United States Patent [19]
`Saino et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,061,787
`Oct. 29, 1991
`
`[54] NOVEL SPERGUALIN-RELATED
`COMPOUNDS AND COMPOSITIONS
`[75] Inventors: Tetsushi Saino, Yono; Tsugio
`Tomiyoshi; Kyuichi Nemoto, both of
`Tokyo; Yoshihisa Umeda, Otsu, all of
`Japan
`[73] Assignees: Nippon Kayaku Kabushiki Kaisha,
`Tokyo; Takara Shuzo Kabushiki
`Kaisha, Kyoto, both of Japan
`[21] Appl. No.: 363,538
`[22] Filed:
`Jun. 8, 1989
`[30]
`Foreign Application Priority Data
`Jun. 24, 1988 [JP]
`Japan .............................. .. 63-154590
`
`[51] Int. Cl.5 ................... .. C07C 237/00; A61K 37/02
`[52] US. Cl. .................................. .. 530/331; 530/323;
`564/158; 564/159; 514/885
`[58] Field of Search .............. .. 564/154, 158; 530/323,
`530/331; 514/885
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,518,532 5/1985 Umezawa et al_ ................ .. 564/159
`4,525,299 6/1985 Urnezawa et al. ................ .. 564/159
`
`OTHER PUBLICATIONS
`Peptide Chemistry, “Spergualin Analogues: Syntheses
`and Their Biological Activities”, 1987, pp. 671-674.
`Nishizawa et a1, Jol. of Antibiotics, vol. 41, No. 11,
`1988, pp. 1629-1643 “Synthesis and Biological Activity
`of Spergualin Analogues 1”.
`
`Primary Examiner-C. Warren Ivy
`Assistant Examiner—Raymond Covington
`Attorney, Agent, or Firm-—Nields &' Lamack
`
`ABSTRACT
`[57]
`The present invention relates to novel spergualin
`related compounds represented by the general formula
`[I]:
`
`wherein X is —(CH2)1_.5 or
`
`Y is a hydrogen atom or a residue obtained by removing
`a hydroxyl group from the carboxyl group of an amino
`acid or a peptide; in is O, 1 or 2 and n is l or 2, with the
`proviso that Y is not a hydrogen atom when n is 2 and
`m is O.
`This compounds are stable and exhibit a high immuno
`suppressive activity.
`
`6 Claims, N0 Drawings
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 1 of 10
`
`

`
`1
`
`NOVEL SPERGUALIN-RELATED COMPOUNDS
`AND COMPOSITIONS
`
`5
`
`BACKGROUND OF THE INVENTION
`Spergualin is an antibiotic produced by microorgan
`isms belonging to the genus Bacillus and many related
`compounds thereof have been synthesized.
`Among the spergualin and its related compounds 10
`which have been already known, highly active com
`pounds are poor in chemical stability, while chemically
`stable compounds are poor in activity. Therefore, it is
`expected to develop spergualin-related compounds
`which are chemically stable and highly active and yet
`has a low toxicity.
`
`15
`
`5,061,787
`2
`leucine, leucylphenylglycine, phenylglycylleucine, gly
`cylglycylglycine, phenylglycylphenylglycylphenylgly
`cine, phenylalanylphenylalanylphenylalanine and leu
`cylleucylleucine.
`Preferred examples of the amino acid and the peptide
`include phenylglycine, phenylalanine, leucine, aspartic
`acid, tryptophan, alanine and di- or tripeptides of them,
`among which phenylglycine tyrosine and leucylleucine
`are more preferred.
`Each of the novel spergualin-related compounds rep
`resented by the general formula [I] forms a salt together
`with an acid. The acid may be any organic or inorganic
`acid, as far as it is non-toxic. Although the inorganic
`acid is not particularly limited, preferred examples
`thereof include hydrochloric, sulfuric, nitric and phos
`phoric acids. Preferred examples of the organic acid
`include acetic, propionic, succinic, fumaric, maleic,
`malic, tartaric, glutaric, citric, benzenesulfonic, toluene
`sulfonic, methanesulfonic, ethanesulfonic, propanesul
`fonic, aspartic and glutamic acids, though the organic
`acid is not particularly limited.
