IV.lETllODS OF TR.'Jr.ATING ACNE
`
`BACKGROUND Olf THE INV.ENTlON
`
`5
`
`Acne is a cotnnJon inflammatory disease in skin areas ·where sebaceous glands are
`largestj most t1un1erous, and most active. In its :mildest fonn, acneis a n1ore or less
`superfi.cial disotder characterized by slight; spouy skin irritati.ons. l:ttsuch cases~ ordinary
`skin hygiene.is typically a satisfactory t;reatment. In the n1ore inflal11n1atory types of acne')
`however, pustules; infected cysts; a11d in extreme cases, cärtaHzing, inflamed and infected
`sacs appear.. Without dTective treattuent, these.le~ions may·becorne extensive and·leave
`, permanent, disfiguring sc;ars.
`
`10
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`15
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`20
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`25
`
`Mkro .. organisn1s, especially Propionibacteriwn acti.es, are strongly ünplicated in
`the pathogenesis of acne. The tnicro",.organisins are · thought to release. n1icrobial ntediators
`of inflan1ma.tion into the dernris or trigger the release>of cytokines from ducta1
`keratinoc)~tes.
`
`Accordingly, the effic.acy ofantibiotics in treating acne is1hougbt tobe due~ in
`significant part, to the direct inhibitory effect of the antihiotics on th~e grovvth an.d
`n1etaboHsn1ofthese m1cro~organisms. Sy.stemicaUy..;administered tetracyclineantibiotics,
`espec:ialiy·niinocycline.hydrochloridC:}are•parti.cularly etiectiveintreatingacne.
`
`The tetracycli11es are. a class öf cnmpounds öf\:v;hich tetracyclineis the parent
`cmnpound. Tetracycline has the toUovving general structure:
`
`00
`
`.J;J ~(C~)z
`OH
`~
`~
`
`CONH2
`
`Structtrre r\
`
`Dr. Reddy's Laboratories, Ltd., et al.
`V.
`Galderma Laboratories, lnc.
`IPR2015-__
`Exhibit 1002
`
`Exh. 1002
`
`

`
`, ·' ... .
`
`The numbering system of the multiple ring nucleus is as follows:
`
`5
`
`10
`
`Structure B
`
`Tetracycline, as well as the 5-0H (oxytetracycline, e.g. Terramycin) and 7-Cl
`(chlorotetracycline, e.g. Aureomycin) derivatives, exist in nature, and are all well known
`antibiotics. Semisynthetic derivatives such as 7-dimethylarnino-tetracycline (minocycline)
`and 6a-deoxy-5-hydroxy-tetracycline (doxycycline) are also known tetracycline
`antibiotics. Natural tetracyclines may be modified without losing their antibiotic
`properties, although certain elements of the structure must be retained to do so.
`
`In addition to the direct antimicrobial activity of tetracyclines, further activities of
`antibiotic tetracyclines have been investigated for possible therapeutic effects on acne.
`
`15
`
`For example, a study by Elewsla et al., J Amer. Acad. Dermatol. , 8:807-81 2
`( 1983) suggests that acne therapy, consisting of orally-administered tetracycline at a total
`daily dose of lOOOmg, may have therapeutic anti-inflammatory effects in addition to
`antimicrobial effects. In particular, it was found that tetracycline inhibited neutroJiPil
`chemotaxis induced by bacterial chemotactic factors.
`
`However, a more recent study performed by Eady et al., J Invest. Dermatol.,
`101:86-91 (1993) found somewhat different results with respect to the effect of
`tetracyclines_on cytokines. The study was designed to determine whether oral acne
`therapy with minocycline or tetracycline altered the cytokine content of open comedones.
`The total daily dose of minocycline administered was 1 OOmg. The total daily dose of
`tetracycline administered was 1 OOOmg. It was found that these therapies up regulated the
`production of bioactive IL-l ex-like material and irnmunochemical IL-1 ~- IL-l is
`considered to be a pro-inflammatory cytokine. The authors speculate that increased levels
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`2
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`Exh. 1002
`
`

