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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with AFRICURE PHARMA, ROW2TECH, NIPER-G, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India as ADVISOR, earlier assignment was
with GLENMARK LIFE SCIENCES LTD, as CONSUlTANT, Retired from GLENMARK in Jan2022 Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 32 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
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and implementation them on commercial scale over a 32 PLUS year tenure till date Feb 2023, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 100 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 100 Lakh plus views on dozen plus blogs, 227 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 38 lakh plus views on New Drug Approvals Blog in 227 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc
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Atosiban, sold under the brand name Tractocile among others, is an inhibitor of the hormones oxytocin and vasopressin. It is used as an intravenousmedication as a labour repressant (tocolytic) to halt premature labor. It was developed by Ferring Pharmaceuticals in Sweden and first reported in the literature in 1985.[5] Originally marketed by Ferring Pharmaceuticals, it is licensed in proprietary and generic forms for the delay of imminent preterm birth in pregnant adult women.
Atosiban is an inhibitor of the hormones oxytocin and vasopressin. It is used intravenously to halt premature labor. Although initial studies suggested it could be used as a nasal spray and hence would not require hospital admission, it is not used in that form. Atobisan was developed by the Swedish company Ferring Pharmaceuticals. It was first reported in the literature in 1985. Atosiban is licensed in proprietary and generic forms for the delay of imminent pre-term birth in pregnant adult women.
Medical uses
Atosiban is used to delay birth in adult women who are 24 to 33 weeks pregnant, when they show signs that they may give birth pre-term (prematurely).[4] These signs include regular contractions lasting at least 30 seconds at a rate of at least four every 30 minutes,[4] and dilation of the cervix (the neck of the womb) of 1 to 3 cm and an effacement (a measure of the thinness of the cervix) of 50% or more.[4] In addition, the baby must have a normal heart rate.[4]
Pharmacology
Mechanism of action
Atosiban is a nonapeptide, desamino-oxytocin analogue, and a competitive vasopressin/oxytocin receptor antagonist (VOTra). Atosiban inhibits the oxytocin-mediated release of inositol trisphosphate from the myometrial cell membrane. As a result, reduced release of intracellular, stored calcium from the sarcoplasmic reticulum of myometrial cells and reduced influx of Ca2+ from the extracellular space through voltage-gated channels occur. In addition, atosiban suppresses oxytocin-mediated release of PGE and PGF from the decidua.[6]
In human preterm labour, atosiban, at the recommended dosage, antagonises uterine contractions and induces uterine quiescence. The onset of uterus relaxation following atosiban is rapid, uterine contractions being significantly reduced within 10 minutes to achieve stable uterine quiescence.
Other uses
Atosiban use after assisted reproduction
Atosiban is useful in improving the pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ET) in patients with repeated implantation failure.[7] The pregnancy rate improved from zero to 43.7%.[8]
First- and second-trimester bleeding was more prevalent in ART than in spontaneous pregnancies. From 2004 to 2010, 33 first-trimester pregnancies with vaginal bleeding after ART with evident uterine contractions, when using atosiban and/or ritodrine, no preterm delivery occurred before 30 weeks.[9]
In a 2010 meta-analysis,[10] nifedipine is superior to β2 adrenergic receptor agonists and magnesium sulfate for tocolysis in women with preterm labor (20–36 weeks), but it has been assigned to pregnancy category C by the U.S. Food and Drug Administration, so is not recommended before 20 weeks, or in the first trimester.[9] A report from 2011 supports the use of atosiban, even at very early pregnancy, to decrease the frequency of uterine contractions to enhance success of pregnancy.[7]
Pharmacovigilance
Following the launch of atosiban in 2000, the calculated cumulative patient exposure to atosiban (January 2000 to December 2005) is estimated as 156,468 treatment cycles. To date, routine monitoring of drug safety has revealed no major safety issues.[11]
Regulatory affairs
Atosiban was approved in the European Union in January 2000 and launched in the European Union in April 2000.[12][4] As of June 2007, atosiban was approved in 67 countries, excluding the United States and Japan.[12] It was understood that Ferring did not expect to seek approval for atosiban in the US or Japan, focusing instead on development of new compounds for use in Spontaneous Preterm Labor (SPTL).[12] The fact that atosiban only had a short duration before it was out of patent that the parent drug company decided not to pursue licensing in the US.[13]
Systematic reviews
In a systematic review of atosiban for tocolysis in preterm labour, six clinical studies — two compared atosiban to placebo and four atosiban to a β agonist — showed a significant increase in the proportion of women undelivered by 48 hours in women receiving atosiban compared to placebo. When compared with β agonists, atosiban increased the proportion of women undelivered by 48 hours and was safer compared to β agonists. Therefore, oxytocin antagonists appear to be effective and safe for tocolysis in preterm labour.[14]
A 2014 systematic review by the Cochrane Collaboration showed that while atosiban had fewer side effects than alternative drugs (such as ritodrine), other beta blockers, and calcium channel antagonists, it was no better than placebo in the major outcomes i.e. pregnancy prolongation or neonatal outcomes. The finding of an increase in infant deaths in one placebo-controlled trial warrants caution. Further research is recommended.[15]
PATENT
WO 2021207870
Atosiban (Atosiban) is an oxytocin and vasopressin V1A combined receptor antagonist, which can be used as a competitive antagonist of cyclic peptide oxytocin receptors in the uterus, decidua and fetal membrane. Atosiban is a disulfide-bonded cyclic polypeptide composed of 9 amino acids. It is a modified oxytocin molecule at positions 1, 2, 4 and 8. The N-terminal of the peptide is 3-mercaptopropionic acid (thiol and [ Cys] 6 thiol forms a disulfide bond), the C-terminal is in the form of an amide, and the second amino acid at the N-terminal is ethylated [D-Tyr(Et)] 2 . Atosiban is generally present in medicines in the form of acetate salt, commonly known as atosiban acetate. Its chemical formula is C 45 H 71 N 11 O 14 S 2 , its molecular weight is 994.19, and its structural formula is as follows:
[0003]
[0004]
In the prior art, atosiban is usually synthesized by a solid-phase peptide synthesis (SPPS) method, an amino resin is used as a starting carrier resin, and protected amino acids are sequentially connected, and the obtained atosiban is oxidized and then cleaved to obtain atosiban. However, the above-mentioned existing process has high cost, generates a large amount of solvent waste, and is not easy to monitor during the cyclization process. In addition, the above-mentioned prior art has deficiencies in the overall yield of crude peptides. Moreover, due to the existence of D-Tyr(Et) in the structure of atosiban, Fmoc-D-Tyr(Et) easily undergoes a racemization reaction during the peptide attachment process, resulting in [Tyr(Et) 2 ]-A The impurity of tosiban, which is similar in polarity to atosiban itself, is difficult to completely remove through purification, thus affecting the quality of atosiban.
[table 0001]
Amino acid name
alphabetic symbols
Glycine
Gly
Ornithine
Orn
Proline
Pro
cysteine
Cys
Asparagine
Asn
Threonine
Thr
Isoleucine
Ile
D-tyrosine (oxyethyl)
D-Tyr(ET)
Table 3 List of intermediates and Fmoc protected amino acids
According to the most preferred embodiment of the present invention, the method of the present invention comprises the following steps:
[0046]
The first step: Fmoc-Gly Rink resin can be directly purchased, which reduces the first step of synthesis and improves the synthesis efficiency;
[0047]
The second step: preparing a deprotection solution: the deprotection solution is a mixture of piperidine/N,N-dimethylformamide, preferably piperidine/N,N-dimethylformamide in a volume ratio of 1/4.
[0048]
The third step: preparation of Fmoc-Orn(Boc)-Gly Rink resin: deprotect the Fmoc-Gly Rink resin obtained in the first step, wash with DMF, add Fmoc-Orn(Boc)-OH in DMF solution, Condensation reaction is carried out under the condition of peptide coupling condensing agent to obtain Fmoc-Orn(Boc)-Gly Rink resin;
[0049]
The fourth step: preparation of Fmoc-Pro-Orn(Boc)-Gly Rink resin: the peptide resin obtained in the fourth step is deprotected and washed, and then reacted with Fmoc-Pro-OH under the condition of a peptide coupling agent to obtain Fmoc-Pro-Orn(Boc)-Gly Rink resin;
[0050]
The fifth step: preparation of Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the fifth step is deprotected and washed, and then reacted with Fmoc-Cys(Trt)-OH under the condition of peptide coupling agent to obtain Fmoc-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin;
[0051]
The sixth step: preparation of Fmoc-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the sixth step is deprotected and washed, and then reacted with Fmoc-Asn-OH under the condition of peptide coupling agent to obtain Fmoc-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin ;
[0052]
The seventh step: preparation of Fmoc-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the seventh step was deprotected and washed, and then reacted with Fmoc-Thr(tBu)-OH under the condition of a peptide coupling agent. Obtain Fmoc-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin;
[0053]
The eighth step: preparation of Fmoc-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the eighth step is deprotected and washed, and then reacted with Fmoc-Ile-OH under the condition of a peptide coupling agent to obtain Fmoc-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn (Boc)-Gly Rink resin;
[0054]
The ninth step: preparation of Fmoc-D-Tyr(RT)-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the ninth step is deprotected and washed, and then reacted with Fmoc-D-Tyr(ET)-OH under the condition of a peptide coupling agent to obtain Fmoc-D-Tyr(RT)-Ile-Thr(tBu )-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin;
[0055]
The tenth step: preparation of Mpa(Trt)-D-Tyr(ET)-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin. The peptide resin obtained in the tenth step is deprotected and washed, and then reacted with Mpa(Trt) under the condition of a peptide coupling agent to obtain Mpa(Trt)-D-Tyr(ET)-Ile-Thr(tBu)-Asn -Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin;
[0056]
The eleventh step: Mpa(Trt)-D-Tyr(ET)-Ile-Thr(tBu)-Asn-Cys(Trt)-Pro-Orn(Boc)-Gly Rink resin in TFA/TIS/EDT/H2O =90/54/10/5 TFA, cleaved for 3 hours, and filtered to obtain crude peptide solution;
[0057]
The twelfth step: sedimentation and washing of the crude peptide solution with methyl tert-butyl ether, centrifugation at 2000 rpm, and vacuum drying to obtain a pale yellow solid powder of atosiban linear crude peptide;
[0058]
The thirteenth step: prepare three solutions for atosiban cyclization: solution A-sodium acetate buffered aqueous solution, solution B-aqueous solution of linear peptide atosiban crude peptide acetic acid, solution C: 30%-60% hydrogen peroxide solution ;
[0059]
The fourteenth step: Mix the above three solutions of A, B, and C at 15-25 ° C, and stir for 1-3 hours after mixing, so that the Mpa at the 1st position and the Cys at the 6th position form a disulfide bond to obtain Cyclized atosiban crude peptide.
[0060]
Step fifteen: Purify crude atosiban by preparative high performance liquid chromatography with a water/acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
[0061]
The sixteenth step: freeze-dry the purified atosiban solution at -50 to -70° C. for 18-48 hours with a freeze dryer.
[0062]
The purity of atosiban obtained by the method of the invention is more than 99.5%, and the total product yield is 55%-65%.
[0063]
The advantage of the method for preparing atosiban of the present invention is:
[0064]
The traditional SPPS synthesis of atosiban usually produces a large amount of waste with high disposal costs. This process adopts high-temperature SPPS process and selects different condensing agent combinations, which is faster than the conventional SPPS process, the product purity can reach more than 99.9%, the purity is better than that of the conventional atosiban process, the impurity content is low, and the product quality is high. The total yield can reach 55%-65%.
Detailed ways
[0065]
The invention will now be described with reference to specific embodiments. It must be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, experimental materials and reagents used in the following examples were obtained from commercial sources.
[0066]
Example 1:
[0067]
Using Rink-Fmoc-Gly resin (40 g, substitution amount 0.61 mmol/g) as the starting material, the stepwise Fmoc-SPPS (solid phase peptide synthesis) method was used to synthesize the peptide. Fmoc deprotection was performed with piperidine in DMF (1:4 v/v). Subsequently, other amino acids in the sequence are connected in the following order, and the coupling reagents are N,N-diisopropylcarbodiimide, 2-(7-benzotriazole)-N,N,N’,N ‘-Tetramethylurea hexafluorophosphate mixed in a volume ratio of 1:1, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(ET)-OH, Mpa(Trt). Coupling and deprotection of amino acids were carried out at 90°C for 2-3 min and monitored with the Kaiser test. The peptide was cleaved with the lysing solution of TFA for 3 hours, precipitated and washed twice with methyl tert-butyl ether, and after centrifugal drying, the atosiban linear crude peptide was cyclized by the method of liquid phase synthesis, and the volume ratio was 1: 2:2 A solution-acetic acid-sodium acetate buffer aqueous solution (concentration is 30g/L), B solution-linear peptide atosiban crude peptide acetic acid aqueous solution and C solution: 60% hydrogen peroxide solution.
[0068]
The crude peptide yield was 85%. Crude atosiban was purified by preparative high performance liquid chromatography with a water/acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified atosiban solution is freeze-dried at -50 to -70° C. for 18 hours with a freeze dryer, the obtained atosiban has a purity of more than 99.5%, and the total product yield is 56%.
[0069]
Example 2:
[0070]
Using Rink-Fmoc-Gly resin (40 g, substitution amount 0.36 mmol/g) as the starting material, the stepwise Fmoc-SPPS (solid phase peptide synthesis) method was used to synthesize the peptide. Fmoc deprotection was performed with piperidine in DMF (1:4 v/v). Subsequently, the other amino acids in the sequence are connected in the following order, and the coupling reagents are N,N-tert-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and Oxyma, which are mixed in a volume ratio of 1:1:1 , Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D- Tyr(ET)-OH, Mpa(Trt). Coupling and deprotection of amino acids were carried out at 90°C for 2-3 min and monitored with the Kaiser test. The peptide was cleaved with the lysing solution of TFA for 3 hours, precipitated with methyl tert-butyl ether and washed twice, and after centrifugal drying, the atosiban linear crude peptide was cyclized by the method of liquid phase synthesis, and the volume ratio was 1: 3:2 solution A-formic acid-sodium formate buffer aqueous solution (concentration 25g/L), solution B-linear peptide atosiban crude peptide formic acid aqueous solution and solution C: 30% hydrogen peroxide solution, and oxygen was introduced.
[0071]
The crude peptide yield was 83%. Crude atosiban was purified by preparative high performance liquid chromatography with a water/acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified atosiban solution is freeze-dried at -50 to -70° C. for 18 hours with a freeze dryer, the obtained atosiban has a purity greater than 99.5%, and the total product yield is 57%.
