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TAMOXIFEN

10540-29-1 CAS

READ ABOUT TITLE AT……….http://www.rsc.org/chemistryworld/sites/default/files/CIIE_Tamoxifen.mp3

Molecular Formula: C₂₆H₂₉NO•C₆H₈O₇
CAS Number: 54965-24-1
Brands: Nolvadex, TAMOXIFEN CITRATE

Chemically, NOLVADEX (tamoxifen citrate) is the trans-isomer of a triphenylethylene derivative. The chemical name is (Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N, N-dimethylethanamine 2 hydroxy-1,2,3- propanetricarboxylate (1:1). The structural and empirical formulas are:

Tamoxifen citrate has a molecular weight of 563.62, the pKa’ is 8.85, the equilibrium solubility in water at 37°C is 0.5 mg/mL and in 0.02 N HCl at 37°C, it is 0.2 mg/mL.

NDA021807 APPR2005-10-29 DARA BIOSCIENCES,

US PATENT  6,127,425

Tamoxifen is an antagonist of the estrogen receptor in breast tissue via its active metabolite, hydroxytamoxifen. In other tissues such as the endometrium, it behaves as an agonist, and thus may be characterized as a mixed agonist/antagonist. Tamoxifen is the usual endocrine (anti-estrogen) therapy for hormone receptor-positive breast cancer in pre-menopausal women, and is also a standard in post-menopausal women although aromatase inhibitors are also frequently used in that setting.

Some breast cancer cells require estrogen to grow. Estrogen binds to and activates the estrogen receptor in these cells. Tamoxifen is metabolized into compounds that also bind to the estrogen receptor but do not activate it. Because of this competitive antagonism, tamoxifen acts like a key broken off in the lock that prevents any other key from being inserted, preventing estrogen from binding to its receptor. Hence breast cancer cell growth is blocked.

Tamoxifen was discovered by pharmaceutical company Imperial Chemical Industries (now AstraZeneca) and is sold under the trade names Nolvadex, Istubal, and Valodex. However, the drug, even before its patent expiration, was and still is widely referred to by its generic name “tamoxifen.

Breast cancer

Tamoxifen is currently used for the treatment of both early and advanced ER+ (estrogen receptor positive) breast cancer in pre- and post-menopausal women.Additionally, it is the most common hormone treatment for male breast cancer. It is also approved by the FDA for the prevention of breast cancer in women at high risk of developing the disease. It has been further approved for the reduction of contralateral (in the opposite breast) cancer.

In 2006, the large STAR clinical study concluded that raloxifene is equally effective in reducing the incidence of breast cancer, but after an average 4-year follow-up there were 36% fewer uterine cancers and 29% fewer blood clots in women taking raloxifene than in women taking tamoxifen, although the difference is not statistically significant.

Nolvadex (tamoxifen) 20 mg tablets

In 2005, the ATAC trial showed that after average 68 months following a 5 year adjuvant treatment, the group that received anastrozole (Arimidex) had significantly better results than the tamoxifen group in measures like disease free survival, but no overall mortality benefit. Data from the trial suggest that anastrozole should be the preferred medication for postmenopausal women with localized breast cancer that is estrogen receptor (ER) positive.Another study found that the risk of recurrence was reduced 40% (with some risk of bone fracture) and that ER negative patients also benefited from switching to anastrozole.

Crystallographic structure of 4-hydroxy-tamoxifen (carbon = white, oxygen = red, nitrogen = blue) complexed with ligand binding domain of estrogen receptor alpha (cyan ribbon)

Tamoxifen

Adequate patent protection is required to develop an innovation in a timely manner. In 1962, ICI Pharmaceuticals Division filed a broad patent in the United Kingdom (UK) (Application number GB19620034989 19620913). The application stated, “The alkene derivatives of the invention are useful for the modification of the endocrine status in man and animals and they may be useful for the control of hormone-dependent tumours or for the management of the sexual cycle and aberrations thereof. They also have useful hypocholesterolaemic activity”.

This was published in 1965 as UK Patent GB1013907, which described the innovation that different geometric isomers of substituted triphenylethylenes had either oestrogenic or anti-oestrogenic properties. Indeed, this observation was significant, because when scientists at Merrell subsequently described the biological activity of the separated isomers of their drug clomiphene, they inadvertently reversed the naming. This was subsequently rectified.

Although tamoxifen was approved for the treatment of advanced breast cancer in post-menopausal women in 1977 in the United States (the year before ICI Pharmaceuticals Division received the Queen’s Award for Technological Achievement in the UK), the patent situation was unclear. ICI Pharmaceuticals Division was repeatedly denied patent protection in the US until the 1980s because of the perceived primacy of the earlier Merrell patents and because no advance (that is, a safer, more specific drug) was recognized by the patent office in the United States. In other words, the clinical development advanced steadily for more than a decade in the United States without the assurance of exclusivity. This situation also illustrates how unlikely the usefulness of tamoxifen was considered to be by the medical advisors to the pharmaceutical industry in general. Remarkably, when tamoxifen was hailed as the adjuvant endocrine treatment of choice for breast cancer by the National Cancer Institute in 1984, the patent application, initially denied in 1984, was awarded through the court of appeals in 1985. This was granted with precedence to the patent dating back to 1965! So, at a time when world-wide patent protection was being lost, the patent protecting tamoxifen started a 17 year life in the United States. The unique and unusual legal situation did not go uncontested by generic companies, but AstraZeneca (as the ICI Pharmaceuticals Division is now called) rightly retained patent protection for their pioneering product, most notably, from the Smalkin Decision in Baltimore, 1996. (Zeneca, Ltd. vs. Novopharm, Ltd. Civil Action No S95-163 United States District Court, D. Maryland, Northern Division, March 14, 1996.)

 

Title: Tamoxifen

CAS Registry Number: 10540-29-1

CAS Name: (Z)-2-[4-(1,2-Diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine

Additional Names: 1-p-b-dimethylaminoethoxyphenyl-trans-1,2-diphenylbut-1-ene

Molecular Formula: C26H29NO

Molecular Weight: 371.51

Percent Composition: C 84.06%, H 7.87%, N 3.77%, O 4.31%

Literature References: Nonsteroidal estrogen antagonist.

Prepn: BE 637389 (1964 to ICI). Identification and separation of isomers: G. R. Bedford, D. N. Richardson, Nature 212, 733 (1966); BE 678807; M. J. K. Harper et al., US 4536516 (1966, 1985 both to ICI). Stereospecific synthesis: R. B. Miller, M. I. Al-Hassan, J. Org. Chem. 50, 2121 (1985). Review of chemistry and pharmacology: B. J. A. Furr, V. C. Jordan, Pharmacol. Ther. 25, 127-205 (1984). Reviews of clinical experience in treatment and prevention of breast cancer: I. A. Jaiyesimi et al., J. Clin. Oncol. 13, 513-529 (1995); C. K. Osborne, N. Engl. J. Med. 339, 1609-1618 (1998).

Properties: Crystals from petr ether, mp 96-98°.

Melting point: mp 96-98°

Derivative Type: Citrate

CAS Registry Number: 54965-24-1

Manufacturers’ Codes: ICI-46474

Trademarks: Kessar (Pharmacia); Nolvadex (AstraZeneca); Tamofène (Aventis); Zemide (Alpharma); Zitazonium (Servier)

Molecular Formula: C26H29NO.C6H8O7

Molecular Weight: 563.64

Percent Composition: C 68.19%, H 6.62%, N 2.49%, O 22.71%

Properties: Fine, white, odorless crystalline powder, mp 140-142°. Slightly sol in water; sol in ethanol, methanol, acetone. Hygroscopic at high relative humidities. Sensitive to uv light. LD50 in mice, rats (mg/kg): 200, 600 i.p.; 62.5, 62.5 i.v.; 3000-6000, 1200-2500 orally (Furr, Jordan).

Melting point: mp 140-142°

Toxicity data: LD50 in mice, rats (mg/kg): 200, 600 i.p.; 62.5, 62.5 i.v.; 3000-6000, 1200-2500 orally (Furr, Jordan)

Derivative Type: (E)-Form

CAS Registry Number: 13002-65-8

Properties: mp 72-74° from methanol.

Melting point: mp 72-74° from methanol

Derivative Type: (E)-Form citrate

Manufacturers’ Codes: ICI-47699

Properties: mp 126-128°.

Melting point: mp 126-128°

CAUTION: Tamoxifen is listed as a known human carcinogen: Report on Carcinogens, Eleventh Edition (PB2005-104914, 2004) p III-239.

Therap-Cat: Antineoplastic (hormonal).

Keywords: Antineoplastic (Hormonal); Antiestrogens; Selective Estrogen Receptor Modulator (SERM).

Synthesis of the E and Z isomers of the antiestrogen Tamoxifen. David W.Robertson and John A. Katzenellenbogen. Journal of Organic Chemistry 1982 , 47, Pages 2387-2393. An early synthesis of Tamoxifen : Production of non stereo specific products. 

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 For easy of understanding the complete synthesis has been broken down into a number of steps.Step 1.  Step 1. This step shows use of a simple friedel-craft acylation involving Anisole(A) and Phenylacetic acid (B). The acylating agent in this process was a mixture of PCl5 / SnCl4. The ketone C was formed in a 78% yield.

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Step 2.

 

 

 

Step 2. 

Alkylation was promoted by treating the ketone C with Sodium hydride (NaH). This removed the acidic protons (located on the position alpha to the carbonyl group) to produce the enolate ion. This could be isolated as the sodium enolate of the ketone treatment of this with ethyl iodide resulted in the formation of compound (D) in a 94% yield. The Ethyl iodide was chosen as the acylating agent probably as it contains the iodide ion , which is an excellent leaving group. It can therefore facilitate an SN2 substitution reaction with relative easy. 

 

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Step 3.

 

 

 

Step 3.  The phenol was deprotected using Lithium ethanthiolate in DMF ( Dimethyl This facilitated the removal of the methyl group and replaced it with a H to form a hydroxl group. Thus forming compound (E) in a 96% yield.

 

This is a key step as it has left a chink in the armour of the molecule. This can then be used to build up a characteristic part of the Tamoxifen molecule. (eg the (diemthylamino)ethyl group can be added easily from here) 

 

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Step 4.

 

 

 

 

Step 4. 

Then product E can be alkylated by treatment with 2-(dimethylamino) ethy chloride. The most facile site of alklation is the OH group on the phenyl ring. This can be interpreted roughly by using HSAB theory. e.g Hard and Soft acid/base theory. The carbon adjacent to the chloride ion of the reactant 2-(dimethylamino)ethyl chloride is made slightly harder due to the process of symbiosis. This can rationalise the formation between the hard oxygen atom to the normally soft carbon atom. In this case the carbon atom has become slightly harder due to the presence of the hard chorine atom. Hence the interaction is favourable by HSAB theory. The above reaction gives product F via a SN2 substitution reaction in 70% yield. 

 

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Step 5.

 

 

Step5. 

F on treatment with PhMgBr forms the tertiary alcohol (G). 

Formation of the Grignard reagent can be achieved via reaction of PhBr + Mg —–> PhMgBr. The Grignard reagent has effectively formed a carbanion species eg C delta negative (-ve). This is due to the presence of the C-Mg bond. the fact that Magnesium is a more electropositive element thus making the Carbon atom the more electronegative element and hence acquiring a negative charge. As a result of the negative nature of the carbon atom it can now attack the delta positive (+ve) Carbon atom of the carbonyl group. 

 

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step 6.

 

 

 

 

Step 6.The dehydration of F was initiated by treatment of methanoic hydrogen chloride. this gives the required structure of Tamoxifen. However it gives a racemic mixture of both cis and trans isomers. 

The ratio of the Cis / Trans isomers was (1.3 / 1). These isomers of Tamoxifen can be separated by Silica gel thin layer chromatography with benzene / triethylamine (9:1) as the developing solvent. Analysis of this technique revealed that the Z (Trans) isomer was more mobile than the E (Cis) isomer.

Synthetic Route 2: A Stereospecific Approach.

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Stereospecific Synthesis of (Z) – Tamoxifen via carbometalation of Alkynylsilanes.