`
`. \ .
`
`of IL-l in comedones destined to become inflamed may enhance resolution and promote
`repair of the damaged follicular epithelium.
`
`Another possible activity of tetrac~clines in acne therapy was investigated by
`Bodokh, 1., et al., Acta. Derm. Venerol., 77:255-259 (1997). Their study was designed to
`evaluate the action of minocycline on sebaceous excretion in acne patients. A 1 OOmg daily
`dose of minocycline was administered. A subclinical increase in seborrhoea was reported.
`The authors propose that minocycline induces an increase in seborrhoea via a reduction in
`ductal obstruction. The mechanism by which the ductal obstruction is reduced is proposed
`to be the reduction in ductal irritation. The authors suggest that the reduction of ductal
`irritation is due to minocycline's direct effect on P. acnes, or minocycline's effect on the
`lipase produced by P. acnes.
`
`Bodokh et a!. also found that during treatment no correlation exists between
`seborrhoea intensity and clinical severity of acne. The authors state that the lack of
`correlation shows that seborrhoea is pathogenic because it is the "culture medium'' of P.
`acnes. Thus, it can be concluded that the authors consider the antimicrobial activity of
`minocycline as a significant therapeutic activity with respect to acne.
`
`Similarly, in a recent clinical study it was found that tetracycline doses lower than_
`antimicrobial doses had no clinical effect on acne. (Cunliffe et al., J Am. Acad
`D~rmatol., 16:591-9 (1987).) In particular, a l OOmg total daily dose ofminocycliPe; and a
`l.Og total daily dose of tetracycline were found to be necessary to successfully treat acne.
`
`The antimicrobial effects of antibiotics are generally directly proportional to the
`dose administered of the antibiotics. Accordingly, in moderate to severe (i.e.
`inflammatory) forms of acne, oral antibiotics are typically administered at high doses. For
`example, in conventional acne therapy, tetracycline is administered at an initial dose of
`500 to 2,000 mg/day, followed by a maintenance dose of250-500 mg/day.
`
`5
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`15
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`20
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`:;
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`3
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`Exh. 1002
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`.. ·"\·, ,
`
`Clearly, the state-of-the-art teaching is that the clinical efficacy of systemically-
`administered tetracyclines in the treatment of acne is due in significant part to the
`antimicrobial effects of the tetracyclines.
`
`5
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`10
`
`The use of tetracycline antibiotics, however, can lead to undesirable side effects.
`For example, the long term administration of antibiotic tetracyclines can reduce or
`eliminate healthy microbial flora, such as intestinal flora, and can lead to the production of
`'
`antibiotic resistant organisms or the overgrowth of yeast and fungi. Other side effects
`include gastrointestinal symptoms such as nausea, vomiting, abdominal pain, diarrhea,
`rashes, and other allergic reactions. Tetracyclines also can cause fetal harm if used during
`pregnancy.
`
`Accordingly, there is a need for an effective treatment of acne which does not
`cause the undesirable side effects produced by the systemically-administered antibiotics
`used in conventional acne therapy. ·
`
`~1}
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`SUMMARY OF INVENTION
`
`The present invention provides a method of treating acne in a human in need
`thereof. The method comprises administering systemically to the human a tetracycline
`compound in an amount that is effective to treat ?tCne but has substantially no
`antimicrobial activity, without administrating a bisphosphonate compound.
`
`j
`
`BIUEF DESCIUPTION OF THE DRAWINGS
`
`Figure 1 shows the photoirritancy factor (PIF) for some tetracycline compounds.
`For structure K, the compounds indicated are as follows:
`
`COL
`
`308
`311
`306
`
`R7
`
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`amino
`palmitamide
`dimethylamino
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`
`4
`
`Exh. 1002
`
`