`Representative examples of the compound repre
`sented by the general formula [I] are shown in Table 1.
`
`SUMMARY OF THE INVENTION
`The inventors of the present invention have exten
`sively studied and have found that a compound repre
`sented by the general formula [I]:
`
`NI-I
`
`OH
`
`wherein X is -(CH2)T~? or
`
`25
`
`TABLE 1
`
`Y is a hydrogen atom or a residue obtained by removing
`a hydroxyl group from the carboxyl group of an amino
`acid or a peptide; in is 0, l or 2 and n is l or 2, with the
`proviso that Y is not a hydrogen atom when n is 2 and
`m is 0, is stable and exhibits a high immunosuppressive
`activity. The present invention has been accomplished
`on the basis of this ?nding.
`
`35
`
`45
`
`No. of
`compound
`
`X
`
`n
`
`in
`
`l
`2
`
`3
`
`4
`s
`6
`
`7
`8
`
`9
`l0
`ll
`12
`13
`14
`
`15
`
`—(CI-I2)3—
`"
`
`"
`
`—CH2-
`"
`"
`
`-(CH2)2_
`"
`
`"
`—'(CI-I2)4-
`"
`—(CH2)4—
`—(CI-I2)5-
`"
`
`"
`
`l
`"
`
`"
`
`"
`"
`
`"
`"
`
`"
`
`'
`l
`'
`"
`
`"
`
`l
`"
`
`"
`
`"
`”
`
`”
`
`"
`l
`"
`'
`
`"
`
`Con?guration
`of hydroxyl
`group
`
`S, R or RS
`"
`
`Y
`
`H
`ph G
`
`"
`
`Lew-Len
`
`’
`
`”
`S, R or RS
`"
`
`H
`ph G
`Leu-Leu
`
`H
`ph G
`
`Leu-Leu
`H
`ph G
`Leu-Leu
`H
`ph G
`
`Leu-Leu
`
`H
`
`DETAILED DESCRIPTION OF THE
`'
`INVENTION
`In the general formula [I], the group Y is a hydrogen
`atom or a residue obtained by removing a hydroxyl
`group from the carboxyl group of an amino acid or a
`peptide. Examples of the amino acid and the peptide
`include the following compounds, the con?guration of
`which may be of S,R type or R,S type except glycine,
`B-alanine and 'y-aminobutyric acid:
`(1) amino acids
`alanine, arginine, ornithine, aspartic acid, asparagine,
`cysteine, cystine, glutamic acid, glutamine, pyro
`glutamic acid, glycine, histidine, lysine, proline, hy
`droxyproline, isoleucine, leucine, methionine, phenylal
`anine, phenyl-substituted phenylalanine, serine, threo
`nine, tryptophan, homoserine, tyrosine, valine, phe
`nylglycine, p-hydroxyphenylglycine, 4-hydroxymeth
`yl-3-hydroxyphenylglycine, B-alanine, 'y-aminobutyric
`acid and 3-amino2-hydroxy-4-phenylbutyric acid.
`(2) peptides
`Di- or tri-peptides obtained by the condensation of
`one or more of the above amino acids are preferable.