Studied for historical reasons rather than synthetic brilliance. This synthesis was the first stereo specific synthesis of (Z) Trans Tamoxifen. Comparison between this synthesis and the previous route I believe can illustrate the development of synthetic approaches to large molecules. In particular the quest for stereo specific reactions. So starting from an alkynylsilane (A) and through a series of reactions we can generate only the (Z) – Trans isomer of Tamoxifen.

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Again for ease of understanding the complete synthesis has been broken down into a number of steps.

Step1.

 

Step1.

 

 

This step contains the vital stereo specific step. Namely the carbometalation of the alkynylsilane.It is this step which establishes the stereochemistry about the double bond. The phenyl (trimethyl silyl) – acetylene was carbometalated with diethylaluminium chloride – titanocene dichloride reactant to produce an organometallic intermediate. This organometallic intermediate was then cleaved with N bromosucciniamide to produce the alkene (B) in 85% yield.

The stereochemistry was assigned as E (Cis) mechanistic evidence suggests that this is linked to some steric reasons.

(Earlier work dedicated to this reaction see : Miller, R.B. Al-Hassan.M.I J.Org.Chem. 1984, 49, 725)

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Step2.

Step 2.

The second step shows the stereo specific replacement of the Br group by a phenyl group. This was achieved by use of Palladium – catalysed coupling of compound (B) with phenyl zinc chloride to form (C) the vinylsilane in a 95% yield.

Step3.

 

Step3.

This step during the synthesis was reported to be tricky and several approaches were attempted before a successful technique was discovered.

 

The objective of this step was to replace the trimethyl Silyl group by a suitable halogen atom (e.g. Bromine or Iodine)

However a facile reaction was reported when (C) was treated with bromine – sodium methoxide at -78�C to produce the vinyl bromide (D) in a yield of 85%

 

Step 4.

 

Step 4.

The vinyl bromide (D) coupled well with a Zinc organometallic species to produce (E) the ethyl triaryl olefin in a yield of 84%.

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Step 5.

 

Step 5.

The formation of (F) Tamoxifen was achieved by demethylation with sodium ethylthoilate in refluxing dimethyl formamide. then reaction of the phenoxide ion with 2-( dimethylamino)ethyl chloride via a SN2 substitution.

Purification of the crude product was achieved via it’s hydrochloride salt ( via a reaction with HCl (g)) then F was regenerated by treatment with dilute base this produced the stereospecific (Z)- Trans isomer in an overall yield of 60%.

a synthesis

Palladium-Catalyzed Fluoride-Free Cross-Coupling of Intramolecularly ActivatedAlkenylsilanes and Alkenylgermanes: Synthesis of Tamoxifen as a Synthetic Application (pages 642–650)Kenji Matsumoto and Mitsuru ShindoArticle first published online: 23 FEB 2012 | DOI: 10.1002/adsc.201100627

• Abstract
• Full Article (HTML)
• PDF(262K)

 

 

http://pubs.rsc.org/en/content/articlelanding/2011/cs/c0cs00129e#!divAbstract

 

 

 

 

EP 0883587 A1  WO1997026234A1)

 

Preparation of Z isomer of Tamoxifen

A solution of bromobenzene (3.92g, 25mmol) in ether (5ml) containing a crystal of iodine was added dropwise to a suspension of magnesium turnings (0.63g, 26mmol) in ether (5ml) at reflux, under nitrogen. After the addition was complete, the reaction mixture was cooled to room temperature and a solution of l- [ 4- ( 2- chloroethoxy)phenyl]-2-phenyl-l-butanone (3.75g, 12.4mmol) in ether (15ml) was added over 1 hour. The resulting mixture was refluxed for 16 hours, then poured into dilute hydrochloric acid (50ml) and extracted with ether (3x40ml) . The combined ether layers were concentrated, the residual oil was dissolved in ethanol (10ml) and refluxed with concentrated hydrochloric acid (5ml) for 4 hours. The organic phase was separated, dried (Na₂S0₄) and evaporated to dryness to give a yellow oil. Η NMR (see Figures 1 to 4 and discussion below) showed this to be a 2:1 mixture of the Z and E isomers. The oil was then dissolved in warm methanol (about 40°C) and allowed to cool to room temperature. The colourless crystals formed proved to be pure Z isomer of 2-chloroethoxy tamoxifen (4.12g, 11.4mmol, 92% yield) . M.p. 107-109°C, m/z 362/364 (chlorine atom present), <S_H 0.92 (3H, t, J = 7.33 Hz, CH₃) , 2.46 (2H, q, J = 7.33 Hz, CH₂CH₃) , 3.72 (2H, t, J = 5.86 Hz, 0CH₂CH₂C1) , 4.09 (2H, t, J = 5.86 Hz, 0CH₂CH₂C1) , 6.55 (2H, d, J = 8.79 Hz, aromatic protons ortho to 0CH₂CH₂C1) , 6.79 (2H, d, J = 8.79 Hz, aromatic protons meta to 0CH₂CH₂C1) , 7.10-7.38 (10H, m, the two remaining C₆H₅ ^,s) (see Figure 5) . The 2-chloroethoxy tamoxifen was reacted with dimethylamine in ethanol, under reflux, to produce the desired Z isomer of tamoxifen.

Analysis of Η NMR data

Figures 1 to 4 represent a mixture of the E- and Z- forms of compound XI described above.

The expansion of the region ό^* 0.80 to 1.05 shows two overlapping triplets corresponding to the CH₃ groups in the

Z- and E- derivatives respectively. The critical point is the ratio of the heights of the peaks at 0.92 (for the Z) and 0.94 (for the E) , which is approximately 2:1. The expansion of the 4.00 to 4.35 region reveals similar information where ratios are 10:6.4 and 5.56:3.43.

Similarly expansion of the region 3.6 to 3.9 shows the ratio to be 2.46:1. All of these measurements suggest an approximate 2:1 ratio.

Referring to Figure 5, this shows almost pure Z- isomer. It should be noted that there is 660 mg of this from an original mixture of a 2:1 ratio mixture of 780 mg which would contain only 520 mg of the Z-isomer.

 

 

 

Z isomer of tamoxifen and 4-hydroxytamoxi en include stereoselective syntheses (involving expensive catalysts) as described in J. Chem. Soc, Perkin Trans I 1987, 1101 and J. Org. Chem. 1990, 55, 6184 or chromatographic separation of an E/Z mixture of isomers as described in J. Chem. Res., 1985 (S) 116, (M) 1342, 1986 (S) 58, (M) 771.

(Z)-tamoxifen (1) as a white solid, mp: 95.8-96.3 ºC. ¹H-NMR (500 MHz, CDCl₃) d 0.92 (3H, t, J 7.3 Hz), 2.29 (6H, s), 2.45 (2H, q, J 7.3 Hz), 2.65 (2H, t, J 5.8 Hz), 3.93 (2H, t, J 5.8 Hz), 6.68 (2H, d, J 9.5 Hz), 6.78 (2H, d, J 9.5 Hz), 7.08-7.28 (10H, m).¹³C-NMR (125 MHz, CDCl₃) d 13.6 (CH₃), 29.0 (CH₂), 45.8 (CH₃), 58.2 (CH₂), 65.5 (CH₂), 113.4 (C), 126.0 (C), 126.5 (CH), 127.8 (CH), 128.1 (C), 129.7 (C), 131.8 (CH), 135.6 (CH), 138.2 (CH), 141.3 (CH), 142.4 (CH), 143.8 (C), 156.7 (C). IR (KBr film) n_max/cm^-1: 3055, 2979, 2925, 2813, 2769, 1606, 1509, 1240, 1035, 707. GC-MS (EI) m/z 371(5%), 58(100%).

 

(Z)-tamoxifen (1) and (E)-tamoxifen (2) in 52% yield. ¹H-NMR (300 MHz, CDCl₃) d 0.91 (Z isomer. 3H, t, J 7.3 Hz), 0.94 (E isomer. 3H, t, J 7.3 Hz), 2.28 (Z isomer. 6H, s), 2.34 (E isomer. 6H, s), 2.42-2.52 (Z and Eisomers. 4H, m), 2.63 (Z isomer. 2H, t, J 5.9 Hz), 2.74 (E isomer. 2H, t, J 5.9 Hz), 3.94 (Z isomer. 2H, t, J 5.9 Hz), 4.07 (E isomer. 2H, t, J 5.9 Hz), 6.68 (Z isomer. 2H, d, J 9.7 Hz), 6.76 (E isomer. 2H, d, J 9.3 Hz), 6.86-7.36 (Z and E isomers. 10H, m). IR (KBr film) n_max/cm^-1: 3081, 3056, 2974, 2826, 2770, 1611, 1509, 1238, 1044. GC-MS (EI) m/z: Z isomer, 371(4%), 72 (24%), 58(100%); E isomer, 371(3%), 72 (24%), 58(100%). (the diastereoisomeric ratio was determined by capillary GC analysis and the configuration of the major diastereoisomer established by comparison of the NMR data of the synthetic mixture with an authentic sample of (Z)-tamoxifen (1).

 

 

nmr

 

 

ir

FTIR

shows the typical spectra’s of pure tamoxifen citrate, PCL, a physical mixture of tamoxifen citrate and PCL and drug-loaded implants. The spectrum of tamoxifen citrate shows characteristic absorption bands at 3027 cm⁻¹ (=C-H stretching), 1507 and 1477 (C=C ring stretching) and 3180 cm ^-1 (-NH2). PCL displays a characteristic absorption band at strong bands such as the carbonyl stretching mode around 1727 cm⁻¹ (C=O), asymmetric stretching 2949 cm⁻¹ (CH ₂ ) symmetric stretching 2865 cm⁻¹ (CH ₂ ). No changes in the spectrum of the physical mixture and drug-loaded microspheres were evident by FTIR spectroscopy. The strong bands such as the carbonyl peak were clear at all points.

Figure 2: Transmission FTIR spectra of (a) tamoxifen-loaded implant, (b) physical mixture of drug+PCL, (c) pure PCL, (d) pure tamoxifen citrate

enlarged view

 

FTIR spectra of A) tamoxifen citrate; B) PLGA; C) mixture of drug and excipients; D) freshly prepared nanoparticles in the formulation (BS-3HS).

Mentions: The pure drug tamoxifen citrate, PLGA-85:15, PVA, a mixture of PLGA and PVA, and a mixture of tamoxifen citrate, PLGA, and PVA; and a freshly prepared formulation were mixed separately with IR grade KBr in the ratio of 1:100 and corresponding pellets were prepared by applying 5.5 metric ton pressure with a hydraulic press. The pellets were scanned in an inert atmosphere over a wave number range of 4000–400 cm−1 in Magna IR 750 series II, FTIR instrument (Nicolet, Madison, WI, USA).

 

dsc

 

DSC thermograms of pure tamoxifen (a), pure PCL (b), physical mixture of drug+PCL (c) and (d) drug-loaded implant. The experiment was carried with crimped aluminum pans and a heating rate of 10ºC/min

 

 

xrd

X-ray diffraction studies of pure drug (a), pure PCL (b), physical mixture of drug+PCL (c) and (d) drug-loaded implant

 

synthesis

J.Chem. Research,1985(S) 116, (M) 1342 and 1986 (S) 58, (M) 0771.

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http://cen.acs.org/articles/91/i45/Improving-Drug-Delivery.html

Nanoparticles are promising cargo ships for targeted drug delivery. But the materials have had limited success treating cancer, because they often can’t penetrate deep into tumors. The nanoparticles are stalled by the extracelluar matrix and compressed blood vessels.

http://cen.acs.org/articles/91/i45/Improving-Drug-Delivery.html

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The plant hormone methyl jasmonate can be used to increase broccoli’s antitumoral properties

Read more

http://www.chemistryviews.org/details/news/5428251/Boosting_Broccoli_Power.html

back to home for more updates

DR ANTHONY MELVIN CRASTO Ph.D

amcrasto@gmail.com

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Pimecrolimus

137071-32-0 ^cas 

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)- 3-{(E)-2-[(1R,3R,4S)-4-Chloro-3-methoxycyclohexyl]- 1-methylvinyl}-8-ethyl-5,6,8,11,12,13,14,15,16,17,

18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy- 14,16-dimethoxy-4,10,12, 18-tetramethyl-15,19-epoxy- 3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1, 7,20,21(4H,23H)-tetrone

The systematic name of pimecrolimus is (lR,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(lE)-2- {(1 R,3R,4S)-4-chloro-3-methoxycyclohexyl} - 1 -methylvinyl] – 17-ethyl- 1,14- dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-ll,28-dioxa-4-aza- tricyclo[22.3.1.0⁴'⁹]octacos-18-ene-2,3,10,16-tetraone.