[0072]
Example 3:
[0073]
Using Rink-Fmoc-Gly resin (40 g, substitution amount 0.36 mmol/g) as the starting material, the stepwise Fmoc-SPPS (solid phase peptide synthesis) method was used to synthesize the peptide. Fmoc deprotection was performed with piperidine in DMF (1:4 v/v). Subsequently, other amino acids in the sequence were connected in the following order, and the coupling reagents were N,N-diisopropylethylamine, 2-(7-benzotriazole)-N,N,N’,N’- Two kinds of tetramethylurea hexafluorophosphate mixed in a 1:1 volume ratio, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc- Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(ET)-OH, Mpa(Trt). Coupling and deprotection of amino acids were carried out at 75°C for 2-3 min and monitored with the Kaiser test. The peptide was cleaved with the lysing solution of TFA for 3 hours, precipitated and washed twice with methyl tert-butyl ether, and after centrifugal drying, the atosiban linear crude peptide was cyclized by the method of liquid phase synthesis, and the volume ratio was 1: 2:3 solution A-sodium phosphate buffered aqueous solution (concentration 15g/L), solution B-linear peptide atosiban crude peptide phosphoric acid aqueous solution and solution C: DMSO aqueous solution (volume 1:1).
[0074]
The crude peptide yield was 80%. Crude atosiban was purified by preparative high performance liquid chromatography with a water/acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified atosiban solution is freeze-dried at -50 to -70 DEG C for 28 hours with a freeze dryer, the obtained atosiban has a purity of more than 99.5%, and the total product yield is 55%.
[0075]
Example 4:
[0076]
Using Rink-Fmoc-Gly resin (40 g, substitution amount 0.36 mmol/g) as the starting material, the stepwise Fmoc-SPPS (solid phase peptide synthesis) method was used to synthesize the peptide. Fmoc deprotection was performed with piperidine in DMF (1:3 by volume). Subsequently, the other amino acids in the sequence were connected in the following order, and the coupling reagents were selected from 2-oxime ethyl cyanoacetate, N,N-diisopropylcarbodiimide, and 1-hydroxybenzotriazole in a volume ratio of 1. :1:1 mix, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH , Fmoc-D-Tyr(ET)-OH, Mpa(Trt). Coupling and deprotection of amino acids were carried out at 80°C for 2-3 min and monitored with the Kaiser test. The peptide was cleaved with the lysing solution of TFA for 3 hours, precipitated with methyl tert-butyl ether and washed twice, and after centrifugal drying, the atosiban linear crude peptide was cyclized by the method of liquid phase synthesis, and the volume ratio was 1: 3:4 solution of A-trifluoroacetic acid-aqueous ammonia solution (concentration of 45 g/L), solution B-aqueous solution of linear peptide atosiban crude peptide trifluoroacetic acid and solution C: saturated aqueous iodine solution.
[0077]
The crude peptide yield was 78%. Crude atosiban was purified by preparative high performance liquid chromatography with a water/acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified atosiban solution is freeze-dried at -50 to -70° C. for 38 hours with a freeze dryer, the obtained atosiban has a purity of more than 99.5%, and the total product yield is 52%.
Atosiban Acetate Injection was first listed in Austria on March 23, 2000 under the trade name: Atosiban, a new type of anti-prematurity drug developed by Ferring GmbH, which is an oxytocin The analog is a competitive antagonist of oxytocin receptors in the uterus, decidua, and fetal membranes. It is a first-line drug recommended by the European Medical Association; it can inhibit the binding of oxytocin and oxytocin receptors, thereby directly inhibiting the effect of oxytocin. In the uterus, it can inhibit uterine contraction; it can also inhibit the hydrolysis of phosphatidylinositol.
Atosiban is a cyclic nonapeptide whose molecular formula is C 43 H 67 N 11 O 12 S 2 ; molecular weight is 994.19; CAS registration number is 90779-69-4; its peptide sequence is as follows:
In the Chinese patents with announcement numbers CN101314613B and CN101696236B, the solid-phase synthesis of atosiban uses Rink Amide AM Resin resin solid-phase coupling stepwise to obtain Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)- Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Resin is directly oxidized in solid phase to generate disulfide bonds, and then cleaved to obtain atosiban. The Rink Amide AM Resin resin used in the prior art needs to be cracked under a strong acid environment, which is not conducive to product stability and has a greater operational risk; Mpr and Cys both have sulfhydryl groups, and the sulfhydryl groups have the ability to capture tBu to generate double tBu impurities, When the peptide resin after solid-phase oxidation is cleaved to remove the protective group and resin, due to the presence of tBu or tBu source Boc protective group, it requires high capture agent, which is not conducive to product quality control and reduces product yield.
The Chinese patent with publication number CN105408344B discloses a method for synthesizing atosiban starting from Fmoc-Orn-Gly-NH2, wherein Fmoc-Orn-Gly-NH2 is connected to trityl through the side chain of ornithine On the base resin, impurities can be effectively controlled. However, using dipeptide and trityl-type resin for coupling, the resin attached to the Orn side chain of the dipeptide increases the steric hindrance of the subsequent Pro coupling and prolongs the coupling time, which is easy to cause missing peptide impurities.
Example 1. Synthesis of Fmoc-Pro-Orn-Gly-NH 2 tripeptide
[0027]
Fmoc-Pro-OH (134.94 g, 400 mmol) and N-hydroxysuccinimide (46.00 g, 400 mmol) were weighed into 1600 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (90.72g, 440mmol) in tetrahydrofuran (320ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 400 ml of tetrahydrofuran, and H-Orn(Boc)-NH 2 (92.92 g, 400 mmol) was dissolved in 300 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate to dryness under reduced pressure, add N-hydroxysuccinimide (46.00 g, 400 mmol) and 1600 ml of tetrahydrofuran to dissolve, and stir at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (90.72g, 440mmol) in tetrahydrofuran (320ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 400 ml of tetrahydrofuran, and H-Gly-NH 2 (29.64 g, 400 mmol) was dissolved in 300 ml of tetrahydrofuran and slowly added dropwise to the above solution, and the reaction was continued at room temperature after dropping, and the monitoring of the raw materials was completed. The reaction was filtered, and the filtrate was concentrated under reduced pressure. Dry, add 1000 mL of 5% TFA/DCM solution to the reaction solution, continue to react for 1 h, and concentrate to dryness to obtain a yellow oil, which is recrystallized from isopropanol to obtain 171.56 g of white solid with a yield of 69%.
[0028]
Example 2. Synthesis of Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.42 mmol/g
[0029]
Trityl resin (37.5 g, 30 mmol, substitution degree: 0.80 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1 , DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 52.14 g of Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.42 mmol/g.
[0030]
Example 3. Synthesis of Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
[0031]
2-CTC Resin resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1 , DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.80 g of Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
[0032]
Example 4. Synthesis of Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
[0033]
4-methyl-trityl resin (33.33 g, 30 mmol, substitution degree: 0.90 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1 , DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.89 g of Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
[0034]
Example 5. Synthesis of Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
[0035]
4-Methoxy-trityl resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1 , DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.69 g of Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
[0036]
Example 6. Synthesis of Atosiban Linear Peptide Resin 1
[0037]
Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 (35.71 g) prepared in Example 2 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod. The ninhydrin test was positive, indicating that Fmoc had been removed.
[0038]
Weigh 17.57g Fmoc-Cys(Trt)-OH and 4.86g HOBt, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.68g DIC (pre-cooled to <0°C), Activated in the solution for about 3 to 5 minutes, the activated solution was added to the reaction column under control, and reacted at 20 to 35 °C for 2 to 3 hours. The ninhydrin test was negative. The reaction solution was removed, and 200 mL of DMF was added to wash the resin. 6 times. After washing, the washing liquid was removed to obtain Fmoc-Cys(Trt)-Pro-Orn (trityl resin)-Gly-NH 2 .
[0039]
Repeat the step of receiving the peptide and remove the Fmoc protective group. According to the amino acid sequence of atosiban, Fmoc-Cys(Trt)-Pro-Orn (trityl resin)-Gly-NH 2 was coupled to Fmoc- Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH give Mpa(Trt)-D-Tyr(Et)-Ile-Thr- Asn-Cys(Trt)-Pro-Orn (trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 48.72g after drying, and the resin weight gain was 89.0%.
[0040]
Example 7. Synthesis of atosiban linear peptide resin 2
[0041]
Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 (30.00 g) prepared in Example 3 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod. The ninhydrin test was positive, indicating that Fmoc had been removed.
[0042]
Weigh 17.57g Fmoc-Cys(Trt)-OH and 13.65g HBTU, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.82g DIEA (pre-cooled to <0°C), put Activated in the solution for about 3 to 5 minutes, the activated solution was added to the reaction column under control, and reacted at 20 to 35 °C for 2 to 3 hours. The ninhydrin test was negative. The reaction solution was removed, and 200 mL of DMF was added to wash the resin. 6 times. After washing, the washing solution was removed to obtain Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2 .
[0043]
Fmoc-D-Tyr(Et)-OH (86.30 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-Ile-OH (26.24 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. The monitoring of the raw materials was completed. After filtration, the solution was concentrated under reduced pressure. , the concentrated solution was added to petroleum ether to separate out the solid, the solid was washed and then dried, recrystallized and dried with isopropanol to obtain 75.60 g of Fmoc-D-Tyr(Et)-Ile-OH with a yield of 75%.
[0044]
Repeat the step of receiving the peptide and removing the Fmoc protective group. According to the amino acid sequence of atosiban, sequentially couple Fmoc-Asn on Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2 -OH, Fmoc-Thr-OH, Fmoc-D-Tyr(Et)-Ile-OH, Mpa(Trt)-OH to give Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt )-Pro-Orn( 2 -CTC Resin)-Gly-NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.77g after drying, and the resin weight gain rate was 87.4%.
[0045]
Example 8. Synthesis of atosiban linear peptide resin 3
[0046]
Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 4 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
[0047]
Weigh 17.57g Fmoc-Cys(Trt)-OH, 13.65g HBTU and 4.05g HOBt, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.82g DIEA (pre-cooled to <0 ℃), activate in the solution for about 3-5min, add the activated solution to the reaction column, react at 20-35 ℃ for 2-3h, the ninhydrin test is negative, remove the reaction solution, add 200mL of DMF The resin was washed 6 times. After washing, the washing liquid was removed to obtain Fmoc-Cys(Trt)-Pro-Orn(4-methyl-trityl resin)-Gly-NH 2 .
[0048]
Mpa(Trt)-OH (69.69 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and HD-Tyr(Et)-OH (41.85 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate under reduced pressure, add the concentrated solution to petroleum ether to precipitate a solid, wash the solid and then dry it, recrystallize and dry with isopropanol to obtain Mpa(Trt)-D-Tyr(Et)-OH 77.98g, yield 72%.
[0049]
Repeat the step of receiving the peptide and removing the Fmoc protective group, according to the amino acid sequence of atosiban, on Fmoc-Cys(Trt)-Pro-Orn (4-methyl-trityl resin)-Gly- NH 2 Fmoc-Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Mpa(Trt)-D-Tyr(Et)-OH were sequentially coupled to obtain Mpa(Trt)-D-Tyr(Et)-Ile-Thr -Asn-Cys(Trt)-Pro-Orn(4-methyl-trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.91g after drying, and the resin weight gain rate was 88.3%.
[0050]
Example 9. Synthesis of atosiban linear peptide resin 4
[0051]
Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 5 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod. The ninhydrin test was positive, indicating that Fmoc had been removed.
[0052]
Fmoc-Asn-OH (70.87 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-Cys(Trt)-OH (79.96 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate under reduced pressure, add the concentrated solution to petroleum ether to precipitate a solid, wash the solid and then dry, recrystallize and dry with isopropanol to obtain Fmoc-Asn-Cys(Trt)-OH 102.17g, yield 73%.
[0053]
Weigh 20.99g Fmoc-Asn-Cys(Trt)-OH and 13.65g HCTU, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.82g DIEA (pre-cool to <0°C) , activate in the solution for about 3-5min, add the activated solution to the reaction column, react at 20-35°C for 2-3h, the ninhydrin test is negative, remove the reaction solution, add 200mL of DMF to wash the resin , wash 6 times. After washing, the washing liquid was removed to obtain Fmoc-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly-NH 2 .
[0054]
Repeat the step of receiving the peptide and removing the Fmoc protective group. According to the amino acid sequence of atosiban, in Fmoc-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly- Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH were sequentially coupled on NH 2 to obtain Mpa(Trt)-D-Tyr(Et)-Ile- Thr-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.28g after drying, and the resin weight gain was 84.0%.
[0055]
Example 10. Synthesis of atosiban crude peptide 1
[0056]
Configure 487.2ml of TFA/DCM=2/98 (V/V) lysis solution, cool to 5-10°C, add 48.72g of peptide resin prepared in Example 6 into the lysis solution, at room temperature (20-35°C) React for 5h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.77g of atosiban linear peptide, dissolve 14.30g of atosiban linear peptide in 0.75L of glacial acetic acid, add 6.75L of water to dilute, add 0.1M/L iodine ethanol solution dropwise until the solution changes color, react at room temperature for 1.0h, That is, the crude atosiban peptide is obtained, and its HPLC spectrum is shown in Figure 1.
[0057]
Example 11. Synthesis of atosiban crude peptide 2
[0058]
Configure TFA/DCM=5/95 (V/V) lysate 448.6ml, cool to 5～10℃, add 42.77g of peptide resin prepared in Example 7 into the lysate, at room temperature (20～35℃) React for 3h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate, dry to obtain a solid, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.21g of atosiban linear peptide, dissolve 14.21g of atosiban linear peptide in 1.5L of glacial acetic acid, add 6L of water to dilute, add 0.1M/L iodoethanol solution dropwise until the solution changes color, react at room temperature for 1.0h, that is The crude atosiban peptide was obtained, and its HPLC chromatogram was similar to that in Figure 1.
[0059]
Example 12. Synthesis of atosiban crude peptide 3
[0060]
Configure 450.5ml of TFA/DCM=20/80(V/V) lysis solution, cool to 5～10℃, add 45.05g of peptide resin prepared in Example 8 into the lysis solution, at room temperature (20～35℃) React for 2h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.63g of atosiban linear peptide, dissolve 14.63g of atosiban linear peptide in 1.5L of glacial acetic acid, add 6L of water to dilute, add 10% hydrogen peroxide solution, and react at room temperature for 1.0h to obtain atosiban Crude peptide, its HPLC chromatogram is similar to Figure 1.
[0061]
Example 13. Synthesis of atosiban crude peptide 4
[0062]
Configure TFA/DCM=1/99 (V/V) lysate 442.7ml, cool to 5～10℃, add 44.27g of peptide resin prepared in Example 9 into the lysate, at room temperature (20～35℃) React for 5h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.13 g of atosiban linear peptide, dissolve 14.13 g of atosiban linear peptide in 1.5 L of glacial acetic acid, add 6 L of water to dilute, add 30% hydrogen peroxide solution, and react at room temperature for 1.0 h to obtain atosiban Crude peptide, its HPLC chromatogram is similar to Figure 1.
[0063]
Example 14. Purification of atosiban crude peptide 1
[0064]
The atosiban crude peptide prepared in Example 10 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.12g , the yield is 64%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is shown in Figure 2.
[0065]
Example 15. Purification of atosiban crude peptide 2
[0066]
The crude atosiban peptide obtained in Example 11 was dissolved in a 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 9.80 g , the yield is 62%, the purity is 99%, and the obtained atosiban peptide HPLC spectrum is similar to Figure 2.
[0067]
Example 16. Purification of atosiban crude peptide 3
[0068]
The crude atosiban peptide obtained in Example 12 was dissolved in a 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.28g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.