`Examples of the peptide include alanylalanine, leucyl
`leucine, valylvaline, phenylalanylphenylalanine, tyro
`syltyrosine, phenylglycylphenylglycine, glycylglycine,
`isoleucylisoleucine, leucylphenylalanine, phenylalanyl
`
`55
`
`65
`
`l6
`
`"
`
`'
`
`17
`
`18
`l9
`20
`
`21
`
`22
`23
`
`24
`25
`26
`
`27
`
`28
`
`"
`
`"
`—(CI-I2)2—
`"
`
`"
`
`-=(CI-I2)3—
`"
`
`"
`—(CHZ)4—
`"
`
`"
`
`"
`
`"
`l
`"
`
`"
`
`"
`"
`
`"
`l
`"
`
`"
`
`"
`
`0
`"
`
`"
`
`"
`"
`
`"
`0
`"
`
`"
`
`"
`
`"
`'
`
`’
`
`"
`"
`
`ph G
`
`_ Leu-Leu
`H
`ph G
`
`Leu-Leu
`
`H
`ph G
`
`"
`S, R or RS
`"
`
`Leu-Lcu
`H
`ph G
`
`"
`
`Leu-Leu
`
`"
`
`"
`
`H
`
`29
`
`30
`
`~
`
`"
`
`"
`
`"
`
`"
`
`"
`
`"
`
`ph G
`
`Leu-Leu
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 2 of 10
`
`

`
`5,061,787
`
`4
`about —50° to 100° C. The inert solvent may be water
`or a hydrophilic organic solvent and examples of the
`solvent include lower alcohols such as methanol and
`ethanol; ketones such as acetone and methyl ethyl ke
`tone; amides such as dimethylformamide and dimethyl
`acetamide; cyclic ethers such as tetrahydrofuran and
`dioxane; lower aliphatic acids such as acetic and trifluo
`roacetic acids; liquid ammonia and liquid hydrogen
`fluoride.
`The spergualin-related compound represented by the
`general formula [I] can be isolated from the reaction
`mixture of the above removal step as follows. When, for
`example, the removal is carried out by catalytic reduc
`tion with palladium black, after the removal of the cata
`lyst from the reaction mixture by ?ltration, the ?ltrate is
`concentrated under a reduced pressure and the residue
`is puri?ed by an ordinary method using CM
`Sephadex ® (Na+) or Sephadex ® LH-ZO (see T,
`Takeuchi et al., J. Antibiotics, 34, 1619 (1981)). Alter
`nately, when the removal is carried out by the use of
`trifluoroacetic acid, the reaction mixture is concen
`trated under a reduced pressure and the residue is puri
`?ed by an ordinary method as described above.
`According to the isolation method as described
`above, the spergualin-related compound represented by
`the general formula [I] is obtained as a hydrochloride.
`The conversion of the hydrochloride into another salt
`may be carried out by dissolving the hydrochloride in
`water, passing the obtained aqueous solution through a
`strongly basic ion exchange resin to collect fractions
`containing an objective compound, neutralizing the
`fractions by the addition of an objective acid or a solu
`tion thereof in water or a hydrophilic organic solvent
`such as methanol, ethanol, acetone, tetrahydrofuran or
`dioxane and evaporating the neutralized solution under
`a reduced pressure to dryness. Alternatively, when the
`neutralized solution contains an organic solvent, the
`neutralized solution is distilled under a reduced pressure
`to remove the solvent and the residue is freeze-dried to
`obtain an objective salt. Further, the conversion may be
`carried out by adding silver hydroxide or an aqueous
`solution of silver oxide to the hydrochloride of the
`compound of the general formula [I] to thereby neutral
`ize the hydrochloric acid, ?ltering out insoluble silver
`chloride, adding an objective acid to the ?ltrate to form
`an objective salt and freeze-drying it.
`The objective salt prepared above is sometimes pres
`ent as a hydrate depending upon the treatment condi
`tions.
`The protected spergualin-related compound repre
`sented by the general formula [II] to be used as a raw
`material in the present invention can be prepared as
`follows:
`(a) a compound represented by the general formula
`[II] wherein R1 and R2 are each an amino-protec
`tive group can be prepared according to the pro
`cess disclosed in EP-A-l05l93 by reacting a di
`protected spermidine represented by the general
`formula [III]:
`
`wherein R1 and R2’ are each an amino-protective
`group, with a reactive derivative of a protected
`
`31
`33
`
`34
`
`34
`
`No. of
`compound
`
`X
`
`n m
`
`-(CH2)3—-
`"
`
`2
`2
`
`2
`
`O
`0
`
`0
`
`Con?guration
`of hydroxyl
`group
`
`'
`
`Y
`
`ph G
`Leu-Leu
`
`ph G
`
`2
`
`0
`
`"
`
`H
`
`36
`
`"
`
`2
`
`O
`
`Leu-Leu
`
`In Table l, phG refers to
`
`and LeuLeu refers to
`
`15
`
`25
`
`35
`
`The compound represented by general formula [I]
`can be prepared by removing protective groups from a
`protected compound represented by the general for
`mula [II]:
`
`NH
`
`OH
`
`45
`
`wherein X is -(CH2)Eor
`
`55
`
`R1 is an amino-protective group; R2 is an amino-protec
`tive group or a residue obtained by removing a hy
`droxyl group from the carboxyl group of an amino acid
`or peptide having a protected amino group (the side
`chain of the residue may be protected); m is 0, l or 2 and
`n is 1 or 2, with the proviso that Y is not a hydrogen
`atom when n is 2 and m is O.