Pimecrolimus is the 32 epichloro derivative of ascomycin.

Pimecrolimus is an immunomodulating agent used in the treatment of atopic dermatitis (eczema). It is currently available as a topical cream, once marketed by Novartis, (however Galderma will be promoting the molecule in Canada in early 2007) under the trade name Elidel.

NMR…http://file.selleckchem.com/downloads/nmr/S500401-Pimecrolimus-NMR-Selleck.pdf

HPLC…….http://file.selleckchem.com/downloads/hplc/S500401-Pimecrolimus-HPLC-Selleck.pdf

http://file.selleckchem.com/downloads/hplc/S500401-Pimecrolimus-HPLC-Selleck.pdf

Pimecrolimus is an immunomodulating agent used in the treatment of atopic dermatitis (eczema). It is available as a topical cream, once marketed by Novartis (however, Galderma has been promoting the compound in Canada since early 2007) under the trade name Elidel.

Pimecrolimus is an ascomycin macrolactam derivative. It has been shown in vitro that pimecrolimus binds to macrophilin-12(also referred to as FKBP-12) and inhibits calcineurin. Thus pimecrolimus inhibits T-cell activation by inhibiting the synthesis and release of cytokines from T-cells. Pimecrolimus also prevents the release of inflammatory cytokines and mediators from mast cells.

Pimecrolimus is a chemical that is used to treat atopic dermatitis (eczema). Atopic dermatitis is a skin condition characterized by redness, itching, scaling and inflammation of the skin. The cause of atopic dermatitis is not known; however, scientists believe that it may be due to activation of the immune system by various environmental or emotional triggers. Scientists do not know exactly how pimecrolimus reduces the manifestations of atopic dermatitis, but pimecrolimus reduces the action of T-cells and mast cells which are part of the immune system and contribute to responses of the immune system. Pimecrolimus prevents the activation of T-cells by blocking the effects of chemicals (cytokines) released by the body that stimulate T-cells. Pimecrolimus also reduces the ability of mast cells to release chemicals that promote inflammation.

Pimecrolimus, like tacrolimus, belongs to the ascomycin class of macrolactam immunosuppressives, acting by the inhibition of T-cell activation by the calcineurin pathway and inhibition of the release of numerous inflammatory cytokines, thereby preventing the cascade of immune and inflammatory signals.^[1] Pimecrolimus has a similar mode of action to that of tacrolimus but is more selective, with no effect on dendritic (Langerhans) cells.^[2] It has lower permeation through the skin than topical steroids or topical tacrolimus^[3] although they have not been compared with each other for their permeation ability through mucosa. In addition, in contrast with topical steroids, pimecrolimus does not produce skin atrophy.^[4] It has been proven to be effective in various inflammatory skin diseases, e.g., seborrheic dermatitis,^[5] cutaneous lupus erythematosus,^[6]oral lichen planus,^[7] vitiligo,^[8] and psoriasis.^[9][10] Tacrolimus and pimecrolimus are both calcineurin inhibitors and function as immunosuppressants.^[11]

Ascomycin macrolactams belong to a new group of immunosuppressive, immunomodulatory and anti-inflammatory agents and include, e.g., ascomycin (FK520), tacrolimus (FK506) and pimecrolimus (ASM 981). The main biological effect of ascomycin macrolactams appears to be the inhibition of the synthesis of both Th1 and Th2-type cytokines in target cells.

As used herein, the term “ascomycin macrolactam” means ascomycin, a derivative of ascomycin, such as, e.g., tacrolimus and pimecrolimus, or a prodrug or metabolite of ascomycin or a derivative thereof.

Ascomycin, also called immunomycin, is a structurally complex macrolide produced by Streptomyces hygroscopicus. Ascomycin acts by binding to immunophilins, especially macrophilin-12. It appears that ascomycin inhibits the production of Th1 (interferon- and IL-2) and Th2 (IL-4 and IL-10) cytokines. Additionally, ascomycin preferentially inhibits the activation of mast cells, an important cellular component of the atopic response. Ascomycin produces a more selective immunomodulatory effect in that it inhibits the elicitation phase of allergic contact dermatitis but does not impair the primary immune response when administered systemically. The chemical structure of ascomycin is depicted below.

Tacrolimus (FK506) is a synthetic derivatives of ascomycin. As a calcineurin inhibitor, it works through the FK-binding protein and inhibits the dephosphorylation of nuclear factor of activated T cells (NFAT), thereby preventing the transport of the cytoplasmic component of NFAT to the cell nucleus. This leads to transcriptional inhibition of proinflammatory cytokine genes such as, e.g., interleukin 2, which are dependent on the nuclear factor of activated NFAT. The chemical structure of tacrolimus is depicted below.

Pimecrolimus, an ascomycin derivative, is a calcineurin inhibitor that binds with high affinity to the cytosolic receptor macrophilin-12, inhibiting the calcium-dependent phosphatase calcineurin, an enzyme required for the dephosphorylation of the cytosolic form of the nuclear factor of the activated T cell (NF-AT). It thus targets T cell activation and proliferation by blocking the release of both TH1 and TH2 cytokines such as IF-g, IL-2, -4, -5, and -10.3 It also prevents the production of TNF-a and the release of proinflammatory mediators such as histamine, hexosaminidase, and tryptase from activated mast cells.3 It does not have general antiproliferative activity on keratinocytes, endothelial cells, and fibroblasts, and in contrast to corticosteroids, it does not affect the differentiation, maturation, functions, and viability of human dendritic cells. The chemical structure of pimecrolimus is depicted below.

Pimecrolimus is an anti-inflammatory compound derived from the macrolactam natural product ascomycin, produced by certain strains of Streptomyces.

In January 2006, the United States Food and Drug Administration (FDA) announced that Elidel packaging would be required to carry a black box warning regarding the potential increased risk of lymph node or skin cancer, as for the similar drug tacrolimus. Whereas current practice by UKdermatologists is not to consider this a significant real concern and they are increasingly recommending the use of such new drugs.^[12]

Importantly, although the FDA has approved updated black-box warning for tacrolimus and pimecrolimus, the recent report of the American Academy of Dermatology Association Task Force finds that there is no causal proof that topical immunomodulators cause lymphoma or nonmelanoma skin cancer, and systemic immunosuppression after short-term or intermittent long-term topical application seems an unlikely mechanism.^[13] Another recent review of evidence concluded that postmarketing surveillance shows no evidence for this systemic immunosuppression or increased risk for any malignancy.^[14] However, there are still some strong debates and controversies regarding the exact indications of immunomodulators and their duration of use in the absence of active controlled trials.^[15] Dermatologists’ and Allergists’ professional societies, the American Academy of Dermatology[1], and the American Academy of Allergy, Asthma, and Immunology, have protested the inclusion of the black box warning. The AAAAI states “None of the information provided for the cases of lymphoma associated with the use of topical pimecrolimus or tacrolimus in AD indicate or suggest a causal relationship.”[2].

ELIDEL® (pimecrolimus) Cream 1% contains the compound pimecrolimus, the immunosuppressant 33-epi-chloro-derivative of the macrolactam ascomycin.

Chemically, pimecrolimus is (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(1E)-2{(1R,3R,4S)-4-chloro-3-methoxycyclohexyl}-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25 dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0 4,9]octacos-18-ene2,3,10,16-tetraone.

The compound has the empirical formula C₄₃H₆₈CINO₁₁ and the molecular weight of 810.47. The structural formula is

Pimecrolimus is a white to off-white fine crystalline powder. It is soluble in methanol and ethanol and insoluble in water.

Each gram of ELIDEL Cream 1% contains 10 mg of pimecrolimus in a whitish cream base of benzyl alcohol, cetyl alcohol, citric acid, mono- and di-glycerides, oleyl alcohol, propylene glycol, sodium cetostearyl sulphate, sodium hydroxide, stearyl alcohol, triglycerides, and water.

The second representative of the immunosuppressive macrolides for topical application – after tacrolimus (Protopic ®) – has 21 October in the trade. Pimecrolimus is approved for short-term and intermittent long-term treatment for patients aged two years who suffer from mild to moderate atopic dermatitis.

Pimecrolimus is a lipophilic derivative of macrolactam Ascomycin. The macrolides inhibit the production and release of pro-inflammatory cytokines by blocking the phosphatase calcineurin.The anti-inflammatory effect unfolds the drug in the skin. Since he is only minimally absorbed to not measurable, it hardly affects the local or systemic immune response. Therefore, the authorization neither restricts nor a maximum daily dose treatable area or duration of therapy.The cream can also be applied on the face, head and neck, and in skin folds, but not simultaneously with other anti-inflammatory topical agents such as glucocorticoids.

In studies in phases II and III patients aged three months and treated a maximum of one year.In two six-week trials involving 186 infants and young children as well as 403 children and adolescents, the verum symptoms and itching decreased significantly better than the cream base. Already in the first week of itching in 44 percent of children and 70 percent of the infants improved significantly. In adults, pimecrolimus was less effective than 0.1 percent betamethasone 17-valerate.

In the long-term treatment the verum significantly reduced the incidence of flares, revealed two studies with 713 and 251 patients. About a half and one year each about twice as many of the small patients were free of acute disease exacerbations than with the cream base (example: 61 versus 34 per cent of children, 70 versus 33 percent of infants older than six months). Moreover, the use of topical corticosteroids decreased significantly.

In a study of 192 adults with moderate to severe eczema half suffered six months no relapses more (24 percent with placebo). In the long-term therapy pimecrolimus was less effective than 0.1 percent triamcinolone acetonide cream and 1 percent hydrocortisone cream in adults.

The new topicum is-apart from burning and irritation at the application site – relatively well tolerated. It is neither kontaktsensibilisierend still phototoxic or sensitizing and does not cause skin atrophy. As in atopic Ekzen but usually a long-term therapy is necessary studies can reveal long-term adverse effects of the immunosuppressant on the skin only beyond one year.Also available from direct comparative studies between tacrolimus and pimecrolimus. They could help to delineate the importance of the two immunosuppressants.

Pimecrolimus (registry number 137071-32-0; Figure 1) is a macro lide having anti-inflammatory, antiproliferative and immunosuppressive properties. This substance is present as an active ingredient in the Elidel ® drug recently approved in Europe and in the USA for topical treatment of inflammatory conditions of the skin such as atopic dermatitis.

Figure 1: structural formula of pimecrolimus

19^th Ed., vol. π, pg. 1627, spray-drying consists of bringing together a highly dispersed liquid and a sufficient volume of hot air to produce evaporation and drying of the liquid droplets. Spray-drying however is often limited to aqueous solutions unless special expensive safety measures are taken. Also, in spite of the short contact time, certain undesirable physical and chemical characteristics of the emerging solids are in particular cases unavoidable. The turbulence present in a spray-drier as a result of the moving air may alter the product in an undesirable manner. Modifications to the spray-drying technique are disclosed in WO 03/063821 and WO 03/063822. [00012] European Patent EP 427 680 Bl discloses a method of synthesizing amorphous pimecrolimus (Example 66a). The method yields amorphous pimecrolimus as a colorless foamy resin.

U.S. Patent No. US 6,423,722 discloses crystalline forms of pimecrolimus, such as form A, form B, etc. US 722 also contend that by performing example 66a from the European Patent EP 427 680 Bl, amorphous pimecrolimus is obtained.