[0069]
Example 17. Purification of atosiban crude peptide 4
[0070]
The crude atosiban peptide obtained in Example 13 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.27g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.
Atosiban is a nonapeptide which contains three non-natural amino acids: D-Tyr(Et), Mpa and Orn, and a pair of disulfide bonds looped between Mpa and Cys, the structural formula is: c[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH2.
By means of competing for oxytocin receptor with oxytocin, Atosiban can inhibit the combination between oxytocin and oxytocin receptor, and directly prevent the oxytocin from acting on uterus, and then inhibit the uterine contraction; as another hand, atosiban can also inhibit the hydrolysis of phosphatidylinositol and then block the generation of messenger and activity of Ca2+, with the decreasing of activity from oxytocin, the contraction of uterine is indirectly inhabited.
At present, there are many reports about synthesis process method in China and abroad A report in China shows that the inventor found a simple process by adopting solid phase oxidation, resulting in a low purity crude product, with low yield and low application value. The aforementioned reports about atosiban synthesis process reveal that most of them adopt the method using Boc solid phase synthetic and cleaving peptide with liquid ammonia, then oxidating with liquid phase oxidation, and purifying. Those respective processes result in “the three wastes” and are too complex for industrial production. See U.S. Pat. No. 4,504,469.
Example 1Preparing the Linear Atosiban Peptide Resin
(i) 6.25 g of Rink Amide resin (substitutability=0.8 mmol/g) is put into a reaction bottle, DMF is added into the bottle and washed twice, then swelled for 30 min with DMF. Fmoc protecting group of Rink Amide resin is removed with 30-40 ml of 20% DBLK, washed for 4 times with DMF, then washed twice with DCM after removal, the product is detected by ninhydrin detecting method, the resin is reddish-brown.
(ii) 4.46 g of Fmoc-Gly-OH and 2.43 g of HOBt dissolved in a suitable amount of DMF, which had been pre-activated with 3.05 ml DIC; the mixture is, added to the reaction bottle, and reacted for 2 h, the resin is negative by ninhydrin detecting method, after the reaction, the product is washed for 4 times with DMF, then washed twice with DCM, if the resin is positive, repeating the above condensation reaction until negative.
(iii) Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(ET)-OH and Mpa(Trt)-OH are coupled orderly.
Example 2Cleaving the Linear Atosiban Peptide Resin
5.15 g of linear atosiban is prepared by washing the linear atosiban peptide resin obtained from Example 1 for 3 times with 30 ml of methanol, adding the dry resin obtained to 150 ml of mixed solution with a volume ratio of TFA:H2O=95:5, reacting for 2 hours at 25° C. and filtering, washing the resin for 3 times with few trifluoroacetic acid, combining the filtrate and pouring into 1500 ml glacial ether, making rest for 2 hours, centrifugally separating the linear atosiban, washing for 3 times, and drying in a vacuum drier, MS: 995.3, HPLC: 91.5%, content: 65.5%, synthesis yield: 68%.
Example 3Oxidizing the Linear Atosiban
2.85 g of atosiban acetate is prepared by dissolving the linear atosiban obtained from Example 2 in 250 ml of 5% acetonitrile aqueous solution, adjusting the pH value to 8 to 9 with 30% ammonia water, adding 0.60 g of H2O2, reacting for 10 min at 25° C., monitoring with HPLC (HPLC: 75.6%), filtering after reaction, purifying filtrate by preparative RP-HPLC (column C18 or C8), transferring salt, and freeze-drying, MS: 994.5, HPLC: 99.4%.
Example 4Oxidizing the Linear Atosiban
3.01 g of atosiban acetate is prepared by dissolving the linear atosiban obtained from Example 2 in 250 ml of 10% acetonitrile aqueous solution, adjusting the pH value to 8 to 9 with 30% ammonia water, adding 0.85 g of H2O2, reacting for 30 min at 25° C., monitoring with HPLC (HPLC: 89.5%), filtering after reaction, purifying filtrate by preparative RP-HPLC (column C18 or C8), transferring salt, and freeze-drying, MS: 994.5, HPLC: 99.6%.
Example 5Oxidizing the Linear Atosiban
2.95 g of atosiban acetate is prepared by dissolving the linear atosiban obtained from Example 2 in 250 ml of 10% acetonitrile aqueous solution, adjusting the pH value to 8 to 9 with 30% ammonia water, adding 0.85 g of H2O2, reacting for 60 min at 25° C., monitoring with HPLC (HPLC: 83.5%), filtering after reaction, purifying filtrate by preparative RP-HPLC (column C18 or C8), transferring salt, and freeze-drying, MS: 994.5, HPLC: 99.4%.
The above is the further detailed description of the invention in conjunction with specific preferred examples, but it should not be considered that the specific examples of the invention are only limited to the these descriptions. For one of ordinary skill in the art, many deductions and replacements can be made without departing from the inventive concept. Such deductions and replacements should fall within the scope of protection of the invention.
A 2013 retrospective study comparing the efficacy and safety of atosiban and nifedipine in the suppression of preterm labour concluded that atosiban and nifedipine are effective in delaying delivery for seven days or more in women presenting with preterm labour.[16] A total of 68.3% of women in the atosiban group remained undelivered at seven days or more, compared with 64.7% in the nifedipine group.[16] They have the same efficacy and associated minor side effects.[16] However, flushing, palpitation, and hypotension were significantly higher in the nifedipine group.[16]
A 2012 clinical trial compared tocolytic efficacy and tolerability of atosiban with that of nifedipine.[17] Forty-eight (68.6%) women allocated to atosiban and 39 (52%) to nifedipine did not deliver and did not require an alternate agent at 48 hours, respectively (p=.03).[17] Atosiban has fewer failures within 48 hours.[17] Nifedipine may be associated with a longer postponement of delivery.[17]
A 2009 randomised controlled study demonstrated for the first time the direct effects of atosiban on fetal movement, heart rate, and blood flow.[18] Tocolysis with either atosiban or nifedipine combined with betamethasone administration had no direct fetal adverse effects.[18]
Atosiban vs. ritodrine
Multicentre, controlled trial of atosiban vs. ritodrine in 128 women shows a significantly better tocolytic efficacy after 7 days in the atosiban group than in the ritodrine group (60.3 versus 34.9%), but not at 48 hours (68.3 versus 58.7%). Maternal adverse events were reported less frequently in the atosiban group (7.9 vs 70.8%), resulting in fewer early drug terminations due to adverse events (0 versus 20%). Therefore, atosiban is superior to ritodrine in the treatment of preterm labour.[19]
Brand names
In India it is marketed under the brand name Tosiban by Zuventus healthcare ltd.
^ Akerlund M, Carlsson AM, Melin P, Trojnar J (1985). “The effect on the human uterus of two newly developed competitive inhibitors of oxytocin and vasopressin”. Acta Obstet Gynecol Scand. 64 (6): 499–504. doi:10.3109/00016348509156728. PMID4061066. S2CID25799128.
^ Lamont, Ronald F; Kam, KY Ronald (March 2008). “Atosiban as a tocolytic for the treatment of spontaneous preterm labor”. Expert Review of Obstetrics & Gynecology. 3 (2): 163–174. doi:10.1586/17474108.3.2.163. ISSN1747-4108.
^ Jump up to:abc Lamont, Ronald F.; Kam, KY Ronald (2008). “Atosiban as a tocolytic for the treatment of spontaneous preterm labor”. Expert Review of Obstetrics & Gynecology. 3 (2): 163–174. doi:10.1586/17474108.3.2.163.
^ Lamont CD, Jørgensen JS, Lamont RF (September 2016). “The safety of tocolytics used for the inhibition of preterm labour”. Expert Opinion on Drug Safety. 15 (9): 1163–73. doi:10.1080/14740338.2016.1187128. PMID27159501. S2CID4937942. It was for this reason and the fact that Tractocile (atosiban) only had a short duration before it was out of patent that the parent drug company decided not to pursue licensing in the USA.
^ Coomarasamy, A; Knox, EM; Gee, H; Khan, KS (November 2002). “Oxytocin antagonists for tocolysis in preterm labour — a systematic review”. Med Sci Monit. 8 (11): RA268–73. PMID12444392.
^ Jump up to:ab de Heus R, Mulder EJ, Derks JB, Visser GH (June 2009). “The effects of the tocolytics atosiban and nifedipine on fetal movements, heart rate and blood flow”. J Matern Fetal Neonatal Med. 22 (6): 485–90. doi:10.1080/14767050802702349. PMID19479644. S2CID35810758.
^ Shim JY, Park YW, YoonBH, Cho YK, Yang JH, Lee Y, Kim A. “Multicentre, parallelgroup, randomised, single-blind study of the safety and efficacy of atosibanversus ritodrine in the treatment of acute preterm labour in Korean women. BJOG 2006Nov;113(11):1228-34.
External links
“Atosiban”. Drug Information Portal. U.S. National Library of Medicine.
28 Mar 2022No recent reports of development identified for phase-I development in Peripheral-T-cell-lymphoma in China (IV, Injection)
26 Jan 2022ZIOPHARM Oncology is now called Alaunos Therapeutics
11 Dec 2021Safety and efficacy data from a phase II trial in Peripheral T-cell lymphoma presented at the 63rd American Society of Hematology Annual Meeting and Exposition (ASH-2021)
Darinaparsin is a small-molecule organic arsenical with potential antineoplastic activity. Although the exact mechanism of action is unclear, darinaparsin, a highly toxic metabolic intermediate of inorganic arsenicals (iAs) that occurs in vivo, appears to generate volatile cytotoxic arsenic compounds when glutathione (GSH) concentrations are low. The arsenic compounds generated from darinaparsin disrupt mitochondrial bioenergetics, producing reactive oxygen species (ROS) and inducing ROS-mediated tumor cell apoptosis; in addition, this agent or its byproducts may initiate cell death by interrupting the G2/M phase of the cell cycle and may exhibit antiangiogenic effects. Compared to inorganic arsenic compounds such as arsenic trioxide (As2O3), darinaparsin appears to exhibit a wide therapeutic window.
Darinaparsin, also know as ZIO-101 and SP-02, is a small-molecule organic arsenical with potential antineoplastic activity. Although the exact mechanism of action is unclear, darinaparsin, a highly toxic metabolic intermediate of inorganic arsenicals (iAs) that occurs in vivo, appears to generate volatile cytotoxic arsenic compounds when glutathione (GSH) concentrations are low. The arsenic compounds generated from darinaparsin disrupt mitochondrial bioenergetics, producing reactive oxygen species (ROS) and inducing ROS-mediated tumor cell apoptosis; in addition, this agent or its byproducts may initiate cell death by interrupting the G2/M phase of the cell cycle and may exhibit antiangiogenic effects.
Darinaparsin is an organic arsenical composed of dimethylated arsenic linked to glutathione, and is being investigated for antitumor properties in vitro and in vivo. While other arsenicals, including arsenic trioxide, have been used clinically, none have shown significant activity in malignancies outside of acute promyelocytic leukemia. Darinaparsin has significant activity in a broad spectrum of hematologic and solid tumors in preclinical models. Here, we review the literature describing the signaling pathways and mechanisms of action of darinaparsin and compare them to mechanisms of cell death induced by arsenic trioxide. Darinaparsin has overlapping, but distinct, signaling mechanisms. We also review the current results of clinical trials with darinaparsin (both intravenous and oral formulations) that demonstrate significant antitumor activity.
[0071] Sterile water (15.5 L) and ethyl alcohol (200 proof, 15.5 L) were charged in a reaction flask prior to the addition of L-glutathione (3.10 kg). While being stirred, the reaction mixture was cooled to 0-5 °C prior to the addition of triethylamine (1.71 L). Stirring was continued until most of the solids were dissolved and the solution was filtered. After filtration, the reaction mixture was cooled to 0-5 °C prior to the addition of chlorodimethylarsine (1.89 kg) over 115 minutes while maintaining the temperature at 0-5 °C. Stirring continued at 0-5 °C for 4 hours before acetone (30.6 L) was added over 54 minutes while maintaining the temperature at 0-5 °C. The suspension was stored at 0-5°C overnight prior to filtration. The solid was collected in a filter funnel, washed successively with ethyl alcohol (200 proof, 13.5 L) and acetone (13.5 L) and dried in suction for 23 minutes. A second similar run was performed and the collected solids from both runs were combined. Ethyl alcohol (200 proof, 124 L) and the combined solids (11.08 kg) were charged in a vessel. The slurry was stirred at ambient temperature for 2 hours before filtration, washing successively with ethyl alcohol (200 proof, 27 L) and acetone (27 L) and dried in suction for 60 minutes. The resulting solid was transferred to drying trays and dried in a vacuum oven at ambient temperature for 66 hours to provide darinaparsin as a solid with the differential scanning calorimetry (DSC) thermogram of Figure 1, with an extrapolated onset temperature at about 191.36° C and a peak temperature at about 195.65° C.
PATENT
WO 2010021928
Step 1
Dimethylchloroarsine. Dimethylarsinic acid, (CH3)2As(O)OH was supplied by the Luxembourg Chemical Co., Tel Aviv, Israel. The product was accompanied by a statement of its purity and was supplied as 99.7% pure. The dimethylarsinic acid was dissolved in water-hydrochloric acid to pH 3. A stream of sulfur dioxide was passed through this solution for about one hour. Dimethylchloroarsine separated as a heavy, colorless oil. The two liquid phases, water/(CH3)2AsCl were separated using a separatory funnel. The chlorodimethylarsine was extracted into diethylether and the ether solution was dried over anhydrous sodium sulfate. The dried solution was transferred to a distillation flask which was heated slowly to evaporate the ether. The remaining liquid, dimethylchloroarsine was purified by distillation. The fraction boiling at 106-109°C was collected. The product, a colorless oil. 1H NMR resonance at 1.65 ppm.
Step 2
SGLU-1: Glutathione (14.0 g, 45.6 mmol) was stirred rapidly in glyme while dimethylchoroarsine (6.5 g, 45.6 mmol) was added dropwise. Pyridine (6.9 g, 91.2 mmol) was then added to the slurry and the mixture was subsequently heated to reflux. The heat was removed immediately and the mixture stirred at room temperature for 4 h. Isolation of the resultant insoluble solid and recrystallization from ethanol afforded 4 as the pyridine hydrochloride complex (75% yield). mp 115-118°C; NMR (D20) δ1.35 (s, 6H), 1.9-4.1 (m’s, 10H), 7.8-9.0 (m, 5H); mass spectrum (m/e) 140, 125, 110, 105, 79, 52, 45, 36.