`'
`The removal of the protective group may be carried
`out by reduction, acidolysis, hydrolysis or the like.
`The removal is generally carried out in an inert sol
`vent at a temperature of from —60° C. to the boiling
`point of the solvent, preferably at a temperature of
`
`65
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 3 of 10
`
`

`
`5,061,787
`5
`amino acid represented by the general formula
`[IV]:
`
`6
`vents may be each used alone or as a mixture thereof
`with water if it is miscible with water.
`The amino-protective group to be used in the present
`invention includes a benzyloxycarbonyl group, substi
`tuted benzyloxycarbonyl groups, such as a p-methox
`ybenzyloxycarbonyl group, and .t-butyloxycarbonyl,
`t-amyloxycarbonyl, formyl, trityl and o-nitrophenyl
`sulfenyl groups.
`The protective group for the carboxyl group of the
`amino acid side chain includes lower alkyl, t-butyl,
`benzyl and substituted benzyl groups; the one for the
`hydroxyl group thereof includes t-butyl and benzyl
`groups; the one for the mercapto group includes benzyl
`and p-methoxybenzyl groups; the one for the imidazole
`group includes benzyloxycarbonyl, benzyl and tosyl
`groups and the one for the guanidine group includes
`nitro, tosyl and t-butyloxycarbonyl groups, though they
`are each not always limited to the above groups.
`The compounds of the present invention as prepared
`above may be formulated into a drug for oral or paren
`teral administration according to an ordinary method, if
`necessary, by the use of a suitable carrier.
`When the drug is an injection, it~is preferred that the
`content of an active ingredient therein be generally 0.1
`to 30% by weight, still preferably 1 to 10% by weight.
`When the drug is one for oral administration, it may
`be in the form of tablet, capsule, powder, granule or dry
`syrup. The content of an active ingredient in the cap
`sule, granule or powder is generally 5 to 100% by
`weight, preferably 25 to 100% by weight.
`Although the dose varies depending upon the age,
`weight and symptom of a patient and the object of
`treatment, it is 1 to 100 mg/kg.day in parenteral admin
`istration, while it is 5 to 1000 mg/kg.day in oral adminis
`tration.
`
`Effect
`1 Stability of the compound of the invention in water
`(1) Method of experiment
`A compound of the present invention was dissolved
`in water to obtain a 0.5 w/w % aqueous solution. This
`aqueous solution was kept at 40: 1° C., sampled at
`intervals of a determined time and subjected to high
`performance liquid chromatography to determine the
`peak area ratio. The residual rate was calculated on the
`basis of this ratio.
`(2) Results of experiment
`The residual rates of the compounds after the lapse of
`a predetermined time are shown in Table 2 by assuming
`the residual rate at the start of the experiment to be
`100%.
`
`wherein P is an amino-protective group different
`from R1 or R2’ and n and m are as de?ned above, to
`obtain a compound represented by the general 10
`formula:
`
`OH
`
`15
`
`wherein P, R] and R2’ are each as de?ned above, 20
`freeing the compound of the amino-protective
`group P and reacting the resulting compound with
`a reactive derivative of an w-guanidino fatty acid
`represented by the general formula [V]:
`
`25
`
`H2N—?—NHX'-(CH2)3—COOH
`
`NH
`
`30
`
`wherein X is as de?ned above,
`or
`(b) a compound represented by the general formula
`[II] wherein R2 is a residue obtained by removing a
`hydroxyl group from the carboxyl group of an
`amino acid or peptide having a protected amino 35
`group (the side chain of the residue may be pro
`tected) can be prepared by reacting a compound
`represented by the formula [I] wherein Y is a hy
`drogen atom with a reactive derivative of a pro
`tected amino acid or peptide represented by the 40
`general formula [VI]:
`
`45
`
`wherein Y is as described above.