The preparation of pimecrolimus was described for the first time in the patent application EP427680 on behalf of Sandoz. Used as raw material in such document is ascomycin (compound identified by registry number 11011-38-4), a natural product obtained through fermentation from Streptomyces strains (such as for example Streptomyces hygroscopicus var ascomyceticus, or Streptomyces hygroscopicus tsukubaensis N°9993). Pimecrolimus is obtained from the ascomycin through a sequence of four steps of synthesis (scheme 1)

Scheme 1 : synthesis process described in EP427680

From a structural point of view, pimecrolimus is the 33-epi-chloro derivative of ascomycin. As described in EP427680, the simultaneous presence – in the structure of ascomycin – of two secondary hydroxyl groups in position 24 and in position 33, requires the protection of the hydroxyl in position 24 before substituting the second hydroxyl in position 33 with an atom of chlorine.

In order to obtain the monoprotection of the hydroxyl in position 24 of ascomycin, such synthesis process provides for the preparation of 24,33-disilyl derivative and the subsequent selective removal of the silyl ester in position 33.

The high ratio between the silylating agent and the substrate and the non-complete selectivity of the subsequent step of deprotection requires carrying out two chromatographic purifications on the column of silica gel (Baumann K., Bacher M., Damont A., Hogenauer K., Steck A. Tetrahedron, (2003), 59, 1075-1087). The general yields of such synthesis process are not indicated in literature; an experiment by the applicant revealed that such yields amount to about 16% molar starting from ascomycin.

Other synthesis processes were recently proposed as alternatives to the synthesis of EP427680.

In particular, the International patent application WO2006040111 on behalf of Novartis provides for the direct substitution of the hydroxyl in position 33 of ascomycin with an atom of chlorine and a second alternative, described in the international patent application WO2006060614 on behalf of Teva, uses – as a synthetic intermediate – a sulfonate derivative in position 33 of ascomycin. Both the proposed synthetic alternatives are not entirely satisfactory in that in WO2006040111 the proposed halogenating agents (chlorophosphorane and N- chlorosuccinimide) are not capable, according to the same authors, of regioselectively substituting the hydroxyl function in position 33, while in WO2006060614 the quality characteristics of the obtained product are, even after chromatographic purification and/or crystallisation, low for a product to be used for pharmaceutical purposes (i.e. purity of 96% as described in the experimental part).

Generally, purified enzymatic systems may be used for the organic synthesis of polyfunctional molecules (Wang Y-F, Wong C-H. J Org Chem (1988) 53, 3127- 3129; Santaniello E., Ferraboschi P., Grisenti P., Manzocchi A. Chem. Rev. (1992), 92(5), 1071-140; Ferraboschi P., Casati S., De Grandi S., Grisenti P., Santaniello E. Biocatalysis (1994), 10(1-4), 279-88); WO2006024582). WO2007103348 and WO2005105811 describe the acylation of rapamycin in position 42 in the presence of lipase from Candida antartica.

…………………….

EP2432791A1

Scheme 2: synthesis of pimecrolimus for enzymatic transesterification of ascomycin.

Scheme 3. Synthesis of pimecrolimus for enzyme-catalyzed alcoholysis from 33,24- diacetate of ascomycin

Example 1

Preparation of the 33-acetyl derivative of ascomvcin (compound I of scheme II)

Lipase from Candida antarctica (CAL B, Novozym 435) [0.140 g (2 U/mg)

FLUKA] was added to a solution of ascomycin (100 mg; 0.126 mmol) in toluene (8 ml) and vinyl acetate (4.5 eq; 0.473 g). The reaction is kept under stirring at the temperature of 30° C for 80 hrs then the enzyme is taken away for filtration and the filtrate is concentrated at low pressure to obtain 105 mg of 33-acetyl ascomycin.

A sample of such intermediate was purified for analytical purposes by chromatography on silica gel (n-hexane/acetone = 8/2 v/v as eluents) and thus crystallised by acetone/water.

The following analysis were carried out on such sample: ¹H-NMR (500MHz) δ:

2.10 (CH₃CO), 3.92 and 4.70 (24CH and 33CH); IR (cm-¹): 3484.245, 2935.287,

1735.331, 1649.741, 1450.039,

1372.278; DSC: endotherm at 134.25° C; [α]_D=-74,0° (c=0.5 CHCl₃).

Spectrum of MS (ESI +): m/z: 856.4 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇iNO₁₃: C 64.80%; H, 8.58%; N, 1.68%;

O, 24.94%

Elementary analysis found: C 64.78%; H, 8.54%; N, 1.59%; O, 24.89%

Preparation of the 24-tgrt-butyldimethylsilylether-33 -acetyl derivative of ascomvcin (intermediate 24-silyl-33-Oac; compound II of scheme 2)

2,6-lutidine (0.29Og; 2.7 mmolels) and tert-butyldimethylsilyl triflate (0.238g; 0.9 mmoles) are added to a solution of 33-acetyl derivative of ascomycin (150 mg;

0.18 mmoles) in dichloromethane (5ml). The reaction is left under stirring at ambient temperature for 30 minutes. After this period the reaction mixture is washed with a solution saturated with sodium bicarbonate (5 ml) and organic phase obtained is washed in sequence with HCl 0.1N (5 ml 3 times) and with a solution at 30% of NaCl (5ml). The organic phase is anhydrified on sodium sulphate, filtered and concentrated to residue under vacuum to obtain 128 mg of product.

Spectrum of MS (ESI +): m/z: 970.5 (M+23; 100.0%)

¹H-NMR (500 MHz) δ: 0.05 and 0.06 ((CHs)₂Si), 0.90 ((CH₃)₃C-Si), 2.10

(CH₃CO), 4.70 (33CH)

IR (cm-’): 3462.948, 2934.450, 1739.236, 1649.937

Elementary analysis calculated for C₅₁H₈₅NOi₃Si: C 64.59%; H, 9.03%; N, 1.48%; O, 21.93%

Elementary analysis found: C 64.50%; H, 9.05%; N, 1.41%; O, 21.88%

DSC= endoderma a 236,43° C. [α]_D=-81,4° (c=0.5 CHCl₃).

Preparation of 24-tert-butyldimethylsilylether of ascomycin (intermediate 24- silyl-33-OH; compound III of scheme 2) n-octan-1-ol (0.035g; 0.265 mmoles) and CAL B (Novozym 435) [0.100 g (2

U/mg) FLUKA] are added to a solution of 24-tert-butyldimethylsilylether-33- acetyl derivative of ascomycin (50 mg; 0.053 mmoles) in tert-butylmethylether (4 ml). The reaction is kept under stirring at the temperature of 40° C for 120 hours.

After this period the reaction mixture is filtered and the filtrate is evaporated to residue under vacuum to obtain a reaction raw product which is purified by chromatography on silica gel: 44 mg of product (0.048 mmoles) are recovered through elution with petroleum ether/acetone 7/3.

The chemical/physical properties of the obtained product match those of a reference sample obtained according to patent EP427680.

Preparation of 24-tert-butyldimethylsilylether-33-epi-chloro ascomycin

(intermediate 24-silyl-33-chloro; compound IV of scheme 2)

A solution of 24-silyl FR520, i.e. 24-silyl ascomycin (165 g; 0.18 moles) in anhydrous toluene (1.4 litres) and pyridine (50 ml) is added to a suspension of dichlorotriphenylphosphorane (99.95g) in anhydrous toluene (1.1 litres), under stirring at ambient temperature (20-25 °C) in inert atmosphere.

After adding, the reaction mixture is heated at the temperature of 60° C for 1 hour.

After this period the temperature of the reaction mixture is taken to 25° C and thus the organic phase is washed in sequence with water (1 time with 1 L) and with an aqueous solution of NaCl at 10% (4 times with 1 L each time), then it is anhydrified on sodium sulphate, filtered and concentrated under vacuum to obtain about 250 g of a moist solid of toluene. Such residue product is retaken with n- hexane (500 ml) and then evaporated to dryness (in order to remove the toluene present). The residue product is diluted in n-hexane (500 ml) under stirring at ambient temperature for about 45 minutes and then the undissolved solid taken away for filtration on buckner (it is the sub-product of dichlorophosphorane).

The filtrate is concentrated at low pressure to obtain 148.6 g of a solid which is subsequently purified by chromatography on silica gel (elution with n- heptane/acetone = 9/1) to obtain 123 g (0.13 moles) of product.

The chemical/physical properties of the obtained product match those described in literature (EP427680).

Preparation of the pimecrolimus from 24-fert-butyldimethylsilylether-33-epi- chloro ascomycin

The intermediate 24-silyl-33 chloro (123g; 0.13 Moles; compound IV of scheme

2) is dissolved under stirring at ambient temperature in a dichloromethane/methanol mixture=l/l=v/v (1.1 litres) then p-toluenesulfonic acid monohydrate (10.11 g) is added.

The reaction is kept under stirring at the temperature of 20-25° C for 72 hours, thus a solution of water (600 ml) and sodium bicarbonate (4.46 g) is added to the reaction mixture. The reaction mixture is kept under stirring at ambient temperature for 10 minutes, the organic phase is then prepared and washed with an aqueous solution at 10% of sodium chloride (600 ml).

The organic phase is anhydrified on sodium sulphate, filtered and concentrated under vacuum to obtain 119 g of raw pimecrolimus. Such raw product is purified by chromatography on silica gel (n-hexane/acetone as eluents) and thus crystallised by ethyl acetate, cyclohexane/water to obtain 66 g (81.5 mmoles) of purified pimecrolimus.

The chemical/physical data obtained matches the data indicated in literature.

Example 2

Preparation of ascomvcin 24.33-diacetate (intermediate 24, 33-diacetate; compound V of scheme 3)

DMAP (4.5 eq; 0.136 g) and acetic anhydride (4.5 eq; 0.114 g) are added to a solution of ascomycin (200 mg; 0.25 mmoles) in pyridine (2.5 ml), under stirring at the temperature of 0° C.

The reaction is kept under stirring for 1.5 hours at the temperature of 0° C then it is diluted with water and it is extracted with ethyl acetate (3 times with 5 ml). The organic extracts are washed with HCl 0.5 N (5 times with 10 ml), anhydrified on

Na₂SO₄ concentrated under vacuum.

The residue product was purified by chromatography on silica gel (n- hexane/acetone 8/2 v/v as eluent) to obtain ascomycin 24,32-diacetate (210 mg;

0.24 mmoles).

We carried out the following analysis on such purified sample:

¹H-NMR (500 MHz) δ: 2.02 and 2.06 (2 CH₃CO), 5.20 and 4.70 (24CH and

33CH);

IR (Cm-¹): 3462.749, 2935.824, 1734.403, 1650.739, 1449.091, 1371.079.

DSC: endothermic peak at 234.10° C ; [α]_D=- 100.0° (C=0.5 CHCl₃).

Spectrum of MS (ESI+): m/z: 898.4 (100.0%; m+23)_.

Elementary analysis calculated for C₄₇H₇₃NO₁₄: C 64.44%; H 8.40%; N 1.60%; O

25.57%

Elementary analysis found: C 64.55%; H 8.44%; N 1.61%; O 25.40%

Preparation of the 24-acetyl ascomycin (intermediate 24-acetate-33-OH; compound VI of scheme 3)

Lipase from Candida antartica (CAL B Novozym 435) [1.1 g (2 U/mg) FLUKA] is added to a solution of ascomycin 33,24-diacetate (500 mg; 0.57 mmol) in

TBDME (25 ml) and n-octan-1-ol (4.5 eq; 0.371 g). The reaction is kept under stirring at 30° C for 100 hours, then the enzyme is taken away for filtration and the obtained filtrate is concentrated under low pressure to obtain 425 mg (0.51 mmoles) of product.

A sample was purified for analytical purposes by chromatography on silica gel (n- hexane/acetone = 7:3 v/v as eluents) and thus crystallised by acetone/water.

We carried out the following analysis on such purified sample: ¹H-NMR

(500MHz) δ: 2.05 (CH3CO); IR (an ¹): 3491.528, 2935.860, 1744.728, 1710.227,

1652.310, 1448.662, 1371.335. DSC: endothermic peak at 134.68° C; [α]_D=-

102.7° (c=0.5 CHCl₃)

Spectrum of MS (ESI +): m/z: 856.4 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇₁NO₁₃: C 64.80%; H, 8.58%; N, 1.68%;

0, 24.94%

Elementary analysis found: C 64.71%; H, 8.49%; N, 1.60%; O, 24.97%

Preparation of the 24-acetyl-33epi-chloro ascomycin (intermediate 24-Acetate-33- chloro; compound VII of scheme 3) Supported triphenylphosphine (0.335 g; 1.1 mmoles) is added to a solution of 24- acetyl ascomycin (400 mg; 0.48 mmoles) in carbon tetrachloride (5 ml). The reaction mixture is kept under reflux for 3 hours then it is cooled at ambient temperature. The obtained suspension is filtered and the filtrate is concentrated to residue under vacuum to obtain 0.45g of reaction raw product which is purified by chromatography on silica gel: 163mg (0.19 mmoles) of product are obtained by elution with petroleum ether/acetone = 90/10.