PATENT
WO 2009075870
Step 1
Example 1. Preparation of Dimethylchloroarsine (DMCA). A 3-neck round-bottom flask (500 mL) equipped with mechanical stirrer, inlet for nitrogen, thermometer, and an ice bath was charged with cacodylic acid (33 g, 0.23 mol) and cone. hydrochloric acid (67 mL). In a separate flask, a solution of SnCl2·2H2O (54 g, 0.239 mol) in cone. hydrochloric acid (10 mL) was prepared. The SnCl2·2 H2O solution was added to the cacodylic acid in HCl solution under nitrogen while maintaining the temperature between 5 °C and 10 °C. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was transferred to a separatory funnel and the upper layer (organic) collected. The bottom layer was extracted with dichloromethane (DCM) (2 × 25 mL). The combined organic extract was washed with 1 N HCl (2 × 10 mL) and water (2 × 20 mL). The organic extract was dried over MgSO4 and DCM was removed by rotary evaporation (bath temperature 80 °C, under nitrogen, atmospheric pressure). The residue was further distilled under nitrogen. Two tractions of DMCA were collected. The first fraction contained some DCM and the second fraction was of suitable quality (8.5 g, 26% yield). The GC analysis confirmed the identity and purity of the product.
Step 2
Example 3. Preparation of S-Dimethylarsinoglutathione (SGLU-1). In a 3 L three-neck flask equipped with a mechanic stirrer, dropping funnel and thermometer under an inert atmosphere was prepared a suspension of glutathione (114.5 g, 0.37 mol) in a 1:1 (v/v) mixture of water/ethanol (1140 mL) and cooled to below 5 °C. The mixture was treated slowly (over 15 min) with triethylamine (63.6 mL, 0.46 mol) while maintaining the temperature below 20 °C. The mixture was cooled to 4 °C and stirred for 15 min and then the traces of undissolved material removed by filtration. The filtrate was transferred in a clean 3 L three-neck flask equipped with a mechanic stirrer, dropping funnel, nitrogen inlet, and thermometer and DMCA (70 g, 0.49 mol) (lot # 543-07-01-44) was added slowly while maintaining the temperature at 3-4°C. The reaction mixture was stirred at 1-4°C for 4 h, and acetone (1.2 L) was added over a period of 1 h. The mixture was stirred for 90 min between 2 and 3°C and the resulting solid was isolated by filtration. The product was washed with ethanol (2 × 250 mL) and acetone (2 × 250 mL) and the wet solids were suspended in ethanol 200 Proof (2000 mL). The product was isolated by filtration, washed with ethanol (2 × 250 mL) and acetone (2 × 250 mL) and dried in vacuum for 2 days at RT to give 115 g (75%) of SGLU-1, HPLC purity > 99.5% (in process testing).
PATENT
WO 2007027344
Example 2 Preparation of S-Dimethylarsinoglutathione A 5 L, three necked round bottom flask was equipped with a mechanical stirrer assembly, thermometer, addition funnel, nitrogen inlet, and a drying tube was placed in a cooling bath. A polyethylene crock was charged with glutathione-reduced (200 g) and deionized water (2 L) and stirred under a nitrogen atmosphere to dissolve all solids. The mixture was filtered to remove any insoluble material and the filtrate was transferred to the 5 L flask. While stirring, ethanol, 200 proof (2 L) was added and the clear solution was cooled to 0-5° C. using an ice/methanol bath. Pyridine (120 g) was added followed by a dropwise addition of Me2AsCl (120 g) over a minimum of 1 hour. The reaction mixture was stirred at 0-5° C. for a minimum of 2 hours prior to removal of the cooling bath and allowing the mixture to warm to room temperature under a nitrogen atmosphere with stirring. The reaction mixture was stirred overnight (>15 hrs) at room temperature under a nitrogen atmosphere at which time a white solid may precipitate. The reaction mixture was concentrated to a slurry (liquid and solid) at 35-45° C. using oil pump vacuum to provide a white solid residue. As much water as possible is removed, followed by two coevaporations with ethanol to azeotrope the last traces of water. The white solid residue was slurried in ethanol, 200 pf. (5 L) under a nitrogen atmosphere at room temperature overnight. The white solid was filtered and washed with ethanol, 200 pf. (2×500 mL) followed by acetone, ACS (2×500 mL). The resulting solid was transferred to drying trays and vacuum oven dried overnight at 25-35° C. using oil pump vacuum to provide pyridinium hydrochloride-free S-dimethylarsinoglutathione as a white solid. melting point of 189-190° C.
PATENT
WO 20060128682
Step 1
Dimethylchloroarsine. Dimethylarsinic acid, (CH3)2As(O)OH was supplied by the Luxembourg Chemical Co., Tel Aviv, Israel. The product was accompanied by a statement of its purity and was supplied as 99.7% pure. The dimethylarsinic acid was dissolved in water-hydrochloric acid to pH 3. A stream of sulfur dioxide was passed through this solution for about one hour. Dimethylchloroarsine separated as a heavy, colorless oil. The two liquid phases, water/(CH3)2AsCl were separated using a separatory funnel. The chlorodimethylarsine was extracted into diethylether and the ether solution was dried over anhydrous sodium sulfate. The dried solution was transferred to a distillation flask which was heated slowly to evaporate the ether. The remaining liquid, dimethylchloroarsine was purified by distillation. The fraction boiling at 106-109° C. was collected. The product, a colorless oil. 1H NMR resonance at 1.65 ppm.
Step 2
Pyridine Hydrochloride Free Synthesis of S-Dimethylarsinoglutathione (GLU) Dimethylarsinoglutathione is made using an adapted of Chen (Chen, G. C., et al. Carbohydrate Res. (1976) 50: 53-62) the contents of which are hereby incorporated by reference in their entirety. Briefly, dithiobis(dimethylarsinoglutamine) is dissolved in dichloromethane under nitrogen. Tetramethyldiarsine is added dropwise to the solution and the reaction is stirred overnight at room temperature under nitrogen and then exposed to air for 1 h. The mixture is then evaporated to dryness and the residue is washed with water and dried to give a crude solid that is recrystallized from methanol to give S-dimethylarsinoglutathione.
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Solasia Pharma K.K. (TSE: 4597, Headquarters: Tokyo, Japan, President & CEO: Yoshihiro Arai, hereinafter “Solasia”) today announced submission of a New Drug Application (NDA) for its new anti-cancer drug darinaparsin (generic name, development code: SP-02) as a treatment for relapsed or refractory peripheral T-cell lymphoma to the Ministry of Health, Labour and Welfare (MHLW). Based on positive results of R&D on darinaparsin, centered primarily on the results of the Asian Multinational Phase 2 Study (study results released in June 2020), Solasia filed an NDA for the drug with the regulatory authority in Japan ahead of anywhere else in the world.
Solasia expects to obtain regulatory approval in 2022 and to also launch in the same year. If approved and launched, darinaparsin would be the third drug Solasia successfully developed and brought to market since its founding and is expected to contribute to the treatment of PTCL.
Mr. Yoshihiro Arai, President and CEO of Solasia, commented as follows: “No standard treatment has been established for relapsed or refractory PTCL as of yet. I firmly believe that darinaparsin, with its novel mechanism of action that differs from those of already approved drugs, will contribute to patients and healthcare providers at clinical sites as a new treatment option for relapsed or refractory PTCL. Since founding, Solasia has conducted R&D on five pipeline drugs. Of the five, we have successfully developed and brought to market two drugs, i.e., began providing them to patients, and today, we submitted an NDA for our first anti-cancer drug. Under our mission to provide patients with ‘Better Medicine for a Brighter Tomorrow’, we will continue aiming to contribute to patients’ treatment and enhanced quality of life. ”
About darinaparsin (SP-02) Darinaparsin, an organoarsenic compound with anticancer activity, is a novel mitochondrial-targeted agent being developed for the treatment of various hematologic and solid tumors. The proposed mechanism of action of the drug involves the disruption of mitochondrial function, increased production of reactive oxygen species, and modulation of intracellular signal transduction pathways. Darinaparsin is believed to exert anticancer effect by inducing cell cycle arrest and apoptosis. Darinaparsin has been granted orphan drug designation in the US and EU. For more information, please visit at https://solasia.co.jp/en/pipeline/sp-02.html
About Asian Multinational Phase 2 Study The Asian Multinational Phase 2 Study was a multinational, multicenter, single-arm, open-label, non-randomized study to evaluate the efficacy and safety of darinaparsin monotherapy in patients with relapsed or refractory PTCL conducted in Japan, Korea, Taiwan, and Hong Kong. (CT.gov Identifier: NCT02653976). Solasia plans to present the results of the study at an international academic conference to be held in the near future.
Pimitespib (TAS-116) is an oral bioavailable, ATP-competitive, highly specific HSP90α/HSP90β inhibitor (Kis of 34.7 nM and 21.3 nM, respectively) without inhibiting other HSP90 family proteins such as GRP94. Pimitespib demonstrates less ocular toxicity.
Formula
C25H26N8O
CAS
1260533-36-5
Mol weight
454.5269
JAPAN APPROVED 2022/6/20, ピミテスピブ
Jeselhy
Taiho. originator
Pimitespib is a specific inhibitor of heat shock protein 90 (Hsp90) subtypes alpha and beta, with potential antineoplastic and chemo/radiosensitizing activities. Upon oral administration, pimitespib specifically binds to and inhibits the activity of Hsp90 alpha and beta; this results in the proteasomal degradation of oncogenic client proteins, which inhibits client protein dependent-signaling, induces apoptosis, and inhibits the proliferation of cells overexpressing HSP90alpha/beta. Hsp90, a family of molecular chaperone proteins that are upregulated in a variety of tumor cells, plays a key role in the conformational maturation, stability, and function of “client” proteins within the cell,; many of which are involved in signal transduction, cell cycle regulation and apoptosis, including kinases, cell-cycle regulators, transcription factors and hormone receptors. As TAS-116 selectively inhibits cytosolic HSP90alpha and beta only and does not inhibit HSP90 paralogs, such as endoplasmic reticulum GRP94 or mitochondrial TRAP1, this agent may have less off-target toxicity as compared to non-selective HSP90 inhibitors.
3-Ethyl-4-fluorobenzonitrile is an important intermediate for the preparation of a variety of new drugs under development, such as TAS-116, a Phase II clinical drug of Taiho Pharmaceuticals for the treatment of gastrointestinal stromal tumors.
Patent WO2005105760 discloses its preparation method. In the method, tetrakis(triphenylphosphine) palladium is used as a catalyst, and 3-bromo-4-fluorobenzonitrile is coupled with tetraethyl tin in a solvent hexamethylphosphoramide for a heating reaction for 15 hours to obtain 3 -Ethyl-4-fluorobenzonitrile. The method uses highly toxic tetraethyl tin, which brings great harm to operators and the environment, and is difficult to carry out industrial production. Meanwhile, the product 3-ethyl-4-fluorobenzonitrile obtained by the preparation method is an oily substance, which is purified by column chromatography with complicated operation, which is unfavorable for industrial production, and the specific purity of the product is not described.
Therefore, looking for a new method for preparing 3-ethyl-4-fluorobenzonitrile with cheap and easy-to-obtain raw materials, safe and simple operation, high product purity and low cost suitable for industrial production, which will speed up the research process of related new drugs under development. , it is of great significance to reduce the production cost of related new drugs.
Example 1 3-Bromo-4-fluorobenzonitrile
3-Bromo-4-fluorobenzaldehyde (250g, 1.23mol) was dissolved in acetonitrile (1.5L), then hydroxylamine sulfonic acid (67g, 1.48mol) was added, and the reaction was refluxed for 4h. TLC showed that the conversion of the raw materials was complete, and the reaction solution was concentrated. To a small volume, add water (2L) and stir for 30min, cool to 5-10°C and continue stirring for 10min, filter, dissolve the filter cake with methyl tert-butyl ether (1.2L), wash twice with 500ml of water, saturated with 200ml Washed with sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, the filtrate was adsorbed with activated carbon (10g), filtered, concentrated under reduced pressure to remove the solvent, added n-heptane (250ml), cooled and stirred in an ice-salt bath for 1h, filtered, reduced Press drying to give 3-bromo-4-fluorobenzonitrile (217 g, 88% yield). 1 H NMR (CDCl 3 ,400MHz):δ7.91(m,1H),7.63(m,1H),7.24(m,1H)。
Example 2 3-Bromo-4-fluorobenzonitrile
Add tetrahydrofuran (100ml) to a 250ml reaction flask, add 3-bromo-4-fluorobenzaldehyde (10g, 49.2mmol) and ammonia (40ml) under stirring, add elemental iodine (25g, 98.5mmol) in batches under cooling to 5°C ), then raised to ambient temperature and reacted for 2 to 3 hours, the reaction was completed, the reaction solution was poured into a 10% aqueous solution of sodium sulfite (200g), extracted twice with methyl tert-butyl ether (100ml), dried over anhydrous sodium sulfate , concentrated under reduced pressure to remove the solvent, added n-heptane (20 ml), cooled to 0-10 °C and stirred for 1 h, filtered, and dried under reduced pressure to obtain 3-bromo-4-fluorobenzonitrile (9.6 g, yield: 97.5 %). The NMR spectrum of this compound was determined and was identical to the product of Example 1.
Example 3 3-ethyl-4-fluorobenzonitrile
3-Bromo-4-fluorobenzonitrile (200 g, 1 mol) and [1,1-bis(diphenylphosphino)ferrocene]palladium(II) chloride dichloromethane complex (4.08 g, 5mmol) was dissolved in THF (1.2L), 1.0M/L diethylzinc n-hexane solution (600mL, 0.6mol) was added at 40-50°C, and the temperature was raised to 50-60°C for 4-5h. TLC showed The raw materials reacted completely. After the reaction solution was cooled to room temperature, it was added to 5% dilute hydrochloric acid (1 L), the layers were separated, the organic layer was washed twice with 500 ml of water, and then concentrated under reduced pressure to remove the solvent. Then n-hexane (600mL) and activated carbon (20g) were added, refluxed for 0.5h, cooled to room temperature, filtered, then added activated carbon (10g) to the filtrate, refluxed for 0.5h, cooled to room temperature, filtered, and cooled to -50°C to -60°C and filtered, and the filter cake was dried under reduced pressure at 10-20°C to obtain 3-ethyl-4-fluorobenzonitrile (112 g, yield: 75%) as an off-white solid, melting point 23.1-27.4°C. 1 H NMR (CDCl 3 , 400MHz): δ 7.50 (m, 2H), 7.09 (m, 1H), 2.69 (q, J=7.6Hz, 2H), 1.24 (t, 3H, J=7.6Hz), HPLC purity 99.6%.
Acylation of 2-fluoro-4-iodopyridine with isobutyric anhydride in presence of BuLi and DIEA in THF at -78 °C gives 1-(2-fluoro-4-iodo-3-pyridinyl)-2-methylpropan-1-one ,
This upon cyclization using hydrazine hydrate at 65 °C gives 4-iodo-3-isopropylpyrazolo[3,4-b]pyridine.
N-Protection of intermediate with PMB-Cl in the presence of base NaH in solvent DMF at 0 °C affords 4-iodo-3-isopropyl-1-(4-methoxybenzyl)pyrazolo[3,4-b]pyridine,
This is coupled with 4-(4-imidazolyl)-1-methylpyrazole in the presence of Cu2O, 4,7-dimethoxy-1,10-phenanthroline, Cs2CO3 and PEG-diamine in solvent NMP or DMSO at 130 °C to furnish 4-[4-(4-pyrazolyl)-imidazol-1-yl]pyrazolo[3,4-b]pyridine derivative .
