRIAL

<img file="MX345035B_D0027.tif" />

greater than about 98% by HPLC). Hemin is heated in organic solvent with a hydrogenation catalyst on carbon under an atmosphere of hydrogen. This reductive step serves to remove the Fe ion from the porphyrin ring, and to reduce the vinyl groups of protoporphyrin IX to ethyl groups (converting protoporphyrin IX to mesoporphyrin IX), as indicated in the following scheme.

<img file="MX345035B_D0028.tif" />

A preferred hydrogenation catalyst is palladium on carbon, used in an amount of about 0.0135 to 0.0165 equivalents, preferably up to about 0.015 equivalents. Other suitable catalysts that can be used,

<img file="MX345035B_D0029.tif" />

include palladium metal particle catalyst, platinum on carbon, platinum metal particle, nickel, or nickel-aluminum, provided that the residual amounts of catalyst in the product meet pharmaceutical specifications. The nickel-aluminum catalyst may be RANEY nickel (RANEY is a registered trademark of WR Grace &amp; Co., New York, New York). A preferred organic solvent is formic acid.

It has been found that pretreatment of the Pd / C catalyst with hydrogen gas prior to adding hemin to the reaction reduces palladium impurities and thus contributes to the overall purity of the final stansoporfin product. Without prehydrogenation of the catalyst prior to the addition of hemin, residual palladium levels of about 50 ppm were detected in the product, which are significantly above product specifications less than about 20 ppm residual palladium. With the pre-hydrogenation step, residual palladium was reduced to non-detectable levels (less than about 5 ppm residual palladium). Consequently, the improved synthesis provides residual palladium levels in the tin (IV) dichloride mesoporphyrin IX product of less than about 20 ppm palladium, preferably less than about 15 ppm palladium, more preferably less than about 10 ppm. palladium, even more preferably less than about 5 ppm palladium. The pre-hydrogenation of the catalyst can be carried out under a hydrogen atmosphere of

<img file="MX345035B_D0030.tif" />

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL approximately 15 to 75 psi (approximately mont-o · -1 ....... te-up to 5 bar; approximately 100,000 to 500,000), preferably up to approximately 30 to 50 psi (approximately 2 to 3.5 bar; about 200,000 to 350,000 Pascais), more preferably up to about 40 psi (about 2.75 bar or 275,000 Pascais). The temperature for pre-hydrogenation of the catalyst can vary between about 25 to 60 ° C, preferably up to about 35 to 50 ° C, more preferably up to about 40 to 45 ° C. The catalyst pre-hydrogenation period can range from about 2 to 48 hours, preferably from about 6 to 24 hours, more preferably from about 8 to 16 hours, even more preferably up to about 12 hours.

Therefore, commonly, the catalyst is added to the chemical reactor first, followed by the formic acid solvent (eg, about 17.5 to 22.5 parts solvent, preferably up to about 20 parts solvent). Before adding the solvent, the hydrogen can be evacuated and the reactor can be filled with a nitrogen atmosphere for safety reasons. Upon completion of the formic acid addition, the nitrogen atmosphere is replaced by a hydrogen atmosphere, for example, at about 40 pounds per square inch (about 2.75 bar or 275,000 Pascais). The temperature is adjusted d spu's at approximately 35 to 50 ° C

<img file="MX345035B_D0031.tif" />

preferably at about 40 to 45 ° C. lasts for about 8 to 24 hours, preferably up to about 12 hours, prior to introduction of the hemin starting material into the reactor. The pre-hydrogenated catalyst slurry is then cooled, followed by the addition of hemin (in solvent) to the reactor. The hydrogen atmosphere is evacuated during the introduction of hemin for safety purposes, leaving only the hydrogen associated with the Pd / C catalyst. The reactor is re-pressurized to about 30 to 35 psi with hydrogen, and the reaction is stirred at about 20 to 25 ° C for about 30 minutes. The reaction is then heated to about 80 to 100 ° C, preferably to about 85 to 90 ° C, with vigorous stirring, and the hydrogen pressure is increased to about 50 to 70 psi (about 3.4 to 4.8 bar or about 340,000 to

480,000 Pascáis), preferably at approximately 55 to 60 psi (approximately 3.8 to 4.2 bar or approximately 380,000 to

420,000 Pascais). The reaction temperature is maintained for about 1 to 3 hours, preferably about 1 to 1.5 hours. The reaction is then cooled to about 40 to 60 ° C, preferably to about 45 to 50 ° C, and the hydrogen pressure is maintained and hydrogenation is continued for about 18 to 48 hours, preferably 20 to 30 hours, longer. preferably up to about 24 hours.

The reaction is cooled and deprived after riorm nt with the

<img file="MX345035B_D0032.tif" />

IMPI

INSTITUTO MEXICANO DE LA Ε »· ΕΙΕΟΑΠ IN» UST »1AL evacuation of hydrogen from the reactor. Diatunidied earth (lUT as HYFLO SUPERCEL, a registered trademark of Celite Corp., Santa Barbara, California), activated carbon (such as DARCO KB, a registered trademark of NORIT Americas, Inc., Marshall, Texas), and solvent were added. to the reactor. The suspension is filtered, and the filter cake is washed with solvent. This treatment serves to remove residual iron and residual palladium from the material.

The filtrate is concentrated by vacuum distillation (which can be done at room temperature, or at other temperatures, such as about 10 to 15 ° C) to remove excess solvent. A precipitant, for example, an ether such as methyl t-butyl ether (MTBE), is then added over a period of at least about 30 seconds for at least about 3 hours, preferably over a period of at least about 30 seconds. 1 hour, to the concentrated solution. When MTBE is added, it can be added in about 17.5 to 22.5 parts, preferably about 20 parts.

The suspension can be cooled to a temperature of about -15 to -30 ° C, preferably about 20 to -25 ° C.

The suspension is filtered and the filter cake rinsed with an organic solvent, such as ethers, including methyl t-butyl ether (MTBE), diethyl ether, or diisopropyl ether. After filtration is complete and the cake is rinsed, the material is then dried in a

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL vacuum oven at a temperature not exceeding approximately 60 ° C, for example, from approximately 45 to 60 ° C.