`
`.. :."\, , ,
`
`5
`
`10
`
`1 5
`
`20
`
`25
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`30
`
`r~1
`~;;J
`r~~~
`t:§j
`
`f=~
`~~J
`~:~~
`~~1
`
`;;!
`:':.'.:-1
`
`~: ,;:
`
`:::ll;:;
`~T.!;
`b:-~
`t~:~
`~~~
`
`For structures L, M, Nor 0 the compounds indicated are as follows:
`
`COL
`
`801
`802
`804
`805
`
`R}
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`acetamido
`dimethylaminoacetamido
`nitro
`amino
`
`For structure P, R7 is hydrogen, R8 is hydrogen and R9 is nitro.
`
`D.ETAILED DESCRIPTION
`
`The present invention provides methods of treating acne. As used herein, the term
`"acne" is a disorder of the skin characterized by papules, pustules, cysts, nodules,
`comedones, and other blemishes or skin lesions. These blemishes and lesions are often
`accompanied by inflammation of the skin glands and pilosebaceous follicles, as well as,
`microbial, especially bacterial, infection.
`
`For the purposes of this specification, acne includes all known types of acne Some
`types of acne include, for example, acne vulgaris, cystic acne, acne atrophica, bromide
`acne, chlorine acne, acne conglobata, acne cosmetica, acne detergicans, epidemic acne,
`acne estivalis, acne fulminans, halogen acne, acne indurata, iodide acne, acne kelgid, acne
`mechanica, acne papulosa, pomade acne, premenstral acne, acne pustulosa, acne rosacea,
`acne scorbutica, acne scrofulosorum, acne urticata, acne varioliformis, acne venenata,
`propionic acne, acne excoriee, gram negative acne, steroid acne, and nodulocystic acne.
`
`•.
`
`The present invention can also be used to treat certain other types of acneiform
`dermal disorders, e.g. perioral dermatitis, seborrheic dermatitis in the presence of acne,
`gram negative folliculitis, sebaceous gland dysfunction, hiddradenitis suppurativa, pseudo-
`folliculitis barbae, or folliculitis.
`
`5
`
`Exh. 1002
`
`

`
`The method comprises the administration of a tetracycline compound to a human
`in an amount which is effective to treat acne, but which has substantially no antimicrobial
`activity.
`
`5
`
`The tetracycline compound can be an antimicrobial or non-antimicrobial
`compound. The tetracycline compound has the general structure indicated above.
`
`10
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`15
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`20
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`25
`
`Some examples of antimicrobial tetracycline compounds include doxycycline,
`minocycline, tetracycline, oxytetracycline, chlortetracycline, demeclocycline, lymecycline
`and their pharmaceutically acceptable salts. Doxyclycline is preferably administered as its
`hyclate salt or as a hydrate, preferably monohydrate.
`
`Non-antimicrobial tetracycline compounds are structurally related to the antibiotic
`tetracyclines, but have had their antibiotic activity substantially or completely eliminated
`by chemical modification. For example, non-antimicrobial tetracycline compounds are
`capable of achieving antibiotic activity comparable to that of tetracycline at concentrations
`at least about ten times, preferably at least about twenty five times, greater than that of
`tetracycline.
`
`Examples of chemically modified non-antimicrobial tetracyclines (CMT's)
`include, 4-de( dimethyl amino )tetracycline (CMT -1 ), tetracyclinonitrile (CMT -2), 6-
`demethyl-6-deoxy-4-de( dimethylamino )tetracycline (CMT-3 ), 7 -chloro-4-
`,f
`de(dimethylamino )tetracycline (CMT-4), tetracycline pyrazole (CMT-5), 4-hydroxy-4-
`de( dimethyl amino )tetracycline (CMT-6), 4-de( dirnethylamino-12a-deoxytetracycline
`(CMT -7), 6-deoxy-Sa-hydroxy-4-de( dimethyl amino )tetracycline (CMT -8), 4-
`de( dimethyl amino )-12a-deoxyanhydrotetracycline (CMT -9), 4-
`de( dimethyl amino )minocycline (CMT-1 0).
`
`Further examples of chemically modified non-antimicrobial tetracyclines include
`Structures C-Z. (See Index of Structures.)
`
`30
`
`6
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`Exh. 1002
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`

`
`\ .
`
`The minimal amount of the tetracycline compound administered to a human is the
`lowest amount capable of providing effective treatment of acne. Effective treatment is a
`reduction or inhibition of the blemishes and lesions associated with acne.
`
`5
`
`The amount of a tetracycline compound that has substantially no antimicrobial
`activity is an amount that does not significantly prevent the growth of microbes, e.g.
`bacteria.
`
`For example, tetracycline compounds that have significant antimicrobial activity
`I 0 may be administered in an amount which is 10-80% of the antimicrobial amount. More
`preferably, the antimicrobial tetracycline compound is administered in an amount which is
`40-70% of the antimicrobial amount.
`
`'. 1...::..
`:··-
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`15
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`20
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`25
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`30
`
`Some examples of antimicrobial amounts of members of the tetracycline family
`include 1 OOmg/day of doxycycline, 200mg/day of minocycline, 250mg of tetracycline four
`times a day, 1 OOOmg/day of oxytetracycline, 600mg/day of demeclocycline and
`600mg/day of lymecycline.
`
`An example of an antimicrobial tetracycline administered in a non-antimicrobial
`amount is doxycycline hyclate administered at a 20 milligram dose twice daily. Such a
`,
`formulation is sold for the treatment of periodontal disease by CollaGenex
`t
`Pharmaceuticals, Inc. ofNewtown, Pennsylvania under the trademark Periostat ®.~
`
`The tetracycline compound may be administered by sustained release. Sustained
`release administration is a method of drug delivery to achieve a certain level of the drug
`over a particular period of time. The level typically is measured by serum concentration.
`Further description of methods of delivering tetracycline compounds by sustained release
`can be found in the patent application, "Controlled Delivery of Tetracycline and
`Tetracycline Derivatives,'' filed on April 5, 2001 and assigned to CollaGenex
`Pharmaceuticals, Inc. of Newtown, Petmsylvania. The aforementioned application is
`incorporated herein by reference in its entirety.
`
`7
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`Exh. 1002
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`