¹H-NMR δ: 2.08 (CH₃CO); 4.60 (33CH); IR (Cm^“1)= 3464.941, 2934.360,

1738.993, 1650.366, 1450.424, 1371.557; DSC: endothermic peak at 231.67° C

[α]_D=-75.2° (c=0.5 CHCl₃)

Spectrum of MS (ESI +): m/z: 874.3 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇₀ClNO₁₂: C 63.40%; H, 8.28%; Cl,

4.16%; N, 1.64%; O, 22.52%

Elementary analysis found: C 63.31%; H, 8.30%; Cl, 4.05%; N, 1.58%; O,

22.42%.

Preparation of pimecrolimus from 24-acetyl-33-epi-chloro ascomycin

A solution of 24-acetyl-33-epi-chloro ascomycin (200 mg; 0.23 mmoles; compound VII) in methanol (2 ml) and HCl 3N (1 ml) is stirred at ambient temperature for 40 hours. After this period, the reaction is neutralised with an aqueous bicarbonate solution, the methanol evaporated under vacuum. The mixture is extracted with dichloromethane (3 times with 5 ml), anhydrified on sodium sulphate, filtered and concentrated to residue to obtain a residue product which is purified by chromatography on silica gel (n-hexane/acetone as eluents) and thus crystallised by ethyl acetate, cyclohexane/water to obtain 78 mg of purified pimecrolimus (0.096 mmoles).

The chemical/physical characteristics of the obtained product matches the data indicated in literature for pimecrolimus.

Example 4 (comparative^*)

Verification of the method of synthesis of pimecrolimus described in EP427680 Imidazole (508 mg) and tert-Butyldimethylsilylchloride (1.125 g) are added in portions to a solution of 2g (2.53 mmoles) of ascomycin in anhydrous N,N- dimethylformamide (40 ml). The reaction mixture is kept under stirring at ambient temperature for 4.5 days. The reaction is thus processed diluting it with ethyl acetate (200 ml) and processing it using water (5 x 100 ml). The organic phase is separated, anhydrified on sodium sulphate, filtered and evaporated to residue under vacuum to obtain a foamy raw product which is subsequently purified by chromatography on silica gel (1:30 p/p): 2.1 g (2.05 mmoles; yields 81% molars) of ascomycin 24,33 disilyl intermediate are obtained by elution with n- hexane/ethyl acetate 3/1. The chemical/physical data of such intermediate matches that indicated in EP427680.

2.1 g (2.05 mmoles) of ascomycin 24,33 disilyl intermediate are dissolved in a solution under stirring at the temperature of 0°C composed of acetonitrile (42 ml) and aqueous HF 40% (23.1 ml). The reaction mixture is kept under stirring at the temperature of 0°C for 2 hours then it is diluted with dichloromethane (30 ml). Then the reaction is washed in sequence with a saturated aqueous solution using sodium bicarbonate (30 ml) and water (30 ml). The separated organic phase is anhydrified on sodium sulphate, filtered and evaporated to residue under vacuum to obtain a foamy residue which is subsequently purified by chromatography on silica gel (1:30 p/p): 839 mg (0.92 mmoles; yields 45% molars) of ascomycin 24 monosilyl intermediate are obtained by elution with dichloromethane/methanol 9/1. The chemical/physical data of such intermediate matches that obtained on the compound III scheme 2 and matches the data of literature indicated in EP427680. A mixture of 839 mg (0.92 mmoles; yields 45% molars) of ascomycin 24 monosilyl intermediate, triphenylphosphine (337 mg) in carbon tetrachloride (36.4 ml) is heated under stirring under reflux for 15 hours. After this period the reaction mixture is evaporated to residue under vacuum to obtain a solid product purified by chromatography on silica gel (1:30 p/p): 535 mg (0.57 mmoles; yields 63% molars) of ascomycin 24 monosilyl intermediate, 33-chloro derivative are obtained by elution with n-hexane/ethyl acetate 2/1. The chemical/physical data of such intermediate matches those we obtained on compound IV scheme 2 and matches the data of literature indicated in EP427680.

535 mg (0.57 mmoles) of ascomycin 24 monosilyl intermediate, 33-chloro derivative are dissolved under stirring at ambient temperature in acetonitrile (16.4 ml) and aqueous HF 40% (0.44 ml). The reaction mixture is kept under stirring at ambient temperature for 45′ and then it is diluted with ethyl acetate (100 ml). The organic phase is thus washed in sequence with an aqueous solution of sodium bicarbonate (70 ml) with water (2 x 70 ml) and thus it is anhydrified on sodium sulphate, filtered and evaporated under vacuum to obtain a solid which is subsequently purified by chromatography on silica gel (1 :30 p/p): 323 mg (0.399 mmoles; yields 70% molars) of pimecrolimus is obtained by elution with n- hexane/ethyl acetate 2/3. The chemical/physical characteristics of the obtained product matches the data indicated in literature regarding pimecrolimus; the overall yield of the process is 16%.

………………………..

POLYMORPHS…….WO2006060615A1

Example 7: Preparation of amorphous pimecrolimus by precipitation [00094] 19,5 g purified pimecrolimus (colorless resin) was dissolved in 217 ml acetone at 4O⁰C and concentrated. Residue: 38,76 g. The residue was diluted with 6 ml distilled water with stirring. Finally 1 ml acetone was added. This solution was added slowly to 2 L chilled distilled water that was stirred efficiently. After the addition had been completed, the suspension was stirred 20 min at O⁰C. Then the solid was filtered and dried at 45⁰C in vacuum oven overnight. Product: 15,65 g yellowish solid. Amorphous (XRD, DSC).

Example 8: Preparation of amorphous pimecrolimus by grinding

[00095] Procedure of grinding: 200 mg of Pimecrolimus sample was ground gently in an agate mortar using a pestle for half a minute. ,

References

• Elidel official homepage
• FDA News
• NPS RADAR
• Article about American Academy of Dermatology speaking out against black box warning
• Report of the Calcineurin Task Force of the ACAAI and AAAAI

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Plerixafor

cas 110078-46-1

CXCR4 chemokine antagonist

Stem cell mobilization [CXCR4 receptor antagonist]

A bicyclam derivate, highly potent & selective inhibitor of HIV-1 & HIV-2.

Bone marrow transplantation; Chronic lymphocytic leukemia; Chronic myelocytic leukemia; Myelodysplastic syndrome; Neutropenia; Sickle cell anemia

Mozobil (plerixafor injection) is a sterile, preservative-free, clear, colorless to pale yellow, isotonic solution for subcutaneous injection. Each mL of the sterile solution contains 20 mg of plerixafor. Each single-use vial is filled to deliver 1.2 mL of the sterile solution that contains 24 mg of plerixafor and 5.9 mg of sodium chloride in Water for Injection adjusted to a pH of 6.0 to 7.5 with hydrochloric acid and with sodium hydroxide, if required.

Plerixafor is a hematopoietic stem cell mobilizer with a chemical name l, 1′-[1,4phenylenebis (methylene)]-bis-1,4,8,11-tetraazacyclotetradecane. It has the molecular formula C₂₈H₅₄N₈. The molecular weight of plerixafor is 502.79 g/mol. The structural formula is provided in Figure 1.

Figure 1: Structural Formula

 

 

Plerixafor is a white to off-white crystalline solid. It is hygroscopic. Plerixafor has a typical melting point of 131.5 °C. The partition coefficient of plerixafor between 1octanol and pH 7 aqueous buffer is < 0.1.

Plerixafor (rINN and USAN, trade name Mozobil) is an immunostimulant used to mobilize hematopoietic stem cells in cancer patients. The stem cells are subsequently transplanted back to the patient. The drug was developed by AnorMED which was subsequently bought by Genzyme.

History

The molecule 1,1′-[1,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane], consisting of two cyclam rings linked at the amine nitrogen atoms by a 1,4-xylyl spacer, was first synthesised by Fabbrizzi et al. in 1987 to carry out basic studies on the redox chemistry of dimetallic coordination compounds.^[1] Then, it was serendipitously discovered by De Clercq that such a molecule, could have a potential use in the treatment of HIV[2] because of its role in the blocking of CXCR4, a chemokine receptor which acts as a co-receptor for certain strains of HIV (along with the virus’s main cellular receptor, CD4).^[2]Development of this indication was terminated because of lacking oral availability and cardiac disturbances. Further studies led to the new indication for cancer patients.^[3]

Indications

Peripheral blood stem cell mobilization, which is important as a source of hematopoietic stem cells for transplantation, is generally performed using granulocyte colony-stimulating factor (G-CSF), but is ineffective in around 15 to 20% of patients. Combination of G-CSF with plerixafor increases the percentage of persons that respond to the therapy and produce enough stem cells for transplantation.^[4] The drug is approved for patients with lymphoma and multiple myeloma.^[5]

Contraindications

Pregnancy and lactation

Studies in pregnant animals have shown teratogenic effects. Plerixafor is therefore contraindicated in pregnant women except in critical cases. Fertile women are required to use contraception. It is not known whether the drug is secreted into the breast milk. Breast feeding should be discontinued during therapy.^[5]

Adverse effects

Nausea, diarrhea and local reactions were observed in over 10% of patients. Other problems with digestion and general symptoms like dizziness, headache, and muscular pain are also relatively common; they were found in more than 1% of patients. Allergies occur in less than 1% of cases. Most adverse effects in clinical trials were mild and transient.^[5][6]

The European Medicines Agency has listed a number of safety concerns to be evaluated on a post-marketing basis, most notably the theoretical possibilities of spleen rupture and tumor cell mobilisation. The first concern has been raised because splenomegaly was observed in animal studies, and G-CSF can cause spleen rupture in rare cases. Mobilisation of tumor cells has occurred in patients with leukaemia treated with plerixafor.^[7]

Phase III clinical development in combination with G-CSF (granulocyte colony-stimulating factor) is under way at Genzyme (which acquired the product through its acquisition of AnorMED in late 2006) in a stem cell mobilization regimen in non-Hodgkin’s lymphoma (NHL). The trials are designed to evaluate the potential of plerixafor in combination with G-CSF, to rapidly increase the number of peripheral blood stem cells capable of engraftment, thereby increasing the proportion of patients reaching a peripheral blood stem cell target and, as a result, reducing the number of apheresis sessions required for patients to collect a target number of peripheral blood stem cells. A phase I safety trial had been under way for the treatment of renal cancer, however, no recent development for this indication has been reported. An IND has been filed in the U.S. seeking approval to initiate clinical evaluation of the drug candidate to help repair damaged heart tissue in patients who have suffered heart attacks. Currently, an investigator-sponsored study is ongoing to evaluate plerixafor as a single agent in allogeneic transplant. AMD-3100, in combination with mitoxantrone, etoposide and cytarabine, is also in phase I/II clinical trials at the University of Washington for the treatment of acute myeloid leukemia (AML).

The University has also been conducting early clinical trials for increasing the stem cells available for transplantation in patients with advanced hematological malignancies, however, no recent developments on this trial have been reported. Genzyme has completed a phase I/II clinical study of plerixafor hydrochloride in combination with rituximab for the treatment of chronic lymphocytic leukemia. The former AnorMED had been developing plerixafor for the treatment of rheumatoid arthritis (RA), but no clinical development has been reported as of late. AnorMED was also developing plerixafor for the treatment of HIV, but discontinued the trials in 2001 due to abnormal cardiac activity and lack of efficacy.