`The condensation in the above processes (a) and (b)
`may be carried out by any method which has been
`ordinarily used for the preparation of a peptide. Exam
`ples of the method include the carbodiimide method
`using dicyclohexylcarbodiimide or l-ethyl-3-(3-dime- 50
`thylaminopropy1)-carbodiimide; the azide method using
`a hydrazide; the mixed anhydride method using ethyl
`chlorocarbonate or isobutyl chlorocarbonate; the active
`caster method using cyanomethyl ester, vinyl ester,
`substituted or unsubstituted phenyl ester, thiophenyl 55
`ester or hydroxysuccinimide ester; the O-acylhydroxya
`mine derivative method using acetoxime or cyclohexa
`none oxime; the N-acyl compound method using car
`bonyldiimidazole; and the carboxylic acid activation
`method using 1,3-thiazolidine-2thione. The solvent to 60
`be used in the condensation may be any one which is
`ordinarily used in the formation of a peptide. Examples
`thereof include ethers such as diethyl ether, tetrahydro
`furan and dioxane; esters such as ethyl acetate; ketones
`such as acetone and methyl ethyl ketone; halogenated 65
`hydrocarbons such as methylene chloride and chloro
`form; amides such as dimethylformamide and dimethyl
`acetamide; and nitriles such as acetonitrile. These sol
`
`TABLE 2
`Residual rate of the compound of the invention in 0.5% aqueous
`solution (%)
`Hours
`72
`
`No.
`of compound
`
`0
`
`'
`24
`
`48
`
`96
`
`168
`
`1
`4
`16
`19
`22
`25
`23
`
`100
`"
`
`"
`"
`"
`"
`
`99.76
`102.50
`93.08
`99.38
`100.05
`98.19
`99.12
`
`101.21
`99.38
`101.12
`99.05
`101.23
`99.33
`100.87
`
`99.98
`99.05
`99.01
`101.20
`98.09
`101.56
`99.46
`
`99.22
`101.10
`98.15
`99.03
`99.87
`99.48
`98.92
`
`99.05
`99.23
`100.91
`99.86
`98.07
`99.15
`99.08
`
`As shown in Table 2, the compounds of the present
`invention are substantially stable even after the lapse of
`one week.
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 4 of 10
`
`

`
`7
`Activity
`The physiological activity of the compound accord
`ing to the present invention will now be experimentally
`illustrated.
`1. Method of experiment
`(a) A skin piece (5X10 mm) of the tail of a male
`WKAH (RTlk) rat (of 7 to 10 weeks of age) was trans
`planted to the back of a male F 344 (RTl 1v1) rat (of 7 to
`10 weeks of age) to carry out the rat skin transplantation
`test.
`A compound of the present invention was intraperito
`neally administered to the latter rat to determine the
`immunosuppressive effect thereof. The effect is shown
`by the survival times of allografts. In this experiment, a
`known compound represented by the general formula
`(I) wherein X is -(CH2)3-, n is 2, m is 0 and Y is H was
`used as a control
`(b) The results of the rat skin transplantation test are
`shown in Table 3.