When prepared using formic acid as the solvent, the resulting product, mesoporphyrin IX, is precipitated as a formate salt; this is the preferred way of isolating mesoporphyrin IX after the hydrogenation step. After further purification steps, the mesoporphyrin IX format is converted to a hydrochloride salt. This step provides further purification of the intermediate product. Furthermore, it has been shown that the presence of proton scavengers such as forma (or other organic anions, such as acetate) during the subsequent tin insertion step results in higher levels of impurities than if said proton scavengers were excluded. Accordingly, it is preferred to replace the format anion of the mesoporphyrin IX format with an anion with a greater ability to take up protons or regulate the pH of the solution during the tin insertion step; such anions include chloride and other halide anions such as bromide or iodide.

When the intermediate product isolated from the hydrogenation step is mesoporphyrin IX format, it is placed in a reaction vessel with diatomaceous earth, activated carbon, and formic acid, (for example, with approximately 10% w / w of diatomaceous earth , about 20% w / w activated carbon, and about 10 parts formic acid) to undergo further purification. The suspension is stirred, for example, at about 20 to 30 ° C, preferably at about 20 to 25 ° C, for about 1.5 to 2.5 hours. The suspension is then filtered, and the filter cake washed with formic acid, for example, about 5 parts formic acid. The resulting filter solution is then concentrated to about 5 to 6 parts by volume. Another container is charged with purified water and 31% hydrochloric acid in order to prepare about 15 parts of about 1N hydrochloric acid. About 6 parts of this HCl solution are transferred into the container containing about 6 parts of the filtrate, preferably at a temperature of about 20 to 25 ° C and for a period of at least about 60 minutes. The solution is seeded with mesoporphyrin IX dichloride (available from previous syntheses) and shaken, preferably for at least about 2 hours. The remaining 9 parts of 1N hydrochloric acid are transferred into the vessel under vigorous stirring, preferably for a period of at least 60 minutes. The suspension is subsequently further stirred at about 20 to 30 ° C, preferably at about 20 to 25 ° C, for about 2 to 3 hours. It is then filtered and rinsed with purified water. The product is dried under a stream of nitrogen.

In the initial processes, the previous stage was carried out by re-dissolving d I format of solid mesoporphyrin IX n

<img file="MX345035B_D0033.tif" />

formic acid, and then adding the formic acid solution to the hydrochloric acid in order to convert the mesoporphyrin IX format to mesoporphyrin IX dichloride. However, the filtration of the mesoporphyrin IX dichloride thus produced was found to be too slow on a pilot plant scale, requiring up to five days to complete, and subsequent drying on the filter takes between approximately two to three weeks. A process improvement was developed; As previously described, the 1N hydrochloric acid solution is added into the formic acid solution of the mesoporphyrin IX format. This has been found to result in mesoporphyrin IX dichloride that can be filtered out more quickly. The addition of the symbol material can also be performed during the procedure, for example, at the beginning of the addition of 1N HCl in the formic acid solution of the mesoporphyrin IX format, or during the addition of 1N HCl in the acid solution. formic of the mesoporphyrin IX format, such as when about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% has been added, or about 90% 1N HCl to formic acid solution of mesoporphyrin IX format. Preferably, as in the process described immediately above, the seed material is added after 40% of the 1N HCl has been added to the mesoporphyrin IX formic acid solution. The addition of the sowing material can

<img file="MX345035B_D0034.tif" />

IMPI

MEXICAN INSTITUTE

M INDUSTRIAL PROPERTY also help in the formation of a product that can be filtered more quickly. Since the mesoporphyrin IX dichloride resulting from these process improvements can be filtered much faster, on the order of a few hours or even minutes rather than days, significant savings in time and cost are achieved. Therefore, in another embodiment, the filtration time of at least about 10 grams of mesoporphyrin IX dichloride is reduced to less than about 90 minutes, less than about 60 minutes, less than about 45 minutes, less than about 35 minutes. , less than about 25 minutes, or less than about 10 minutes. In a further embodiment, the filtration time of at least about 1000 grams of mesoporphyrin IX dichloride is reduced to less than about 1 day, less than about 12 hours, less than about 6 hours, less than about 4 hours, less of about 3 hours, or less than about 2 hours.

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

<img file="MX345035B_D0035.tif" />

<img file="MX345035B_D0036.tif" />

Conversion of mesoporphyrin IX hydrochloride to stansoporfin (tin (IV) mesoporphyrin IX) via tin (II) salt treatment

The mesoporphyrin hydrochloride IX is then treated with a tin (II) salt, such as SnCl.₂ in an organic solvent, such as acetic acid, under oxidation conditions, which produces the desired product, tin (IV) dichloride mesoporphyrin IX (stansoporfin). For example, mesoporphyrin IX dichloride and tin (II) chloride are placed in a container, and acetic acid is added at about 20 to 30 ° C, preferably at about 20 to 25 ° C. suspended reagents are stirred for at least about 30 minutes. With

<img file="MX345035B_D0037.tif" />

IMPI

INSTITUTO MBXICANO Di LA INDUSTRIAL PROPERTY vigorous stirring, the mixture is heated under an inert atmosphere (such as nitrogen or argon) until reflux.

<img file="MX345035B_D0038.tif" />

Once reflux has started, an atmosphere of about 6% oxygen in nitrogen is introduced into the headspace of the vessel. The gas can be from about 3% to about 22% oxygen; s prefers about 6% to minimize explosion hazards. The mixture is refluxed for about 100 to 130 hours. It has been found that the use of 6% oxygen in the nitrogen atmosphere in the headspace instead of steam injection or bubbling of the gas mixture through the liquid is advantageous in increasing the production of tin (IV) dichloride. mesoporphyrin IX. Tin (II) can enter the porphyrin ring to complex with nitrogens, and can also leave the porphyrin ring. However, the tin (IV) that is not

IMPI already bound to the nitrogens of the porf¡riTra ~ TTO ~ ring can-e - errtra-r --- to the ring to form a complex with the nitrogens. In order to generate tin (IV) mesoporphyrin IX, the tin (II) ion must enter the porphyrin ring, and then undergo oxidation to tin (IV) in situ. Excessively rapid oxidation of the tin (II) ion will cause the insertion reaction to stop, which can significantly reduce yields. Consequently, proper control of the oxidation rate is necessary. The introduction of oxygen into the mixture through the interface between the solvent and the oxygen / nitrogen headspace atmosphere provides this control and leads to a reasonable reaction rate with good yield of the final product.