`
`. '
`
`'
`
`For example, 40 milligrams of doxycycline may be administered by sustained
`release over a 24 hour period.
`
`5
`
`The antimicrobial tetracycline compound can be administered in an amount which
`results in a serum tetracycline concentration which is 10-80% of the minimum
`antimicrobial serum concentration. The minimun1 antimicrobial serum concentration is
`the lowest concentration known to exert a significant antimicrobial effect.
`
`10
`
`Some examples of the approximate minimum antimicrobial serum concentrations
`of members of the tetracycline family follow.
`
`A single dose of two 1 OOmg minocycline HCl tablets administered to adult humans
`results in minocycline serwn levels ranging from 0.74 to 4.45 J.tg/ml over a period of an
`hour. The average level is 2.24 J.tg/rnl.
`
`Two hundred and fifty milligrams of tetracycline HCl administered every six hours
`over a twenty-four hour period produces a peak plasma concentration of approximately 3
`J.tg/ml. Five hundred milligrams of tetracycline HCl administered every six hours over a
`twenty-four hour period produces a serum concentration level of 4 to 5 J.tg/ml.
`
`15
`
`20
`
`The minimum antimicrobial serum concentration of doxycycline is 1 J.tg/mf.
`Accordingly, a serum concentration of doxycycline in the range of about 0.1 to about 0.8
`J.tg/ml is 10-80% of the minimum antimicrobial serum concentration of doxycycline. The
`25 more preferred range is about 0.4 to about 0.7 J.tg/ml.
`
`To achieve this serum concentration, doxycycline can be administered by sustained
`release, or by intermittent administration.
`
`30
`
`In another embodiment, the tetracycline compound can be administered in an
`an1ount which results in a serum concentration between about 0.1 and 10.0 J.tg/ml, more
`
`8
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`Exh. 1002
`
`

`
`preferably between 0.3 and 5.0 J..tg/ml. For example, doxycycline is administered in an
`amount which results in a serum concentration between about 0.1 and 0.8 J..tg/ml, more
`preferably between 0.4 and 0.7 J..tg/ml.
`
`Non-antimicrobial tetracycline compounds can be used in higher amounts than
`antimicrobial tetracyclines, while avoiding the indiscriminate killing of microbes, and the
`emergence of resistant microbes. For example, 6-demethyl-6-deoxy-
`4-de(dimethylamino)tetracycline (CMT-3) may be administered in doses of about 40 to
`about 200 mg/day, or in amounts that result in serum levels of about 1.55ug/ml to about 10
`ullml.
`
`The actual preferred amounts of tetracycline compound in a specified case will
`vary according to the particular compositions formulated, the mode of application, and the
`particular sites and subject being treated.
`
`5
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`10
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`15
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`20
`
`The tetracycline compounds can be in the form of pharmaceutically acceptable
`salts of the compounds. The term "pharn1aceutically acceptable salt" refers to a salt
`prepared from tetracycline compounds and pharmaceutically acceptable non-toxic acids or
`bases. The acids may be inorganic or organic acids of tetracycline compounds. Examples
`of inorganic acids include hydrochloric, hydrobromic, hydroiodic, sulfuric, and phosphoric
`acids. Examples of organic acids include carboxylic and sulfonic acids. The radic~l of the
`organic acids may be aliphatic or aromatic. Some examples of organic acids inchlde
`formic, acetic, phenylacetic, propionic, succinic, glycolic, glucuronic, maleic, furoic,
`glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic),
`25 methanesulfonic, ethanesulfonic, panthenoic, benzenesulfonic, stearic, sulfanilic, alginic,
`as tartaric, citric, gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriate
`organic bases may be selected, for example, from N,N-dibenzylethylenediamine,
`chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-
`methylglucamine ), and procaine.
`
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`Exh. 1002
`
`