`
`15
`
`20
`
`TABLE 3
`Mean survival time (days)
`Dose (mg/kg)
`3.0
`6.0
`
`0
`
`7.0 i 0.6
`
`7.6 i 1.1
`
`8.5 i 0.7
`
`69 i 0.7
`
`7.7 i 0.8
`
`10.3 i 1.0
`
`—
`6.4 i 0.5
`
`9.6 i 0.5
`8.5 i- 0.6
`
`17.0 i 4.0
`14.0 ;L 2.8
`
`Control
`n = 2,
`m = O,
`Y = H
`Compound 1
`(S-type)
`Compound 13
`(S-type)
`(RS-IYPB)
`
`12.0
`
`9.3 i 2.5
`
`14.5 i 0.8
`
`-
`—
`
`35
`
`40
`
`45
`
`EXAMPLE
`10-{N-(7-Guanidinoheptanoyl)-'y-amino-B-(S)hydrox
`ybutyryl}- 1 ,5, IO-triazadecane trihydrochloride
`(Compound 1)
`6.7 g (9.3 mmol) of 10-{N-(7-guanidinoheptanoyl)-y
`amino-B-(S)-hydroxybutyryl}-1,5-dibenzyloxycarbo
`ny1-l,5,10-triazadecane hydrochloride was dissolved in
`60 ml of methanol, followed by the addition of 1.3 g of
`acetic acid and 0.9 g of palladium black. The catalytic
`reduction was carried out at a room temperature under
`normal pressure for 4 hours.
`The reaction mixture was ?ltered to remove the cata
`lyst and the ?ltrate was concentrated under reduced
`pressure to give 5.9 g of an oil (the yield was quantita
`tive). This oil was dissolved in 60 ml of distilled water,
`placed on a column packed with 500 ml of Sephadex ®
`C-25 (Na+) and subjected to gradient elution between
`2500 ml of distilled water and 2500 ml of a 1.0 M aque
`ous solution of sodium chloride to collect fractions
`containing an objective compound. The fractions were
`combined and dryed under reduced pressure, followed
`by the addition of methanol. The obtained mixture was
`?ltered to remove an insoluble sodium chloride. The
`isolation of the objective compound from the obtained
`oil was carried out as follows. In order to remove a
`trace amount of sodium chloride remaining in the oil,
`the oil was dissolved in 20 ml of methanol, placed on a
`column packed with 300 ml of Sephadex ® LH-2O and
`eluted with methanol to obtain fractions containing the
`objective compound. The fractions were combined and
`concentrated under reduced pressure to obtain an oil.
`This oil was dissolved in 35 ml of distilled water and
`?ltered to remove an insoluble material. The ?ltrate was
`
`5,061,787
`8
`freeze-dried to give 3.77 g of the title compound (yield:
`55.9%).
`NMR(D2O, external TMS) 8: l.5~3.1 (m, 18H),
`3.2~4.l (m, 12H), 4.4~4.8 (m, H)
`IR(KBr) v(cm“1)=332O, 2930, 1640, 1545, 1460,
`1365, 1170, 1080 [<1]1>20+2.9° (c=,1.02, H2O)
`Referential Example
`Synthesis of 10-{N-(7-guanidinoheptanoyl)-'y-amino
`B-(S)-hydroxybutyryl}-1,5-dibenzyloxycarbonyl
`1,5,10-triazadecane hydrochloride
`(1)
`10-(7-N-tert-butyloxycarbonyl-B-(S)-hydrox
`ybutyryl)-1,5-dibenzyloxycarbonyl-1,5,10-triazadecane
`2.63 g (12.0 mmol) of 'y-N-tert-butyloxycarbonylB
`(S)-hydroxybutyric acid and 2.76 g (18.0 mmol) of N
`hydroxybenzotriazole were dissolved in a mixture of 50
`ml of dichloromethane and 30 ml of tetrahydrofuran.
`The obtained solution was cooled with ice, followed by
`the addition of 3.96 g (18.0 mmol) of dicyclohexylcar
`bodiimide. The obtained mixture was reacted under
`cooling with ice for 15 minutes. A solution of 5.4 g (14.4
`mmol) of 1,5-dibenzyloxycarbonyl~1,5,lO-triazadecane
`hydrochloride and 1.6 g (15.8 mmol) of triethylamine in
`dichloromethane was added to the reaction mixture
`obtained above under cooling with ice to carry out the
`reaction at a room temperature overnight. The reaction
`mixture was ?ltered to remove a precipitate and the
`?ltrate was concentrated under reduced pressure. The
`obtained oily residue was dissolved in 200 ml of ethyl
`acetate and washed with distilled water twice. The
`organic layer was dried over anhydrous sodium sulfate
`and ?ltered to remove the drying agent. The ?ltrate was
`concentrated under reduced pressure to give 11.0 g of a
`pale yellow oil.