The reaction mixture can be optionally mastered during the tin insertion step by reducing the temperature to about 50 to 70 ° C, preferably about 55 to 60 ° C, stirring a sample and returning the reaction to reflux.

After the tin insertion step, the reaction mixture is cooled, and WFI grade water (water for injection) is added. The suspension is then filtered and the filter cake is washed with WFI water. The filter cake is placed under vacuum for a minimum of 4 hours to remove residual water.

IMPI

INSTITUTO .MEXICANO DE LA PKOPIEDA »INDUSTRIAL

Conversion of mesoporphyrin IX dichloride to stansoporfin (tin (IV) mesoporphyrin IX) via tin (II) oxide treatment

SnO, AcOH

Tin can also be inserted into the mesoporphyrin IX ring by treating mesoporphyrin IX dichloride with tin (II) oxide. This reaction can proceed to completion in as little as two hours, compared to the four days to three weeks required for tin insertion using the tin (II) salt method described above. A solution / suspension of dichloride of

INSTITUTO mexicana M LA PROPERTY INDUSTRIAL mesoporphyrin IX in a suitable solvent, for example, formic acid or acetic acid, is added to a solution / suspension of tin (II) oxide in a suitable solvent, for example, acetic acid or formic acid. An illustrative procedure is described below and also in the Examples.

Mesoporphyrin IX dichloride is dissolved / suspended in formic acid at room temperature. Since the solution or suspension will be dark purple in color, it is advantageous to powder mesoporphyrin IX dichloride into as fine a powder as possible to aid dissolution.

Tin (II) oxide is suspended in acetic acid at room temperature and stirred. After prolonged stirring, the suspension of tin oxide in a gel, which has been observed not to adversely affect the reaction. The g I separates once the addition of mesoporphyrin dichloride IX begins. The amount of tin (II) oxide is about two equivalents to about six equivalents per equivalent of mesoporphyrin IX dichloride; preferably, about four equivalents of tin (II) oxide are used per equivalent of mesoporphyrin IX dichloride. (In this reaction, the equivalent ratio is the same as the molar ratio.)

The tin (II) oxide solution is maintained at a temperature of about 25-115 ° C, preferably about 50-75 ° C, most preferably about

<img file="MX345035B_D0039.tif" />

60-65 ° C. The mesoporphyrin IX dichloride solution is then added over a period of about three to nine hours, preferably over a period of about six hours. The mesoporphyrin IX dichloride solution can be at room temperature during the addition, or it can be maintained at a temperature of about 50-75 ° C, such as about 60-65 ° C, during the addition. The reaction mixture is maintained at about 25-115 ° C, preferably about 50-75 ° C, more preferably about 60-65 ° C, for an additional about 2 to 48 hours, preferably about 16 to 30 hours. additional hours, more preferably for about an additional 18 to 24 hours, such as for about an additional 18 hours or for an additional about 24 hours. After the additional reaction time, the suspension is cooled to room temperature (about 20-25 ° C), stirred or stirred for at least about five minutes, preferably for at least about one hour, and filtered.

General metal insertion into porphyrins using metal oxides

The procedure used for inserting tin into porphyrin rings using metal oxides can also be applied to inserting other metals using metal oxides.

MÍX1CANC INSTITUTE

DE LA CKOFIEDAL V&gt; va, i «Yes ¿rf / INDUSTRIAL

Particularly useful metal oxides are metal oxides where the metal cation of the metal oxide is in an intermediate oxidation state. The method can be used for porphyrin compounds or salts thereof, including, but not limited to, a mesoporphyrin or a salt thereof, mesoporphyrin IX or a salt thereof, mesoporphyrin IX dichloride, a protoporphyrin or a salt thereof, a hematoporphyrin or a salt thereof, or a deuteroporphyrin or a salt thereof, to produce the metallized porphyrin compound (or a salt thereof).

Metal oxides that can be used include, but are not limited to, tin oxide, zinc oxide, copper oxide, cadmium oxide, cobalt oxide, chromium oxide, iron oxide, aluminum oxide, titanium oxide , nickel oxide, manganese oxide, silver oxide, gold oxide, vanadium oxide, platinum oxide, antimony oxide, arsenic oxide, tin (II) oxide, zinc (II) oxide, copper (I), copper (II) oxide, cadmium (II) oxide, cobalt (II) oxide, Cobalt (III) oxide, Cobalt (IV) oxide, CO3O4, Chromium (II) oxide, Chromium (III) oxide, Chromium (IV) oxide, Chromium (V) oxide, Chromium (VI) oxide , iron (II) oxide, iron (III) oxide, Fe₃OR₄, aluminum (III) oxide, titanium (II) oxide, titanium (III) oxide, titanium (IV) oxide, nickel (II) oxide, manganese (II) oxide, manganese (III) oxide, manganese (IV) oxide, manganese (V) oxide, silver (I) oxide, silver (II) oxide, gold (I) oxide, gold (III) oxide, vanadium (II) oxide, oxide d

IMPI iNsnnno Mexican OT LA FOF1WAD INDUSTRIAL vanadium (III), vanadium (IV) oxide, vanadium (V) oxide, platinum (II) oxide, platinum (IV) oxide, antimony (III) oxide, antimony oxide (IV), antimony oxide (V), arsenic oxide (III), or arsenic (V).

Other porphyrin and tetrapyrrole compounds can also be metallized using the procedures described herein, including, but not limited to, porphyrins such as deuteroporphyrins and deuteroporphyrin acid IX 2,4-bis (ethylene glycol) (8,13-bis ( 1,2-d ih id roxieti 1) -3,7,12,17-tetramethyl-21H, 23H-porphine2,18-dipropionic). Additional porphyrin compounds that can be metallized using the procedures described herein include, but are not limited to, coproporphyrins, cytoporphyrins, ethioporphyrins, hematoporphyrins, mesoporphyrins, phylloporphyrins, protoporphyrins, pyrroporphyrins, rhodoporphyrins, uroporphyrins, and phytoporphyrins. A complete list of porphyrin computations is given at World-Wide-Web.chem. qmul.ac.uk/iupac/tetrapirrole/; the porphyrins described therein are incorporated herein by reference as the porphyrins that can be metallized using the procedures described herein.