N-Deprotection of PMB-protected pyrazolo[3,4-b]pyridine derivative by using TFA and anisole gives free pyrazolo[3,4-b]pyridine ,
This on condensation with 3-ethyl-4-fluorobenzonitrile in the presence of Cs2CO3 in DMF at 95 °C yields 4-(pyrazolo[3,4-b]pyridin-1-yl)benzonitrile .
Finally, partial hydrolysis of nitrile by means of aqueous NaOH and H2O2 in DMSO/EtOH gives the Pimitespib TAS-116 .
The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure–activity relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e (TAS-116), is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16e analogue 16d demonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.
Compound 1 in the present invention is 3-ethyl-4- {3-isopropyl-4- (4- (1-methyl-1H-pyrazol-4-yl) -1H-imidazole-1-yl) -1H-. Pyrazolo [3,4-b] pyridin-1-yl} benzamide (formula below). Compound 1 is known to have HSP90 inhibitory activity and exhibit excellent antitumor activity. Compound 1 can be synthesized based on the production methods described in Patent Documents 1 and 2.
[0013]
[hua 1]
Patent Document 1: International Publication No. 2012/093708 Patent Document 2: International Publication No. 2011/004610
Comparative Example 1 3-Ethyl-4- {3-isopropyl-4- (4- (1-methyl-1H-pyrazole-4-yl) -1H-imidazole-1-yl) -1H-pyrazolo [3, 4-b] Pyridine-1-yl} Synthesis of type I crystals of benzamide 3-Ethyl-4 obtained according to the production method described in International Publication No. 2012/093708 and International Publication No. 2011/004610. -{3-Isopropyl-4- (4- (1-methyl-1H-pyrazole-4-yl) -1H-imidazole-1-yl) -1H-pyrazolo [3,4-b] pyridin-1- A white solid (3.58 g) of yl} benzamide was added to ethanol (7.84 mL) and stirred at room temperature for 2 hours. After sampling, it was washed with ethanol (7.84 mL) and dried under reduced pressure at 70 to 80 ° C. for 20 hours to obtain type I crystals (yield: 2.40 g, yield: 61.2%, purity: 98.21%). rice field. Further, as shown in FIG. 1, the type I crystal has a diffraction angle (2θ) of 8.1 °, 10.9 °, 12.1 °, 14.0 °, and 14.9 in the powder X-ray diffraction spectrum. °, 16.2 °, 17.7 °, 20.2 °, 21.0 °, 21.5 °, 22.6 °, 24.3 °, 25.4 ° 26.4 °, 27.0 ° , 28.3 °, 30.2 °, 30.9 °, 31.5 °, 32.7 °, 34.7 °, 35.4 ° and 36.6 ° showed characteristic peaks.
Synthesis of Test Compound The following synthesis example compounds (Synthesis Examples 1 to 3) were synthesized according to the method described in WO2011 / 004610.
Originator University of California at San Francisco; University of Michigan; University of Washington
Developer University of California at San Francisco; University of Michigan; University of Washington; ViewPoint Therapeutics
Class Small molecules; Sterols
Mechanism of Action Amyloid inhibitors; Protein aggregation inhibitors; Protein folding inhibitors
28 Jun 2020 No recent reports of development identified for research development in Presbyopia in USA (Ophthalmic, Drops)
28 Dec 2019 No recent reports of development identified for preclinical development in Cataracts in USA (Ophthalmic, Drops)
01 Apr 2016 ViewPoint Therapeutics receives SBIR grant from National Eye Institute for VP1 001 development in Cataracts (ViewPoint Therapeutics website)
We previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.
Investigators are exploring chemical compounds to restore transparency to the crystalline lens.
Surgery is an effective but costly means of managing cataracts, and, like all surgical interventions, it carries risks. Moreover, a substantial number of people worldwide, particularly in developing countries, lack access to cataract surgery. The World Health Organization estimates that 65.2 million people globally are blind or visually impaired from cataracts.1 The development of an eye drop that restores transparency and flexibility to the crystalline lens would therefore be a game-changer as a less expensive, noninvasive option for treating a leading cause of blindness.
RESEARCH RESULTS
The crystalline lens is composed of epithelial and fiber cells. One of the major lens proteins in the fiber cells is alpha-crystallin, a chaperone protein thought to maintain homeostasis of the crystalline lens, thereby preserving its transparency and flexibility. As a person ages, alpha-crystallin proteins become prone to misfolding, causing them to clump together and form insoluble high-molecular-weight protein aggregates, which can lead to cataract formation.
Compound 29 (full name, 5-cholesten-3beta,25-diol), also known as VP1-001 (ViewPoint Therapeutics) and as 25-hydroxy-cholesterol, is an oxysterol, a derivative of cholesterol. Usha Andley, PhD, FARVO, is an investigator conducting research on this chemical compound’s use as a treatment for cataracts.2,3 Another oxysterol being investigated for this purpose is lanosterol. In a recent study, neither oxysterol was effective at reducing opacities of in vitro cultured lenses treated with various reagents to induce opacification in vitro.4 In an interview with ME, however, Dr. Andley stated that VP1-001 appears to be more effective than lanosterol at reducing lens opacity. VP1-001 differs from lanosterol in terms of solubility, she said, allowing VP1-001 to penetrate the eye better.
The goal of the research being conducted by Dr. Andley and her colleagues, she said, is twofold:
No. 1: To ensure that the compound is not toxic to the cornea; and
No. 2: To show that the compound is capable of reducing the tendency of alpha-crystallins to aggregate and perhaps reverse their aggregation.
In proof-of-concept studies, VP1-001 was incorporated into an eye drop formulation of 8% cyclodextrin. Dr. Andley and colleagues administered the drops three times per week in a mouse model for 2 to 4 weeks. According to Dr. Andley, the compound seemed to increase the stability of the alpha-crystallin protein so that it increased the soluble fraction of proteins from mouse and human lens cataracts. The compound also increased the solubility of two other lens crystallins, beta- and gamma-crystallin, in the mouse lens, and it seemed to reduce the abundance of high-molecular-weight aggregates in the lens.2,3
ViewPoint Therapeutics is currently developing this technology for use in humans. According to Dr. Andley, who is working with the company, ViewPoint Therapeutics is using different model systems for in vitro and protein-binding studies in an attempt to improve on earlier results with the chemical compound. Their research has identified new compounds, nonsterol ligands for alpha-crystallin, that exhibited in vitro activity and efficacy similar to or better than those of VP1-001 in mouse models of cataracts.5 The discovery of these nonsterols supports the hypothesis that pharmacologic chaperones targeting alpha-crystallin can prevent or reverse cataracts, Dr. Andley said.
CHALLENGES AND FUTURE DIRECTIONS
In studies to date, Dr. Andley and her colleagues have treated mice in one eye with the VP1-001 formulation, and the contralateral untreated eyes have served as controls. They then compared the two eyes after the conclusion of treatment (Figure). She is looking forward to conducting masked and randomized studies that include baseline measurements of lens opacity. According to Dr. Andley, this research will begin this year. Animal testing, however, can advance understanding only so far. Human testing of safety and efficacy will be a major step forward in the research on VP1-001 and newer variants.
Figure | Representative slit-lamp images from aged wild-type mouse lenses show the extent of opacity treated with vehicle (left) or drug (right). Mice were treated topically with the drug in one eye and vehicle in the contralateral eye three times per week for 2 weeks. Slit-lamp examinations were performed on conscious, live mice. Mouse 1 and 2 were treated with VP1-001.
A major challenge in the development of a pharmacologic treatment for cataract is how to determine if a chemical compound can be maintained in the lens at a sufficient concentration and for an adequate duration to achieve the desired outcome. A second challenge relates to detecting change. Dr. Andley and her colleagues are seeking a more quantitative method by which to assess the extent of lenticular opacity before and after treatment. They have a modified Lens Opacities Classification System, she said, but it is less objective than methods such as Scheimpflug photography. A goal, then, is to develop a more standardized, objective way of measuring results.
In addition to age-related cataract, Dr. Andley suggested, a topical agent could be advantageous for the treatment of congenital cataracts that form because of a mutation in alpha A crystallin or alpha B crystallin. Retaining the crystalline lens instead of extracting it would allow pediatric eyes to develop normally, she noted.
The idea of an eye drop formulation to treat cataracts may seem like science fiction, but Dr. Andley expects it to become a more realistic possibility within the next few years. The utility of such a chemical compound could extend beyond cataracts to the treatment of presbyopia. The hypothesis, she said, is that softening the crystalline lens would improve accommodative amplitude.
2. Makley LN, McMenimen KA, DeVree BT, et al. Pharmacological chaperone for α-crystallin partially restores transparency in cataract models. Science. 2015;350(6261):674-677.
3. Molnar KS, Dunyak BM, Su B, et al. Mechanism of action of VP1-001 in cryAB(R120G)-associated and age-related cataracts. Invest Ophthalmol Vis Sci. 2019;60(10):3320-3331.
4. Daszynski DM, Santhoshkumar P, Phadte AS, et al. Failure of oxysterols such as lanosterol to restore lens clarity from cataracts. Sci Rep. 2019;9(1):8459.
5. Dunyak B, Su B, Molnar K, et al. Discovery of non-sterol aB-crystallin ligands as potential cataract therapeutics. Invest Ophthalmol Vis Sci. 2019;60(9):5691.
Vutrisiran Vutrisiran Sodium is a sodium salt of an siRNA derivative targeting transthyretin (TTR) covalently linked to a triantennary GalNAc3 complex at the 3’ end of the sense strand. The siRNA moiety is composed of a duplex oligonucleotide of sense strand consisting of chemically modified 21 nucleotide residues and antisense strand consisting of chemically modified 23 nucleotide residues each.
Vutrisiran is a double-stranded small interfering ribonucleic acid (siRNA) that targets wild-type and mutant transthyretin (TTR) messenger RNA (mRNA).7 This siRNA therapeutic is indicated for the treatment of neuropathies associated with hereditary transthyretin-mediated amyloidosis (ATTR), a condition caused by mutations in the TTR gene.2 More than 130 TTR mutations have been identified so far,3 but the most common one is the replacement of valine with methionine at position 30 (Val30Met).2 The Val30Met variant is the most prevalent among hereditary ATTR patients with polyneuropathy, especially in Portugal, France, Sweden, and Japan.2
TTR mutations lead to the formation of misfolded TTR proteins, which form amyloid fibrils that deposit in different types of tissues. By targeting TTR mRNA, vutrisiran reduces the serum levels of TTR.6,7 Vutrisiran is commercially available as a conjugate of N-acetylgalactosamine (GalNAc), a residue that enables the delivery of siRNA to hepatocytes.5,7 This delivery platform gives vutrisiran high potency and metabolic stability, and allows for subcutaneous injections to take place once every three months.8 Another siRNA indicated for the treatment of polyneuropathy associated with hereditary ATTR is patisiran.2 Vutrisiran was approved by the FDA in June 2022.
Schematic illustrations of the working mechanisms of miRNA (a) and siRNA (b)
Structures of chemical modifications and analogs used for siRNA and ASO decoration. According to the modification site in the nucleotide acid, these structures can be divided into three classes: phosphonate modification, ribose modification and base modification, which are marked in red, purple and blue, respectively. R = H or OH, for RNA or DNA, respectively. (S)-cEt-BNA (S)-constrained ethyl bicyclic nucleic acid, PMO phosphorodiamidate morpholino oligomer
Representative designs for the chemical modification of siRNA. The sequences and modification details for ONPATTRO®, QPI-1007, GIVLAARI™ and inclisiran are included. The representative siRNA modification patterns developed by Alnylam (STC, ESC, advanced ESC and ESC+) and arrowhead (AD1-3 and AD5) are shown. Dicerna developed four GalNAc moieties that can be positioned at the unpaired G–A–A–A nucleotides of the DsiRNA structure. 2′-OMe 2′-methoxy, 2′-F 2′-fluoro, GNA glycol nucleic acid, UNA unlocked nucleic acid, SS sense strand, AS antisense strand
siRNA delivery platforms that have been evaluated preclinically and clinically. Varieties of lipids or lipidoids, siRNA conjugates, peptides, polymers, exosomes, dendrimers, etc. have been explored and employed for siRNA therapeutic development by biotech companies or institutes. The chemical structures of the key component(s) of the discussed delivery platforms, including Dlin-DMA, Dlin-MC3-DMA, C12-200, cKK-E12, GalNAc–siRNA conjugates, MLP-based DPC2.0 (EX-1), PNP, PEI, PLGA-based LODER, PTMS, GDDC4, PAsp(DET), cyclodextrin-based RONDEL™ and dendrimer generation 3 are shown. DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate, DPC Dynamic PolyConjugates, MLP membrane-lytic peptide, CDM carboxylated dimethyl maleic acid, PEG polyethylene glycol, NAG N-acetylgalactosamine, PNP polypeptide nanoparticle, PEI poly(ethyleneimine), LODER LOcal Drug EluteR, PLGA poly(lactic-co-glycolic) acid, PTMS PEG-PTTMA-P(GMA-S-DMA) poly(ethylene glycol)-co-poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl))] ethane methacrylate-co-poly(dimethylamino glycidyl methacrylate), GDDC4 PG-P(DPAx-co-DMAEMAy)-PCB, where PG is guanidinated poly(aminoethyl methacrylate) PCB is poly(carboxybetaine) and P(DPAx-co-DMAEMAy) is poly(dimethylaminoethyl methacrylate-co-diisopropylethyl methacrylate), PEG-PAsp(DET) polyethylene glycol-b-poly(N′-(N-(2-aminoethyl)-2-aminoethyl) aspartamide), PBAVE polymer composed of butyl and amino vinyl ether, RONDEL™ RNAi/oligonucleotide nanoparticle delivery
Vutrisiran SodiumVutrisiran Sodium is a sodium salt of an siRNA derivative targeting transthyretin (TTR) covalently linked to a triantennary GalNAc3 complex at the 3’ end of the sense strand. The siRNA moiety is composed of a duplex oligonucleotide of sense strand consisting of chemically modified 21 nucleotide residues and antisense strand consisting of chemically modified 23 nucleotide residues each.C530H672F9N171Na43O323P43S6 : 17289.77 [1867157-35-4 , Vutrisiran]
REF
Nucleic Acids Research (2019), 47(7), 3306-3320.
Drug Metabolism & Disposition (2019), 47(10), 1183-1201.
The present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof a combination of a benzoxazole derivative transthyretin stabilizer or a pharmaceutically acceptable salt or prodrug thereof and an additional therapeutic agent for the treatment of transthyretin amyloidosis. Particularly, the present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent for the treatment of transthyretin amyloidosis.
The present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof a combination of a benzoxazole derivative transthyretin stabilizer or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent. Particularly, the present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent. The compositions and methods of the invention are useful in stabilizing transthyretin, inhibiting transthyretin misfolding, proteolysis, and treating amyloid diseases associated thereto.
Transthyretin (TTR) is a 55 kDa homotetrameric protein present in serum and cerebral spinal fluid and which functions as a transporter of L-thyroxine (T4) and holo-retinol binding protein (RBP). TTR has been found to be an amyloidogenic protein that, under certain conditions, can be transformed into fibrils and other aggregates which can lead to disease pathology such as polyneuropathy or cardiomyopathy in humans.