`
`"'' '"'' .
`
`Preferably, the tetracycline compounds have low phototoxicity, or is administered
`in an amount that results in a serum level at which the phototoxicity is acceptable.
`Phototoxicity is a chemically-induced photosensitivity. Such photosensitivity renders skin
`susceptible to damage, e.g. sunburn, blisters, accelerated aging, erythemas and eczematoid
`lesions, upon exposure to light, in particular ultraviolet light. The preferred amount of the
`tetracycline compound produces no more phototoxicity than is produced by the
`administration of a 40mg total daily dose of doxycycline.
`
`Phototoxicity can be evaluated in terms of a photoirritancy factor (PIF), as
`described in the examples. A PIF value of about 1.0 indicates that a compound is
`considered to have no measurable phototoxicity.
`
`5
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`10
`
`The low phototoxic derivatives preferably have PIF values no greater than about 5,
`preferably no greater than about 2, more preferably no greater than about 1.5, most
`preferably no greater than about 1.2, and optimally about 1.
`
`t~~
`
`15
`
`Some antimicrobial tetracyclines having low phototoxicity include, for example,
`minocycline and tetracyline.
`
`20
`
`Some non-antimicrobial tetracyclines having low phototoxicity include, but are not
`limited to, tetracycline compounds having the general formulae:
`
`STRUCTUREK
`
`25 wherein: R7, R8, and R9 taken together in each case, have the following meanings:
`
`R7
`
`hydrogen
`hydrogen
`hydrogen
`
`30
`
`and
`
`R9
`
`ammo
`palmitamide
`dimethylamino
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`
`10
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`Exh. 1002
`
`

`
`. ".
`
`STRUCTUREL
`STRUCTUREN
`
`STRUCTUREM
`STRUCTURE 0
`
`5
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`10
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`15
`
`wherein: R 7, R8, and R9 taken together in each case, have the following meanings:
`
`R7'
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`and
`
`R8
`
`hydrogen
`hydrogen
`hydrogen
`hydrogen
`
`R9
`
`acetamido
`dimethylaminoacetamido
`nitro
`ammo
`
`STRUCTUREP
`
`f""
`
`wherein: R7, R8, and R9 taken together are, respectively, hydrogen, hydrogen and nitro.
`
`20
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`25
`
`30
`
`The tetracycline compounds are administered vvithout administering a
`bisphosphonate compound. Bisphosphonates compounds are related to inorganic
`pyrophosphonic acid. The bisphosphonates include, as non-limiting examples,
`alendronate ((4-arnino-1- hydroxybutylidene) bisphosphonic acid), clodronate
`( dichloromethane diphosphonic acid), etidronate ( ( 1-hydroxyethylidene) di phospb'anic
`acid) and pamidronate ((3-amino-1- hydroxypropylidene) bisphosphonic acid); also
`risedronate ((-hydroxy-2-(3-pyridinyl)ethylidene] bisphosphonic acid), tiludronate, i.e.,
`tiludronic acid (((4-chlorophenyl) thio] methylene] bisphosphonic acid) and zolendronate.
`
`The tetracycline compounds are administered systemically. For the purposes of
`this specification, "systemic administration" means administration to a human by a
`method that causes the compounds to be absorbed into the bloodstream.
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`11
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`Exh. 1002
`
`