Purification of tin (IV) dichloride mesoporphyrin IX: trituration with hot acid

At this point, the tin (IV) dichloride m soporphyrin IX

<img file="MX345035B_D0040.tif" />

njalITUTO MtXICANL from pkofieoai: raw INOUSTU1AL is crushed with hot acid in order to remove imnujL &amp; z-as — The material is re-suspended in hydrochloric acid (approximately

0.5 N to 2.0 N, preferably 1N) and the temperature was raised to approximately 75 to 100 ° C or approximately 80 to 100 ° C, preferably approximately 85 to 95 ° C, more preferably approximately 85 to 90 ° C , for approximately one to two hours with moderate agitation. The suspension is then cooled to about 20 to 30 ° C, preferably to about 20 to 25 ° C, and filtered; the filter cake is rinsed with purified water and dried on the filter under a stream of nitrogen.

Purification of stannous (IV) dichloride mesoporphyrin IX: treatment at high pH

The material from the hot acid grinding stage is combined with diatomaceous earth, activated carbon, water, and ammonium hydroxide. The temperature is adjusted to about 20 to 30 ° C, preferably about 20 to 25 ° C, and is stirred, preferably about 1 to 2 hours. A sample is taken to ensure that the pH is at or above about 9. The mixture is stirred, preferably for about 1 to 2 more hours. The mixture is filtered afterwards. Any material that remains in the filter is rinsed with water;

subsequently.

IΜ PI «« WnOMUCANv 1M LA «OWEDMP WAISTHIAI filter cake is discarded

<img file="MX345035B_D0041.tif" />

Re-acidification of stannous (IV) dichloride mesoporphyrin IX

The filtrate is then transferred into a mixture of acetic acid and 31% hydrochloric acid, and the mixture is adjusted to about 20 to 30 ° C, preferably up to about 20 to 25 ° C. The resulting suspension is stirred, preferably for about 15 minutes, sampled to ensure that the pH is less than or equal to about 1, and then stirred again, preferably for about 1 to 2 additional hours. The suspension is then filtered, and the filter cake rinsed with water, followed by removal of residual water under vacuum.

At this stage, the filter cake is sampled for residual starting material, mesoporphyrin dichloride IX. If the level is above about 0.1%, the high pH treatment is repeated followed by re-acidification as necessary (eg, a 1, 2, or 3-fold addition).

Additional hot acid trituration of tin (IV) dichloride mesoporphyrin IX

The filter cake from the top to the top

<img file="MX345035B_D0042.tif" />

suspended in a mixture of about _d £ LS_4iaxtes_ £ .n_p £ s or ______ of WFI grade water and about one part by weight of 31% HCl, at about 20 to 30 ° C, preferably at about 20 to 25 ° C. Under moderate agitation, the mixture is adjusted to about 80 to 100 ° C, preferably up to about 85 to 90 ° C, for about 6 to 48 hours, preferably about 12 to 24 hours, more preferably about 16 to 18 hours. hours, followed by cooling to about 20 to 30 ° C, preferably to about 20 to 25 ° C, for at least about 1 hour. The suspension is filtered, the filter cake rinsed with an aqueous solution of hydrochloric acid (for example, approximately 1 part of 31% HCl to 25 parts of WFI grade water, w / w), and rinsed under a stream of nitrogen (ao below about 50 ° C).

The final hot acid treatment serves to restore the shape of the stansoporfin to monomer. In neutral solution, stansoporfin is in a monomer-dimer equilibrium; strong acid treatment shifts the equilibrium markedly towards the monomer form.

Development work on the synthesis of stansoporfin indicates that for optimal results, the hydrogenation catalyst will be pre-hydrogenated prior to the introduction of the hemin starting material; the isolation of mesoporphyrin IX dichloride from d I format dm soporphyrin IX in formic acid proc d rá by means of the

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL adding the HCI solution to the formic acid solution; the presence of proton scavengers will be avoided during the tin insertion stage; and the introduction of oxygen during the tin insertion step will proceed through the introduction of the oxygen / nitrogen mixture to the upper reaction space, instead of bubbling or vapor injection of the gas through the solution. Considering these optimal parameters, other variables such as temperature, reaction time, reagent concentration, and order of reagent addition can be manipulated to some degree, for example, concentration and reaction time can be varied within about 50 up to 200% of the indicated values, or within approximately 75 to 150% of the indicated values, and the temperature can be varied from approximately 5 to 10 ° C of the indicated values, to the extent that variation does not result in large scale stansoporfin synthesis at a level less than high purity as defined herein. The purification and precipitation steps can be repeated as necessary in order to maintain the high purity of the large-scale preparation of stansoporfin.

Therapeutic use of stansoporfin for treatment or prevention of infantile hyperbilirubinemia and other conditions

Stansoporfin as Produced by Invention

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US 4,668,670; and WO 94/28906). Additional methods for using stansoporfin are described in US 4,692,440 (to increase the rate of heme excretion), WO 89/02269 (to counteract the toxicity of cancer therapy), US 4,782,049 (to treat psoriasis), and other publications. Treatment or prevention of infantile hyperbilirubinemia is accomplished by dissolving stansoporfin in a pharmaceutically acceptable vehicle. The stansoporfin is preferably provided in a solution that can be adjusted in its pH to maintain a suitable pH. The pH regulators that can be used include phosphate, gluconate citrate, lactate, tartrate, glycinate, glycylglycinate, bicarbonate, carbonate, maleate, or acetate, with sodium, potassium, magnesium, calcium, or aluminum present as the cation. Histidine and imidazole can also be used as pH regulators. Phosphate buffers are preferred, in particular sodium phosphate buffers. The pH regulators must be pharmaceutically acceptable for use as an injectable agent in neonates. The pH of the solution for administration is preferably between about 7.0 to 8.0, more preferably about 7.2 to 7.9, even more preferably about 7.4. The osmolarity of the solution is preferably at or near the physiological osmolarity; a preferred range is between approximately 280 mOsm / L and 310

INSTITUTO MEXICANO DE LA MOHEDAL. ' INDUSTRIAL mOsm / L. Stansoporfin is preferably administered by injection, more preferably by intramuscular injection. Stansoporfin is administered in an amount sufficient to treat or prevent infantile hyperbilirubinemia, commonly about 4.5 mg / kg of the newborn; United States Patent Application No. (Attorney Case No. 606952000200) filed on October 4, 2007, and International Patent Application (Patent Cooperation Treaty) No. (Proxy Case No. 606952000240) filed on October 4, 2007, which claim the priority of the Application for Provisional Patent of the United States of North America No. 60 / 849,509, filed October 4, 2006, describe a method of treating infantile hyperbilirubinemia using doses of stansoporfin, such as 1.5 mg / kg of the newborn or 3.0 mg / kg of the newborn.