US Patent Nos. 7,214,695; 7,214,696; 7,560,488; 8, 168.683; and 8,653,119 each of which is incorporated herein by reference, discloses benzoxazole derivatives which act as transthyretin stabilizers and are of the formula
or a pharmaceutically acceptable salt thereof; wherein Ar is 3,5-difluorophenyl, 2,6-difluorophenyl, 3,5-dichlorophenyl, 2,6-dichlorophenyl, 2-(trifluoromethyl)phenyl or 3-(trifluoromethyl)phenyl. Particularly, 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis) of the formula
is disclosed therein. Tafamidis is an orally active transthyretin stabilizer that inhibits tetramer dissociation and proteolysis that has been approved in certain jurisdictions for the treatment of transthyretin polyneuropathy (TTR-PN) and is currently in development for the treatment of transthyretin cardiomyopathy (TTR-CM). US Patent No. 9,249, 112, also incorporated herein by reference, discloses polymorphic forms of the meglumine salt of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis meglumine). US Patent No. 9,770,441 discloses polymorphic forms of the free acid of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis), and is also incorporated by reference herein.
Summary of the Invention
The present invention provides pharmaceutical compositions and methods comprising the compound 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent. Particular embodiments of this invention are pharmaceutical compositions and methods comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agents selected from the group consisting of agents that lower plasma levels of TTR such as an antisense therapy, TTR gene editing therapy, transcriptional modulators, translational modulators, TTR protein degraders and antibodies that bind and reduce TTR levels; amyloid reduction therapies such as anti amyloid antibodies (either TTR selective or general), stimulators of amyloid clearance, fibril disruptors and therapies that inhibit amyloid nucleation; other TTR stabilizers; and TTR modulators such as therapeutics which inhibit TTR cleavage. Particularly, the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and
methods comprising a polymorphic form of tafamidis free acid or a polymorphic form of tafamidis meglumine salt with one or more additional therapeutic agents.
The present invention also provides a method of treating or preventing transthyretin amyloidosis in a patient, the method comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of 2-(3,5-dichlorophenyl)-1,3-benzoxazole- 6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agents.
A particular embodiment of the present method of treatment is the method comprising a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent are administered orally. Additional embodiments of this invention are methods of treatment as described above wherein the 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent are administered parenterally (intravenously or subcutaneously). Further embodiments of this invention are methods of treatment wherein the 2-(3,5-dichlorophenyl)-1, 3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally and the one or more additional therapeutic agent is administered either orally or parenterally. Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR 5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR 5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR
cardiomyopathy, the method comprising administering to a patient in need thereof a therapeutically effective amount of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agents.
Exemplary RNAi agents that reduce the expression of TTR include patisiran and vutrisiran.
The ter s “antisense polynucleotide agent”, “antisense oligonucleotide”, “antisense compound”, and “antisense agent” as used interchangeably herein, refer to an agent comprising a single-stranded oligonucleotide that specifically binds to the target nucleic acid molecules via hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) and inhibits the expression of the targeted nucleic acid by an antisense mechanism of action, e.g., by RNase H. In some embodiments, an antisense agent is a nucleic acid therapeutic that acts by reducing the expression of a target gene, thereby reducing the expression of the polypeptide encoded by the target gene. Exemplary antisense agents that reduce the expression of TTR include inotersen and Ionis 682884/ ION-TTR-LRx (see, e.g., WO2014179627 which is incorporated by reference in its entirety). Further antisense agents that reduce the expression of TTR are provided, for example in WO2011139917 and WO2014179627, each of which is incorporated by reference in its entirety.
Tissues targeted by siRNA and miRNA therapeutics currently being investigated at the clinical stage. The corresponding therapeutic names are shown beside the tissues
Alnylam announces 3-month extension of review period for new drug application for vutrisiran to treat ATTR amyloidosis.
Alnylam Pharmaceuticals, Inc., a RNAi therapeutics company, announced that the FDA has extended the review timeline of the New Drug Application (NDA) for vutrisiran, an investigational RNAi therapeutic in development for the treatment of transthyretin-mediated (ATTR) amyloidosis, to allow for the review of newly added information related to the new secondary packaging and labelling facility.
Alnylam recently learned that the original third-party secondary packaging and labelling facility the Company planned to use for the vutrisiran launch was recently inspected and the inspection requires classification for the FDA to take action on the vutrisiran NDA. The inspection observations were not directly related to vutrisiran. In order to minimize delays to approval, Alnylam has identified a new facility to pack and label vutrisiran and submitted an amendment to the NDA for review by the FDA. The updated Prescription Drug User Fee Act (PDUFA) goal date to allow for this review is July 14, 2022. No additional clinical data have been requested by the FDA.
////////////Vutrisiran sodium, APPROVALS 2022, FDA 2022, FDA APPROVED, AMVUTTRA, 2022/6/13, ブトリシランナトリウム , ALN 65492, Votrisiran, siRNA
Difluprednate is a topical corticosteroid used for the symptomatic treatment of inflammation and pain associated with ocular surgery.
Difluprednate is a corticosteroid, It is chemically a butyrate ester of 6(alpha),9(alpha)-difluoro prednisolone acetate. Accordingly, difluprednate is sometimes abbreviated DFBA, for difluoroprednisolone butyrate acetate.
Difluprednate is a topical corticosteroid indicated for the treatment of infammation and pain associated with ocular surgery. It is a butyrate ester of 6(α), 9(α)-difluoro prednisolone acetate. Difluprednate is abbreviated DFBA, or difluoroprednisolone butyrate acetate. It is indicated for treatment of endogenous anterior uveiti.
Approval
On June 24, 2008, the US Food and Drug Administration (FDA) approved difluprednate for the treatment of post-operative ocular inflammation and pain.[1] It is marketed by Alcon under the tradename Durezol.
WO/2022/118271DIFLUPREDNATE FOR REDUCING THE ADVERSE EFFECTS OF OCULAR INFLAMMATION
SYN 1
Synthetic Reference
Process for preparation of Difluprednate from sterol fermentation product; Ding, Kai; Xu, Feifei; Assignee Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Peop. Rep. China; East China University of Science and Technology; 2014; Patent Information; Aug 06, 2014; CN; 103965277; A
SYN 2
Synthetic Reference
Preparation method of Difluprednate; Tian, Yuan; Zhou, Shengan; Guo, Bin; Xu, Zhiguo; Assignee Guangzhou Renheng Pharmaceutical Technology Co., Ltd., Peop. Rep. China 2017; Patent Information; May 10, 2017; CN; 106632561; A
SYN3
Synthetic Reference
Shailesh, Singh; Bharat, Suthar; Jain, Ashish; Gaikwad, Vinod; Kulkarni, Kuldip. Process for preparing difluprednate. Assignee Ajanta Pharma Ltd., India. IN 2013MU02535. (2015).
SYN4
Synthetic Reference
Sun, Hongbin; Chen, Bo. Method for preparation of Difluprednate. Assignee China Pharmaceutical University, Peop. Rep. China. CN 103509075. (2014).
Embodiment 1:4, pregnant steroid-17 α of 9 (11)-diene, 21-dihydroxyl-3,20-diketone-21-acetic ester (formula III compound)
10g hydrocortisone-21 acetic ester (formula II compound) is joined in 250mL eggplant type bottle, add 50mL N, dinethylformamide and 8.8mL pyridine, slowly heat up and make material dissolution complete, slowly cooling afterwards, slowly be added dropwise to 4.4mL methylsulfonyl chloride, add rear solution to be yellow completely.Be warming up to 85 ℃ of stirrings, the reaction solution thick one-tenth that can slowly become sticky is faint yellow, adds slightly some DMFs and makes reaction solution dilution, can normally stir, and keeps this thermotonus one hour, and reaction solution slowly becomes grey black during this period.TLC follows the tracks of (sherwood oil: ethyl acetate=1: 1) show that reaction finishes.Stop heating, treat that the backward reaction solution of slow cooling adds 200mL methyl alcohol, stir 1min, reaction flask is placed in to crystallization under ice-water bath.Suction filtration after 1h, makes water and methanol wash filter cake, crude product productive rate 100%.With methyl alcohol-methylene dichloride mixed solvent system recrystallization, obtain sterling, M.P.231-235 ℃, productive rate 90%. 1H-NMR(300MHz，CDCl 3)：δ(ppm)5.75(1H，s，4-H)，5.55(1H，s，11-H)，5.07(1H，d，J＝5Hz，21-H)，4.84(1H，d，J＝5Hz，21-H)，2.15(3H，s，H-21-OAc)，1.31(3H，s，19-CH 3)，0.65(3H，s，18-CH 3)，0.66-2.90(m，17H，backbone).
By 9.4g4, pregnant steroid-17 α of 9 (11)-diene, 21-dihydroxyl-3,20-diketone-21-acetic ester (formula III compound) and 10g4-Dimethylamino pyridine add in 1000mL eggplant-shape bottle, add again 50mL diethylene glycol dimethyl ether and 260mL methylene dichloride, heated and stirred makes dissolution of solid, slowly adds 32mL butyryl oxide slightly after cooling, is warming up to 80 ℃ of return stirrings.After 23h, TLC follows the tracks of, and raw material primitive reaction is complete, stops heating and stirs.Vacuum concentration is removed methylene dichloride.After being down to room temperature, add frozen water in reaction flask, white solid standing to be separated out.Suction filtration, saturated sodium bicarbonate aqueous solution washing leaching cake, dries under infrared lamp, obtain 4,9 (11)-diene-17 α, 21-dihydroxyl-3,20-ketone-21-acetic ester 17 iophenoxic acid esters (formula IV compound) sterling 10.65g, M.P220-224 ℃, productive rate 95.9%. 1H-NMR(500MHz，CDCl 3)：δ(ppm)5.75(1H，s，4-H)，5.54(1H，m，11-H)，4.87(1H，d，J＝4.8Hz，O＝C-CH 2-O，21-H)，4.64-4.91(2H，ABq，J＝16.6Hz，21-H)，2.75(2H，m，2-H)，0.70(3H，s，18-CH 3)，0.95(3H，t，J＝4.4Hz)，1.34(3H，s，18-CH 3)，1.66(2H，m，-CH 2CH 3)，2.17(3H，s，O＝C-CH 3)，2.32(2H，t，J＝4.3Hz，O＝C-CH 2)，? 13C-NMR(75MHz，CDCl 3)：δ(ppm)199.1，198.9，173.4，170.4，169.1，144.1，124.1，118.5，94.5，66.9，48.2，46.3，40.9，37.5，36.4，34.2，33.8，32.7，32.2，32.1，30.6，26.2，24.5，20.5，18.3，13.7，13.6；ESI-MS?m/z：457.2[M+H +]，479.2[M+Na +]；HRMS?for?C 27H 36O 6+Na +?calcd?479.2410，found479.2402.
10g4, pregnant steroid-17 α of 9 (11)-diene, 21-dihydroxyl-3,20-diketone-21-acetic ester 17 iophenoxic acid esters add in 250mL eggplant type bottle, then add 80mL methylvinyl acetate, slowly drip while stirring the 1mL vitriol oil.Be warming up to 80 ℃ of stirring reactions, solution is thin out yellow clarification slowly.(sherwood oil: ethyl acetate=3: 1), raw material reaction is complete produces new point to TLC after 30min.Stop heating, wait to be cooled to 50 ℃, add 1mL triethylamine, be stirred to and be down to room temperature.Add water in reaction solution, ethyl acetate aqueous layer extracted three times, saturated common salt water washing organic phase twice, anhydrous sodium sulfate drying.After 30min, steam organic solvent and obtain brown color oily matter.Column chromatography is purified and is obtained 3,5,9 (11) pregnant steroid-3 of triolefin, 17 α, 21 trihydroxy–3,20-diketone-3,21-diacetate esters 17 iophenoxic acid esters, productive rate 90%. 1H-NMR(300MHz，CDCl 3)：δ(ppm)5.74(1H，s，4-H)，5.53(1H，s，11-H)，5.45(1H，s，6-H)，4.64-4.91(2H，ABq，J＝16.6Hz，21-H)，2.17(3H，s，-COCH 3)，1.17(3H，s，19-CH 3)，0.96(3H，t，J＝7.5Hz)，0.70(3H，s，18-CH 3).
14g4, 9 (11)-diene-6 α-fluoro-17 α, 21-dihydroxyl-3, 20-diketone-21-acetic ester 17 iophenoxic acid esters (formula VII) and 9 (11)-diene-6 β-fluoro-17 α, 21-dihydroxyl-3, the mixture of 20-diketone-21-acetic ester 17 iophenoxic acid esters (formula VI) adds in dry three-necked bottle, add while stirring 400mL acetum, under room temperature, slowly pass into anhydrous hydrogen chloride gas (98% vitriol oil is added dropwise in 37% concentrated hydrochloric acid solution and makes) until saturated, be stirred to raw material and be dissolved into yellow solution completely, continue to stir 2h, TLC monitoring reacts completely, stop stirring, in reaction solution, add the aqueous solution, after separating out solid, suction filtration, saturated sodium bicarbonate aqueous solution washing, dry, be weighed as 13g, productive rate is 93%. 1H?NMR(300MHz，CDCl 3)：δ(ppm)6.10(s，1H)，5.61(s，1H)，5.41-5.16(m，1H)，4.64-4.91(2H，ABq，J＝16.6Hz，21-H)，2.82(dd，J＝28.3，15.7Hz，3H)，2.50(s，2H)，2.32(t，J＝7.4Hz，2H)，2.17(s，3H)，1.96(s，5H)，1.66(d，J＝7.4Hz，2H)，1.46(s，2H)，1.33(s，3H)，0.96(s，3H)，0.71(s，3H).
13g 6 α-fluoro-4; 9; (11)-diene-pregnant steroid-3,20-22 ketone-17-butyric ester-20-acetic ester is dissolved in and fills 300mL1, in the eggplant type bottle of 4 dioxane; add while stirring 40mL 0.46mol/L high chloro acid solution; under room temperature, stir after several minutes, add 14g N-succinimide in reaction system, under nitrogen protection, stir; raw material dissolves gradually, and it is faint yellow that reaction solution is.(the sherwood oil: ethyl acetate=12: 5) monitoring, raw material primitive reaction is complete, adds 10%Na of TLC after 2h 2sO 3unnecessary N-succinimide is fallen in aqueous solution cancellation, and checks (it is blue that test paper no longer becomes) with starch-kalium iodide test paper.Add water in reaction flask, ethyl acetate extraction three times, twice of saturated common salt water washing organic phase, anhydrous sodium sulfate drying organic phase, after 30min, be spin-dried for organic phase, obtain faint yellow oily matter, column chromatography purification (sherwood oil: ethyl acetate=12: 1) obtain white solid 6 α-fluoro-9 α-bromo-11 beta-hydroxies-4-alkene-pregnant steroid-3, the about 14g of 20-diketone-17-butyric ester-20-acetic ester, productive rate is 89%. 1H-NMR(300MHz，CDCl 3)：δ(ppm)5.93(1H，d，J＝4.5，4-H)，5.06(1H，m，6-H)，4.64-4.91(2H，ABq，J＝16.6Hz，21-H)，2.17(3H，s，-COCH 3)，1.84(3H，s，18-CH 3)，0.96(3H，t，J＝7.5Hz)，1.02(3H，s，19-CH 3)，4.72(1H，s，11-H)；ESI-MS?m/z：593.3，595.3[M+Na +].
