Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment

MONOCLONAL ANTIBODIES Comments Off

Mar 232016

The number of antibiotic-resistant bacterial infections has increased dramatically over the past decade. To combat these pathogens, novel antimicrobial strategies must be explored and developed. We previously reported a strategy based on hapten-modified cell wall analogues to induce recruitment of endogenous antibodies to bacterial cell surfaces. Cell surface remodeling using unnatural single d-amino acid cell wall analogues led to modification at the C-terminus of the peptidoglycan stem peptide. During peptidoglycan processing, installed hapten-displaying amino acids can be subsequently removed by cell wall enzymes. Herein, we disclose a two-step dipeptide peptidoglycan remodeling strategy aimed at introducing haptens at an alternative site within the stem peptide to improve retention and diminish removal by cell wall enzymes. Through this redesigned strategy, we determined size constraints of peptidoglycan remodeling and applied these constraints to attain hapten–linker conjugates that produced high levels of antibody recruitment to bacterial cell surfaces.

Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment

Jonathan M. Fura, Sean E. Pidgeon, Morgan Birabaharan, and Marcos M. Pires^*

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States

ACS Infect. Dis., Article ASAP

DOI: 10.1021/acsinfecdis.6b00007

Publication Date (Web): February 02, 2016

*(M.M.P.) E-mail: map311@lehigh.edu.

ACS Editors’ Choice – This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

http://pubs.acs.org/doi/full/10.1021/acsinfecdis.6b00007

SEE OTHER PUBLICATIONS

Illumination of growth, division and secretion by metabolic …

femsre.oxfordjournals.org

Figure 1.

A new metabolic cell-wall labelling method reveals peptidoglycan …

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Novel dipeptide PG labelling strategy.

Keywords:, antibiotics, bacterial, surface, remodeling, d-amino acids, Dipeptide-Based Metabolic Labeling, Bacterial Cells, Endogenous Antibody Recruitment

///

BRIVARACETAM

Uncategorized Comments Off

Mar 222016

BRIVARACETAM, UCB-34714

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

(2S)-2-[(4R)-2-Oxo-4-propyl-1-pyrrolidinyl]butanamide

1-Pyrrolidineacetamide, α-ethyl-2-oxo-4-propyl-, (αS,4R)-

CAS 357336-20-0

Molecular Formula:	C₁₁H₂₀N₂O₂
Molecular Weight:	212.2887 g/mol

UNII-U863JGG2IA

UCB; For the treatment of partial onset seizures related to epilepsy, Approved February 2016

Brivaracetam, the 4-n-propyl analog of levetiracetam, is a racetam derivative with anticonvulsant properties.^[1]^[2] Brivaracetam is believed to act by binding to the ubiquitous synaptic vesicle glycoprotein 2A (SV2A).^[3] Phase II clinical trials in adult patients with refractory partial seizures were promising. Positive preliminary results from stage III trials have been recorded,^[4]^[5] along with evidence that it is around 10 times more potent^[6] for the prevention of certain types of seizure in mouse models than levetiracetam, of which it is an analogue.

On 14 January 2016, the European Commission,^[7] and on 18 February 2016, the USFDA^[8] approved brivaracetam under the trade name Briviact (by UCB). The launch of this anti-epileptic is scheduled for the first quarter of that year. Currently, brivaracetam is still not approved in other countries like Australia, Canada and Switzerland.

Brivaracetam was approved by European Medicine Agency (EMA) on Jan 14, 2016 and approved by the U.S. Food and Drug Administration (FDA) on Feb 18, 2016. It was developed and marketed as Briviact^® by UCB in EU/US.

Brivaracetam is a selective high-affinity synaptic vesicle protein 2A ligand, as an adjunctive therapy in the treatment of partial-onset seizures with or without secondary generalization in adult and adolescent patients from 16 years of age with epilepsy.

Briviact^® is available in three formulations, including film-coated tablets, oral solution and solution for injection/infusion. And it will be available as 10 mg, 25 mg, 50 mg, 75 mg and 100 mg film-coated tablets, a 10 mg/ml oral solution, and a 10 mg/ml solution for injection/infusion. The recommended starting dose is either 25 mg twice a day or 50 mg twice a day, depending on the patient’s condition. The dose can then be adjusted according to the patient’s needs up to a maximum of 100 mg twice a day. Briviact can be given by injection or by infusion (drip) into a vein if it cannot be given by mouth.

European Patent No. 0 162 036 Bl discloses compound (S)-α-ethyl-2-oxo-l- pyrrolidine acetamide, which is known under the International Non-proprietary Name of Levetiracetam.

Levetiracetam

Levetiracetam is disclosed as a protective agent for the treatment and prevention of hypoxic and ischemic type aggressions of the central nervous system in European patent EP 0 162 036 Bl. This compound is also effective in the treatment of epilepsy.

The preparation of Levetiracetam has been disclosed in European Patent No. 0 162 036 and in British Patent No. 2 225 322.

International patent application having publication number WO 01/62726 discloses 2-oxo-l -pyrrolidine derivatives and methods for their preparation. It particularly discloses compound (2S)-2-[(4R)-2-oxo-4-propyl-pyrrolidin-l-yl] butanamide known under the international non propriety name of brivaracetam.

Brivaracetam

International patent application having publication number WO 2005/121082 describes a process of preparation of 2-oxo-l -pyrrolidine derivatives and particularly discloses a process of preparation of (2S)-2-[(4S)-4-(2,2-difluorovinyl)-2-oxo-pyrrolidin-l- yl]butanamide known under the international non propriety name of seletracetam.

Seletracetam

Kenda et al., in J. Med. Chem. 2004, 47, 530-549, describe processes of preparation of 2-oxo-l -pyrrolidine derivatives and particularly discloses compound 1-((1S)-I- carbamoyl-propyl)-2-oxo-pyrrolidone-3-carboxylic acid as a synthetic intermediate.

WO2005028435

CLIPS

Find better ways to make old and new epilepsy drugs. J. Surtees and co-inventors disclose alternative processes for making active pharmaceutical ingredients (APIs) that are used to treat epilepsy and seizures. One compound that can be prepared by their processes is the established drug levetiracetam (1, Figure 1), marketed under the trade name Keppra. Because 1 is now off-patent, there is obvious interest in new drugs.

The inventors also claim that seletracetam (2) and brivaracetam (3) (Figure 2) can be prepared by their processes. These drugs are apparently much more active than 1.

All of the drugs are used as single isomers, so a stereoselective synthesis is desirable. The inventors describe two routes for preparing the molecules; the first, shown in Figure 1, is the synthesis of 1 by the reaction between pyrrolidone (4) and chiral bromo amide 5 in the presence of a base. GC analysis showed that the conversion is 40.3% and that the product contains 51% of the (S)-enantiomer and 49% of the (R)-isomer. No details of their separation are given, although the use of chiral HPLC is discussed.

The same reaction is used to prepare derivative 6 of 1. Compound 7 is prepared from the corresponding hydroxy ester and then condensed with 4 to give 6. Chiral HPLC showed that the product is a mixture of 89.3% (S)-enantiomer 6 and 10.7% of its (R)-isomer.

The inventors do not describe the detailed preparation of 2, but they report that acid 8 is prepared in 41% yield from pyrrolidone 9 and acid 10 in the presence of NaH (Figure 2). Ammonolysis of 8 produces 2; no reaction details are provided.

In a reaction similar to the preparation of 8, acid 11 is prepared from 10 and pyrrolidone 12. The product is isolated in 77% yield and can be converted to 3 by ammonolysis. Again, no details are provided for this reaction.

The second route for preparing the substituted pyrrolidones does not start with simple pyrrolidones and is the subject of additional claims. The route involves a cyclization reaction, shown in Figure 3. The preparation of enantiomer 13 begins with the reaction of racemic salt 14 and optically pure bromo ester 15. This step produces intermediate 16, isolated as a yellow oil. The crude material is treated with 2-hydroxypyridine (2-HP) to cyclize it to 17. This ester is hydrolyzed to give acid 18. Conversion to 13 is carried out by adding ClCO₂Et, followed by reaction with liquid NH₃ in the presence of K₂CO₃. The overall yield of 13 is 32%.

This route is also used to prepare levetiracetam (1) by treating 5 with the HCl salt of amino ester 19 to give 20, recovered as its HCl salt in 49% yield. The salt is basified with Et₃N and treated with 2-HP to cyclize it to 1, initially isolated as an oil. GC analysis showed 100% conversion, and chiral HPLC showed that the product contains 98.6% (S)-isomer and 1.4% (R)-isomer.

The inventors also prepared 1 and its (R)-enantiomer 21 by using a similar reaction scheme with alternative substrates to 5. Figure 4 outlines the route, which starts from protected hydroxy amide 22 and amino ester 23. When the reaction is carried out in the presence of Cs₂CO₃, the product is (R)-enantiomer24, which is used without purification to prepare 21 by treating it with 2-HP. Chiral HPLC showed that the product is 94% (R) and 6% (S).

When the reaction between 22 and 23 is run with K₂CO₃, the product is (S)-enantiomer 25. This is used to prepare 1, but the product contains only 79% (S)-isomer.

The inventors do not comment on the apparent stereoselectivity of the carbonate salts in the reaction of 22 with 23. This is an intriguing finding and worthy of investigation. (UCB S.A. [Brussels]. US Patent 8,338,621

SYNTHESIS

PATENT

WO2005028435

Example 1: Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide 1.1 Synthesis of (2S)-2-aminobutyramide free base

1800 ml of isopropanol are introduced in a 5L reactor. 1800 g of (2S)-2- aminobutyramide tartrate are added under stirring at room temperature. 700 ml of a 25% aqueous solution of ammonium hydroxide are slowly added while maintaining the temperature below 25°C. The mixture is stirred for an additional 3 hours and then the reaction is allowed to complete at 18°C for 1 hour. The ammonium tartrate is filtered. Yield : 86%.

1.2 Synthesis of 5-hydroxy-4-n-propyl-furan-2-one

Heptane (394 ml) and morpholine (127.5 ml) are introduced in a reactor. The mixture is cooled to 0°C and glyoxylic acid (195 g, 150 ml, 50w% in water) is added. The mixture is heated at 20°C during 1 hour, and then valeraldehyde (148.8 ml) is added . The reaction mixture is heated at 43°C during 20 hours. After cooling down to 20^CC, a 37 % aqueous solution of HCl (196.9 ml) is slowly added to the mixture, which is then stirred during 2 hours.

After removal of the heptane phase, the aqueous phase is washed three times with heptane. Diisopropyl ether is added to the aqueous phase. The organic phase is removed, and the aqueous phase further extracted with diisopropyl ether (2x). The diisopropyl ether phases are combined, washed with brine and then dried by azeotropic distillation. After filtration and evaporation of the solvent, 170g of 5- hydroxy-4-n-propyl-furan-2-one are obtained as a brown oil. Yield: 90.8 %

1.3 Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide and (2S)-2-((4S)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide

(S, R) (S, S) The (2S)-2-aιninobutyrarnide solution in isopropanol containing 250 g obtained as described here above is dried by azeotropic distillation under vacuum. To the dried (2S)-2-am obutyraιnide solution is added 5-hydroxy-4-n-propyl-furan-2-one (290 g) between 15°C and 25 °C; the mixture is heated to 30 °C and kept for at least 2 hours at that temperature. Acetic acid (1, 18 eq.), Pd/C catalyst (5 w/w%; Johnson Matthey 5% Pd on carbon – type 87L) are then added and hydrogen introduced into the system under pressure. The temperature is kept at 40 °C maximum and the H₂ pressure maintained between 0,2 bar and 0,5 bar followed by stirring for at least 20 hours following the initial reaction. The solution is then cooled to between 15 °C and 25 °C and filtered to remove the catalyst. The solution of product in isopropanol is solvent switched to a solution of product in isopropyl acetate by azeotropic distillation with isopropyl acetate. The organic solution is washed with aqueous sodium bicarbonate followed by a brine wash and then filtered. After recristallisation, 349 g of (2S)-2-((4R)-2- oxo-4-n-propyl-l-pyrrolidinyl)butanamide and (2S)-2-((4S)-2-oxo-4-n-propyl-l- pyιτolidinyl)butanamide are obtained (Yield: 82.5%).