United States Patent No. 6,818,763, United States Patent Application Publication 2004/0210048, and United States Patent Application No. 11 / 096,359 are specifically incorporated by referenced herein in its entirety.

The following examples are intended to illustrate the invention, and are not intended to limit the invention in any way.

EXAMPLES

Example 1

Illustrative Synthesis of High Purity Stansoporfin

Initial conversion of hemin to mesoporphyrin IX

A 200 L glass lined vessel, with pressure measured at 150 psi, is charged with 0.6 kg of 5% palladium on carbon and 73 kg of formic acid. With vigorous stirring, the reactor is pressurized with hydrogen to 60-65 psi and heated to 40-45 ° C for a minimum time of 12 hours. With moderate stirring, the reaction is cooled to 20-25 ° C, the hydrogen atmosphere is evacuated, and the reactor charged with 6.0 kg of hemin (DMF grade) and 73 kg of formic acid. The reactor is pressurized to 30-35psi with hydrogen and stirred at 20-25 ° C for 30 minutes.

With vigorous stirring the reaction is heated to 85-90 ° C. The hydrogen pressure is then increased to 55-60 psi. Pressure and temperature are maintained for a period of 1-1.5 hours.

The reaction is cooled to 45-50 ° C and hydrogenation is continued at 55-60psi for 24 hours. The reaction is cooled down to 20-25 ° C, sprinkled and sampled.

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The reaction is heated to 45-50 ° C, pressurized to 55 '* ~ Τ ~~ η-ΓΤτ — irm: ι · ~ ι-- · β · ι jtlmi-— 60 psi with hydrogen and stirred for 6 hours plus. The reaction is then cooled to 20-25 ° C, depressurized, and re-sampled.

Hydrogen is evacuated from the container, which is loaded with 3.0 kg of HYFLO SUPERCEL, 2.3 kg of DARCO KB and 42 kg of formic acid. The suspension is filtered, and the filter cake is rinsed with 122 kg of formic acid.

A portion of the filtrate is transferred to a 200 L glass lined vessel, cooled to 10-15 ° C and distilled under vacuum to remove formic acid. Once the residual volume has dropped to 25-35L, the rest of the filtrate is transferred and the distillation continues until a residual volume of 25-30L.

The reaction temperature is adjusted to 20-25 ° C and 89 kg of methyl tert-butyl ether is added for a minimum time of 1 hour. The resulting suspension is stirred at 20-25 ° C for 2 hours before cooling to -25 to -20 ° C over a period of 4 hours.

The suspension is filtered and rinsed with 12 kg of methyl t-butyl ether. The intermediate product is dried in a vacuum oven at 60 ° C or less.

Purification of mesoporphyrin IX format with diatomaceous earth and activated carbon; converting mesoporphyrin IX format to mesoporphyrin IX dichloride

<img file="MX345035B_D0043.tif" />

The intermediate product is transferred to a 50 L glass-lined vessel with 10% w / w DARCO KB, 20% w / w HYFLO SUPERCEL, and 10 parts formic acid. The suspension is stirred at 20-25 ° C for a period of 1.5-2.5 hours.

The suspension is filtered in a second 50L glass-lined container. The filter cake is rinsed with 5 parts of formic acid and discarded. The filtrate solution is vacuum distilled to a residual volume of 5-6 parts.

A third container is loaded with purified water and 31% hydrochloric acid to prepare 15 parts of 1N hydrochloric acid. Six parts of the filtrate solution are transferred into the reactor at 20-25 ° C for a minimum time of 60 minutes.

The solution is seeded with mesoporphyrin dichloride IX and stirred for a minimum time of 2 hours. With vigorous stirring, the remaining 9 parts of 1N hydrochloric acid are transferred into the container for a minimum time of 1 hour.

The resulting suspension is stirred at 20-25 ° C for a period of 2-3 hours before isolation by means of filtration. The filter cake is rinsed with 4 parts of purified water. The mesoporphyrin IX dichloride intermediate is dried on the filter under a stream of nitrogen.

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<img file="MX345035B_D0044.tif" />

Conversion of mesoporphyrin IX dichloride to stannous (IV) dichloride mesoporphyrin IX (stansoporfin)

A 50 L glass-lined container is loaded with 1.57 kg of mesoporphyrin dichloride IX, 1862 kg of stannous (II) chloride, and 40.9 kg of acetic acid at 20-25 ° C. With moderate stirring, the suspension is kept at 20-25 ° C for a minimum of 30 minutes.

With vigorous stirring, under nitrogen, the mixture is heated to reflux (ca. 115 ° C). Once reflux has been obtained, 6% oxygen is introduced into the nitrogen atmosphere into the headspace of the vessel. The reaction mixture is refluxed for a period of 100-130 hours.

The reaction mixture is cooled to 55-60 ° C and sampled for residual mesoporphyrin; While waiting for results, the reaction mixture is heated to reflux. Once complete, the reaction is cooled to 60-70 ° C and charged with 15.7 kg of WFI grade water (water for injection). The temperature of the suspension is adjusted to 20-25 ° C for 30 minutes and stirred for a period of 1 hour.

The suspension is filtered, and the container and cake are rinsed with 6.3 kg of WFI water. At the end of the washing, the cake is placed under vacuum for a minimum time of 4 hours to remove residual water.

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A 50 L glass-lined container is loaded with the ΜΙ · Π · Ι "ww" in "i wet filter cake, 22.4 kg of purified water, and 3.7 kg of 31% hydrochloric acid at 20-25 ° C. With moderate agitation, the temperature of the mixture is adjusted to 85-90 ° C for a period of 1-2 hours, followed by cooling to 20-25 ° C. The suspension was filtered and the filter cake rinsed with 6.3 kg of purified water. The product is dried on the filter under a stream of nitrogen and packed.

Purification of stannous (IV) dichloride mesoporphyrin IX (stansoporfin) at high pH with diatomaceous earth and activated carbon

A 50 L glass-lined container is loaded with 1,448 kg of tin (IV) dichloride mesoporphyrin IX, 0.194 kg of HYFLO SUPERCEL, 0.066 kg of DARCO KB, 14.5 kg of WFI water, and 1.0 kg of ammonium hydroxide 26 Be . The temperature of the reaction mixture is adjusted to 20-25 ° C and stirred for a period of 1-2 hours. A sample was taken to verify that the pH is&gt; 9. The mixture is then stirred for an additional 1-2 hours. The mixture is filtered through a glass receiver. The cake was rinsed with 2.9 kg of water and discarded.