`This oil was placed on a column placed with Silica
`gel ® 60 (mfd. by Merck & Co.) and eluted with chlo~
`roform and a chloroform/methanol (20:1 v/v) mixture
`successively to give 7.0 g of an oil (yield: 94.85%).
`NMR (CD3OD) 8=0.9~2.0 (m, 15H), 14.2 (s, 9H),
`2.1 ~2.5 (d, 2H, J=5Hz), 2.6~9.0 (b, 4H), 2.8~3.5 (m,
`10H), 3.7~4.3 (m, H), 5.03 (s, 2H), 5.07 (s, 2H), 7.28 (s,
`10H).
`TLC (chloroformzmethanol: 10:1, v/v) Rf = 0.42
`(2)
`10-('y-amino-B-(S)-hydroxybutyryl)-1,5-diben
`zyloxycarbonyl-1,5,10-triazadecane hydrochloride
`7.0 g (11.38 mmol) of 10¢('y-N-tert-buty1oxycarbonyl
`B-(S)-hydroxybutyryl)-1,5-dibenzy1oxycarbonyl1,5,10
`triazadecane was dissolved in 10 ml of dichloromethane
`to give a solution. 10 ml of a 4N solution of hydrochlo
`ric acid in dioxane was added to the solution under
`cooling with ice to carry out the reaction at a room
`temperature for 3 hours.
`The reaction mixture was concentrated‘ under re
`duced pressure to give an oil. This oil was washed with
`100 m1 of n-hexane and concentrated under reduced
`pressure to give 6.3 g of an oil (the yield was guantita
`tive).
`TLC (ch1oroform:methanol:17%
`monia=6:2.5:0.5 v/v) Rf =0.48
`(3) 10-{N-(7-guanidinoheptanoyD-y-amino-B-(S)hy
`droxybutyryl}-1,5-dibenzyloxycarbonyl-1,5,10-triaza
`decane hydrochloride
`2.92 g (13.05 mmol) of 7-guanidinoheptanoic acid
`hydrochloride was dissolved in 30 m1 of dimethylform
`amide to give a solution. 1.8 g (15.64 mmol) of N
`hydroxysuccinimide and 3.23 g (15.64 mmol) of N,N'
`dicyclohexylcarbodiimide were added to the solution
`
`aqueous am
`
`65
`
`NOVARTIS EXHIBIT 2027
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`Page 5 of 10
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`

`
`9
`under cooling with ice to carry out the reaction at a
`room temperature overnight. The precipitate was ?l
`tered off and the ?ltrate was used as such in the follow
`ing step.
`6.3 g (11.38 mmol) of oily 10-(‘y-arnino-B-(S)-hydr0x
`ybutyryl)1,5-dibenzy1oxycarbonyl-1,5,10-triazadecane
`hydrochloride was dissolved in 40 ml of dimethylform
`amide to give a solution. 1.38 g (13.66 mmol) of triethyl
`amine was added to the solution under cooling with ice,
`followed by the addition of the above solution of an
`ester of 7-guanidinoheptanoic acid hydrochloride with
`N-hydroxysuccinimide in dimethylformamide. The ob
`tained mixture was reacted at a room temperature over
`night and concentrated under reduced pressure to ob
`tain an oily residue. This residue was washed with 100
`ml of n-hexane twice and concentrated under reduced
`
`10
`
`15
`
`5,061,787
`
`10
`pressure to obtain a residue. 150 ml of distilled water
`was added to the residue. The mixture was ?ltered to
`remove an insoluble material and the ?ltrate was con
`centrated under reduced pressure to give 145 g of an
`oil.
`This oil was column-chromatographed over Silica
`gel 60 mfd. by Merck & Co.) and,developed with a
`chloroform/methanol/ 17% aqueous ammonia (6:2.5:0.5
`v/v) mixture to give 6.7 g of an oil (yield: 81.7%).