1.4 Preparation of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide The chromatographic separation of the two diastereoisomers obtained in 1.3 is performed using of (CHIRALPAK AD 20 um) chiral stationary phase and a 45/55 (volume /volume) mixture of n-heptane and ethanol as eluent at a temperature of 25 + 2°C. The crude (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide thus obtained is recristallised in isopropylacetate, yielding pure (2S)-2-((4R)-2-oxo-4-n-propyl-l- pyrrolidinyl)butanamide (Overall yield: 80%) .

Example 2: Synthesis of (2S)-2-((4R)-2-oxo-4-n-propyl-l-pyrrolidinyl)butanamide

Example 1 is repeated except that in step 1.1 a solution of (2S)-2- aminoburyramide.HCl in isopropanol is used (27.72 g, 1.2 equivalent), which is neutralised with a NHs/isopropanol solution (3,4-3,7 mol/L). The resulting ainmonium chloride is removed from this solution by filtration and the solution is directly used for reaction -with 5-hydroxy-4-n-propyl-furan-2-one (23.62 g, 1.0 equivalent) without intermediate drying of the (2S)-2-aminobutyramide solution. Yield after separation of the two diastereoisomers and recristallisation: approximately 84%.

Ref ROUTE1

1. WO0162726A2.

2. WO2005028435A1 / US2007100150A1.

3. J. Med. Chem. 1988, 31, 893-897.

4. J. Org. Chem. 1981, 46, 4889-4894.

PATENT

https://www.google.com/patents/WO2007031263A1?cl=en

Example 3-Synthesis of brivaracetam (I)

3.a. Synthesis of (S) and (R) 2-((R)-2-oxo-4-propyl-pyrrolidin-l-yl)-butyric acid methyl ester fVIaa^*) and (Wlab)

(VIaa) (VIab) A slurry of 60% sodium hydride suspension in mineral oil (0.94g, 23.4 mmol) in tetrahydrofuran (30 mL) is cooled at 0°C under a nitrogen atmosphere. A solution of substantially optically pure (R)-4-propyl-pyrrolidin-2-one (Ilia) (2g, 15.7 mmol) in tetrahydrofuran (2 mL) is added over a 15 minutes period. The reaction mixture is stirred 10 min at 0°C then a solution of methyl-2-bromo-butyric acid methyl ester (V) (3.69g, 20.4 mmol) in tetrahydrofuran (2mL) was added over a 20 minutes period. The reaction mixture is stirred at O⁰C until maximum conversion of starting material and the reaction mixture is then allowed to warm to room temperature and diluted with water (20 mL). Tetrahydrofuran is removed by evaporation and the residue is extracted with isopropyl acetate (20 ml + 10 mL). The combined organic layers are dried on anhydrous magnesium sulfate and evaporated to afford 3g (13.2 mmol, 86 %) of a mixture of epimers of compound (Via), as a mixture respectively of epimer (VIaa) and epimer (VIab). ¹H NMR(400 MHz, CDCI3) of the mixture of epimers (VIaa) and (VIab) : δ = 4.68

(dd, J= 10.8, J= 5.1, 2×1 H) ; 3.71 (s, 2x3H); 3.60 (t app, J= 8.2, IH); 3.42 (t app, J= 8.7, IH); 313 (dd, J= 9.2, J = 6.8, IH); 2.95 (dd, J= 9.2, J= 6.8, IH); 2.56 (dd, J= 16.6, J = 8.7, 2xlH); 2.37 (dm, 2xlH); 2.10 (m, 2xlH); 2.00 (m, 2xlH); 1.68 (m, 2xlH); 1.46 (m, 2x2H); 1.36 (m, 2x2H); 0.92 (m, 2x6H).

¹³C NMR (400 MHz, CDCl₃) of the mixture of epimers (VIaa) and (VIab) : δ =

175.9; 175.2; 171.9; 55.3; 52.4; 49.8; 49.5; 38.0; 37.8; 37.3; 36.9; 32.5; 32.2; 22.6; 22.4; 21.0; 14.4; 11.2; 11.1

HPLC (GRAD 90/10) of the mixture of epimers (VIaa) and (VIab): retention time= 9.84 minutes (100 %)

GC of the mixture of epimers (VIaa) and (VIab): retention time = 13.33 minutes (98.9 %)

MS of the mixture of epimers (VIaa) and (VIab) (ESI) : 228 MH+

3.b. Ammonolysis of compound of the mixture of (VIaa) and (VIab)

(VIaa) (VIab) (I) (VII)

A solution of (VIaa) and (VIab) obtained in previous reaction step (1.46g, 6.4 mmol) in aqueous ammonia 50 % w/w (18 mL) at 0⁰C is stirred at room temperature for 5.5hours. A white precipitate that appears during the reaction, is filtered off, is washed with water and is dried to give 0.77g (3.6 mmol, yield = 56 %) of white solid which is a mixture of brivaracetam (I) and of compound (VII) in a 1 :1 ratio.

¹H NMR of the mixture (I) and (VII) (400 MHz, CDCI3) : δ = 6.36 (s, broad, IH); 5.66 (s, broad, IH); 4.45 (m, IH); 3.53 (ddd, J= 28.8, J= 9.7, J= 8.1, IH); 3.02 (m, IH); 2.55 (m, IH); 2.35 (m, IH); 2.11 (m, IH); 1.96 (m, IH); 1.68 (m, IH); 1.38 (dm, 4H); 0.92 (m, 6H). 13c NMR of the mixture (I) and (VII) (400 MHz, CDCl₃) : δ = 176.0; 175.9; 172.8;

172.5; 56.4; 56.3; 50.0; 49.9; 38.3; 38.1; 37.3; 37.0; 32.3; 32.2; 21.4; 21.3; 21.0; 20.9; 14.4; 10.9; 10.8

HPLC (GRAD 90/10) of the mixture of (I) and (VII) retention time= 7.67 minutes (100 %)

Melting point of the mixture of (I) and (VII) = 104.9⁰C (heat from 40⁰C to 120⁰C at 10°C/min)

Compounds (I) and (VII) are separated according to conventional techniques known to the skilled person in the art. A typical preparative separation is performed on a 11.7g scale of a 1 :1 mixture of compounds (I) and (VII) : DAICEL CHIRALPAK® AD 20 μm, 100*500 mm column at 30⁰C with a 300 mL/minutes debit, 50 % EtOH – 50 % Heptane. The separation affords 5.28g (45 %) of compound (VII), retention time = 14 minutes and 5.2Og (44 %) of compounds (I), retention time = 23 minutes.

¹H NMR of compound (I) (400 MHz, CDCl₃): δ = 6.17 (s, broad, IH); 5.32 (s, broad, IH); 4.43 (dd, J= 8.6, J= 7.1, IH); 3.49 (dd, J= 9.8, J= 8.1, IH); 3.01 (dd, J= 9.8, J= 7.1, IH); 2.59 (dd, J= 16.8, J= 8.7, IH); 2.34 (m, IH); 2.08 (dd, J= 16.8, J= 7.9, IH); 1.95 (m, IH); 1.70 (m, IH); 1.47-1.28 (m, 4H); 0.91 (dt, J= 7.2, J= 2.1, 6H)

HPLC (GRAD 90/10) of compound (I) : retention time = 7.78 minutes

¹H NMR of compound (VII) (400 MHz, CDCl₃): δ = 6.14 (s, broad, IH); 5.27 (s, broad, IH); 4.43 (t app, J = 8.1, IH); 3.53 (t app, J = 9.1, IH); 3.01 (t app, J = 7.8, IH); 2.53 (dd, J = 16.5, J = 8.8, IH); 2.36 (m, IH); 2.14 (dd, J = 16.5, J = 8.1, IH); 1.97 (m, IH); 1.68 (m, IH); 1.43 (m, 2H); 1.34 (m, 2H); 0.92 (m, 6H)

3c. Epimerisation of compound of (2RV2-((R)-2-oxo-4-propyl-pyπOlidin-l-ylV butyramide (VID

Compound (VII) (200 mg, 0.94 mmol) is added to a solution of sodium tert- butoxide (20 mg, 10 % w/w) in isopropanol (2 mL) at room temperature. The reaction mixture is stirred at room temperature for 18h. The solvent is evaporated to afford 200 mg

(0.94 mmol, 100 %) of a white solid. Said white solid is a mixture of brivaracetam (I) and of (VII) in a ratio 49.3 / 50.7.

HPLC (ISO80): retention time= 7.45 min (49.3%) brivaracetam (I); retention time= 8.02 minutes (50.7%) compound (VII).

Route 2

Reference：ROUTE 2

1. WO2007031263A1 / US2009318708A1.

PATENT

http://www.google.com/patents/WO2007065634A1?cl=en

(scheme 3).

Scheme 3

scheme 4.

5h. Synthesis of brivaracetam and (V) A suspension of (Id) and (Ie) (0.6 g, 2.3 mmol) in MIBK (10 mL) is heated at

120°C for 6 hours. The resulting solution is concentrated and separated on chromatography column (Silicagel 600.068-0.200 mm, cyclohexane/EtOAc : 10/90) to give 0.13 g of brivaracetam (0.6 mmol, 26 %, ee = 94 %) and (V).

¹H NMR (400 MHz, CDCl₃): δ = 6.17 (s, broad, IH); 5.32 (s, broad, IH); 4.43 (dd, J= 8.6, J= 7.1, IH); 3.49 (dd, J= 9.8, J= 8.1, IH); 3.01 (dd, J= 9.8, J= 7.1, IH); 2.59 (dd, J= 16.8, J= 8.7, IH); 2.34 (m, IH); 2.08 (dd, J= 16.8, J= 7.9, IH); 1.95 (m, IH); 1.70 (m, IH); 1.47-1.28 (m, 4H); 0.91 (dt, J= 7.2,J= 2.1, 6H).

HPLC (method 90/10) : Retention time = 7.78 minutes Chiral HPLC : Retention time = 9.66 minutes (97%) MS (ESI): 213 MH⁺

Route 3

Reference：1. WO2007065634A1 / US2009012313A1.

References

von Rosenstiel P (Jan 2007). “Brivaracetam (UCB 34714)”. Neurotherapeutics 4 (1): 84–7. doi:10.1016/j.nurt.2006.11.004.PMID 17199019.
Malawska B, Kulig K (Jul 2005). “Brivaracetam UCB”. Current Opinion in Investigational Drugs 6 (7): 740–746. PMID 16044671.
Rogawski MA, Bazil CW (Jul 2008). “New molecular targets for antiepileptic drugs: alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels”. Current Neurology and Neuroscience Reports 8 (4): 345–352. doi:10.1007/s11910-008-0053-7. PMC 2587091.PMID 18590620.
Clinical trial number NCT00464269 for “Double-blind, Randomized Study Evaluating the Efficacy and Safety of Brivaracetam in Adults With Partial Onset Seizures” at ClinicalTrials.gov
Rogawski MA (Aug 2008). “Brivaracetam: a rational drug discovery success story”. British Journal of Pharmacology 154 (8): 1555–7.doi:10.1038/bjp.2008.221. PMC 2518467. PMID 18552880.
Matagne A, Margineanu DG, Kenda B, Michel P, Klitgaard H (Aug 2008). “Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A”. British Journal of Pharmacology 154 (8): 1662.doi:10.1038/bjp.2008.198. PMID 18500360.
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/003898/human_med_001945.jsp&mid=WC0b01ac058001d124
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm486827.htm

Brivaracetam

Names

IUPAC name

(2S)-2-[(4R)-2-oxo- 4-propylpyrrolidin-1-yl] butanamide

Identifiers

Jmol interactive 3D

Properties

C₁₁H₂₀N₂O₂

212.15 g/mol

Pharmacology

ATC code

N03AX23

Legal status

Investigational

Routes of
administration

Oral

Nearly 100%

<20%

Hydrolysis, CYP2C8-mediated hydroxylation

Biological half-life

8 hrs

Excretion

>75% renal

Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

//////BRIVARACETAM, UCB, 2016 FDA, UCB-34714

CCCC1CC(=O)N(C1)C(CC)C(=O)N

FDA approves new treatment for inhalation anthrax, Anthim (obiltoxaximab) ETI-204

MONOCLONAL ANTIBODIES Comments Off

Mar 222016

Anthim (obiltoxaximab)

ETI-204

March 21, 2016

Release

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm491470.htm

On Friday, March 18, the U.S. Food and Drug Administration approved Anthim (obiltoxaximab) injection to treat inhalational anthrax in combination with appropriate antibacterial drugs. Anthim is also approved to prevent inhalational anthrax when alternative therapies are not available or not appropriate.