A second 50 L glass-lined container is loaded with 38.2 kg of acetic acid and 2.6 kg of 31% HCl. The temperature is set at 20-25 ° C. The filtrate from the glass receiver is transferred

Λ. » χ Mexican institute οε the INDUSTRIAL mohtoah inside the second 50 L container for a minimum time of 45 minutes at 20-25 ° C. The glass receiver and the transfer apparatus are rinsed with 2.1 kg of WFÍ water in the container. The resulting suspension is stirred at 20-25 ° C for 15 minutes before taking a sample to verify that the pH is &lt;1. The suspension is then stirred for a further 1-2 hours.

The suspension is filtered, and the container and cake are rinsed with 1.3 kg of WFI water. At the end of the washing, the cake is placed under vacuum for a minimum time of 4 hours to remove residual water.

A sample of the filter cake is taken for testing. If the residual starting material (mesoporphyrin dichloride IX) is at an acceptable level, the reaction proceeds to the next stage, otherwise the entire treatment is repeated (i.e. the filter cake is dissolved again using ammonium hydroxide as before).

Low pH stannous (IV) dichloride mesoporphyrin IX (stansoporfin) treatment to fix the monomer form

The wet filter cake is returned to a 50 L glass-lined vessel which is then loaded with 20.4 kg of WFI water and 10.2 kg of 31% hydrochloric acid at 20-25 ° C. With moderate agitation, the temperature of the The mixture is adjusted to 8590 ° C for a period of 16-18 hours, followed by cooling to

INDUSTRIAL

20-25 ° C for a minimum time of 1 hour. The suspension —as ---— filtered and the filter cake rinsed with a premixed solution of 0.5 kg of 31% hydrochloric acid in 12.8 kg of WFI water. The product is dried on the filter at &lt;50 ° C under a stream of nitrogen and packed.

Example 2

Alternative Tin Insertion Stage Using Tin (II) Oxide as a Tin Source

The insertion of tin into mesoporphyrin IX to produce stansoporfin can be carried out via an alternate synthetic route using tin (II) oxide as the reagent for introducing tin.

A dark 100 ml three-necked round bottom flask equipped with a Ciais n head magnetic stir bar, addition funnel, thermometer, condenser, and nitrogen sparger was charged with 8.4 g of tin (II) oxide, and 200 ml of acetic acid, at 20-25 ° C, to form a gray suspension. The suspension was heated to 60-65 ° C under nitrogen.

A separate 250 ml single neck round bottom flask equipped with a stir bar was charged with 10 gd mesoporphyrin IX dichloride, and 50 ml of formic acid. The mixture was stirred at 20-25 ° C for 30 minutes to effect dissolution, resulting in approximately 60 ml of a

<img file="MX345035B_D0045.tif" />

dark purple suspension / solution at 20-25 ° C. (Due to the colored solution, it is difficult to visually detect complete dissolution; mesoporphyrin dichloride IX will be completely ground before addition of formic acid).

The mesoporphyrin IX dichloride solution was charged to the addition funnel and added dropwise to the suspension / solution of tin (ll) oxide / acetic acid over a period of 6 hours, so long as the temperature of the suspension / tin (ll) oxide / acetic acid solution at 60-65 ° C. The volume in the flask was increased from 200 ml to 260 ml; the appearance of the reaction changed from a gray suspension (or white gel), to a purple suspension, to a red suspension.

After the addition was complete, the reaction was stirred under a nitrogen atmosphere at 60-65 ° C for 18-24 hours. Then 100 ml of water were added dropwise over 20-40 minutes, while maintaining the temperature at 60-65 ° C. The resulting red suspension (approximately 360 ml) was cooled to 2025 ° C for 30 minutes and stirred for a minimum time of 1 hour, followed by filtration under reduced pressure (total filtration time was approximately 10-20 minutes) . The filter cake was rinsed with two 20 ml portions of water. The filtrate volume of approximately 400 ml was a cherry colored solution; the filter cake mass was about 40-50 g and was also cherry colored.

The wet filter cake was carefully separated by n pieces and charged to the reaction flask with 1.00 ml of 1N HCl. The resulting cherry-colored suspension was heated at 85-95 ° C for 1 hour. The suspension was then cooled to 20-25 ° C and filtered under reduced pressure (total filtration time was approximately 20-30 minutes); the filtrate was dark cherry to brown in color. The cherry-colored filter cake was rinsed with two 20 ml portions of water, dried under a stream of nitrogen, and further dried under high vacuum at 80-90 ° C for 24 hours. In several repetitions of the synthesis, the yield of the product ranged from 16.5-21.2 g (70-90%).

Example 3

High Purity Stansoporfin Analysis Made by Illustrative Synthesis Using Tin (II) Chloride as Tin Source

Batches of this nsoporfin were prepared using the illustrative synthesis essentially as outlined above in Example 1, as well as in prior methods (see US 6,818,763 and US 2004/0210048).

Basic HPLC analysis was run using a C-18 column (Zorbax Extend C-18, 4.6 x 150 mm, 3.5 pm particle size, or the equivalent). The detector is set at 400 nm. Solvents (acetonitrile, methanol, and water) are HPLC grade. The mobile phase is 16% acetonitrile: 40% methanol: 44% 0.5M ammonium acetate, pH 5.15.

(The ammonium acetate solution is to prepare the solution of 38.5 g of ammonium acetate in 440 mL of H₂Or, and adjusting the pH to 5.15 with acetic acid. Both ammonium acetate and acetic acid are reagent grade. 160 mL of acetonitrile and 400 mL of methanol are added later; the mobile phase solution is mixed, filtered and degassed before use). The flow rate is 1.0 ml / minute. Stansoporfin samples and standards are prepared for injection at a concentration of 0.04 mg / mL in 1N NaOH. Since stansoporfin and related compounds are sensitive to light, solutions that rely on stansoporfin, starting materials, or impurity standards will be kept in opaque containers, and handling and analysis will be conducted under reduced light conditions. Samples and standards will be used 12 hours after preparation. 5 pL d analyte solution is injected and takes a 10-minute run time. The retention time of stansoporfin is typically about 4.8 minutes. The column temperature is kept at 60 ° C. After analysis, the column was washed with 80% methanol and 20% water for at least 1 hour at 1.0 mL / min.