`
`For example, the tetracyclines compounds can be administered orally by any
`method known in the art. For example, oral administration can be by tablets, capsules,
`pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the like.
`
`Additionally, the tetracycline compounds can be administered enterally or
`parenterally, e.g., intravenously, intramuscularly, or subcutaneously, as injectable
`solutions or suspensions. Administration can also be intranasally, in the form of, for
`example, an intranasal spray; or transderrnally, in the form of, for example, a patch.
`
`For the pharmaceutical purposes described above, the tetracycline compounds of
`the invention can be formulated per se in pharmaceutical preparations optionally with a
`suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the
`art. These preparations can be made according to conventional chemical methods.
`
`Examples of carriers and excipients include starch, milk, sugar, certain types of
`clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable
`fats or oils, gums and glycols.
`
`In the embodiment in which the tetracycline compound is a non-antimicrobial
`tetracycline compound, administration can include topical application. Particular non-
`antimicrobial tetracycline compounds have only limited biodistribution, e.g. CMT~5. In
`such cases, topical application is the preferred method of administration of the co~ pound.
`
`Carrier compositions deemed to be suited for topical use include gels, salves,
`lotions, creams, ointments and the like. The non-antimicrobial tetracycline compound can
`also be incorporated with a support base or matrix or the like which can be directly applied
`to skin.
`
`5
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`20
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`25
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`Topical application of non-antimicrobial tetracycline compounds in amounts of up
`to about 25% (w/w) in a vehicle are effective in treating acne while not inducing
`
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`Exh. 1002
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`significant toxicity in the human. Amounts of from about 0.1% to about 10% are
`preferred.
`
`5
`
`Combined or coordinated topical and systemic administration of the tetracycline
`compounds is also contemplated under the invention. For example, a non-absorbable non-
`antimicrobial tetracycline compound can be administered topically, while a tetracycline
`compound capable of substantial absorption and effective systemic distribution in a human
`can be administered systemically.
`
`10
`
`The tetracycline compounds are prepared by methods known in the art. For
`example, natural tetracyclines may be modified without losing their antibiotic properties,
`although certain elements of the structure must be retained. The modifications that may
`and may not be made to the basic tetracycline structure have been reviewed by Mitscher in
`The Chemistry ofTetracyclines, Chapter 6, Marcel Dekker, Publishers, New York ( 1978).
`1 5 According to Mitscher, the substituents at positions 5-9 of the tetracycline ring system
`may be modified without the complete loss of antibiotic properties. Changes to the basic
`ring system or replacement of the substituents at positions 1-4 and 10-12, however,
`generally lead to synthetic tetracyclines with substantially less or effectively no
`antimicrobial activity.
`
`ei1
`
`20
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`25
`
`Further methods of preparing the tetracycline compounds are described in. the
`!
`examples.
`
`EXAMPLES
`
`The following examples serve to provide further appreciation of the invention but
`are not meant in any way to restrict the effective scope of the invention.
`
`Preparation of Compounds
`
`30
`
`EXAMPLE 1
`4-Dedimethylamino-7 -dimethy lam ino-6-demethyl-6-deoxy -9-ni trotetrac ycline sulfate
`
`13
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`Exh. 1002
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`

`
`To a solution of one millimole of 4-dedimethylamino-7 -dimethylamino-6-
`demethyl-6-deoxytetracycline in 25 ml of concentrated sulfuric acid at 0°C was added
`1.05 mmole of potassium nitrate. The resulting solution was stirred at ice bath
`temperature for 15 minutes and poured in one liter of cold ether with stirring. The
`precipitated solid was allowed to settle and the majority of solvent decanted. The
`remaining material was filtered through a sintered glass funnel and the collected solid was
`'
`washed well with cold ether. The product was dried in a vacuum desiccator overnight.
`
`EXAMPLE2
`9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline
`sulfate
`
`5
`
`10
`
`l;J
`
`15
`
`To a solution of300 mg of the 9-nitro compound from example 1, in 30 ml of
`ethanol was added 50 mg ofPt02. The mixture was hydrogenated at atmospheric pressure
`until the theoretical amount ofhydrogen was absorbed. The system is flushed with
`nitrogen, the catalyst Pt02 is filtered and the filtrate added dropwise to 300 ml of ether.
`The product that separates is filtered and dried in a vacuum desiccator.
`
`EXAMPLE3
`9-Acetamido-4-ded imethylamino-7 -dimethylamino-6-demethy 1-6-deoxytetracycline
`~
`sulfate
`
`To a well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-7-
`dimethylamino-6-demethyl-6-deoxytetracycline sulfate from example 2, in 2.0 ml of 1.3-
`dimethyl-2-imidazolidinone, 500 mg of sodium bicarbonate was added followed by 0.21
`ml of acetyl chloride. The mixture is stirred at room temperature for 30 minutes, filtered
`and the filtrate was added dropwise to 500 ml of ether. The product that separated was
`filtered and dried in a vacuum desiccator.
`
`20
`
`25
`
`30
`
`14
`
`" ... , . '
`
`.l
`
`Exh. 1002
`
`