Inhalational anthrax is a rare disease that can occur after exposure to infected animals or contaminated animal products, or as a result of an intentional release of anthrax spores. It is caused by breathing in the spores of the bacterium Bacillus anthracis. When inhaled, the anthrax bacteria replicate in the body and produce toxins that can cause massive and irreversible tissue injury and death. Anthrax is a potential bioterrorism threat because the spores are resistant to destruction and can be spread by release in the air.

“As preparedness is a cornerstone of any bioterrorism response, we are pleased to see continued efforts to develop treatments for anthrax,” said Edward Cox, M.D., M.P.H, director of the Office of Antimicrobial Products in FDA’s Center for Drug Evaluation and Research.

Anthim is a monoclonal antibody that neutralizes toxins produced by B. anthracis. Anthim was approved under the FDA’s Animal Rule, which allows efficacy findings from adequate and well-controlled animal studies to support FDA approval when it is not feasible or ethical to conduct efficacy trials in humans.

Anthim’s effectiveness for treatment and prophylaxis of inhalational anthrax was demonstrated in studies conducted in animals based on survival at the end of the studies. More animals treated with Anthim lived compared to animals treated with placebo. Anthim administered in combination with antibacterial drugs resulted in higher survival outcomes than antibacterial therapy alone.

The safety of Anthim was evaluated in 320 healthy human volunteers. The most frequently reported side effects were headache, itching (pruritus), upper respiratory tract infections, cough, nasal congestion, hives, and bruising, swelling and pain at the infusion site.

Anthim carries a Boxed Warning alerting patients and health care providers that the drug can cause allergic reactions (hypersensitivity), including a severe reaction called anaphylaxis. Anthim should be administered in settings where patients can be monitored and treated for anaphylaxis. However, given that anthrax is a very serious and often deadly condition, the benefit of Anthim for treating anthrax is expected to outweigh this risk.

Anthim was developed by Elusys Therapeutics, Inc. of Pine Brook, New Jersey, in conjunction with the U.S. Department of Health and Human Services’ Biomedical Advanced Research and Development Authority.

Obiltoxaximab is a monoclonal antibody designed for the treatment of exposure to Bacillus anthracis spores (etiologic agent ofanthrax).^[1]

This drug was developed by Elusys Therapeutics, Inc.

Infographic: What You Should Know About Anthrax

ANTHIM (obiltoxaximab) Data

The efficacy of ANTHIM for treatment and prophylaxis of inhalational anthrax was demonstrated in multiple studies in the cynomolgus macaque and NZW rabbit models of inhalational anthrax. These studies tested the efficacy of ANTHIM compared to placebo and the efficacy of ANTHIM in combination with antibacterial drugs relative to the antibacterial drugs alone. The primary endpoint was survival following challenge with B. anthracis.

Two studies in NZW rabbit and two studies in cynomolgus macaques evaluated treatment with ANTHIM 16mg/kg IV single dose compared to placebo in animals with systemic anthrax. Treatment with ANTHIM alone resulted in statistically significant improvement in survival relative to placebo in both species. Survival rates were 93% and 62% with ANTHIM compared to 0 placebo survivors in rabbits, and 47% and 31-35% survival with ANTHIM compared to 6% or 0% placebo survival in macaques.

ANTHIM administered in combination with antibacterial drugs (levofloxacin, ciprofloxacin and doxycycline) for the treatment of systemic inhalational anthrax disease resulted in higher survival outcomes than antibacterial therapy alone in multiple studies where ANTHIM and antibacterial therapy was given at various doses and treatment times.

ANTHIM administered as prophylaxis resulted in higher survival outcomes compared to placebo in multiple studies where treatment was given at various doses and treatment times. ANTHIM administered as prophylaxis resulted in higher survival outcomes compared to placebo in multiple studies where treatment was given at various doses and treatment times. In one study, cynomolgus macaques were administered ANTHIM 16 mg/kg at 18 hours, 24 hours or 36 hours after exposure. Survival was 6/6 (100%) at 18 hours, 5/6 (83%) at 24 hours, and 3/6 (50%) at 36 hours. Another cynomolgus macaque study evaluated ANTHIM 16 mg/kg administered 72, 48 or 24 hours prior to exposure. Survival was 100% at all three time points (14/14, 14/14, 15/15, respectively) at day 56 (end of study).

Elusys Therapeutics

Elusys Therapeutics, Inc., a private company based in Pine Brook, NJ, is focused on the development of antibody therapeutics for the treatment of infectious disease.

In November 2015, Elusys was awarded a $45M delivery order from the U.S. government to produce ANTHIM® for the U.S. Strategic National Stockpile (SNS), the U.S. government’s repository of critical medical supplies for public health emergency preparedness. Elusys has received grants and contracts from the USG totaling over $240 million to support ANTHIM’s development.

In March 2016, ANTHIM (obiltoxaximab) Injection, the company’s monoclonal antibody (mAb) anthrax antitoxin, received approval from the U.S. Food and Drug Administration (FDA) for the treatment of adult and pediatric patients with inhalational anthrax due toBacillus anthracis in combination with appropriate antibacterial drugs, and for prophylaxis of inhalational anthrax due to B. anthracis when alternative therapies are not available or not appropriate. ANTHIM should only be used for prophylaxis when its benefit for prevention of inhalational anthrax outweighs the risk of hypersensitivity and anaphylaxis. The effectiveness of ANTHIM is based solely on efficacy studies in animal models of inhalational anthrax. There have been no studies of the safety or pharmacokinetics (PK) of ANTHIM in the pediatric population. Dosing in pediatric patients was derived using a population PK approach. ANTHIM does not have direct antibacterial activity. ANTHIM should be used in combination with appropriate antibacterial drugs. ANTHIM is not expected to cross the blood-brain barrier and does not prevent or treat meningitis.

Company	Elusys Therapeutics Inc.
Description	High-affinity humanized mAb against the Bacillus anthracis protective antigen that inhibits binding of anthrax toxins
Molecular Target	Bacillus anthracis protective antigen
Mechanism of Action	Antibody
Therapeutic Modality	Biologic: Antibody

Latest Stage of Development	Approved
Standard Indication	Anthrax
Indication Details	Treat and prevent anthrax infection; Treat anthrax infection
Regulatory Designation	U.S. – Fast Track (Treat and prevent anthrax infection); U.S. – Orphan Drug (Treat and prevent anthrax infection)

References

Statement On A Nonproprietary Name Adopted By The USAN Council – Obiltoxaximab, American Medical Association.

Obiltoxaximab
Monoclonal antibody
Type	?
Source	Chimeric (mouse/human)
Target	Bacillus anthracis anthrax
Clinical data
Trade names	Anthim
Identifiers
CAS Number	1351337-07-9
ATC code	none
ChemSpider	none
Chemical data
Formula	C₆₄₄₄H₉₉₉₄N₁₇₃₄O₂₀₂₂S₄₄
Molar mass	145.5 kg/mol

///////////Anthim, obiltoxaximab, fda 2016, Orphan Drug,

Phytochemical compounds or their synthetic counterparts? A detailed comparison of the quantitative environmental assessment for the synthesis and extraction of curcumin

PROCESS, spectroscopy, SYNTHESIS Comments Off

Mar 212016

Green Chem., 2016, 18,1807-1818
DOI: 10.1039/C6GC00090H, Paper

Elisabetta Zerazion, Roberto Rosa, Erika Ferrari, Paolo Veronesi, Cristina Leonelli, Monica Saladini, Anna Maria Ferrari
LCA of the synthesis of curcumin and its direct conventional and microwave assisted extractions fromCurcuma longa L. were compared.

Phytochemical compounds or their synthetic counterparts? A detailed comparison of the quantitative environmental assessment for the synthesis and extraction of curcumin

Elisabetta Zerazion,^a Roberto Rosa,*^b Erika Ferrari,^c Paolo Veronesi,^b Cristina Leonelli,^b Monica Saladini^c and Anna Maria Ferrari^a

http://pubs.rsc.org/en/Content/ArticleLanding/2016/GC/C6GC00090H?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+rss%2FGC+%28RSC+-+Green+Chem.+latest+articles%29#!divAbstract

*Corresponding authors

^aDipartimento di Scienze e Metodi dell’Ingegneria, Università degli Studi di Modena e Reggio Emilia, via Amendola 2, 42100 Reggio Emilia, Italy

^bDipartimento di Ingegneria “Enzo Ferrari”, Università degli Studi di Modena e Reggio Emilia, via Pietro Vivarelli 10, 41125 Modena, Italy
E-mail: roberto.rosa@unimore.it
Fax: +390592056243
Tel: +390592056224

Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Modena e Reggio Emilia, via Campi 103, 41125 Modena, Italy

Green Chem., 2016,18, 1807-1818

DOI: 10.1039/C6GC00090H

Natural compounds represent an extremely wide category to be exploited, in order to develop new pharmaceutical strategies. In this framework, the number of in vitro, in vivo and clinical trials investigating the therapeutic potential of curcumin is exponentially increasing, due to its antioxidant, anti-inflammatory and anticancer properties. The possibility to obtain this molecule by both chemical synthesis and extraction from natural sources makes the environmental assessments of these alternative production processes of paramount importance from a green chemistry perspective, with the aim, for both industries and academia, to pursue a more sustainable development. The present work reports detailed and quantitative environmental assessments of three different curcumin production strategies: synthesis, conventional Soxhlet-based extraction (CE) and microwave-assisted extraction (MAE). The chemical synthesis of curcumin, as recently optimized by the authors, has been firstly evaluated by using the EATOS software followed by a complete “cradle to the grave” study, realized by applying the Life Cycle Assessment (LCA) methodology. The life cycles of CE and MAE were then similarly assessed, considering also the cultivation of Curcuma longa L., the production of the dried rhizomes as well as their commercialization, in order to firstly investigate the widely claimed green character of MAE with respect to more conventional extraction procedures. Secondly, the results related to the two different extraction strategies were compared to those obtained by the chemical synthesis of curcumin, with the aim to determine its greenest preparation procedure among those investigated. This work represents the first example of an environmental assessment comparison between different production strategies of curcumin, thus smoothing the way towards the highly desirable establishment of environmentally friendly rankings, comprising all the existing alternatives to the chemical synthesis of a target chemical compound.