By blocking CXCR4, a specific cellular receptor, plerixafor triggers the rapid movement of stem cells out of the bone marrow and into circulating blood. Once in the circulating blood, the stem cells can be collected for use in stem cell transplant. In terms of use for cardiac applications, there is clinical evidence that the presence of stem cells circulating in the bloodstream or directly injected into the hearts of patients who have suffered a heart attack may result in improved cardiac function.

Chemical properties

Plerixafor is a macrocyclic compound and a bicyclam derivative.^[4] It is a strong base; all eight nitrogen atoms accept protons readily. The two macrocyclic rings form chelate complexes with bivalent metal ions, especially zinc, copper and nickel, as well as cobalt and rhodium. The biologically active form of plerixafor is its zinc complex.^[8]

Synthesis

Three of the four nitrogen atoms of the macrocycle 1,4,8,11-tetraazacyclotetradecan are protected with tosyl groups. The product is treated with 1,4-dimethoxybenzene or 1,4-bis(brommethyl)benzene and potassium carbonate in acetonitrile. After cleaving of the tosyl groups with hydrobromic acid, plerixafor octahydrobromide is obtained.^[9]

SEE   CHINESE JOURNAL OF MEDICINAL CHEMISTRY    2010 20 (6): 511-513   ISSN: 1005-0108   CN: 21-1313/R

DOWNLOAD………http://download.bioon.com.cn/upload/201207/24113552_9395.pdf

http://www.zgyhzz.cn/qikan/epaper/zhaiyao.asp?bsid=14753

( 1 ) BASE FORM
0155g ( 8016% ), m p 129 ~ 131 e 。
1H-NM R
( CDC l3 ) D: 7.28( s, 4H, A r-H ), 3.55 ( br s, 4H,A r-CH2 ), 2.82 ~ 2.52( m, 32H, NCH2, NHCH2 ),
1.86 ~ 1.68 ( m, 8H, CCH2C )。 ESI-M S m /z:
503.55 [M + H]+ 。

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SEE

http://doc.sciencenet.cn/upload/file/2011531154034454.pdf

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http://www.google.com/patents/US5756728

U.S. Pat. No. 5,021,409 is directed to a method of treating retroviral infections comprising administering to a mammal in need of such treatment a therapeutically effective amount of a bicyclic macrocyclic polyamine compound. Although the usefulness of certain alkylene and arylene bridged cyclam dimers is generically embraced by the teachings of the reference, no arylene bridged cyclam dimers are specifically disclosed.

WO 93/12096 discloses the usefulness of certain linked cyclic polyamines in combating HIV and pharmaceutical compositions useful therefor. Among the specifically disclosed compounds is 1,1′- 1,4-phenylenebis-(methylene)!-bis-1,4,8,11 tetraazacyclotetradecane (and its acid addition salts), which compound is a highly potent inhibitor of several strains of human immune deficiency virus type 1 (HIV-1) and type 2 (HIV-2).

European Patent Appln. 374,929 discloses a process for preparing mono-N-alkylated polyazamacrocycles comprising reacting the unprotected macrocycle with an electrophile in a non-polar, relatively aprotic solvent in the absence of base. Although it is indicated that the monosubstituted macrocycle is formed preferentially, there is no specific disclosure which indicates that linked bicyclams can be synthesized by this process.

U.S. Pat. No. 5,047,527 is directed to a process for preparing a monofunctionalized (e.g., monoalkylated)cyclic tetramine comprising: 1) reacting the unprotected macrocycle with chrominum hexacarbonyl to obtain a triprotected tetraazacyloalkane compound; 2) reacting the free amine group of the triprotected compound prepared in 1) with an organic (e.g., alkyl) halide to obtain a triprotected monofunctionalized (e.g., monoalkylated) tetraazacycloalkane compound; and 3) de-protecting the compound prepared in 2) by simple air oxidation at acid pH to obtain the desired compound. In addition, the reference discloses alternative methods of triprotection employing boron and phosphorous derivatives and the preparation of linked compounds, including the cyclam dimer 1,1′- 1,4-phenylenebis-(methylene)!-bis-1,4,8,11-tetraazacyclotetradecane, by reacting triprotected cyclam prepared as set forth in 1) above with an organic dihalide in a molar ratio of 2:1, and deprotecting the resultant compound to obtain the desired cyclam dimer.

J. Med. Chem., Vol. 38, No. 2, pgs. 366-378 (1995) is directed to the synthesis and anti-HIV activity of a series of novel phenylenebis(methylene)-linked bis-tetraazamacrocyclic analogs, including the known cyclam dimer 1,1′- 1,4-phenylenebis-(methylene)!-bis-1,4,8,11-tetraazacyclotetradecane. The cyclam dimers disclosed in this reference, including the afore-mentioned cyclam dimer, are prepared by: 1) forming the tritosylate of the tetraazamacrocycle; 2) reacting the protected tetraazamacrocycle with an organic dihalide, e.g., dibromo-p-xylene, in acetonitrile in the presence of a base such as potassium carbonate; and 3) de-protecting the bis-tetraazamacrocycle prepared in 2) employing freshly prepared sodium amalgam, concentrated sulfuric acid or an acetic acid/hydrobromic acid mixture to obtain the desired cyclam dimer, or an acid addition salt thereof.

Although the processes disclosed in U.S. Pat. No. 5,047,527 and the J. Med. Chem. reference are suitable to prepare the cyclam dimer 1,1′- 1,4-phenylene bis-(methylene)!-bis-1,4,8,11-tetraazacyclotetradecane, they involve the use of cyclam as a starting material, a compound which is expensive and not readily available. Accordingly, in view of its potent anti-HIV activity, a number of research endeavors have been undertaken in an attempt to develop a more practical process for preparing 1,1′- 1,4-phenylenebis-(methylene)!-bis-1,4,8,11-tetraazacyclotetradecane.

EXAMPLE 1

a) Preparation of the 1,4-phenylenebis-methylene bridged hexatosyl acylic precursor of formula III

To a 4-necked, round-bottom flask, equipped with a mechanical stirrer, heating mantle, internal thermometer and addition funnel, is added 43.5 g (0.25 mol) of N,N’-bis(3-aminopropyl) ethylenediamine and 250 ml of tetrahydrofuran. To the resultant solution is added, over a period of 30 minutes with external cooling to maintain the temperature at 20° C., 113.6 g (0.8 mol) of ethyl trifluoroacetate. The reaction mixture is then stirred at room temperature for 4 hours, after which time 52.25 ml. (0.3 mol) of diisopropylethylamine is added. The resultant reaction mixture is warmed to 60° C. and, over a period of 2 hours, is added a solution of 33.0 g (0.125 mol) of α,α’-dibromoxylene in 500 ml. of tetrahydrofuran. The reaction mixture is then maintained at a temperature of 60° C., with stirring, for an additional 2 hours after which time a solution of 62.0 g. (1.55 mol) of sodium hydroxide in 250 ml. of water is added. The resultant mixture is then stirred vigorously for 2 hours, while the temperature is maintained at 60° C. A solution of 152.5 g. (0.8 mol) of p-toluenesulfonyl-chloride in 250 ml. of tetrahydrofuran is then added, over a period of 30 minutes, while the temperature is maintained at between 20° C. and 30° C. The reaction is then allowed to proceed for another hour at room temperature. To the reaction mixture is then added 1 liter of isopropyl acetate, the layers are separated and the organic layer is concentrated to dryness under vacuum to yield the desired compound as a foamy material.

b) Preparation of the hexatosyl cyclam dimer of formula IV

To a 4-necked, round-bottom flask, equipped with a mechanical stirrer, heating mantle, internal thermometer and addition funnel, is added 114.6 g. (0.10 mol) of the compound prepared in a) above and 2.5 liters of dimethylformamide. After the system is degassed, 22.4 g. (0.56 mol) of NaOH beads, 27.6 g (0.2 mol) of anhydrous potassium carbonate and 5.43 g. (0.016 mol) of t-butylammonium sulfate are added to the solution, and the resultant mixture is heated to 100° C. and maintained at this temperature for 2.5 hours. A solution of 111.0 g (0.3 mol) of ethyleneglycol ditosylate in 1 liter of dimethylformamide is then added, over a period of 2 hours, while the temperature is maintained at 100° C. After cooling the reaction mixture to room temperature, it is poured into 4 liters of water with stirring. The suspension is then filtered and the filter cake is washed with 1 liter of water. The filter cake is then thoroughly mixed with 1 liter of water and 2 liters of ethyl acetate. The solvent is then removed from the ethyl acetate solution and the residue is re-dissolved in 500 ml. of warm acetonitrile. The precipitate that forms on standing is collected by filtration and then dried to yield the desired compound as a white solid.

c) Preparation of 1,1′- 1,4-phenylenebis-(methylene)!-bis-1,4,8,11-tetraazacyclotetradecane

In a 4-necked, round-bottom flask, equipped with a mechanical stirrer, heating mantle, internal thermometer and addition funnel, is added 26.7 g.(0.02 mol) of the compound prepared in b) above, 300 ml. of 48% hydrobromic acid and 1 liter of glacial acetic acid. The resultant mixture is then heated to reflux and maintained at reflux temperature, with stirring, for 42 hours. The reaction mixture is then cooled to between 22° C. and 23° C. over a period of 4 hours, after which time it is stirred for an additional 12 hours. The solids are then collected using suction filtration and added to 400 ml. of deionized water. The resultant solution is then stirred for 25 to 30 minutes at a temperature between 22° C. and 23° C. and filtered using suction filtration. After washing the filter pad with a small amount of deionized water, the solution is cooled to between 10° C. and 15° C. 250 g. of a 50% aqueous solution of sodium hydroxide is then added, over a period of 30 minutes, while the temperature is maintained at between 5° C. and 15° C. The resultant suspension is stirred for 10 to 15 minutes, while the temperature is maintained at between 10° C. and 15° C. The suspension is then warmed to between 22° C. and 23° C. and to the warmed suspension is added 1.5 liters of dichloromethane. The mixture is then stirred for 30 minutes, the layers are separated and the organic layer is slurried with 125 g. of sodium sulfate for 1 hour. The solution is then filtered using suction filtration, and the filtrate is concentrated under reduced pressure (40°-45° C. bath temperature, 70-75 mm Hg) until approximately 1.25 liters of solvent is collected. To the slurry is then added 1.25 liters of acetone, and the filtrate is concentrated under reduced pressure (40°-45° C. bath temperature, 70-75 mm Hg) until approximately 1.25 liters of solvent is collected. The slurry is then cooled to between 22° C. and 23° C. and the solids are collected using suction filtration. The solids are then washed with three 50 ml. portions of acetone and dried in a vacuum oven to obtain the desired compound as a white solid.

EXAMPLE 2

The following is an alternate procedure for the preparation of the 1,4-phenylenebis-methylene bridged hexatosyl acyclic precursor of formula III.

To a 3-necked, round-bottomed flask, equipped with a mechanical stirrer, heating mantle, internal thermometer and addition funnel, is added 3.48 g. (20 mmol) of N,N’-bis-(3-aminopropyl)ethylenediamine and 20 ml. of tetrahydrofuran. To the resultant solution is added, over a period of 20 minutes with external cooling to maintain the temperature at 20° C., 5.2 ml. (42 mmol) of ethyl trifluoroacetate. The reaction mixture is then stirred at room temperature for 1 hour, after which time a solution of 2.64 g. (10 mmol) of α,α’-dibromoxylene in 20 ml. of tetrahydrofuran is added. The resultant reaction mixture is then stirred at room temperature for 4 hours. A solution of 4.8 g. (120 mmol) of sodium hydroxide in 20 ml. of water is then added and the resultant mixture is warmed to 60° C. and maintained at this temperature, with vigorous stirring, for 2 hours. Over a period of 20 minutes, 13.9 g. (73 mmol) of p-toluenesulfonylchloride is then added portionwise, while the temperature is maintained at 20° C. The reaction is then allowed to proceed for another hour at room temperature. To the reaction mixture is then added 100 ml. of isopropyl acetate, the layers are separated and the organic layer is washed with saturated sodium bicarbonate aqueous solution. The solution is then condensed to 40 ml., cooled to 4° C. and kept at that temperature overnight. The resultant suspension is filtered and the solid is washed with 10 ml. of isopropyl acetate. The solvents are then removed from the filtrate to yield the desired compound as a brown gel.