HPLC analysis for quantification of impurities was performed using an ACE 5 C-18 column, 4.6 x 250 mm, particle size 5 pm, with detection at 400 nm. Protection of light sensitive samples and standards was practiced as previously described. The mobile phases used are A: 30% methanol, 70% water with 0.02M ammonium acetate pH, 9 .... 1, and B; 8QX methanol, 20% water with 0.02M ammonium acetate, pH 9.1 (mobile phase A is prepared by dissolving 3.0 g of ammonium acetate in 1400 mL of water, adjusting the pH to 9.1 with NH₄OH, and adding 600 mL of methanol; mobile phase B is prepared by dissolving 3.0 g of ammonium acetate in 400 mL of water, adjusting the pH to 9.1 with NH₄OH, and adding 1600 mL of methanol; mobile phases are mixed, filtered and degassed before use). The samples are dissolved in 0.5% v / v TEA in water at a concentration of approximately 0.2 mg / mL. Samples and standard solutions must be used within 12 hours of preparation.

The analysis is performed using the following gradient conditions:

<td>Time</td><td>%TO</td><td>% B</td>

<td>OR</td><td>1OO</td><td> 0</td>

<td> 50</td><td> 70</td><td> 30</td>

<td> 65</td><td> 70</td><td> 30</td>

<td> 90</td><td> 0</td><td> 100</td>

<td> 110</td><td> 0</td><td> 100</td>

<td> 111</td><td> 100</td><td> 0</td>

<td> 120</td><td> 100</td><td> 0</td>

where the concentrations change linearly between the points shown.

Table 1 contains a comparison of the HPLC analysis of the current synthesis product, column C, compared to the

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<img file="MX345035B_D0046.tif" />

Analysis of products from previous syntheses in column A and column B. The detected peaks are listed in order of retention time relative to stansoporfin, with the retention time of stansoporfin set to 1. The batch analyzed in column A was produced in a quantity of 1.1 kg; the batch analyzed in column C was also produced in a quantity of 1.1 kg.

Table 1. Analysis of various stansoporfin preparations

<td>Time of Relative retention</td><td>TO</td><td>B</td><td>c</td>

<td> 0.33</td><td> 0.06%</td><td></td><td></td>

<td> 0.51</td><td> 0.05%</td><td> 0.05%</td><td> 0.07%</td>

<td> 0.55</td><td></td><td></td><td> 0.06%</td>

<td> 0.73</td><td> 0.14%</td><td> 0.05%</td><td> 0.06%</td>

<td> 0.76</td><td> 0.07%</td><td></td><td> 0.05%</td>

<td> 0.83</td><td> 0.05%</td><td></td><td></td>

<td> 0.84</td><td> 0.05%</td><td></td><td></td>

<td> 0.92</td><td> 0.26%</td><td> 0.06%</td><td></td>

<td> 0.95</td><td> 0.30%</td><td></td><td> 0.05%</td>

<td> 0.96</td><td></td><td> 0.22%</td><td></td>

<td> 1</td><td> 98%</td><td> 99%</td><td> 100%</td>

<td> 1.05</td><td> 0.09%</td><td></td><td></td>

<td> 1.26</td><td> 0.06%</td><td></td><td></td>

As seen from Table 1, the current stansoporfin synthesis in column C resulted in a material that resulted in a product of high purity, overall purity&gt; 99% and containing no impurities at or above 0.1%. .

Example 4

Analysis of High Purity Stansoporfin Prepared by Illustrative Synthesis Using Tin (II) Oxide as Source of

Tin

Three batches of stansoporfin were made using the cap

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<img file="MX345035B_D0047.tif" />

insertion of tin as described in Example 2. Analysis of the three batches indicated that the purity of the stansoporfin produced was 99.7%, 99.7%, and 99.6% (the total content of stansoporfin was 96.4%, 99.1% , and 97.2%, respectively).

HPLC analysis was performed on a Zorbax Extend C column

18, 4.6x150mm, 5pm thick. The eluents used were: A:

80% 0.05 M Ammonium Acetate, pH 5.15 with Acetic Acid: 20% Acetonitrile; B: 90% Methanol: 10% Acetonitrile. The temperature used was 40 ° C. A flow rate of 1.2 ml / min was used, with detection at 400 nm. The retention time of stansoporfin was 8.8 min, while that of mesoporphyrin IX was 23.1 min, with the use of the following gradient listed in Table 2.

Table 2

<td>Time</td><td>TO</td><td>B</td>

<td> 0.0</td><td> 60</td><td> 40</td>

<td> 10.0</td><td> 25</td><td> 75</td>

<td> 30.0</td><td> 25</td><td> 75</td>

<td> 31.0</td><td> 60</td><td> 40</td>

<td> 40.0</td><td> 60</td><td> 40</td>

More extensive analyzes of two batches of stansoporfin produced using the tin oxide insertion method were conducted. These analyzes are detailed in Table 3 (weight d

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<img file="MX345035B_D0048.tif" />

batch 0.840 kg) and Table 4 (batch weight 1.364 kg) below (where a / a indicates the area ratio of the HPLC peaks).

Table 3

<td>Proof</td><td>Method</td><td>Results</td>

<td>Total Purity</td><td>HPLC</td><td>Total impurities &lt;1% a / a; impurity at RRt 0.72 = 0.06% a / a; no other impurity&gt; 0.05% a / a</td>

<td>Water content</td><td>Karl Fischer, colorimetric</td><td>Trace &lt;1% w / w</td>

<td>Residual Solvents- Acetone</td><td>Chromatographic (GC-upper space)</td><td>Not detected &lt;0.1% p / p</td>

<td>Organic content - formic acid + acetic acid</td><td>HPLC</td><td>0.1% w / w</td>

<td>Inorganic content - palladium and iron</td><td>Inductively Coupled Plasma Optical Emission Spectroscopy</td><td>Pd = 5 ppm Fe = 5 ppm</td>

<td>Inorganic content - tin free</td><td>Differential Impulse Polarography</td><td>&lt;0.1% tin free</td>