/////Phytochemical compounds, synthesis, extraction, curcumin

A pot-economical and diastereoselective synthesis involving catalyst-free click reaction for fused-triazolobenzodiazepines

PROCESS, SYNTHESIS Comments Off

Mar 212016

Green Chem., 2016, Advance Article
DOI: 10.1039/C6GC00497K, Communication

Xiaofeng Zhang, Sanjun Zhi, Wei Wang, Shuai Liu, Jerry P. Jasinski, Wei Zhang
A pot-economical synthesis involving two [3 + 2] cycloadditions for diastereoselective synthesis of novel triazolobenzodiazepine-containing polycyclic compounds

A pot-economical and diastereoselective synthesis involving catalyst-free click reaction for fused-triazolobenzodiazepines

http://pubs.rsc.org/en/Content/ArticleLanding/2016/GC/C6GC00497K?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+rss%2FGC+%28RSC+-+Green+Chem.+latest+articles%29#!divAbstract

A pot-economical and diastereoselective synthesis involving catalyst-free click reaction for fused-triazolobenzodiazepines

Xiaofeng Zhang,^a Sanjun Zhi,^b Wei Wang,^c Shuai Liu,^a Jerry P. Jasinski^d and Wei Zhang*^a

*Corresponding authors

^aCentre for Green Chemistry and Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, USA
E-mail: wei2.zhang@umb.edu

^bJiangsu Key Laboratory for the Chemistry of Low-Dimensional Materials, Huaiyin Normal University, Huaian, PR China

^cSchool of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, PR China

^dDepartment of Chemistry, Keene State College, Keene, USA

Green Chem., 2016, Advance Article

DOI: 10.1039/C6GC00497K

A pot-economical synthesis involving sequential [3 + 2] cycloadditions of an azomethine ylide and an azide–alkyne (click reaction) has been developed for diastereoselective synthesis of novel triazolobenzodiazepine-containing polycyclic compounds. A new example of catalyst-free click chemistry of non-strained alkynes is also disclosed

/////A pot-economical, diastereoselective synthesis, catalyst-free click reaction, fused-triazolobenzodiazepines

Cycloaddition of epoxides and CO2 catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

PROCESS Comments Off

Mar 212016

Cycloaddition of epoxides and CO2 catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

Green Chem., 2016, Advance Article
DOI: 10.1039/C6GC00370B, Paper

Xu Jiang, Faliang Gou, Fengjuan Chen, Huanwang Jing
Bisimidazole-functionalized cobaltoporphyrin acted as efficient bifunctional catalysts to facilitate the synthesis of cyclic carbonates from epoxides and CO₂.

see

http://pubs.rsc.org/en/Content/ArticleLanding/2016/GC/C6GC00370B?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+rss%2FGC+%28RSC+-+Green+Chem.+latest+articles%29#!divAbstract

Cycloaddition of epoxides and CO₂ catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

Xu Jiang,^a Faliang Gou,^a Fengjuan Chen^a and Huanwang Jing*^a^b

*Corresponding authors

^aState Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering Lanzhou University, Gansu 730000, PR China

^bState Key Laboratory of Coal Conversion, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001, P R China
E-mail: hwjing@lzu.edu.cn

Green Chem., 2016, Advance Article

DOI: 10.1039/C6GC00370B

A series of innovative bisimidazole-functionalized porphyrin cobalt(III) complexes have been devised, synthesized and characterized using NMR, MS and elemental analysis. These homogeneous catalysts were applied to the cycloaddition of epoxides and carbon dioxide without organic solvent and co-catalyst. It was found that the performance of the catalysts deeply relies on their structural features. The alkoxyl chain length of the linkage and the imidazole position relative to the phenyl rings of porphyrin evidently affects the catalyst activities. [5,15-Di(3-((8-imidazolyloctyl)oxy)phenyl)porphyrin] cobalt(III) chloride (J-m8) and [5,15-di(2-((6-imidazolylhexyl)oxy)phenyl)porphyrin] cobalt(III) chloride (J-o6) demonstrated excellent activity under optimal reaction conditions. Synchronously, a preliminary kinetic investigation of this reaction was carried out using three catalysts and illustrated the activation energies of cyclic carbonate formation. Furthermore, a tri-synergistic catalytic mechanism has been carefully proposed in light of the features of the new catalysts and experimental results.

References [1] L. Jin, H. Jing, T. Chang, X. Bu, L. Wang and Z. Liu, J. Mol. Catal. A: Chem., 2007, 261, 262. [2] X. Jiang, F. Gou and H. Jing, J. Catal., 2014, 313, 159. [3] B. Li, L. Zhang, Y. Song, D. Bai and H. Jing, J. Mol. Catal. A: Chem., 2012, 363– 364, 26.

///Cycloaddition of epoxides, CO2 catalyzed, bisimidazole-functionalized porphyrin cobalt(III) complexes

Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

PROCESS Comments Off

Mar 212016

Chemical Research in Chinese Universities Vol.32 No.1 February 2016 2016 Vol. 32 (1): 0-0 [Abstract] ( 20 ) [HTML 1KB] [PDF 2198KB] ( 11 )

A novel spherical cellulose adsorbent with amide and sulphinate groups was used for a first reduction of trichloroacetic acid(TCAA) and a subsequent adsorption of generated species, haloacetic acids. The removal mechanism involved TCAA reduction by sulphinate groups and the adsorption of the haloacetic acids through electrostatic interaction with amide group. Investigation of product formation and subsequent disappearance reveals that the reduction reactions proceed viasequential hydrogenolysis, and transform to acetate ultimately. Adsorption of haloacetic acids was ascertained by low chloride mass balances(89.3%) and carbon mass balances(75.1%) in solution. The pseudo-first-order rate constant for TCAA degradation was (0.93±0.12) h^-1. Batch experiments were conducted to investigate the effect of pH value on the reduction and adsorption process. The results show that the reduction of TCAA by sulphinate groups requires higher pH values while the electrostatic attraction of haloacetic acids by amino group is favorable in more acidic media.

Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

LIN Chunxiang^1,3, TIAN Chen¹, LIU Yifan^1,3, LUO Wei¹, ZHU Moshuqi¹, SU Qiaoquan¹, LIU Minghua^1,2,3

1. College of Environment & Resources, Fuzhou 350108, P. R. China;
2. State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, P. R. China;
3. Key Laboratory of Eco-materials Advanced Technology(Fuzhou University), Fujian Province University, Fuzhou 350108, P. R. China

LIU Minghua E-mail: mhliu2000@fzu.edu.cn

Cite this article:

LIN Chunxiang,TIAN Chen,LIU Yifan等. Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent[J]. CHEMICAL RESEARCH IN CHINESE UNIVERSITIES, 2016, 32(1): 95-99.

LIN Chunxiang, TIAN Chen, LIU Yifan, LUO Wei, ZHU Moshuqi, SU Qiaoquan, LIU Minghua
Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

2016 Vol. 32 (1): 95-99 [Abstract] ( 9 ) [HTML 1KB] [PDF 0KB] ( 12 )
doi: 10.1007/s40242-016-5304-6

/////Trichloroacetic Acid Removal, Reductive Spherical Cellulose Adsorbent

Trioxacarcin A

cancer Comments Off

Mar 182016

Trioxacarcin A, DC-45A

CAS No. 81552-36-5

Molecular FormulaC₄₂H₅₂O₂₀
Average mass876.850 Da
17′-[(4-C-Acetyl-2,6-dideoxyhexopyranosyl)oxy]-19′-(dimethoxymethyl)-10′,13′-dihydroxy-6′-methoxy-3′-methyl-11′-oxospiro[oxirane-2,18‘-[16,20,22]trioxahexacyclo[17.2.1.0^2,15.0^5,14.0^7,12.0^17,21 ]docosa[2(15),3,5(14),6,12]pentaen]-8′-yl 4-O-acetyl-2,6-dideoxy-3-C-methylhexopyranoside

(1S,2R,3aS,4S,8S,10S,13aS)-13a-(4-C-Acetyl-2,6-dideoxy-alpha-L-xylo-hexopyranosyloxy)-2-(dimethoxymethyl)-10,12-dihydroxy-7-methoxy-5-methyl-11-oxo-4,8,9,10,11,13a-hexahydro-3aH-spiro[2,4-epoxyfuro[3,2-b]naphtho[2,3-h]-1-benzopyran-1,2′-oxiran]-8-yl 4-O-acetyl-2,6-dideoxy-3-C-methyl-alpha-L-xylo-hexopyranoside
Kyowa Hakko Kirin INNOVATOR

Trioxacarcin B

Trioxacarcin B; Antibiotic DC 45B1; DC-45-B1; Trioxacarcin A, 14,17-deepoxy-14,17-dihydroxy-; AC1MJ5N1; 81534-36-3;

Molecular Formula:	C₄₂H₅₄O₂₁
Molecular Weight:	894.86556 g/mol

Trioxacarcin C

(CAS NO.81781-28-4):C₄₂H₅₄O₂₀
Molecular Weight: 878.8662 g/mol
Structure of Trioxacarcin C :

The trioxacarcins are polyoxygenated, structurally complex natural products that potently inhibit the growth of cultured human cancer cells

Natural products that bind and often covalently modify duplex DNA figure prominently in chemotherapy for human cancers. The trioxacarcins are a new class of DNA- modifying natural products with antiproliferative effects. The trioxacarcins were first described in 1981 by Tomita and coworkers (Tomita et al. , J. Antibiotics, 34( 12): 1520- 1524, 1981 ; Tamaoki et al., J. Antibiotics 34( 12): 1525- 1530, 1981 ; Fujimoto et al. , J. Antibiotics 36(9): 1216- 1221 , 1983). Trioxacarcin A, B, and C were isolated by Tomita and coworkers from the culture broth of Streptomyces bottropensis DO-45 and shown to possess anti-tumor activity in murine models as well as gram-positive antibiotic activity. Subsequent work led to the discovery of other members of this family. Trioxacarcin A is a powerful anticancer agent with subnanmolar IC70 values against lung (LXFL 529L, H-460), mammary (MCF-7), and CNS (SF-268) cancer cell lines. The trioxacarcins have also been shown to have antimicrobial activity {e.g., anti-bacterial and anti-malarial activity) (see, e.g. , Maskey et al., J. Antibiotics (2004) 57:771 -779).

trioxacarcin A

An X-ray crystal structure of trioxacarcin A bound to N-7 of a guanidylate residue in a duplex DNA oligonucleotide substrate has provided compelling evidence for a proposed pathyway of DNA modification that proceeds by duplex intercalation and alkylation (Pfoh et al, Nucleic Acids Research 36( 10):3508-3514, 2008).

All trioxacarcins appear to be derivatives of the aglycone, which is itself a bacterial isolate referred to in the patent literature as DC-45-A2. U.S. Patent 4,459,291 , issued July 10, 1984, describes the preparation of DC-45-A₂ by fermentation. DC-45-A2 is the algycone of trioxacarcins A, B, and C and is prepared by the acid hydrolysis of the fermentation products trioxacarcins A and C or the direct isolation from the fermentation broth of Streptomyces bottropensis.

Based on the biological activity of the trioxacarcins, a fully synthetic route to these compounds would be useful in exploring the biological and chemical activity of known trioxacarcin compounds and intermediates thereto, as well as aid in the development of new trioxacarcin compounds with improved biological and/or chemical properties.

PAPER

Component-Based Syntheses of Trioxacarcin A, DC-45-A1, and Structural Analogs
T. Magauer, D. Smaltz, A. G. Myers, Nat. Chem. 2013, 5, 886–893. (Link)

Component-based syntheses of trioxacarcin A, DC-45-A1 and structural analogues

Nature Chemistry5,886–893(2013)

doi:10.1038/nchem.1746

PAPER

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

PAPER

PATENT

http://www.google.com/patents/EP2550285A1?cl=en

(S)-9-Hvdrox v- 10-methoxy-5-(4-methoxybenzylox v)- 1 -(methoxymethox y)-3- methyl-8-oxo-5,6.7.8-tetrahvdroanthracene-2-carbaldehvde. Potassium osmate dihydrate (29 mg, 0.079 mmol, 0.05 equiv) was added to an ice -cooled mixture of (S,£)-9-hydroxy- 10- methoxy-4-(4-methoxybenzyloxy)-8-(methoxymethoxy)-6-methyl-7-(prop- l -enyl)-3,4- dihydroanthracen-l -one (780 mg, 1.58 mmol, 1 equiv), 2,6-lutidine (369 μί, 3.17 mmol, 2.0 equiv), and sodium periodate ( 1.36 g, 6.33 mmol, 4.0 equiv) in a mixture of tetrahydrofuran (20 mL) and water ( 10 mL). After 10 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 1.5 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed with aqueous sodium chloride solution (50 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (20% ethyl acetate- hexanes) to provide 498 mg of the product, (5)-9-hydroxy- 10-methoxy-5-(4- methoxybenzyloxy)- l -(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8-tetrahydroanthracene-2- carbaldehyde, as an orange foam (65%). Ή NMR (500 MHz, CDC1₃): 15.17 (s, 1 H), 10.74 (s, 1 H), 7.66 (s, 1 H), 7.27 (d, 2H, 7 = 8.5 Hz), 6.86 (d, 2H, 7 = 8.6 Hz), 5.30-5.18 (m, 3H), 4.63 (d, 1H,7= 11.1 Hz), 4.52 (d, 1H,7 = 12.0 Hz), 3.86 (s, 3H), 3.79 (s, 3H), 3.62 (s, 3H), 3.22 (m, 1H), 2.75 (s, 3H), 2.63 (m, 1H), 2.54 (m, 1H), 2.08 (m, 1H). ^I3C NMR (125 MHz, CDC1₃): 204.9, 193.2, 163.2, 161.7, 159.2, 144.4, 141.7, 137.0, 130.1, 129.4, 120.7, 117.9, 113.8, 110.0, 102.8, 70.4, 67.2, 62.9, 58.3, 55.2, 32.3, 26.3, 22.2. FTIR, cm^-1 (thin film): 2936 (m), 2907 (m), 1684 (s), 1611 (s), 1377 (s), 1246 (s). HRMS (ESI): Calcd for

(C₂₇H2808+K)⁺: 519.1416; Found 519.1368. TLC (20% ethyl acetate-hexanes): R,= 0.17 (CAM).