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see

Synthesis and structure-activity relationships of phenylenebis(methylene)linked bis-tetraazamacrocycles that inhibit HIV replication. Effects of macrocyclic ring size and substituents on the aromatic linker
J Med Chem 1995, 38(2): 366

http://pubs.acs.org/doi/abs/10.1021/jm00002a019

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see

New bicyclam-AZT conjugates: Design, synthesis, anti-HIV evaluation, and their interaction with CXCR-4 coreceptor
J Med Chem 1999, 42(2): 229

http://pubs.acs.org/doi/full/10.1021/jm980358u

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CN 102584732

 http://www.google.com/patents/CN102584732B?cl=en

[0003]

[0004] plerixafor (trade name Mozobil ™) was developed by the U.S. company Genzyme chemokine receptor 4 (CXCR4) antagonist specificity. The drug is a hematopoietic stem (progenitor) cell activator, and can stimulate hematopoietic stem cell proliferation and differentiation into functional blood circulation.

[0005] As the non-Hodgkin’s lymphoma (NHL) and multiple myeloma (Korea) most of the cases and the progress of cases to alleviate the need for autologous peripheral blood stem cell transplantation, and plerixafor joint G-CSF can significantly improve the number of patients with ⑶ 34 + cells, about 60% of the patient’s peripheral blood can ⑶ 34 + cells increased to ensure that the NHL and MM patients with autologous hematopoietic stem cell transplantation success.

[0006] U.S. FDA approval on December 15, 2008 its listing, clinical studies showed that the drug can greatly increase the number of white blood cells of patients and to promote hematopoietic stem cells from bone marrow to the blood flow, and granulocyte colony-stimulating factor (G-CSF ) have a synergistic effect; has been used in multiple myeloma and Hodgkin’s lymphoma patients with stem cell transplantation in clinical trials.

[0007] About plerixafor or synthetic analogs have some at home and abroad reported in the literature, there are J.0rg.Chem.2003, 68,6435-6436; J.Med Chem.1995, 38 (2): 366-378; J.SynthCommun.1998 ,28:2903-2906; Tetrahedron, 1989,45 (1) :219-226; Chinese Journal of Pharmaceuticals 2007,38 (6); World Patent W09634860A1; W09312096A1; U.S. Patent US5047527, US5606053, US5801281, US5064956, Chinese patent CN1466579A.

[0008] J.Med Chem.1995, 38 (2) = 366-378 relates to a preparation method comprises the following steps: a) forming a salt of trimethoxy benzene tetraaza macrocycles; 2) reacting the protected tetrazole hetero macrocycle in acetonitrile under the presence of a base such as potassium carbonate as dibromo-p-xylene is reacted with an organic dihalide; 3) using freshly prepared sodium amalgam, concentrated sulfuric acid or acetic acid / hydrobromic acid mixture deprotected target product.

[0009] US 5047527 relates to preparation of the cyclic four monofunctional amine, the method comprising: a) reacting the unprotected macrocycle of reaction with chromium hexacarbonyl to obtain protection tetraazadecalin three compounds; 2) 3 Protection of the free amino compound with an organic halide to obtain three-protected monofunctional tetraaza naphthenic compounds; 3) simple air oxidation, deprotection to obtain the desired product. [0010] J.Synth Commun.1998 ,28:2903-2906 describes an improved method for synthesizing intermediates Plerixafor, the method using phosphor protection, deprotection to give a smooth 1,1 ‘- [1,4 - phenylene bis (methylene)] _ two _1, 4,8,11 – tetraazacyclododecane fourteen burn.

[0011] US 5606053 relates to a process for preparing dimers 1, I ‘- [1,4 - phenylene bis (methylene)] – two -1,4,8,11 – tetraazacyclododecane-tetradecane method. The preparation of compounds include: 1) the four-amine as the starting material, obtained by acylation of toluene Juan acyclic intermediates and three xylene sulfonate and toluene sulfonate and toluene intermediates; 2) and xylene sulfonate and intermediates trimethylbenzene toluenesulfonic acid intermediates after alkylation separation dibromo xylene, toluene sulfonate and then obtain a non-cyclic dimers of six toluenesulfonic acylated; 3) six isolated bridged acyclic toluenesulfonic acid dimer form is reacted with ethylene glycol ditosylate three equivalents of cyclization; 4) deprotection to obtain the objective product was purified by hydrobromic acid and acetic acid.

[0012] US 5801281 relates to preparation of dimer 1, I ‘- [1,4 _-phenylene bis (methylene)] – two _1, 4,8,11

[0013] – tetraazacyclo tetradecane, comprising: a) reacting the acyclic tetraamine with 3 equivalents of ethyl trifluoroacetate, the reaction; 2) with 0.5 equivalents of the tri-dibromo-p-xylene-protected acyclic alkylation of the amine obtained form four non-cyclic dimers; 3) hydrolysis to remove the six trifluoroacetyl compound group; 4) acylation of the compound toluenesulfonic bridged tetraamine dimer; 5) B Juan xylene glycol ester cyclization; 6) and glacial acetic acid mixed with hydrobromic acid deprotection was the target product.

Under the [0014] US 5064956 discloses a multi-alkylated single-ring nitrogen of the compound prepared, the method involves reacting the unprotected macrocycle in an aprotic, relatively non-polar solvent in presence of alkali electrophilic reagent. Not mentioned in this document similar to the embodiment Seclin dimer synthesis.

[0015] Through the open Plerixafor synthetic route research and meta-analysis of the literature, mainly in the following four synthetic routes:

[0016] Route One, is 1,4,8,11 – tetraazacyclododecane cyclotetradecane as raw material, NI, N4, N8 three protected with 1,4 – bis (halomethyl) benzene-bridged deprotection to obtain the finished product. The following reaction scheme, wherein R is p-toluenesulfonyl group, a methanesulfonyl group, a trifluoroacetyl group, a tert-butoxycarbonyl group and the like:

[0017]

[0018] Route II is di (2 – aminopropyl) ethylenediamine as raw material, the ring and the reaction with 1,4 – bis (halomethyl) benzene-bridged, and then deprotection Bullock Suffolk.

[0019] Route 3 to 1,4,8,11 – tetraazacyclododecane cyclotetradecane as raw material, under anhydrous, anaerobic conditions, after the ring protection with 1,4 – bis (halomethyl ) benzene bridging, and then deprotection plerixafor. Synthesis scheme below, wherein R is P, Ni, etc.;

[0021] line four, based on acrylate as starting material, first with ethylene diamine as raw material by Michael addition of the amine solution, then with malonate cyclization 1,4,8,11 – Tetraaza _5, 7,12 – three oxo cyclotetradecane by α, α ‘- dibromo-p-xylene bridging, the final deprotection plerixafor. Reaction Roadmap follows:

[0022]

[0023] The above synthesis route and the existing methods have the following disadvantages:

[0024] In an intermediate of the synthesis route, the existing technology, the need for column purification of the intermediates, low yield.

[0025] route to protect the stability of the two because of the strong, leading to the final deprotection step difficult, long production cycle, low yield, and finished organic residues can not be achieved within the standard limits.

Higher dry anaerobic demands [0026] Route 3 on, harsh reaction conditions, deprotection is not complete, intermediates need to repeatedly purified, low yield, after repeated recrystallization, finished monohetero difficult to control in 0.1% less.

[0027] Anhydrous ethylene diamine route and need four anhydrous THF, more stringent requirements on the process, and to use dangerous borane dimethyl sulfide, while the second step is only about 35% lower yield. Selectivity of the reaction is not high shortcomings, so do not be the most economical and reasonable synthetic route.

[0028] We prepared by Plerixafor prepared by methods disclosed above may Plerixafor single impurity of 0.1% or less is difficult to achieve, it is difficult to meet the quality requirements of the injection material, the same techniques can not reach the European Quality of ICH guidelines of the relevant technical requirements, low yield, high cost required for each step of the intermediate column to afford a large amount of solvent, time consuming, and the greater the elution solvent toxicity, is not suitable for industrial production.

(I) Preparation of 1,4,8 _ tris (p-toluenesulfonyl) -1,4,8,11 – tetraazacyclododecane-tetradecane: the raw 1,4,8,11 – tetraazacyclododecane cyclotetradecane suspended in methylene chloride, in the role of acid binding agent, at a temperature 10 ~ 30 ° C, p-toluenesulfonyl chloride and 3 ~ 8h, filtered, and the filtrate was collected and concentrated to dryness to obtain a residue; will have The residue of said C ^ C3 alkyl group in a mixed solvent of alcohol and an aprotic solvent, purification, crystallization segment greater than 95% purity of 1,4,8 – tris (p-toluenesulfonyl) _1, 4,8,11 – tetraaza cyclotetradecane;

[0032] (2) Preparation of 1,1 ‘- [1,4 - (phenylene methylene)] – two – [4,8,11 - tris (p-toluenesulfonyl)] -1,4, 8,11 – tetraazacyclododecane-tetradecane: A (I) the resulting 1,4,8 – tris (p-toluenesulfonyl) _1, 4,8,11 – tetraazacyclododecane-tetradecane, α, α two bromo-p-xylene in place of anhydrous acetonitrile, was added acid-binding agent, the reaction was refluxed under nitrogen for 5 to 24 hours; After the reaction was cooled to room temperature, the reaction mixture was then collected by filtration and the filter cake was purified to obtain a mixed solvent I , I, – [1,4 - (phenylene methylene)] – two – [4,8,11 - tris (p-toluenesulfonyl)] _1, 4,8,11 – tetraazacyclododecane ten four alkyl;

[0033] (3) Synthesis Plerixafor: A (2) the resultant I, 1′-[1,4 _ (phenylene methylene)] – two – [4,8,11 - tris (p-toluene sulfonyl)] -1,4,8,11 – tetraazacyclododecane myristic acid solution was added to the mixture, stirred and dissolved, the reaction was warmed to reflux for 10 to 24 hours, cooled, filtered, and filter cake was collected; the filter cake was dissolved in purified water, adjusted with sodium hydroxide solution or potassium hydroxide solution to the PH-12, filtered, and the filtrate was extracted with a halogenated solvent, and the organic layer was dried over anhydrous sodium sulfate and then filtered, the filtrate was concentrated under reduced pressure P Le Suffolk crude;

[0034] (4) Purification Plerixafor: Plerixafor the crude was dissolved into a solvent and heated to reflux to dissolve, filtered, and the crystallization solvent is added dropwise at 40 ~ 45 ° C crystallization 30min, filtered and the filtrate then cooled to 20 ~ 25 ° C crystallization I hour at O ​​~ 5 ° C crystallization three hours, filtered, and the filter cake was dried Plerixafor.

Plerixafor Preparation: 6 [0075] Implementation

[0076] The starting material 1,4,8,11 – tetraazacyclo tetradecane (5g, 25mmol) was suspended in dichloromethane (50g) was added N, N-diisopropylethylamine (7.5ml) , a solution of p-toluenesulfonyl chloride (10.8g, 56.5mmol) and methylene chloride (50g) in a solution of, at 25 ~ 30 ° C reaction temperature 3h, filtered, and the filtrate was collected and concentrated to dryness and to the residue in methanol (30g), toluene (IOg) was heated to reflux, filtered, and the filtrate was cooled to 40 ° C crystallization 30min, filtered to remove impurities little over protection, and the filtrate was added methyl tert-butyl ether (30g), stirring rapidly cooled to O ~ 5 ° C crystallization 3h, filtered, and dried to give 1,4,8 – tris (p-toluenesulfonyl) -1, 4,8,11 – tetraazacyclododecane-tetradecane (9.6g, 61.9%), purity of 97.2%.

[0077] The 4,8 _ tris (p-toluenesulfonyl) _1, 4,8,11 – tetraazacyclododecane-tetradecane (9g, 13.6mmol) α, α ‘- dibromo-p-xylene (1.81 g, 6.8mmol) in dry acetonitrile was placed (90ml) was added potassium carbonate (15.0g, 108.5mmol), the reaction was refluxed under nitrogen for 5 hours. Cooled to room temperature and filtered to collect the filter cake, was added anhydrous methanol (10ml), ethyl acetate (30ml), dichloromethane (IOml) hot melt, whereby the cooling crystallization, filtration, and dried under reduced pressure to obtain white solid (16. lg, 83%), purity 97.5%.