<td>Inorganic content - tin</td><td>Inductively Coupled Plasma Optical Emission Spectroscopy</td><td>144,500 ppm</td>

<td>Inorganic content - chloride</td><td>Elemental analysis</td><td>104 100 ppm</td>

Table 4

<td>Proof</td><td>Method</td><td>Results</td>

<td>Total Purity</td><td>HPLC</td><td>Total impurities &lt;1% a / a; impurity at RRt 0.72 = 0.06% a / a; no other impurity&gt; 0.05% a / a</td>

<td>Water content</td><td>Kart Fischer, colorimetric</td><td>Trace &lt;1% w / w</td>

<td>Residual Solvents-Acetone</td><td>Chromatographic (GC-headspace)</td><td>Not detected &lt;0.1% p / p</td>

<td>Organic content - formic acid + acetic acid</td><td>HPLC</td><td>Not detected &lt;0.1% w / w</td>

<td>Inorganic content - palladium and iron</td><td>Inductively Coupled Plasma Optical Emission Spectroscopy</td><td>Pd = 5 ppm Fe = 67 ppm</td>

<td>Inorganic content - tin free</td><td>Differential Impulse Polarography</td><td>&lt;0.1% tin free</td>

<td>Inorganic content - tin</td><td>Inductively Coupled Plasma Optical Emission Spectroscopy</td><td>165000 ppm</td>

<td>Inorganic content - chloride</td><td>Elemental analysis</td><td>103 300 ppm</td>

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<img file="MX345035B_D0049.tif" />

Example 5

Stability of High Purity Stansoporfin Preparations

The long-term stability of the compound was studied under two different storage conditions: 25 ° C (+/- 2 ° C) and 60% relative humidity (+/- 5%); and 40 ° C (+/- 2 ° C) and 75% relative humidity (+/- 5%). The primary packaging for the compound was a 4-mil polyethylene bag and the secondary packaging for the compound was a 4-mil polyethylene bag. The double-bagged stansoporfin was stored in a high-density polyethylene drum (CurTec®).

Table 5 and Table 6 show the stability data for the batch described in Table 3, under the conditions of 25 ° C / 60% RH and 40^andC / 75% RH, respectively. Table 7 and Table 8 show the stability data for the batch described in Table 4, under the conditions of 25 ° C / 60% RH and 40 ° C / 75% RH, respectively. The data for the zero month time point were taken from the batch release analysis (the zero time point represents the actual date when the samples were placed in the stability test chambers). The samples were analyzed approximately 3 months and approximately 6 months after the samples were placed under the storage conditions.

<img file="MX345035B_D0050.tif" />

Table 5

<td>Proof</td><td>0 months</td><td>3 months</td><td>6 months</td>

<td>Appearance</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td>

<td>HPLC purity</td><td> 0.3</td><td> 0.22</td><td> 0.24</td>

<td>HPLC purity (peak impurity at retention time 0.720.73)</td><td> 0.06%</td><td> &lt;0.05%</td><td> 0.07%</td>

<td>HPLC test (w / w, solvent-free anhydrous base)</td><td> 100.7%</td><td> 99.8%</td><td> 98.4%</td>

<td>HPLC test (w / w as is)</td><td> 100.4%</td><td> 99.6%</td><td> 98.2%</td>

<td>Water content (Karl Fischer, colorimetric)</td><td>Trace &lt;1%</td><td> &lt;1% (0.1%)</td><td> &lt;1% (0.1%)</td>

Table 6

<td>Proof</td><td>0 months</td><td>3 months</td><td>6 months</td>

<td>Appearance</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free visual evidence of contamination</td>

<td>HPLC purity</td><td> 0.3</td><td> 0.28</td><td> 0.26</td>

<td>HPLC purity (peak impurity at retention time 0.720.73)</td><td> 0.06%</td><td> &lt;0.05%</td><td> 0.067%</td>

<td>HPLC test (w / w, solvent-free anhydrous base)</td><td> 100.7%</td><td> 101.2%</td><td> 99.8%</td>

<td>HPLC test (w / w as is)</td><td> 100.4%</td><td> 100.9%</td><td> 99.6%</td>

<td>Water content (Karl Fisch r, colorimetric)</td><td>Trace &lt;1%</td><td> &lt;1% (0.2%)</td><td> &lt;1% (0.1%)</td>

<img file="MX345035B_D0051.tif" />

Table 7

<td>Proof</td><td>0 months</td><td>3 months</td><td>6 months</td>

<td>Appearance</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td>

<td>HPLC purity</td><td> 0.22</td><td> 0.29</td><td> 0.19</td>

<td>HPLC purity (peak impurity at retention time 0.720.73)</td><td> 0.06%</td><td> &lt;0.05%</td><td> 0.05%</td>

<td>HPLC test (w / w, solvent-free anhydrous base)</td><td> 102.3%</td><td> 102.1%</td><td> 98.5%</td>

<td>HPLC test (w / w as is)</td><td> 102.3%</td><td> 102.0%</td><td> 98.4%</td>

<td>Water content (Karl Fischer, colorimetric)</td><td>Trace &lt;1%</td><td> &lt;1% (0.1%)</td><td> &lt;1% (0.1%)</td>

Table 8

<td>Proof</td><td>0 months</td><td>3 months</td><td>6 months</td>

<td>Appearance</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td><td>Red powder free from visual evidence of contamination</td>

<td>HPLC purity</td><td> 0.22</td><td> 0.29</td><td> 0.24</td>

<td>HPLC purity (peak impurity at retention time 0.720.73)</td><td> 0.06%</td><td> &lt;0.05%</td><td> 0.06%</td>

<td>HPLC test (w / w, solvent-free anhydrous base)</td><td> 102.3%</td><td> 101.1%</td><td> 97.8%</td>

<td>HPLC test (w / w as is)</td><td> 102.3%</td><td> 101.0%</td><td> 97.7%</td>

<td>Water content (Karl Fischer, colorim 'tric)</td><td>Trace &lt;1%</td><td> &lt;1% (0.1%)</td><td> &lt;1% (0.1%)</td>

IMPI 'Mtnuiu MEXICANO os u nomoAo

The descriptions of all publications, patents, patent applications, and published patent applications referred to herein by an identifying citation are incorporated herein by reference in their entirety.

Although the above invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it is apparent to those of skill in the art that certain minor changes and modifications will be made. Therefore, the description and examples will not be construed as limiting the scope of the invention.