100mg 6 α-fluoro-9 β, 11 beta epoxides-4-alkene-pregnant steroid-3,20-diketone-17-butyric ester-20-acetic ester drops in the Plastic Bottle of tetrafluoroethylene, adds 2mL methylene dichloride to dissolve, and stirs at-20 ℃.1mL Olah reagent with under 1mL methylene dichloride low temperature, mix after, be slowly added dropwise in reaction system, maintain low temperature and stir 2 hours, TLC monitoring reaction finishes.Reaction flask shifts out low-temp reaction groove, is slowly added dropwise to the 1mol/L NaOH aqueous solution by excessive HF cancellation, is adjusted to pH7～8.Add chloroform in reaction system, extraction, organic layer is used respectively aqueous hydrochloric acid and the saturated common salt water washing of 3mol/L, anhydrous sodium sulfate drying, after standing 30min, steams except organic solvent, column chromatography is further purified and is obtained white solid powder 6 α, 9 α-fluoro-11 beta-hydroxies-4-alkene-pregnant steroid-3,20-diketone-17-butyric ester-20-acetic ester, productive rate 90%. 1H-NMR(300MHz，CDCl 3)：δ(ppm)?6.11(1H，d，J＝4.5Hz，4-H)，5.27(1H，m，6-H)，4.64-4.91(2H，ABq，J＝16.6Hz，21-H)，2.17(3H，s，-COCH 3)，4.40(1H，d，J＝4.5Hz，11-H)，1.02(3H，s，18-CH 3)，0.96(3H，t，J＝7.5Hz)，1.52(3H，s，19-CH 3)；ESI-MS?m/z：533.3[M+Na +]
40mg 6 α, 9 α-fluoro-11 beta-hydroxies-4-alkene-pregnant steroid-3,20-diketone-17-butyric ester-20-acetic ester is dissolved in 3mL dioxane, adds 28mgDDQ, and 100 ℃ of return stirrings heat up.TLC monitoring reaction (sherwood oil: ethyl acetate=12: 8) after 13h, generate the larger product of polarity, steam except organic solvent dioxane, obtain brown color oily matter, add a small amount of methylene dichloride lysate, suction filtration, elimination solid residue, filtrate is washed with sodium bicarbonate aqueous solution after adding a small amount of methylene dichloride again, steams except organic phase rear pillar Chromatographic purification, obtain white solid powder 6 α, 9 α-fluoro-11 beta-hydroxies-Isosorbide-5-Nitrae-diene-pregnant steroid-3,20-diketone-17-butyric ester-20-acetic ester, be title molecule difluprednate, productive rate 70%. 1h-NMR (300MHz, CDCl 3): δ (ppm) 7.20 (1H, d, J=4.5Hz, 1-H), 6.43 (1H, s, 4-H), 6.38 (1H, d, J=6Hz, 2-H), 5.36 (1H, m, 6-H), 4.64-4.91 (2H, ABq, J=16.6Hz, 21-H), 4.43 (1H, d, J=4.5Hz, 11-H), 2.27 (2H, m ,-CH 2-CH 3), 2.17 (3H, s, O=C-CH 3), 1.55 (3H, s, 19-CH 3), 1.02 (3H, s, 18-CH 3), 0.93 (3H, t, J=4.5Hz, 0=C-CH 2cH 2cH 3); ESI-MS m/z:509.3[M+H +]; HRMS for C 27h 35o 7f 2+ H +calcd 509.2351, found 509.2356.M.P.188-190 ℃ (literature value M.P.190-194 ℃); [α] d22=+30.1 ° of (literature values [α] d22=+31.7 °).
Claims (6)
Hide Dependent
1. a method of preparing difluprednate, as following reaction formula:
Specifically comprise the following steps:
(1) by hydrocortisone-21-acetic ester (formula II compound):
Carry out dehydration reaction, generate formula III compound:
(2) formula III compound is carried out to butyric acid esterification, obtains formula IV compound:
(3) formula IV compound is carried out to the reaction of enolization esterifying reagent, obtains formula V compound:
(4) formula V compound is reacted with fluoro reagent and obtains formula VI and formula VII compound:
(5) by formula VI compound, through configuration reversal, reaction obtains formula VII compound;
(6) formula VII compound is reacted with N-bromo-succinimide and water, obtains formula VIII compound:
(7) formula VIII compound epoxidation under alkaline condition is obtained to formula IX compound:
(8) formula IX compound is reacted with fluorination reagent and obtains formula X compound:
(9) dehydrogenation of formula X compound oxidation is obtained to formula I compound (difluprednate).
2. method as claimed in claim 1, is characterized in that, in step (2), formula III compound is obtained to formula IV compound through fourth esterification, and the fourth esterifying reagent adopting is butyryl oxide or butyryl chloride; The alkaline catalysts adopting is pyridine, triethylamine or DMAP; The solvent adopting is methylene dichloride, diethylene glycol dimethyl ether, 1, the mixture of the optional solvents in 2-ethylene dichloride, dioxane, trichloromethane, DMF, methyl-sulphoxide, N,N-dimethylacetamide or above-mentioned solvent.
3. method as claimed in claim 1, is characterized in that, in step (3), formula IV compound is obtained to formula V compound through enolization esterification, and the enolization esterifying reagent adopting is diacetyl oxide, Acetyl Chloride 98Min., methylvinyl acetate or vinyl-acetic ester; The catalyzer adopting is the vitriol oil or tosic acid; The solvent adopting is the mixture of the optional solvents in methylene dichloride, chloroform, toluene, methylvinyl acetate, vinyl-acetic ester or above-mentioned solvent.
4. method as claimed in claim 1, is characterized in that, in step (4), formula V compound is obtained to formula VI compound and formula VII compound through fluoridizing, and the fluoro reagent adopting is Selectfluor or Accufluor; The solvent adopting is the mixture of the optional solvents in methylene dichloride, chloroform, toluene, acetonitrile or above-mentioned solvent.
5. method as claimed in claim 1, it is characterized in that, in step (8), formula IX compound is obtained to formula X compound through fluoridizing open loop, the fluorination reagent adopting is aqueous hydrogen fluoride solution, hydrogen fluoride pyridine solution (Olah reagent) or hydrogen fluoride triethylamine solution; The solvent adopting is methylene dichloride, chloroform, 1, the mixture of the optional solvents in 2-ethylene dichloride, tetrahydrofuran (THF), toluene or above-mentioned solvent; Range of reaction temperature is-50～50 ℃.
6. a key intermediate compound for synthetic difluprednate, shown in IV compound:
Difluprednate ophthalmic emulsion 0.05% is also being studied in other ocular inflammatory diseases, including a phase 3 study evaluating difluprednate for the treatment of anterior uveitis[2][3]
Ulcerative colitis (UC) is a disease characterized by chronic inflammation of the rectal and colonic mucosa, affecting the innermost lining in the first stage. The disease is recurrent, with both active and inactive stages that differ in pathology, symptoms and treatment. The underlying cause of UC is not understood, nor is it known what triggers the disease to recur between its inactive and active forms (Irvine, EJ (2008) Inflamm Bowel Dis 14(4): 554-565). Symptoms of active UC include progressive loose stools with blood and increased frequency of bowel movements. Active mucosal inflammation is diagnosed by endoscopy.
The stools contain pus, mucous and blood and are often associated with abdominal cramping with urgency to evacuate (tenesmi). Diarrhoea may have an insidious onset or, more rarely, start quite suddenly. In severe cases the symptoms may include fever and general malaise. In severe stages, deep inflammation of the bowel wall may develop with abdominal tenderness, tachycardia, fever and risk of bowel perforation. Furthermore, patients with UC may suffer extra intestinal manifestations such as arthralgia and arthritis, erythema nodosum, pyoderma gangrenosum and inflammation in the eyes. In the case of remission or inactive UC, patients are usually free of bowel symptoms.
The extent of inflamed and damaged mucosa differs among patients with UC. UC that affects only the rectum is termed ulcerative proctitis. The condition is referred to as distal or left sided colitis when inflammatory changes are present in the left side of the colon up to the splenic flexure. In extensive UC the transverse colon is also affected, and pancolitis designates a disease involving the entire colon.
Active mucosal inflammation is diagnosed by endoscopy and is characterized by a loss of vascular patterning, oedema, petechia, spontaneous bleeding and fibrinous exudates. The endoscopic picture is that of continuous inflammation, starting in the rectum and extending proximally to a variable extent into the colon. Biopsies obtained at endoscopy and subjected to histological examination help to diagnose the condition. Infectious causes, including Clostridium difficile, camphylobacter, Salmonella and Shigella, may mimic UC and can be excluded by stool cultures.
The medical management of UC is divided into treatment of active disease and maintenance of remission.
The treatment of patients with active UC aims to reduce inflammation and promote colon healing and mucosal recovery. In milder cases the disease may be controlled with conventional drugs including sulphasalazine, 5 -aminosalicylic acid (5-ASA) (Sutherland, L., F. Martin, S. Greer, M. Robinson, N. Greenberger, F. Saibil, T Martin, J. Sparr, E. Prokipchuk and L. Borgn (1987) Gastroenterology 92: 1894-1898) and glucocorticosteroids (GCS) (Domenech, E., M. Manosa and E. Cabre (2014). Dig Dis 32( 4): 320-327).
GCS are generally used to treat disease flare-ups and are not recommended for maintenance of remission since there are significant side effects in long-term use, and the possible development of steroid dependent disease. Glucocorticoid drugs act non-selectively, so in the long run they may impair many healthy anabolic processes. As a result, maintenance treatment with systemic GCS is not advised (Prantera, C. and S.
For patients who become refractory to GCS and suffer from severe or moderately severe attacks of UC, the addition of immunomodulatory agents such as cyclosporine, 6-mercaptopurine and azathioprine may be used. However, immunomodulators are slow-
acting and the induction of remission in these patients is often temporary (Khan, KJ, MC Dubinsky, AC Ford, TA Ullman, NJ Talley and P. Moayyedi (2011) Am J Gastroenterol 106(4): 630-642).
Further treatment options for UC include biologic agents (Fausel, R. and A. Afzali (2015) Ther Clin Risk Manag 11: 63-73). The three TNF-α inhibitors currently approved for the treatment of moderate to severe UC are infliximab, adalimumab, and golimumab. All three carry potential risks associated with their use, and should be avoided in certain patients, eg those with uncontrolled infections, advanced heart failure, neurologic conditions and in patients with a history of malignancy, due to a potential risk of accelerating the growth of a tumor. Other potential adverse effects of TNF-α inhibitor therapy include neutropenia, hepatotoxicity, serum sickness, leukocytoclastic vasculitis, rash including psoriasiform rash, induction of autoimmunity, and injection or infusion site reactions, including anaphylaxis, convulsions, and hypotension.
All three TNF-α inhibitor agents and their related biosimilar/derivative counterparts may be used to induce and maintain clinical response and remission in patients with UC.
Combination therapy with azathioprine is also used for inducing remission.
However, more than 50% of patients receiving TNF-α inhibitor agents fail to respond to induction dosing, or lose response to the TNF-α inhibitor agents over time (Fausel, R. and A. Afzali (2015) Ther Clin Risk Manag 11 : 63-73).
Vedolizumab, an a4b7 integrin inhibitor, was recently approved for the treatment of UC. In the GEMINI 1 trial, vedolizumab was found to be more effective than placebo for inducing and maintaining clinical response, clinical remission, and mucosal healing (Feagan, BG, P. Rutgeerts, BE Sands, S. Hanauer, JF Colombel, WJ Sandbom, G. Van Assche, J. Axler, HJ Kim, S. Danese, I. Fox, C. Milch, S. Sankoh, T. Wyant, J. Xu, A. Parikh and GS Group (2013) “Vedolizumab as induction and maintenance therapy for ulcerative colitis.” N Engl J Med 369(8): 699-710.).
Ulcerative colitis patients, who are chronically active and refractory to known treatments pose a serious medical challenge and often the only remaining course of action is
colectomy. A total colectomy is a potentially curative option in severe UC, but is a life-changing operation that entails risks as complications, such as pouch failure, pouchitis, pelvic sepsis, infertility in women, and nocturnal faecal soiling, may follow. Therefore, surgery is usually reserved for patients with severe refractory disease, surgical or other emergencies, or patients with colorectal dysplasia or cancer.
An emerging third line treatment for UC is cobitolimod (Kappaproct/DIMS0150), a modified single strand deoxyribonucleic acid (DNA)-based synthetic oligonucleotide of 19 bases in length. Cobitolimod has the sequence 5′- G*G*A*ACAGTTCGTCCAT*G*G*C-3′ (SEQ ID NO:1), wherein the CG dinucleotide is unmethylated.
Cobitolimod functions as an immunomodulatory agent by targeting the Toll-like receptor 9 (TLR9) present in immune cells. These immune cells (ie, B-cells and plasmacytoid dendritic cell (pDCs) reside in high abundance in mucosal surfaces, such as colonic and nasal mucosa. The immune system is the key mediator of the changes of UC. The mucosa of the colon and rectum of patients with UC is chronically inflamed and contains active immune cells. Cobitolimod may be topically administered in the region of inflammation, which places the drug in close contact with a high number of intended target cells, ensuring that the drug will reach an area rich in TLR9 expressing cells.The activation of these cells by cobitolimod induces various cytokines,
The clinical efficacy of cobitolimod has been demonstrated in the “COLLECT” (CSUC-01/10 ) clinical trial, which involved the administration to patients of 30 mg doses of cobitolimod, at 4 week intervals and also in the “CONDUCT” (CSUC- 01/16 ) clinical trial, which involved testing different dosage regimes. The details of the “COLLECT” trial were published in Journal of Crohn’s and Colitis (Atreya et al. J Crohn’s Colitis, 2016 May 20) and are summarized in Reference Example 1. The details of the “CONDUCT” clinical trial were published in The Lancet Gastroenterology and Hepatology (Atreya et al 2020. Lancet Gastroenterol Hepatol. 2020 Dec;5(12): 1063-1075) and are summarized in Reference Example 2. Overall, data on cobitolimod support a positive benefit-risk
assessment for patients with chronic UC which is in an active phase (occasionally referred to herein as “chronic active UC”). Cobitolimod is safe and well tolerated and has been shown to be effective to induce clinical response and remission in patients with chronic UC which is in an active phase, as well as symptomatic and endoscopic remission in patients with treatment refractory, moderate to severe chronic UC which is in an active phase. Despite the clinical trial results obtained this far, there still remains a need for additional effective dosages of cobitolimod which exhibit both good efficacy and safety.