[0078] The intermediate obtained above (5g, 3.5mmol) was added to glacial acetic acid (25ml) and concentrated hydrochloric acid (25ml) was stirred until dissolved in the mixed solution was heated to reflux for 24 hours, cooled, collected by filtration cake. The filter cake was dissolved in purified water (20ml), adjusting the PH value of the solution with sodium hydroxide to 12, filtered, and the filtrate was extracted with dichloromethane (50mlX3), the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain sand Bullock Fu crude (1.4g, 79.5%), purity 98.6%.

[0079] The crude Plerixafor (1.4g) is placed in tetrahydrofuran (14g), heated to reflux to dissolve, filtered, and added dropwise n-hexane (42g), and 40 ~ 45 ° C crystallization 30min, filtered little solid, The filtrate was rapidly cooled to 20 ~ 25 ° C crystallization I hour and then at O ​​~ 5 ° C crystallization three hours, filtered, 45 ° C and dried under reduced pressure to obtain the finished Plerixafor (1.2g, 85.7%), purity 99.93 %, the largest single miscellaneous 0.04%.

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http://www.google.com/patents/US8420626?cl=en

wherein, n is 0 or 1, Ts is tosyl radical, P is trifluoroacetyl or p-tosyl radical;
To the NaOH solution of the starting material 7 is dropwise added ether solution of tosyl chloride. The system is stirred over night. A white solid is formed and filtrated. The filter cake is washed with water and ethyl ether, respectively, recrystallized to give a white solid intermediate of formula 8. To the dried acetonitrile solution of the compound of formula 8 is slowly dropwise added dried acetonitrile solution of 1,2-di-p-tosyloxypropane under reflux state, refluxed for 2-4 days, stood until room temperature. A white solid is precipitated and filtrated. The filter cake is washed with water and ethyl acetate, respectively, recrystallized to give a white solid compound of formula 9. The compound of formula 9 is dissolved in 90% concentrated sulfuric acid, allowed to react at 100° C. for 24-48 hours, stood until room temperature. To the reaction solution are dropwise added successively ethanol and ethyl ether. A white solid is precipitated, filtrated, dried, and dissolved in NaOH solution. The aqueous phase is extracted with chloroform. The chloroform phase is combined, concentrated, recrystallized to give a white solid compound of formula 10. To the chloroform solution of the compound of formula 10 and triethylamine is dropwise added chloroform solution of tosyl chloride. The mixture is allowed to react at room temperature over night, concentrated and column separated (eluant: dichloromethane/methanol system) to give a white solid compound of formula 11 (protective group is tosyl); or to the methanol solution of the compound of formula 10 is dropwise added ethyl trifluoroacetate. The mixture is allowed to react at room temperature over night, concentrated and column separated (eluant: ethyl acetate) to give a white solid compound of formula 11 (protective group is trifluoroacetyl);

Pharmacokinetics

Following subcutaneous injection, plerixafor is absorbed quickly and peak concentrations are reached after 30 to 60 minutes. Up to 58% are bound to plasma proteins, the rest mostly resides in extravascular compartments. The drug is not metabolized in significant amounts; no interaction with the cytochrome P450 enzymes or P-glycoproteins has been found. Plasma half life is 3 to 5 hours. Plerixafor is excreted via the kidneys, with 70% of the drug being excreted within 24 hours.^[5]

Pharmacodynamics

In the form of its zinc complex, plerixafor acts as an antagonist (or perhaps more accurately a partial agonist) of the alpha chemokine receptor CXCR4 and an allosteric agonist ofCXCR7.^[10] The CXCR4 alpha-chemokine receptor and one of its ligands, SDF-1, are important in hematopoietic stem cell homing to the bone marrow and in hematopoietic stem cell quiescence. The in vivo effect of plerixafor with regard to ubiquitin, the alternative endogenous ligand of CXCR4, is unknown. Plerixafor has been found to be a strong inducer of mobilization of hematopoietic stem cells from the bone marrow to the bloodstream as peripheral blood stem cells.^[11]

Interactions

No interaction studies have been conducted. The fact that plerixafor does not interact with the cytochrome system indicates a low potential for interactions with other drugs.^[5]

Legal status

Plerixafor has orphan drug status in the United States and European Union for the mobilization of hematopoietic stem cells. It was approved by the U.S. Food and Drug Administration for this indication on December 15, 2008.^[12] In Europe, the drug was approved after a positive Committee for Medicinal Products for Human Use assessment report on 29 May 2009.^[7] The drug was approved for use in Canada by Health Canada on December 8, 2011.^[13]

Research

Small molecule cancer therapy

Plerixafor was seen to reduce metastasis in mice in several studies.^[14] It has also been shown to reduce recurrence of glioblastoma in a mouse model after radiotherapy. In this model, the cancer surviving radiation are critically depended on bone marrow derived cells for vasculogenesis whose recruitment mediated by SDF-1 CXCR4 interaction is blocked by plerixafor.^[15]

Use in generation of other stem cells

Researchers at Imperial College have demonstrated that plerixafor in combination with vascular endothelial growth factor (VEGF) can produce mesenchymal stem cells andendothelial progenitor cells in mice.^[16]

Other uses

Blockade of CXCR4 signalling by plerixafor (AMD3100) has also unexpectedly been found to be effective at counteracting opioid-induced hyperalgesia produced by chronic treatment with morphine, though only animal studies have been conducted as yet.^[17]

Plerixafor

Systematic (IUPAC) name
1,1′-[1,4-Phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane]
Clinical data
AHFS/Drugs.com	Consumer Drug Information
MedlinePlus	a609018
Pregnancy cat.	D (US)
Legal status	℞-only (US)
Routes	Subcutaneous injection
Pharmacokinetic data
Protein binding	Up to 58%
Metabolism	None
Half-life	3–5 hours
Excretion	Renal
Identifiers
CAS number	110078-46-1
ATC code	L03AX16
PubChem	CID 65015
IUPHAR ligand	844
DrugBank	DB06809
ChemSpider	58531
UNII	S915P5499N
Synonyms	JM 3100, AMD3100
Chemical data
Formula	C28H54N8
Mol. mass	502.782 g/mol

http://www.google.com/patents/CN102653536A?cl=en

(Plerixafor), chemical name: 1, I ‘- [I, 4_ phenylene ni (methylene)] – ni -1,4,

8,11 – tetraazacyclo tetradecane, its molecular structure is as follows:

[0004]

Synthesis of domestic and foreign literature in general, all require 1,4,8,11 – tetraazacyclo-tetradecane for 3 protection (eg of formula I), of the three methods are used to protect the p-toluenesulfonamide chloride, trifluoroacetic acid ko ko cool, tert-butyl carbonate ni. Use of p-toluenesulfonamide-protected deprotection step into strict step because deprotecting reagent (such as hydrobromic acid / glacial acetic acid, concentrated sulfuric acid, etc.) side reactions often occur.The use of trifluoroacetic acid ko ko ester protecting, since the trifluoromethyl group strongly polar ko, resulting fourth-NH unprotected decrease in activity, usually not fully reflect the subsequent reaction, thereby further into ー is introduced after deprotection difficult to remove impurities 1,4,8,11 – tetraazacyclo-tetradecane.

[0006] tert-butyl carbonate ni selective protection of the amino group is widely used (polyamines, amino acids, p printed tidic chains, etc.), but to use it for 1,4,8,11 – tetraazacyclo tetradecane rarely reported, abroad it for 1,4,8,11 – tetraazacyclo tetradecane protection coverage, we use the t-butyl carbonate brother attempted 3 protection, he was surprised to find that in certain conditions, the three protection up to 90% (see Figure I), with high selectivity, significantly higher than the reported domestic Boc protected

Selectivity of the reaction (see table below).

[0007]

[0008] 2 by three protection product with quite different polarity protection products, flash column chromatography using silica gel column to separate the protector 3 of sufficient purity, and deprotection conditions milder (only hydrochloric acid solution), in a certain extent reduce the incidence of side effects, so capable of synthesizing high purity products.

[0009]

SUMMARY OF THE INVENTION

xample I: 3Boc protection 1,4,8,11 _ tetraazacyclo Preparation tetradecane

[0048] 1,4,8,11 taken tetraazacyclo tetradecane _ 10g (0.05mol), and acetone – water (2: l) 50ml, tris ko amine 10. 119g (0. Lmol), ni ko isopropyl amine 3. 225g (0. 025mol), at room temperature was added dropwise tert-butyl carbonate, brother 38. 194g (0. 175mol), dropwise at room temperature after stirring for 24 hours, HPLC monitoring of the reaction. After completion of the reaction 50 ° C under reduced pressure to dryness to give a pale yellow oil, 150g on a silica gel column, and eluted with ko acid esters ko collecting ko ko acid ester liquid evaporated to dryness under reduced pressure to give a white foam 23. 12g, yield of 92.36%. 1HNMR (400MHz, CDCl3, 6 ppm): 1. 74 (2H, q, 5. 5);

I. 96 (2H, q, 6. 5); 2. 66 (2H, t, 5. 5); 2. 82 (2H, t, 5. 5); 3. 33 (4H, m); 3. 34 (2H, m); 3. 37 (2H, m), 3. 43 (4H, m).

[0049] Implementation Example 2: 6Boc protection Bullock Suffolk Preparation

[0050] Take 3Boc protection 1,4,8,11 _ tetraazacyclo tetradecane 20. 03g (0. 04mol), dissolved in anhydrous ko nitrile 400ml, anhydrous potassium carbonate 20g, aa ‘ni chlorine ni toluene 3.5012g (0.02mol), sodium iodide 75mg, at reflux for 24 hours under nitrogen, TLC monitoring of the reaction. After completion of the reaction, cooled to room temperature, filtered, the filter cake was washed with 200ml of ko nitrile, nitrile ko combined solution was evaporated to dryness under reduced pressure to give the protected Bullock 6Boc Suffolk 21. 20g, yield of 96.06%. Alcohol with ko – a mixed solvent of water and recrystallized to give a white solid. [0051] Implementation Example 3: Bullock Suffolk • 8HC1 • 3H20 Preparation of compounds

[0052] Protection Bullock Suffolk take 6Boc 20g, add methanol 200ml, stirring to dissolve, concentrated hydrochloric acid was added dropwise at room temperature, 60ml, was stirred at room temperature after the addition was complete 48 inches, TLC monitoring of the reaction. After completion of the reaction, filtration, the filter cake was dried 50 ° C under reduced pressure to give a white solid 13. 54g, yield of 88.04%.

[0053] Implementation Example 4: Preparation of Suffolk Bullock…………Plerixafor BASE

[0054] Take Bullock Suffolk • 8HC1 • 3H20 compound 13. 54g, add water 40ml ultrasound to dissolve after stirring constantly with 50% sodium hydroxide solution to adjust the pH to 12 and filtered, the filter cake 50 ° C minus pressure and dried to give a white solid 7. 24g, yield 90.24 V0o

1H NMR (400MHz, CDCl3, 6 ppm): 1. 75 (4H, bs); 1. 87 (4H, bs); 2. 95-2. 51 (32H, m); 3. 54 (4H, s); 4. 23 (4H, bs); 7. 30 (4H, s). 

IR (KBr) 3280,2927,2883,2805,1458,1264,1117 cm,

NEW PATENT…………….WO-2014125499

Improved and commercially viable process for the preparation of high pure plerixafor base

Process for the preparation of more than 99.8% pure plerixafor base by HPLC. Also claims solid forms of plerixafor base and composition comprising the same. Appears to be the first filing from the assignee on this API. FDA Orange book lists US6987102 and US7897590, expire in July 2023.

References

External links

• MeSH JM+3100

http://pubs.rsc.org/en/content/articlehtml/2012/dt/c2dt31137b

http://www.thno.org/v03p0047.htm

SEE ALSO……….http://www.scipharm.at/download.asp?id=1427

SEE…………..https://www.academia.edu/5549712/2011531154034454SCHEME 15 IS SYNTHESIS OF PLEXIXAFOR

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