86% yield

[00457] (S)-l,9-Dihvdroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde. A solution of B-bromocatecholborane (418 mg, 2.10 mmol, 2.0 equiv) in dichloromethane (15 mL) was added to a solution of (S)-9-hydroxy-10- methoxy-5-(4-methoxybenzyloxy)-l-(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (490 mg, 1.05 mmol, 1 equiv) in dichloromethane (15 mL) at -78 °C. After 50 min, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (25 mL) and dichloromethane (100 mL). The cooling bath was removed, and the partially frozen mixture was allowed to warm to 23 °C. The biphasic mixture was diluted with 0.2 M aqueous sodium hydroxide solution (100 mL). The layers were separated. The aqueous layer was extracted with dichloromethane (100 mL). The organic layers were combined. The combined solution was washed sequentially with 0.1 M aqueous hydrochloric acid solution (100 mL), water (2 x 100 mL), then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide 380 mg of the product, (S)-\ ,9- dihydroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde, as a yellow foam (86%). Ή NMR (500 MHz, CDCI3):

15.89 (brs, 1H), 12.81 (br s, 1H), 10.51 (s, 1H), 7.27-7.26 (m, 3H), 6.86 (d, 2H, J = 9.2 Hz), 5.14 (app s, 1H),4.62 (d, \H,J= 11.0 Hz), 4.51 (d, 1H,7= 11.0 Hz), 3.85 (s, 3H), 3.80 (s, 3H), 3.21 (m, 1H), 2.73 (s, 3H), 2.62 (m, 1H), 2.54 (m, 1H), 2.07 (m, 1H). ^I3C NMR (125 MHz, CDCI3): 204.4, 192.7, 166.6, 164.3, 159.3, 144.4, 142.7, 137.9, 130.4, 130.2, 129.4, 114.9, 114.2, 113.9, 113.8, 109.4, 70.4, 67.1,62.8, 55.3, 31.8, 26.5. FTIR, cm^-1 (thin film): 3316 (brw), 2938 (m), 1678 (m), 1610 (s), 1514 (m), 1393 (m), 1246 (s). HRMS (ESI): Calcd for (C₂₅H₂₄0₇+Na)⁺ 459.1414; Found 459.1354. TLC (50% ethyl acetate-hexanes): R = 0.30 (CAM).

[00458] (5)-2,2-Di-/erf-butyl-7-methoxy-8-(4-methoxybenzyloxy)-5-methyl- 1 1 -oxo- 8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de \ 1 ,3,21dioxasiline-4-carbaldehyde. Όι^‘-tert- butyldichlorosilane (342 μL·, 1.62 mmol, 1.8 equiv) was added to a solution of (5)-l ,9- dihydroxy- 10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (380 mg, 0.90 mmol, 1 equiv), hydroxybenzotriazole (60.8 mg, 0.45 mmol, 0.50 equiv) and diisopropylethylamine (786 μί, 4.50 mmol, 5.0 equiv) in dimethylformamide (30 mL). The reaction flask was heated in an oil bath at 55 °C. After 2 h, the reaction flask was allowed to cool to 23 °C. The reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution (100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed sequentially with water (2 x 100 mL) then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate- hexanes) to provide 285 mg of the product, (S)-2,2-di-/<?ri-butyl-7-methoxy-8-(4- methoxybenzyloxy)-5-methyl- 1 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de] [ 1 ,3,2]dioxasiline-4- carbaldehyde, as a yellow foam (56%). The enantiomeric compound (/?)-2,2-di-½ri-butyl-7- methoxy-8-(4-methoxybenzyloxy)-5-methyl- l 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 – i/e][ l ,3,2]dioxasiline-4-carbaldehyde has been prepared using the same route by utilizing R- (4-methoxybenzyloxy)cyclohex-2-enone as starting material. Ή NMR (500 MHz, CDCI3): 10.84 (s, 1 H), 7.37 (s, 1 H), 7.25 (d, 2H, J = 8.8 Hz), 6.85 (d, 2H, = 8.7 Hz), 5.20 (app s, 1 H), 4.62 (d, 1 H, 7 = 10.0 Hz), 4.51 (d, 1H, J = 1 1.4 Hz), 3.88 (s, 3H), 3.78 (s, 3H), 3.03 (m, 1H), 2.73 (s, 3H), 2.57-2.53 (m, 2H), 2.07 (m, 1H), 1.16 (s, 9H), 1.14 (s, 9H). ¹³C NMR (125 MHz, CDCl₃): 195.6, 190.9, 160.5, 159.2, 150.4, 145.7, 140.4, 134.0, 133.9, 130.3, 129.4, 1 19.5, 1 16.6, 1 15.8, 1 15.3, 1 13.8, 70.4, 67.8, 62.9, 55.2, 34.0, 26.0, 26.0, 22.5, 21.3, 21.1. FTIR, cm^“1 (thin film): 2936 (m), 2862 (m), 1682 (s), 1607 (s), 1371 (s), 1244 (s) 1057 (s). HRMS (ESI): Calcd for (C₃3H₄o0₇Si+H)⁺ 577.2616; Found 577.2584. TLC (10% ethyl acetate-hexanes): R/ = 0.19 (CAM). Alternative Routes to (4S,6S)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy) cyclohex-2-enone.

Alternative Route 1.

[00459] (25,45,55)-2,4-Bis(ferf-butyldimethylsilyloxy)-5-hvdroxycvclohexanone. Dess- Martin periodinane (6.1 1 g, 14.4 mmol, 1.1 equiv) was added to a solution of diol (5.00 g, 13.3 mmol, 1 equiv) in tetrahydrofuran (120 mL) at 23 °C (Lim, S. M.; Hill, N.; Myers, A. G. J. Am. Chem. Soc. 2009, 131, 5763-5765). After 40 min, the reaction mixture was diluted with ether (300 mL). The diluted solution was filtered through a short plug of silica gel (-5 cm) and eluted with ether (300 mL). The filtrate was concentrated. The bulk of the product was transformed as outlined in the following paragraph, without purification. Independently,

an analytically pure sample of the product was obtained by flash-column chromatography (20% ethyl acetate-hexanes) and was characterized by Ή NMR, ^{l 3}C NMR, IR, and HRMS. TLC: (17% ethyl acetate-hexanes) R = 0.14 (CAM); Ή NMR (500 MHz, CDCI3) δ: 4.41 (dd, 1 H, 7 = 9.8, 5.5 Hz), 4.05 (m, l H), 4.00 (m, 1H), 2.81 (ddd, 1 H, 7 = 14.0, 3.7, 0.9 Hz), 2.52 (ddd, 1 H, 7 = 14.0, 5.3, 0.9 Hz), 2.29 (br s, 1 H), 2.18 (m, 1H), 1.98 (m, 1 H), 0.91 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.1 1 (s, 3H), 0.09 (s, 3H), 0.04 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 207.9, 73.9, 73.3, 70.5, 43.3, 39.0, 25.7, 25.6, 18.3, 17.9, -4.7, -4.8, -4.9, -5.4; FTIR (neat), cm^“‘ : 3356 (br), 2954 (m), 2930 (m), 2857 (m), 1723 (m), 1472 (m). 1253 (s), 1 162 (m), 1 105 (s), 1090 (s), 1059 (s), 908 (s), 834 (s), 776 (s), 731 (s); HRMS (ESI): Calcd for (C|₈H380₄Si2+H)⁺ 375. 2381 , found 375.2381.

[00460] (4 ,6 )-4.6-Bis(fcr/-butyldimethylsilyloxy)cvclohex-2-enone. Trifluoroacetic anhydride (6.06 mL, 43.6 mmol, 3.3 equiv) was added to an ice-cooled solution of the alcohol ( 1 equiv, see paragraph above) and triethylamine ( 18.2 mL, 131 mmol, 9.9 equiv) in dichloromethane (250 mL) at 0 °C. After 20 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 18 h, the reaction flask was cooled in an ice bath at 0 °C, and the product solution was diluted with water ( 100 mL). The cooling bath was removed and the reaction flask was allowed to warm to 23 °C. The layers were separated. The aqueous layer was extracted with dichloromethane (2 x 200 mL). The organic layers were combined. The combined solution was washed with saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (6% ethyl acetate-hexanes) to provide 3.02 g of the product, (4S,65)-4,6-bis(/eri-butyldimethylsilyloxy)cyclohex-2-enone, as a colorless oil (64% over two steps). TLC: (20% ethyl acetate-hexanes) R = 0.56 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.76 (dd, 1 Η, / = 10.1 , 3.6 Hz), 5.88 (d, 1 H, 7 = 10.1 Hz), 4.66 (ddd, 1 H, 7 = 5.6, 4.1 , 3.6 Hz), 4.40 (dd, 1 H, 7 = 8.1 , 3.7 Hz), 2.26 (ddd, 1 H, / = 13.3, 8.0, 4.1 Hz), 2.1 1 (ddd, 1 H, J = 13.2, 5.6, 3.8 Hz), 0.91 (s, 9H), 0.89 (s, 9H), 0.12 (s, 3H), 0. 1 1 (s, 3H), 0. 10 (s, 3H), 0.10 (s, 3H); ¹³C NMR ( 125 MHz, CDC1₃) δ: 197.5, 150.3, 127.0, 71 .0, 64.8, 41.6, 25.7, 25.7, 18.3, 18.1 , -4.7, -4.8, -4.8, -5.4; FTIR (neat), cm^-1 : 3038 (w), 2955 (m), 2930 (m), 1705 (m), 1472 (m), 1254 (m), 1084 (m), 835 (s), 777 (s), 675 (s); HRMS (ESI): Calcd for (C,8H360₂Si2+Na)⁺ 379. 2095, found 379. 2080.

[00461] (4S,6S)-6-(/er/-Butyldimethylsilyloxy)-4-hydroxycvclohex-2-enone. Tetra- j- butylammonium fluoride ( 1 .0 M solution in tetrahydrofuran, 8.00 mL, 8.00 mmol, 1 .0 equiv) was added to an ice-cooled solution of the enone (2.85 g, 8.00 mmol, 1 equiv) and acetic acid (485 ί, 8.00 mmol, 1 .0 equiv) in tetrahydrofuran (80 mL) at 0 °C. After 2 h, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 22 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (300 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 300 mL). The organic layers were combined. The combined solution was washed sequentially with saturated aqueous sodium bicarbonate solution ( 100 mL) then saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (25% ethyl acetate-hexanes) to provide 760 mg of the product, (4S,6S)-6-(ferNbutyldimethylsilyloxy)-4-hydroxycyclohex-2-enone, as a white solid (39%). TLC: (20% ethyl acetate-hexanes) R/ = 0.20 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.87 (dd, 1 Η, 7 = 10.2, 3.2 Hz), 5.95 (dd, 1H, J = 10.3, 0.9 Hz), 4.73 (m, 1 H), 4.35 (dd, 1 H, 7 = 7.6, 3.7 Hz), 2.39 (m, 1 H), 2. 13 (ddd, 1 H, J = 13.3, 6.2, 3.4 Hz), 1.83 (d, 1 H, J = 6.2), 0.89 (s, 9H), 0.10 (s, 3H), 0. 10 (s, 3H); ¹³C NMR ( 125 MHz, CDCb) δ: 197.3, 150.0, 127.5, 70.9, 64.2, 41 .0, 25.7, 18.2, -4.8, -5.4; FTIR (neat), cm^“1 : 2956 (w), 293 1 (w), 2858 (w), 1694 (m); HRMS (ESI): Calcd for (C |₂H220₃Si+H)⁺ 243.141 1 , found 243. 1412.