In the COLLECT study, which involved administration of a relatively low (30mg) dose of cobitolimod, topical administration of cobitolimod was performed using a spray catheter device, administered during an endoscopy. This is an invasive medical procedure which is necessarily carried out by a medical professional. Further, before the topical administration of the cobitolimod to the patients, the colon of each patient was cleaned to remove faecal matter. That was done to enable the cobitolimod to reach the intestinal epithelial cells within the colon and to enable the endoscopist to view the colonic mucosa. Thus, it is well known in the art that oligonucleotides such as cobitolimod bind to organic matter such as faeces.
As noted above, patients suffering from chronic ulcerative colitis, who are in an active disease state and refractory to known treatments pose a serious medical challenge and often the only remaining course of action is colectomy. For this reason, patients will tolerate medical intervention which requires both colonic cleaning to remove faecal matter and topical administration via spray catheter, despite the inconvenience and discomfort involved in such invasive procedures. However, it would be therapeutically desirable to provide a topical treatment for ulcerative colitis patients which does not require colonic cleaning to remove faecal matter and which, preferably, can be self-administered by the patient.
PATENTS
WO2001074344
WO2005080568
WO2007004977
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WO2007050034
EP2596806
WO2018206722
WO2018206713
WO2018206711
WO2020099585
WO2021037764
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InDex Pharmaceuticals Holding AB (publ) announced that the company has entered an agreement for services with global clinical research organisation (CRO) Parexel Biotech for the phase III study CONCLUDE. The study will evaluate the efficacy and safety of the drug candidate cobitolimod for the treatment of moderate to severe left-sided ulcerative colitis.
“We are excited to advance cobitolimod into phase III, which is the final stage of development before applying for market approval. After the successful collaboration in our recent phase IIb study CONDUCT, we are very pleased to collaborate once again with Parexel Biotech as our clinical development partner”, says Peter Zerhouni, CEO of InDex Pharmaceuticals. “Parexel Biotech is a leading global CRO with considerable experience managing phase III studies in inflammatory bowel disease, which will ensure an efficient execution of the study.”
CONCLUDE is a randomised, double-blind, placebo-controlled, global phase III study to evaluate cobitolimod as a novel treatment for patients with moderate to severe left-sided ulcerative colitis. The induction study will include approximately 400 patients, and the primary endpoint will be clinical remission at week 6. Patients responding to cobitolimod in the induction study will be eligible to continue in a one-year maintenance study, where they will be treated with either cobitolimod or a placebo. Apart from the dosing 250 mg x 2, which was the highest dose and the one that showed the best efficacy in the phase IIb study CONDUCT, the phase III study will also evaluate a higher dose, 500 mg x 2, in an adaptive study design. This higher dose has the potential to provide even better efficacy than what was observed in the phase IIb study.
“We are pleased to partner with InDex Pharmaceuticals on phase III clinical trial CONCLUDE to evaluate a potential new therapy for patients with moderate to severe ulcerative colitis,” said Jim Anthony, Senior Vice President and Global Head, Parexel Biotech. “Our collaboration with InDex Pharmaceuticals demonstrates our commitment to designing innovative solutions that draw from our global clinical experience and therapeutic expertise to fulfil unmet medical needs on behalf of patients worldwide.”
///////////COBITOLIMOD, WHO 10066, IDX 0150, DIMS 0150, Kappaproct
Arimoclomol maleate is in a phase III clinical trials by Orphazyme for the treatment of Niemann-Pick disease type C (NP-C). It is also in phase II clinical studies for the treatment of amyotrophic lateral sclerosis (ALS).
Arimoclomol (INN; originally codenamed BRX-345, which is a citrate salt formulation of BRX-220) is an experimental drug developed by CytRx Corporation, a biopharmaceutical company based in Los Angeles, California. In 2011 the worldwide rights to arimoclomol were bought by Danish biotech company Orphazyme ApS.[1] The European Medicines Agency (EMA) and U.S. Food & Drug Administration (FDA) granted orphan drug designation to arimoclomol as a potential treatment for Niemann-Pick type C in 2014 and 2015 respectively.[2][3]
Fig. 1 Structures of (±)-bimoclomol (1) and (R)-(+)-arimoclomol (2).
The present disclosure provides an optimized four-step process for preparing an ultra-pure composition comprising arimoclomol citrate, i.e. N-{[(2R)-2-hydroxy-3-piperidin-l-ylpropyl]oxy}pyridine-3-carboximidoyl chloride 1-oxide citrate. The optimized process comprises a plurality of optimized sub-steps, each contributing to an overall improved process, providing the ultra-pure composition comprising arimoclomol citrate. The ultra-pure composition comprising arimoclomol citrate meets the medicines agencies’ high regulatory requirements. An overview of the four-steps process is outlined below:
Step 1: Overview of process for preparing ORZY-01
Step 2: Overview of process for preparing ORZY-03
Step 4: Overview of process for preparing BRX-345 (ORZY-05)
The previously reported two-step synthesis of ORZY-01 as shown below includes a 2 hour reflux in step 1A, followed by purification of intermediate compound (V) to increase the batch quality.
(R,Z)-3-(N’-(2-hydroxy-3-(piperidin-1-yl)propoxy)carboximidoyl chloride)pyridine-1-oxide1 – (R)-(+)-Arimoclomol – 2 A solution of (R,Z)-3-(N’-(2-hydroxy-3-(piperidin-1-yl)propoxy)carbamimidoyl)pyridine-1-oxide 12 (205 mg, 0.70 mmol) in conc. hydrochloric acid (1.1 mL, 13.9 mmol) and water (3 mL) was cooled to -5 °C for 15 minutes. Sodium nitrite (63 mg, 0.91 mmol) in water (0.5 mL) was then added dropwise to the reaction mixture and the reaction was stirred at -5 °C for 2.5 hours. The reaction mixture was made alkaline with NaOH (7 M, 3 mL). An additional 10 mL of water was added followed by DCM (30 mL) containing EtOAc (5 mL) and the organics were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by FCC on Biotage Isolera using Biotage SNAP 10 g Si cartridge eluting with gradient elution 0-30% MeOH:DCM both containing 0.1% Et3N to afford the title compound (160 mg, 73% yield) as a colourless semi-solid. Analytical data was consistent with literature values. See ESI section SFC traces for specific enantiomeric ratios of 2 synthesised under the various methodologies quoted in the text. Optical rotation was not determined as it was determined in the ultimate product of this 2·citrate and comparative run times on SFC. 1H NMR (600 MHz, CDCl3) δ: 8.63 (t, J = 1.4 Hz, 1H), 8.16 (ddd, J = 6.4, 1.6, 0.9 Hz, 1H), 7.66 – 7.62 (m, 1H), 7.25 (dd, J = 8.0, 6.6 Hz, 1H), 4.26 (qd, J = 11.3, 5.2 Hz, 2H), 4.07 (dd, J = 9.2, 4.7 Hz, 1H), 2.62 (s, 2H), 2.47 – 2.31 (m, 4H), 1.65 – 1.51 (m, 4H), 1.42 (s, 2H); 13C NMR (151 MHz, CDCl3) δ: 140.3, 137.7, 133.1, 132.5, 125.7, 123.9, 78.7, 64.9, 60.9, 54.8, 25.8, 24.0.
(R)-(+)- Arimoclomol citrate – 2·citrate (R,Z)-3-(N’-(2-hydroxy-3-(piperidin-1-yl)propoxy)carboximidoyl chloride)pyridine-1-oxide (159 mg, 0.51 mmol) was dissolved in acetone (3 mL) and citric acid (97 mg, 0.51 mmol) was added. The reaction mixture was left to stir at room temperature for 18 hours. After this time the mixture was sonicated and the precipitate was filtered, rinsed with cold acetone (1 mL) and dried under vacuum to afford the title compound (165 mg, 64% yield) as a white amorphous solid. Analytical data was consistent with literature values. m.p. 161-162 °C, Acetone (lit. 163-165 °C, EtOH); [α]D 20 +8.0 (c=1, H2O); IR νmax (neat): 3423, 3228, 2949, 2868, 1722, 1589, 1483, 1433, 1307, 1128, 972, 829 cm-1; 1H NMR (600 MHz, d6-DMSO) δ: 8.54 (t, J = 1.5 Hz, 1H), 8.39 – 8.35 (m, 1H), 7.72 – 7.68 (m, 1H), 7.55 (dd, J = 8.0, 6.5 Hz, 1H), 4.28 (ddd, J = 17.6, 13.3, 7.4 Hz, 3H), 3.35 (br. s, 2H), 3.13 – 2.74 (m, 6H), 2.59 (d, J = 15.2 Hz, 2H), 2.56 – 2.51 (m, 2H), 1.77 – 1.61 (m, 4H), 1.48 (s, 2H); 13C NMR (151 MHz, d6-DMSO) δ: 176.6, 171.3, 140.5, 136.4, 132.7, 131.5, 126.8, 123.3, 77.8, 71.4, 63.8, 58.7, 53.1, 44.0, 30.7, 23.0, 21.9; HRMS (m/z TOF MS ES+) for C14H20ClN3O3 [M+H]+ calc. 314.1271, observed 314.1263; SFC er purity R:S >99:1
Procedure for the conversion of (R)-(+)-Bimoclomol 1 into (R)-(+)-Arimoclomol 2 To a solution of (R)-(+)-bimoclomol (61 mg, 0.21 mmol) in acetone (2 mL) was added benzenesulfonic acid (33 mg, 0.21 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated in vacuo. Separately to a suspension of hydrogen peroxide-urea adduct (39 mg, 0.41 mmol) in acetonitrile (6 mL) at -5°C (ice-salt bath) was added trifluoroacetic anhydride (58 μL, 0.41 mmol) dropwise. A suspension of (R)-(+)-bimoclomol, 1, benzenesulfonic acid salt, as made above, in acetonitrile (3 mL) was then added dropwise to this solution. The reaction mixture was stirred for 18 hours, whilst slowly warming to room temperature. Aqueous Na2S2O5 solution (0.5 M, 1 mL) was added and the reaction mixture stirred for 1 hour. The reaction mixture was made alkaline with NaOH (7 M) and extracted with DCM (2 x 30 mL). The combined organics were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by FCC on a Biotage Isolera using Biotage SNAP 10g Si cartridge eluting with gradient elution 0-35% MeOH in DCM to afford the title compound (35 mg, 55% yield) as a colourless semi-solid. Analytical data of the products was consistent with literature and/or previous samples synthesised above.
Arimoclomol is believed to function by stimulating a normal cellular protein repair pathway through the activation of molecular chaperones. Since damaged proteins, called aggregates, are thought to play a role in many diseases, CytRx believes that arimoclomol could treat a broad range of diseases.
Arimoclomol has been shown to extend life in an animal model of ALS[11] and was well tolerated in healthy human volunteers in a Phase I study. CytRx is currently conducting a Phase II clinical trial.[12]
Arimoclomol also has been shown to be an effective treatment in an animal model of Spinal Bulbar Muscular Atrophy (SBMA, also known as Kennedy’s Disease).[13]
Arimoclomol was discovered by Hungarian researchers, as a drug candidate to treat insulin resistance[14][15] and diabetic complications such as retinopathy, neuropathy and nephropathy. Later, the compound, along with other small molecules, was screened for further development by Hungarian firm Biorex, which was sold to CytRx Corporation, who developed it toward a different direction from 2003.
^ Kieran D, Kalmar B, Dick JR, Riddoch-Contreras J, Burnstock G, Greensmith L (April 2004). “Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice”. Nat. Med. 10 (4): 402–5. doi:10.1038/nm1021. PMID15034571. S2CID2311751.
^ Kalmar B, Greensmith L, Malcangio M, McMahon SB, Csermely P, Burnstock G (December 2003). “The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury”. Exp. Neurol. 184 (2): 636–47. doi:10.1016/S0014-4886(03)00343-1. PMID14769355. S2CID5316222.
^ Rakonczay Z, Iványi B, Varga I, et al. (June 2002). “Nontoxic heat shock protein coinducer BRX-220 protects against acute pancreatitis in rats”. Free Radic. Biol. Med. 32 (12): 1283–92. doi:10.1016/S0891-5849(02)00833-X. PMID12057766.
^ Kalmar B, Burnstock G, Vrbová G, Urbanics R, Csermely P, Greensmith L (July 2002). “Upregulation of heat shock proteins rescues motoneurones from axotomy-induced cell death in neonatal rats”. Exp. Neurol. 176 (1): 87–97. doi:10.1006/exnr.2002.7945. PMID12093085. S2CID16071543.
TIC10 (ONC-201) is a potent, orally active, and stable tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inducer which acts by inhibiting Akt and ERK, consequently activating Foxo3a and significantly inducing cell surface TRAIL. TIC10 can cross the blood-brain barrier.
ONC-201, also known as TIC10, is a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.
Akt/ERK Inhibitor ONC201 is a water soluble, orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) and extracellular signal-regulated kinase (ERK), with potential antineoplastic activity. Upon administration, Akt/ERK inhibitor ONC201 binds to and inhibits the activity of Akt and ERK, which may result in inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway as well as the mitogen-activated protein kinase (MAPK)/ERK-mediated pathway. This may lead to the induction of tumor cell apoptosis mediated by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL death receptor type 5 (DR5) signaling in AKT/ERK-overexpressing tumor cells. The PI3K/Akt signaling pathway and MAPK/ERK pathway are upregulated in a variety of tumor cell types and play a key role in tumor cell proliferation, differentiation and survival by inhibiting apoptosis. In addition, ONC201 is able to cross the blood-brain barrier.
Herein, we present a copper-catalyzed tandem reaction of 2-aminoimidazolines and ortho-halo(hetero)aryl carboxylic acids that causes the regioselective formation of angularly fused tricyclic 1,2-dihydroimidazo[1,2-a]quinazolin-5(4H)-one derivatives. The reaction involved in the construction of the core six-membered pyrimidone moiety proceeded via regioselective N-arylation–condensation. The presented protocol been successfully applied to accomplish the total synthesis of TIC10/ONC201, which is an active angular isomer acting as a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL): a sought after anticancer clinical agent.
ONC201 is the founding member of a novel class of anti-cancer compounds called imipridones that is currently in Phase II clinical trials in multiple advanced cancers. Since the discovery of ONC201 as a p53-independent inducer of TRAIL gene transcription, preclinical studies have determined that ONC201 has anti-proliferative and pro-apoptotic effects against a broad range of tumor cells but not normal cells. The mechanism of action of ONC201 involves engagement of PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt/ERK inactivation, and consequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animals models, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors confirmed that ONC201 is exceptionally well-tolerated and established the recommended phase II dose of 625 mg administered orally every three weeks defined by drug exposure comparable to efficacious levels in preclinical models. Clinical trials are evaluating the single agent efficacy of ONC201 in multiple solid tumors and hematological malignancies and exploring alternative dosing regimens. In addition, chemical analogs that have shown promise in other oncology indications are in pre-clinical development. In summary, the imipridone family that comprises ONC201 and its chemical analogs represent a new class of anti-cancer therapy with a unique mechanism of action being translated in ongoing clinical trials.
////////////IMIPRIDONE, TIC 10, ONC 201, NSC 350625, OP 10, Fast Track Designation, Orphan Drug Designation, Rare Pediatric Disease Designation, PHASE 3, GLIOMA, CHIMERIX
DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO
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