`
`EXAMPLE 4
`4-Dedimethy I amino-7 -dime thy lamino-6-demethyl-6-deoxy-9-diazoni um tetracycline
`sulfate
`
`To a solution of 0.5 g of 9-amino-4-dedimethylamino-7-dimethylamino-6-
`demethyl-6-deoxytetracycline sulfate, from example 2, in 10 ml of 0.1 N hydrochloric acid
`in methanol cooled in an ice bath, 0.5 ml ofn-butyl nitrite was added. The solution was
`'
`stirred at ice bath temperature for 30 minutes and then poured into 250 ml of ether. The
`product that separated was filtered, washed with ether and dried in a vacuum desiccator.
`
`EXAMPLE S
`9-Azi do-4-dedimeth y I amino-7 -d imethy lan1 ino-6-demeth y 1-6-deoxytetracycline
`sulfate
`
`To a solution of 0.3 mmole of 4-dedimethylamino-7-dimethy1amino-6-demethyl-6-
`deoxy-9-diazoniumtetracycline sulfate, from example 4, 10 ml of 0.1 N methanolic
`hydrogen chloride was added 0.33 mmole of sodium azide. The mixture was stirred at
`room temperature for 1.5 hours. The reaction mixture was then poured into 200 ml of
`ether. The product that separated was filtered and dried in a vacuum desiccator.
`
`EXAMPLE 6
`9-Am ino-8-chloro-4-dedimethylamino-7 -dimethy larnino-6-demethy 1-6-deoxy-
`tetracycline sulfate
`
`5
`
`10
`
`15
`
`20
`
`25
`
`One gram of 9-azido-4-dedimethylamino-7 -dimethy lamino-6-demeth y 1-6-
`deoxytetracycline hydrochloride, from example 4, was dissolved in 10 ml of concentrated
`
`sulfuric acid saturated with HCL at 0°C. The mixture was stirred at ice bath temperature
`for 1.5 hours and then slowly added dropwise to 500 ml of cold ether. The product that
`separated was filtered, washed with ether and dried in a vacuum desiccator.
`
`30
`
`15
`
`Exh. 1002
`
`

`
`EXAMPLE?
`4-Dedimethy !amino-7 -dimeth y lamino-6-demethyl-6-deoxy-9-ethoxythi ocarbon y 1 thio-
`tetracycline sulfate
`
`A solution of 1.0 mmole of 4-dedimethylamino-7-dimethylamino-6-demethyl-6-
`deoxy-9-diazoniumtetracycline sulfate, from example 4, in 15 ml of water was added to a
`solution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water. The mixture was
`stirred at room temperature for one hour. The product separated and was filtered and dried
`in a vacuum desiccator.
`
`EXAMPLE SA
`General Procedure for Nitration
`
`To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml of concentrated
`sulfuric acid at 0°C was added 1 mmole of potassium nitrate with stirring. The reaction
`solution was stirred for 15 minutes and then poured into 100 g of chopped ice. The
`aqueous solution was extracted 5 times with 20 ml of butanol each time. The butanol
`extracts were washed three times with 10 ml of water each time, and concentrated in
`vacuo to a volume of25 ml. The light yellow crystalline solid which precipitated was
`fi ltered, washed with 2 ml of butanol and dried in vacuo at 60°C for 2 hours. This solid
`was a mixture of the two mononitro isomers.
`
`EXAMPLE8B
`4-Dedimethylamino-6-deoxy-9-nitrotetracycline
`
`5
`
`10
`
`15
`
`20
`
`25
`
`To 980 mg of the nitration product from 4-dedimethylamino-6-deoxytetracycline
`(a mixture ofthe 2 isomers) in 25 ml of methanol was added enough triethylamine to
`dissolve the solid. The filtered solution (pH 9.0) was adjusted to pH 5.2 with concentrated
`sulfuric acid. A crystalline yellow solid (236 mg.) was obtained (29% yield). The
`30 material at this point was quite pure and contained only small amounts of the 7-isomer.
`Final purification was accomplished by liquid partition clu-omatography using a
`
`16
`
`Exh. 1002
`
`