82″:.

[00462] (45.6S)-6-(fgrf-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. Triphenylmethyl tetrafluoroborate ( 16 mg, 50 μπιοΐ, 0.050 equiv) was added to a solution of 4-methoxybenzyl-2,2,2-trichloroacetimidate (445 μΙ_, 2.5 mmol, 2.5 equiv) and alcohol (242 mg, 1 .0 mmol, 1 equiv) in ether ( 10 mL) at 23 °C. After 4 h, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 15 mL) and ethyl acetate (50 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL). The organic layers were combined. The combined solution was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 297 mg of the product, (4S,6S)-6-(im-butyldimethylsilyloxy)-4-(4- methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%).

Alternative Route 2.

[00463] (5)-?erf-Butyl(4-(4-methoxybenzyloxy)cvclohexa- 1.5-dienyloxy)dimethylsilane. rerr-Butyldimethylsilyl trifluoromethanesulfonate (202 iL, 0.94 mmol, 2.0 equiv) was added to an ice-cooled solution of triethylamine (262 μί, 1.88 mmol, 4.0 equiv) and enone ( 109 mg, 0.47 mmol, 1 equiv) in dichloromethane (5.0 mL). After 30 min, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 10 mL), water (30 mL), and dichloromethane (40 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous ammonium chloride solution (20 mL) then saturated aqueous sodium chloride solution (20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography with triethylamine-treated silica gel (5% ethyl acetate-hexanes), to provide 130 mg of the product, (5)-ierr-butyl(4-(4- methoxybenzyloxy)cyclohexa- l ,5-dienyloxy)dimethylsilane, as a colorless oil (80%). Ή

NMR (500 MHz, CDC1₃): 7.27 (d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.6 Hz), 5.96 (dd, 1 H, J = 9.9, 3.5 Hz), 5.87 (d, 1 H, 7 = 9.6 Hz), 4.94 (m, l H), 4.46 (s, 2H), 4.14 (m, 1 H), 3.81 (s, 3H), 2.49 (m, 2H), 0.93 (s, 9H), 0. 16 (s, 3H), 0.15 (s, 3H). ^{, 3}C NMR ( 125 MHz, CDC1₃): 159.1 , 147.5, 130.9, 129.2, 128.6, 128.1 , 1 13.8, 101.4, 70.2, 69.0, 55.3, 28.5, 25.7, 18.0, ^1.5, -4.5. FTIR, cm^-1 (thin film): 2957 (m), 2931 (m), 2859 (m), 1655 (w), 1613 (w), 1515 (s), 1248 (s), 1229 (s), 1037 (m), 910 (s). HRMS (ESI): Calcd for (C₂oH3o0₃Si+H)⁺ 347.2037; Found 347.1912. TLC (20% ethyl acetate-hexanes): R = 0.74 (CAM).

OP B OPMB DM 00 ,,Α,,

c Ύ’^“ -ietone ii ·η- ) ‘”OH

OTBS 82 ^Q

[00464] (4S,6S)-6-Hvdroxy-4-(4-methoxybenzyloxy)cvclohex-2-enone. A solution of dimethyldioxirane (0.06 M solution in acetone, 2.89 mL, 0.17 mmol, 1.2 equiv) was added to an ice-cooled solution of (S)-ieri-butyl(4-(4-methoxybenzyloxy)cyclohexa- l ,5- dienyloxy)dimethylsilane (50 mg, 0.14 mmol, 1 equiv). After 10 min, the reaction mixture was partitioned between dichloromethane ( 15 mL) and 0.5 M aqueous hydrochloric acid ( 10 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous sodium bicarbonate solution ( 10 mL) then water ( 10 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 30 mg of the product, (4S,6S)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%). Ή NMR (500 MHz, CDC1₃): 7.28 (d, 2H, J = 8.2 Hz), 6.89 (m, 3H), 6.09 (d, 1 H, J = 10.1 Hz), 4.64 (m, 2H), 4.53 (d, 1 H, 7 = 1 1 .4 Hz), 4.24 (m, 1 H), 3.81 (s, 3H), 3.39 (d, 1 H, 7 = 1.4 Hz), 2.67 (m, 1 H), 1 .95 (ddd, 1 H, 7 = 12.8, 12.8, 3.6 Hz). ^{I 3}C NMR ( 125 MHz, CDC1₃): 200.4, 159.5, 146.6, 129.7, 129.4, 127.8, 1 14.0, 71.6, 69.8, 68.9, 55.3, 35.1 . FTIR, cm^-1 (thin film): 3474 (br), 2934 (m), 2864 (m), 1692 (s), 1613 (m), 1512 (s), 1246 (s), 1059 (s), 1032 (s). HRMS (ESI): Calcd for (C,₄H_l6O₄+Na)⁺ 271.0941 ; Found 271.0834. TLC (50% ethyl acetate-hexanes): R/ = 0.57 (CAM).

[00465] (45,65)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. rerr-Butyldimethychlorosilane (26 mg, 0.18 mmol, 1.5 equiv) was added to an ice- cooled solution of (45,65)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone (29 mg, 0.12 mmol, 1 equiv) and imidazole (24 mg, 0.35 mmol, 3 equiv) in dimethylformamide (0.5 mL). After 45 min, the reaction mixture was partitioned between water (15 mL), saturated aqueous sodium chloride solution (15 mL), and ethyl acetate (20 mL). The layers were separated. The organic layer was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 29 mg of the product, (4S,6S)-6-(rm-butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cyclohex-2- enone, as a colorless oil (87%).

Glycosylation experiments

[00466] Glycosylation experiments demonstrate that the chemical process developed allows for the preparation of synthetic, glycosylated trioxacarcins. Specifically, the C4 or CI 3 hydroxyl group may be selectively glycosylated with a glycosyl donor (for example, a glycosyl acetate) and an activating agent (for example, TMSOTf), which enables preparation of a wide array of trioxacarcin analogues.

Selective Glycosylation of the C4 Hydroxyl Group

[00467] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 19.9 mg, 88 μιτιοΐ, 1.1 equiv) was added to a vigorously stirring, biphasic solution of differentially protected trioxacarcin precursor (60 mg, 80 μιτιοΐ, 1 equiv) in dichloromethane ( 1.1 mL) and pH 7 phosphate buffer (220 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 3 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (50 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μιτι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 33 mg of the product as a yellow-green powder (65%).

[00468] Trimethylsilyl triflate ( 10% in dichloromethane, 28.3 μί, 16 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected trioxacarcin precursor (33 mg, 52 μπιοΐ, 1 equiv), 1 -0- acetyltrioxacarcinose A ( 14.1 mg, 57 μιτιοΐ, 1.1 equiv), and powdered 4- A molecular sieves (-50 mg) in dichloromethane (1 .0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between

dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 20 mg of the product as a yellow-green powder (47%). TLC: (5% methanol-dichloromethane) R = 0.40 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 7.47 (s, 1H), 5.38 (d, 1H, J = 3.6 Hz), 5.35 (app s, 1 H), 5.26 ppm (d, 1 H, 7 = 4.0 Hz), 4.84 (d, 1 H, J = 4.0 Hz), 4.78 (dd, 1 H, 7 = 12.3, 5.2 Hz), 4.75 (s, 1H), 4.71 (s, 1 H), 4.52 (q, 1H, J = 6.6 Hz), 3.86 (s, 1 H), 3.83 (s, 3H), 3.62 (s, 3H), 3.47 (s, 3H), 3.15 (d, l H, y = 5.3 Hz), 3.05 (d, 1 H, 7 = 5.3 Hz), 2.60 (s, 3H), 2.58 (m, 1H), 2.35 (m, 1 H), 2.14 (s, 3H), 1.96 (dd, 1 H, 7 = 14.6, 4.1 Hz), 1.62 (d, 1 H, 7 = 14.6 Hz), 1.26 (s, 1 H), 1.23 (d, 3H, J = 6.6 Hz), 1.08 (s, 3H), 0.95 (s, 9H), 0.24 (s, 3H), 0.16 (s, 3H); ‘³C NMR ( 125 MHz, CDC1₃) 6: 202.8, 170.5, 163.2, 151.8, 144.4, 142.4, 135.2, 126.6, 1 16.8, 1 15.2, 1 15.1 , 108.3, 104.0, 100.3, 98.6, 98.3, 74.6, 73.4, 69.8, 69.5, 69.5, 68.9, 69.5, 69.5, 68.9, 68.4, 62.9, 62.7, 57.2, 56.8, 50.7, 38.8, 36.8, 26.0, 25.9, 21.1 , 20.6, 18.6, 17.0, -4.2, -5.3; FTIR (neat), cm^“‘ : 2953 (w), 2934 (w), 2857 (w), 1749 (w), 1622 (m), 1570 (w), 1447 (w), 1391 (m), 1321 (w), 1294 (w), 1229 (m), 1 159 (m), 1 121 (s), 1084 (s), 1071 (m), 1020 (m), 995 (s), 943 (s), 868 (m), 837 (m), 779 (m); HRMS (ESI): Calcd for (C₄oH₅₄0i₆Si+Na)⁺ 841.3073, found

841.3064.

Glycosylation of a Cycloaddition Coupling Partner

[00469] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 14.3 mg, 63 μπιοΐ, 1.2 equiv) was added to a vigorously stirring, biphasic solution of differentially protected aldehyde (37 mg, 52 μιτιοΐ, 1 equiv) in dichloromethane (870 μί) and water (175 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 2 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (40 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 28 mg of the product as a yellow powder (91 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.37 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1H), 7.30 (s, 1 H), 5.45 (m, 1H), 4.68 (dd, 1H, / = 10.3, 4.2 Hz), 3.97 (s, 3H), 3.31 (brs, 1H), 2.72 (s, 3H), 2.51-2.45 (m, 1H), 2.41-2.37 (m, 1H), 1.15 (s, 9H), 1 , 13 (s, 9H), 0.88 (s, 9H), 0.15 (s, 3H), 0.1 1 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 194.6, 191 , 160.5, 150.2, 146, 140.8, 135.8, 134, 1 19.6, 1 16.2, 1 15.4, 1 14.7, 72.7, 63.7, 62.4, 38.8, 29.9, 62.4, 38.8, 63.7, 62.4, 38.8, 63.7, 62.4, 38.8, 29.9, 26.2, 26.1 , 26, 22.7, 21.4; FTIR (neat), cm^“1 : 3470 (br, w), 2934 (w), 2888 (w), 1684 (s), 1607 (s), 1560 (w), 1472 (m), 1445 (w), 1392 (m), 1373 (s), 1242 (s), 1 153 (s), 1 1 19 (w), 1074 (m), 1044 (s), 1013 (s), 982 (w), 934 (m), 907 (w), 870 (m), 827 (s), 795 (s), 779 (s), 733 (s), 664 (s); HRMS (ESI): Calcd for (C₃iH₄₆0₇Si₂+H)⁺ 587.2855, found 587.2867.

[00470] Trimethylsilyl triflate (10% in dichloromethane, 25.9 μί, 14 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected aldehyde (28 mg, 48 μηιοΐ, 1 equiv), 1-0- acetyltrioxacarcinose A (12.9 mg, 52 μπιοΐ, 1.1 equiv), and powdered 4-A molecular sieves (-50 mg) in dichloromethane ( 1.0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 80→98% acetonitrile in water, flow rate: 15 mL/min) to provide 15 mg of the product as a yellow powder (41 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.29 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1 H), 7.32 (s, 1 H), 5.43 (d, 1 H, J = 3.9 Hz), 5.32 (m, 1H), 4.74 (s, 1 H), 4.67 (dd, 1 H, J = 12.3, 5.0 Hz), 4.54 (q, 1H, J = 6.6 Hz), 3.91 (s, 1H), 3.88 (s, 3H), 2.72 (s, 3H), 2.59 (ddd, 1 H, J = 13.8, 5.0, 3.2 Hz), 2.34 (m, 1H), 2.14 (s, 3H), 1.97 (dd, 1H, J = 14.2, 4.2 Hz), 1.71 (d, 1 Η, / = 14.6 Hz), 1.22 (d, 3H, J = 6.3 Hz), 1.15 (s, 9H), 1.15 (s, 9H), 1.08 (s, 3H), 0.93 (s, 9H), 0.23 (s, 3H), 0.13 (s, 3H); ¹³C NMR (125 MHz, CDC1₃) δ: 193.9, 191.0, 170.5, 146.4, 140.9, 134.0, 132.4, 1 19.8, 1 16.8, 1 15.8, 1 15.0, 1 10.8, 99.6, 74.6, 71.5, 70.4, 68.9, 62.9, 62.7, 39.1 , 36.9, 26.2, 26.1 , 26.1 , 25.9, 24.1 , 22.7, 21.5, 21.3, 21.1 , 18.7, 16.9, -4.1 , -5.3; FTIR (neat), cm^-1 : 3524 (br, w), 2934 (m), 2861 (m), 1749 (m), 1686 (s), 1607 (s), 1560 (m), 1474 (m), 1447 (m), 1424 (w), 1375 (s), 1233 (s), 1 159 (s), 1 1 17 (m), 1080 (m), 1049 (s), 1015 (s), 997 (s), 937 (m), 883 (m), 872 (m), 827 (s), 797 (m), 781 (m), 737 (w), 677 (w), 667 (m); HRMS (ESI): Calcd for (C₄₀H₆₀O, ,Si₂+H)⁺773.3747, found 773.3741.

General Glycosylation Procedure of the C13 Hydroxyl Group

[00471] Crushed 4-A molecular sieves (-570 mg / 1 mmol sugar donor) was added to a stirring solution of the sugar acceptor (1 equiv.) and the sugar donor (30.0 equiv.) in dichloromethane ( 1.6 mL / 1 mmol sugar donor) and diethylether (0.228 mL / 1 mmol sugar donor) at 23 °C. The bright yellow mixture was stirred for 90 min at 23 °C and finally cooled to -78 °C. TMSOTf (10.0 equiv.) was added over the course of 10 min at -78 °C. After 4 h, a second portion of TMSOTf (5.0 equiv.) was added at -78 °C and stirring was continued for 1 h. The last portion of TMSOTf (5 equiv.) was added. After 1 h, triethylamine (20 equiv.) was added and the reaction the product mixture was filtered through a short column of silica gel deactivated with triethylamine (30% ethyl acetate-hexanes initially, grading to 50% ethyl acetate-hexanes). H NMR analysis of the residue showed minor sugar donor remainings and that the sugar acceptor had been glycosylated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→100% acetonitrile in water, flow rate: 15 mL/min) to provide the glycosylation product as a bright yellow oil

Three Specific Compounds Prepared by the General Glycosylation Procedure for the CI 3 Hydroxyl Group:

[00472] 10% yield; TLC: (50% ethyl acetate-hexane) R = 0.58 (UV, CAM); Ή NMR (600 MHz, CDC1₃) δ: 7.43 (s, 1 H), 5.84 (t, J = 3.6 Hz, 1 H), 5.29 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.01 (q, J = 6.6 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.73 (s, 1 H), 3.88 (s, OH), 3.77 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 3.03 (app q, J = 5.4 Hz, 2H), 2.84 (d, J = 6.0 Hz, 1 H), 2.77 (d, J = 6.0 Hz, 1 H), 2.72 (t, J = 6.6 Hz, 2H), 2.58 (s, 3H), 2.36 (s, 3H), 2.33 (t, J = 3.0 Hz, 2H), 2.23 (s, 3H), 2.1 1 -2.06 (m, 2H), 1.08 (d, J = 6.0 Hz, 3H). ^‘

[00473] 81 % yield, TLC: (50% ethyl acetate-hexane) R = 0.30 (UV, CAM); Ή NMR (600 MHz, CDCI3) δ: 7.46 (s, 1 H), 7.28 (d, J = 9 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2 H), 5.83 (dd, J = 3.6, 1.8 Hz, 1 H), 5.30 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.19 (m, 1 H), 5.00 (q, J = 6.0 Hz, 1 H), 4.96 (dd, J = 12.0, 4.8 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.74 (s, l H), 4.70 (d, y = 10.8 Hz, 1 H), 4.59 (d, J = 10.8 Hz, 1 H), 3.86 (s, OH), 3.83 (s, 3H), 3.80 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 2.81 (d, J = 6.0 Hz, 1 H), 2.73-2.68 (m, 1 H), 2.70 (d, J = 6.0 Hz, 1 H), 2.59 (s, 3H), 2.35 (s, 3H), 2.33-2.28 (m, 2H), 2.22 (s, 3H), 2.19- 2.1 3 (m, 1 H), 1 .08 (d, J = 6.0 Hz, 3H), 0.97 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H); HRMS (ESI): Calcd for (C₄9H₆₂0₁₈Si+H)⁺ 967.3778, found 967.3795; HRMS (ESI): Calcd for (C ¾₂0,₈Si+Na)⁺ 989.3598, found 989.3585.

[00474] Compound Detected by ESI Mass Spectrometry: Calculated Mass for

[C52H₇| N₃02i Si-Hr^l = 1 100.4277, Measured Mass = 1 100.4253.

PATENT

US 4511560

https://www.google.com/patents/US4511560

The physico-chemical characteristics of DC-45-A and DC-4-5-B₂ according to this invention are as follows:

(1) DC-45-A

(1) Elemental analysis: H:5.74%, C:55.11%

(2) Molecular weight: 877

(3) Molecular formula: C₄₂ H₅₂ O₂₀

(4) Melting point: 180° C.±3° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 1 (in 50% methanol)

(6) Infrared absorption spectrum: As shown in FIG. 2 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-15.3° (c=1.0, ethanol)

(8) PMR spectrum (in CDC]₃ ; ppm): 1.07 (3H,s); 1.10 (3H, d, J=6.8); 1.24 (3H,d, J=6.5); many peaks between 1.40-2.30; 2.14 (3H,s); 2.49 (3H,s); 2.63 (3H,s); many peaks between 2.30-2.80; 2.91 (1H,d, J=5.6); 3.00 (1H,d, J=5.6); 3.49 (3H,s); 3.63 (3H,s); 3.85 (3H, s); many peaks between 3.60-4.00; 4.18 (1H,s); 4.55 (1H,q, J=6.8); many peaks between 4.70-4.90; 5.03 (1H, q, J=6.5); 5.25 (1H,d, J=4.0); 5.39 (1H, d, J=4.0); 5.87 (1H, m); 7.52 (1H,s); 14.1 (1H,s)

(9) CMR spectrum (in CDCl₃ ; ppm): 210.9; 203.8; 170.3; 162.1; 152.5; 145.2; 142.3; 135.3; 126.7; 117.0; 114.2; 108.3; 105.3; 99.7; 97.2; 93.7; 85.1; 79.0; 74.6; 71.1; 69.6; 69.3; 68.8; 67.9; 66.3; 64.0; 62.8; 57.3; 55.9; 36.5; 32.2; 28.0; 25.7; 20.9; 20.2; 17.0; 14.7

(10) Solubility: Soluble in methanol, ethanol, water and chloroform; slightly soluble in acetone and ethyl acetate, and insoluble in ether and n-hexane

(2) DC-45-B₂

(1) Elemental analysis: H: 6.03%, C: 54.34%

(2) Molecular weight: 879

(3) Molecular formula: C₄₂ H₅₄ O₂₀

(4) Melting point: 181°-182° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 5 (in 95% ethanol)

(6) Infrared absorption spectrum: As shown in FIG. 6 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-10° (c=0.2, ethanol)

(8) PMR spectrum (in CDCl₃ ; ppm): 1.07 (3H,s); many peaks between 1.07-1.5; many peaks between 1.50-2.80; 2.14 (3H,s); 2.61 (3H, broad s); 2.86 (1H, d, J=5.7); 2.96 (1H, d, J=5.7); 3.46 (3H,s); 3.63 (3H, s); 3.84 (3H, s); many peaks between 3.65-4.20; many peaks between 4.40-5.00; many peaks between 5.10-5.50; 5.80 (1H, broad s); 7.49 (1H, d, J=1.0); 14.1 (1H, s)

(9) CMR spectrum (in CDCl₃ ; ppm): 202.8; 170.2; 163.1; 151.8; 144.8; 142.9; 135.4; 126.5; 116.8; 114.9; 107.3; 104.6; 101.5; 99.6; 98.0; 94.4; 74.4; 72.5; 71.4; 70.4; 69.1; 68.8; 68.3; 67.9; 67.5; 66.4; 62.9; 62.7; 56.8; 56.5; 48.0; 36.7; 32.3; 25.7; 20.8; 20.3; 18.2; 16.9; 15.5

(10) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform; slightly soluble in benzene, ether and water; and insoluble in n-hexane.

//////

CC1C(C(CC(O1)OC2CC(C(=O)C3=C(C4=C5C(=C(C=C4C(=C23)OC)C)C6C7C(O5)(C8(CO8)C(O6)(O7)C(OC)OC)OC9CC(C(C(O9)C)(C(=O)C)O)O)O)O)(C)O)OC(=O)C

Already 13 EMA GMP Non-compliance Reports in 2016 published

regulatory Comments Off

Mar 172016

EudraGMDP is the central database for GMP and GDP compliance. Inspections which have been performed by any of the EU member state inspectorates are published in the database. Please get the details about the GMP non-compliance findings at 11 manufacturers in Europe and abroad.

http://www.gmp-compliance.org/enews_05224_Already-13-EMA-GMP-Non-compliance-Reports-in-2016-published_15159,S-QSB_n.html

EudraGMDP is the central database for GMP and GDP compliance. Inspections which have been performed by any of the EU member state inspectorates are published in the database. If the manufacturing or distribution site has been found in compliance with GMP and or GDP then a certificate is issued in the database as reference for other inspectorates. This information is also available to the public. A negative outcome will lead to a GMP or GDP Non-Compliance Report. In 2016 no GDP Non-Compliance Reports have been published until today but already 13 GMP Non-Compliance Reports until March 15th.

Among the companies concerned there are 5 Chinese, 3 French, 2 Spanish manufacturers as well as one each from Sweden, Romania and Poland. All Non-Compliance Report were either issued in 2016 (12) or updated in 2016 (1).

The GMP non compliance findings reveal severe deviations from EU GMP. In addition some companies are involved in falsification and data manipulations – a serious trend which can be observed in many international inspections (e.g. those performed by FDA, WHO). Data Integrity and falsification issues are highlighted in the findings below.

MINSHENG GROUP SHAOXING PHARMACEUTICAL CO. LTD, China

Overall, 18 deficiencies were observed during the inspection, including 2 Critical and 4 Major deficiencies: [Critical 1]Falsification of source of API (Thiamphenicol): Repackaging, relabeling and selling of purchased API from a non-GMP company (Zhejiang Runkang Pharmaceutical Co.Ltd.) as if manufactured in-house; [Critical 2] Praziquantel manufactured according to CP process/grade was released as USP process/grade without a full traceability of the testing activities ; [Major 1] The maintenance and the cleaning operations of the manufacturing line used for the production of Praziquantel (API) were found deficient; [Major 2] The pipes design of some equipment used for the manufacturing of Praziquantel, the handling of change related to these equipment and the instruction used for the transfer of the intermediate solution using nitrogen were found deficient ; [Major 3] The hoses used for unloading of solvent were not identified, had no cleaning status and were stored on a dirty floor of an area not mentioned in the general layout of the site; [Major 4] There was no procedure in place for audit trail and there was no effective audit trail in place to determine any change or deletion of the chromatographic raw data. The audit trial function including the administrator profiles was enabled for all the QC staff.

DESARROLLOS FARMACÉUTICOS BAJO ARAGÓN, S.L., Spain

The manufacturer has not established a quality management system including adequate controls to ensure the accuracy and completeness of the critical records data.

S.C. IRCON SRL, Romania

During inspection a number of 34 deficiencies were found, out of which 4 were critical and 10 major. Critical deficiencies are related to the quality management system, qualification/validation activities, manufacturing and material management documents and quality control laboratories activity.