`
`-..
`
`\
`diatomaceous earth packed column and the so.lvent system: chloroform: butanol: 0.5 M
`phosphate buffer (pH 2) (16:1 :10).
`
`EXAMPLE9
`4-Dedirnethylamino-6-deoxy-7 -ni trotetracycline
`
`The methanol filtrate from example 8 was immediately adjusted to pH 1.0 with
`concentrated sulfuric acid. The light yellow crystalline solid, which was obtained as the
`sulfate salt. A purified free base was obtained by adjusting an aqueous solution of the
`sulfate salt (25 mg/ml) to pH 5.2 with 2 N sodium carbonate.
`
`EXAMPLE 10
`9-Amino-4-dedimethylamino-6-deoxytetracycline
`
`To a solution of 300 mg of the 9-nitro compound, prepared in example 8, in 30 ml
`of ethanol was added 50 mg of Pt02. The mixture was hydrogenated at atmospheric
`pressure until the theoretical amount of hydrogen was absorbed. The system is flushed
`with nitrogen, the Pt02 catalyst is filtered and the filtrate added dropwise to 300 ml of
`ether. The solid that separates is filtered and dried in a vacuum desiccator.
`
`EXAMPLE 11
`9-Acetamido-4-dedimethy lamino-6-deoxytetracycline sulfate
`
`To well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-6-
`deoxytetracycline sulfate, from example 10, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone
`was added 500 mg of sodium bicarbonate followed by 0.21 ml of acetyl chloride. The
`mixture was stirred at room temperature for 30 minutes, filtered and the filtrate was added
`dropwise to 500 ml of ether. The solid that separated was filtered and dried in a vacuum
`desiccator.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`17
`
`Exh. 1002
`
`

`
`···\ .
`
`EXAMPLE12
`4-Dedimethylamino-6-deoxy-9-diazoniumtetra.cycline sulfate
`
`To a solution of 0.5 g of 9-amino-4-dedimethylamino-6-deoxytetracycline sulfate,
`from example 10, in 10 ml ofO.lN hydrochloric acid in methanol cooled in an ice bath
`was added 0.5 ml of n-butyl nitrite. The solution was stirred at ice bath temperature for 30
`minutes and the poured into 250 ml of ether. The solid that separated was filtered, washed
`with ether and dried in a vacuum desiccator.
`
`EXAMPLE 13
`9-Azido-4-dedimethy lamino-6-deoxytetracycline sulfate
`
`To a solution of 0.3 mmole of 4-dedimethylamino-6-deoxy-9-
`diazoniumtetracycline sulfate, of example 12, 10 ml of 0. 1 N methanolic hydrogen
`chloride was added 0.33 mmole of sodium azide. The mixture was stirred at room
`temperature tor 1.5 hours. The reaction mixture was then poured into 200 ml of ether.
`The solid that separated was filtered and dried in a vacuum desiccator.
`
`EXAMPLE14
`9-Amino-8-chloro-4-dedimethylamino-6-deoxytetracycline sulfate
`
`1
`One gram of 9-azido-4-dedimethylamino-7 -dimethylamino-6-deoxytetracycline
`hydrochloride, from example 13, was dissolved in 10 ml of concentrated sulfuric acid
`saturated with HCL at 0°C. The mixture was stirred at ice bath temperature for 1.5 hours
`and then slowly added dropwise to 500 ml of cold ether. The solid that separated was
`filtered, washed and ether and dried in a vacuum desiccator.
`
`EXAMPLE 15
`4-Dedimethylamino-6-deoxy-9-ethoxythiocarbonylthiotetracycline sulfate
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`18
`
`Exh. 1002
`
`

`
`A solution of 1.0 mmole of 4-dedimethylamino-6-deoxy-9-diazoniumtetracycline
`sulfate, from example 12, in 15 ml of water was added to a solution of 1.15 mmole of
`potassium ethyl xanthate in 15 ml of water. The mixture was stirred at room temperature
`for one hour. The solid that separated was filtered and dried in a vacuum desiccator.
`
`EXAMPLE 16
`9-Dime thy lamino-4-dedimethy lalnino-6-deoxytetracycl i ne sulfate
`
`To a solution of 100 mg. of the 9-amino compound from example 10, in 10 ml of
`ethylene glycol monomethyl ether is added 0.05 m1 of concentrated sulfuric acid, 0.4 mi.
`of a 40% aqueous formaldehyde solution and 100 mg of a 10% palladium on carbon
`catalyst. The mixture is hydrogenated under atmospheric pressure and room temperature
`for 20 minutes. The catalyst was filtered and the filtrate was evaporated to dryness