Agila Specialties Polska Sp. z o.o, Poland

29 major deficiencies were found in Agila Specialties which pose a risk of microbial and particulate contamination and could not assure the sterility of the final product. Most of these are related to: 1.) design and qualification of HVAC, laminar air flow system and clean areas, 2.) cleaning and maintenance of clean areas. 3.) manufacturing and batch releasing in the conditions not complying with GMP requirements 4.) change control. In December, 2014 the HVAC system of vials and prefilled syringes lines was significantly modified. Since January till July 2015, 49 batches were manufactured in that area without qualification after the change. During the inspection it was found that: 1) pressure differential between clean areas B and C grade were usually below 10 Pa (effective to < 0 Pa) and alarm (generated electronically, non-validated after the change of the system) has triggered at 0 Pa and after reversing the flow; 2.) laminar air flow system did not comply with requirements given in Annex 1; 3.) test of maximum permitted number of particles “in operation” does not perform properly; 4.) technical condition of clean areas and equipment show lack of proper and regular maintenance. In clean areas A/B grade contamination were found on the arm of the filling machine for prefilled syringes and difficult to clean equipment placed without proper SOP. In grade C e.g. crumbling insulation of pipes, peeling teflon on the ports of tanks and pumps, lack of labelling and mixed clean and dirty equipment, chipped glass accessories was found; 5.) the filtration process was not fully validated and during routine process a pressure difference to be used across the filter was not recorded; 6.) lack of confirmation of A grade in a lyophilizer working in a nitrogen atmosphere; 7.) design, installation and use of nitrogen system did not guarantee tightness and can cause contamination of the clean medium.

HUBEI HONGYUAN PHARMACEUTICAL CO., LTD. , China

This inspection was performed in the framework of the CEP dossier for the manufacture of Metronidazole R1-CEP 2007-309-Rev 01. The inspection identified in total 24 deficiencies to EU GMP. One of them was categorized as critical and related to the Company’s Quality Assurance System for production of Metronidazole. 10 deficiencies were categorized as major and were related to: QA, Documentation, Supplier Qualification, Data Integrity, Out-of-Specification handling, Quality Control, Computerised System validation, Change Control.

HUBEI HONGYUAN PHARMACEUTICAL CO., LTD. (Facility 428) , China

The Company’s facility at No. 428 Yishui North Road, Fengshan Town, Luotian County, Huanggang City, Hubei Province, China was subject to a spot check, because this site is mentioned as an intermediate manufacturing site in CEP 2001-450 Metronidazole. The Company clearly stated in their introduction that the site does not follow EU GMP. The following observations were made and together categorized as critical: a. The manufacturing site and it’s equipment was found in a devastated state. b. Huge layers of dust and product indicated that no cleaning was applied to either the facility or the equipment, leading to an extreme risk of cross-contamination. c. The extremely bad shape of the facility and the equipment showed that no maintenance was in place. d. Almost none of the products seen was labelled. e. No batch manufacturing documentation could be seen. Reference: EU GMP Part II was found not implemented at the facility.

SAS JARMAT « LABORATOIRE ADP », France

As a preliminary note, the starting materials repacked by the site were intended for pharmaceutical compounding activity in community pharmacies. The site did not distribute to the industry. Overall, 21 deficiencies were found, including 3 critical deficiencies and 5 major deficiencies: [Critical 1] Important risks of confusion in the repacking operations were identified. [Critical 2] Important risks of cross contamination in the repacking operations by substances of high pharmacological activity or toxicity were identified. [Critical 3] The active substances and excipients batches were not analysed as per the pharmacopoeial specifications. [Major 1] The release of active substances batches was deficient, notably in the absence of batch production records. [Major 2] Several risks of contamination in the sampling operations, notably cross contamination, were identified. [Major 3] The management of active substance suppliers was deficient, notably in the absence of written confirmation. [Major 4] Several risks of contamination in the repacking operations, notably cross contamination, were identified. [Major 5] The transmission of information to pharmacies was incomplete and confusing, notably regarding the analyses actually performed by the site. The inspection’s observations also apply to excipients, which are repacked and distributed under the same conditions as the active substances.

Svenska Bioforce AB, Sweden

During the inspection, 42 deficiencies were found. None of the deficiencies was critical but 17 were major. The 17 major deficiencies related to batch certification, Product Quality Review, change management system, deviation handling system, management responsibility, training, premises and equipment, documentation, line clearance, quality control, complaint handling, and cleaning validation. Re-inspection after implementation of CAPA is required in order to verify that the Pharmaceutical Quality System meets the requirements according to EU-GMP.

CARGILL FRANCE, France

Overall, 14 observations were made, including 1 critical deficiency and 4 major deficiencies: [Critical] The management of semi-finished batches and of the mixing operations was deficient and conformity of the final batches to specifications, notably Ph.Eur. specifications, could not be guaranted. [Major 1] The site had been manufacturing an active substance without ANSM authorisation. [Major 2] The change control related to the suppression of one filtration step in the active substance manufacturing process was deficient. [Major 3] The manufacturing of the active substance had not been made using master production instructions and no batch production records had been established. [Major 4] No review of batch production records of critical process steps had been done before release of the active substance for distribution. 7 observations are related to lack of traceability, risks of contamination induced by the absence of cleanliness in the production environment, very bad condition of the production equipment and insufficient equipment cleaning procedures. The inspection’s observations also apply to the manufacture of pharmaceutical excipients and starting materials that are intended to be used as ingredients in cosmetics and medical devices, which are manufactured under the same conditions as the active substance.

FARMA MEDITERRANIA, S.L., Spain

Critical deficiencies a) Lack of an effective pharmaceutical quality assurance system b) Release of batches of medicinal products produced without completing all of the manufacturing protocols, without being checked quality assurance unit and without the approval of the technical director. c) Use in quality control a non-qualified chromatographic equipment, with operating faults and with an unvalidated computerized management system. As a result, the integrity, reliability, up-to-dateness, originality and authenticity of the data that are obtained cannot be guaranteed. d) Transfer of some of the final analytical quality controls of medicinal products to a third party, without appropriately transferring the control methods and without the authorization of the relevant health authority e) Manufacture of medicinal products using procedures that have not been appropriately validated or have not been periodically revalidated. f) Acceptance of results of repeated analytical controls and sterility tests of finished medicinal products without having undertaken an in-depth investigation to determine the root cause of a previously result obtained which was out of specifications. g) Although a visual inspection of injectable medicinal products reveals a high number of critical quality defects (the presence of visible particles) non deviations are opened and is not investigated. c) Do not do any quality control on a statistical sample of units of injectable medicinal products that have passed the visual inspection. Major deficiencies a) Do not do the annual quality product review of medicinal products manufactured. b) Deviations in the manufacturing processes are not investigated suitably and in-depth. c) The simulation of the aseptic manufacturing process is not performed every six months and samples used in the simulation are not incubated at the right temperature. c) The air treatment system in manufacturing areas is not properly qualified, as it is only checked when it is “at rest” but not “in operation”. e) Medicinal products are manufactured without full compliance with conditions established in the marketing authorisation dossier and/or without carrying out all the established process controls. f) Manufacturing and quality control documents of each batch of medicinal products manufactured are not filed correctly. g) The facilities have been modified considerably without the authorization of the relevant health authority h) Test of growth promotion of culture media, which are used in the sterility testing, in the simulation of the aseptic manufacturing process or in the environmental control of critical manufacturing areas, is not carried out. h) Do not analyse all of the specification parameters for raw materials used in the manufacturing.

Chengdu Okay Pharmaceutical Co. Ltd., China

Overall, 21 deficiencies were observed during the inspection, including 5 critical and 10 major deficiencies. The critical deficiencies were observed in QC Dept. including calculation of impurities of Diosmin and there were no records of standard (used as a reference) for testing in-house standard. Also the data integrity was not guaranteed. In manufacturing Dept. presented measuring methods were inadequate to the results. The condition in clean area was not acceptable for final product. Critical deficiences: Testing of the final product: There was incorrectly way of calculation the impurities and Diosmin content. There were no records of prepared in-house HPLC standard. There was no confirmation of the conditions HPLC analysis. Computerized systems – documentation and control: There was found in HPLC system that the method was changed, without any savings of previous method. There were no logins and passwords to the HPLC system and no procedure for granting permission to access to the HPLC system. There was no register of persons authorized to access the HPLC system. On the same computer station there were two different HPLC software. Manufacturing documentation: Presented measuring methods of pH during the inspection time were inadequate to the results recorded in the batch report. Premises: Crude Diosmin drying was carried out in an area which did not provide the appriopriate coditions during the discharge from the dryer. Qualification of equipment: Some data of HVAC system qualification had been falsified. The major deficiencies were observed among others: in the warehouse, in the manufacturing documentation and in the production area.

Dongying Tiandong Pharmaceutical Co., Ltd., China

This serious Non-Compliance Report refers to a manufacturing site for Heparin. French Inspectors found 2 critical and 3 major deviations. Heparin manufacturing sites were involved in one of the largest counterfeit scandal ever. Therefore it is worrying that critical deviations in Heparin manufacturing have been found again. Read more in our GMP News Chinese Heparin Manufacturer again involved in Falsification and GMP Non-Compliance.

THERAVECTYS – VILLEJUIF, France

Here a manufacturing site for Investigational Medicinal Products (IMPs) is concerned. Overall 45 deficiencies, including 5 critical deficiencies and 17 major deficiencies have been detected. The following critical deviations in sterile production are listed in the agency report:

1) The implementation of exemption SOP for manufacturing operations which is not compliant to GMP principles, for example, Media Fill Test were performed with unqualified equipment.
2) The lack of sample area for incoming materials and their systematic use in quarantine status for manufacturing operations.
3) Appropriate measures in terms of monitoring locations, alert and action limits rationale, were not set for particle and microbiological monitoring in clean rooms grade A and B.
4) No protocol for clean rooms’ qualification was established and clean rooms classification didn’t fulfill ISO14644 requirements.
5) Some analytical methods and process were not validated for the clinical trial EudraCT : 2015-000845-21

All Non-Compliance Reports with the detailed address of the facilities and the product concerned can be found in the EudraGMDP Database.

///////////// 13 EMA, GMP Non-compliance Reports, 2016 published, EudraGMDP, central database

ECA releases Version 18 of GMP Guideline Manager

regulatory Comments Off

Mar 172016

How to access ten thousand pages of GMP Guidelines from FDA, EMA, ICH, PIC/S, ICH, WHO and many other organisations worldwide? You can print them or purchase hundreds of booklets. Or, alternatively, you can take advantage of a software tool developed by the ECA Academy, allowing you to access to the most comprehensive GMP Guideline Database

http://www.gmp-compliance.org/eca_mitt_05241_n.html

How to access ten thousand pages of GMP Guidelines from FDA, EMA, ICH, PIC/S, ICH, WHO and many other organisations worldwide? You can print them or purchase hundereds of booklets. But this will cause a huge amount of paper. And it will be more than difficult to find a specific regulatory requirement in this comprehensive library.

For that reason the ECA Academy has started to set up the largest GMP Guideline Database of its kind worldwide already 18 years ago. And every year a new release is published with all updates. A software was developed to structure the Guidelines in two so called “Guideline Trees”.

1. Guideline Tree structured according to the issuing authorities (e.g. EU, FDA, ICH)

2. Guideline Tree structured according to GMP topics (e.g. GMP for Medicinal Products, GMP for APIs, sterile production, validation etc)

In addition to the two structured libraries the software also allows you to search for certain key words. You may search the entire database for a keyword like “validation”. But you can also limit the search to certain areas (e.g. search in FDA Guidelines only).

If you have no CD drive on your computer you can also access the GMP Guideline Manager via the ECA WebApp. When you register you will receive the login details to access the ECA members area. The same login details will work for the ECA WebApp. With this service you can access the full GMP Guideline Database from your smartphone or tablet via the internet.

The GMP Guideline Manager can not be purchased – it is only available for ECA Members at no costs! This service is unique and not offered by any other organisation. By participating in any of the ECA conferences or courses you become member of the ECA free of charge for 2 years. If you can not attend an ECA course you can also apply for ECA membership for only 190,- Euro via our webpage.

Please find more information about the GMP Guideline Manager 18.0 here.

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