`NMR (CD3OD) 8=0.9~2.0 (M, 12H), 2.0~2.5 (m,
`4H), 2.6~3.5 (m, 12H), 3.5~9.0 (b, 9H), 3.6~4.2 (m,
`H), 5.03 (s, 2H), 5.07 (s, 2H), 7.27 (s, 10H).
`IR(KBr) v(cm—1)=3290, 2930, 1640, 1535, 1420,
`1360, 1245, 1135, 1075, 1015.
`TLC (chl0roform:methano1:17%
`monia=6:2.5:0.5 v/v) Rf=0.48.
`
`aqueous am
`
`lO-{N-(S-Guanidinopentanoyl)-'y-amino
`10~{N-(5-GuanidinopentanoyD-y-amino-a
`hydroxybutyryU-l,S-dibenzyloxycarbonyl- a-hydroxybutyryU-l.5,l0-triazadecane
`No. of
`compound 1,5-10-triazadecane hydrochloride
`trihydrochloride
`4
`NMR (CD3OD)
`NMR (D20, external TMS)
`8 = 0.9~2.5 (m, 14H), 2.6~
`5 = 1.6~3.1 (m, 14H), 3.3
`3.6 (m, 1211), 3.s~4.2 (m,
`4.1 (m, 1211), 4.5~4.9
`H), 3.6~9.0 (b, 9H), 5.03
`(m, H).
`(s, 2H), 5.05 (s, 2H),
`7.27 (s, 1011).
`IR (KBr)
`v (cm-1) = 3300, 2940, 1640,
`1535, 1455, 1425, 1365,
`1215, 1150, 1080, 1020,
`TLC (chloroform:methanol:17%
`aqueous ammonia = 6:2.5:0.5 v/v)
`Rf = 0.39.
`
`IR (KBr)
`v (cm-1) = 3290, 2940, 1640,
`1540, 1450, 1365, 1260,
`1225, 1165.
`
`IO-{N-[4-(4-Guanidinophenyl)butyryl]-'y
`amino-B-hydroxybutyry1}-1,5-di-
`benzyloxycarbony1-1,5,IO-triazadecane
`No. of
`compound hydrochloride
`
`10-{N-[4-(4-Guanidinopheny1)butyryl]
`‘y-amino-B-hydroxybutyryl}-l,5,10
`triazadecane trihydrochloride
`
`16
`
`NMR (CD3OD)
`5 = 0.9~3.7 (m, 24H), 3.5~
`9.0 (b, 9H), 3.8~4.4 (m,
`H), 5.03 (s, 2H), 5.07
`(s, 2H), 7.0~7.5 (1n, 41-1),
`7.3 (s, 10H)
`IR (KBr)
`v (cm-1) = 3330, 2940, 1645,
`1540, 1425, 1355, 1250,
`1140, 1080.
`TLC (chlorofonn:methanol:17%
`aqueous ammonia = 6:1.5-.0.25 v/v)
`Rf = 0.23
`
`NMR (D20, external TMS)
`3 = 1.7~3.3 (m, 14H), 3.3~
`4.0 (m, 10H), 4.3~4.8
`(m, H), 7.6~8.0 (m, 4H)
`
`IR (KBr)
`v (cm-1) = 3290, 2940, 1630,
`1545, 1515, 1440, 1255,
`1170, 1085.
`
`10-{N-(6-GuanidinohexanoyD-B-amino-a
`hydroxypropanoyl}-l,5-dibenzy1oxy-
`_
`carbonyl-1,5,10-triazadecane
`No. of
`compound hydrochloride
`19
`NMR (CD3OD)
`as = 1.0~2.0(m, 1211), 2.0~
`2.5 (1'11, 211), 2.8~3.6 (m,
`1211), 3.9~4.3 (m, 11),
`4.o~9.0 (b, 91-1). 5.03 (s,
`21-1), 5.07 (s, 2H), 7.28
`(s, 10H)
`IR (KBr)
`V (cm*‘) = 3320, 2940, 2470,
`1650, 1530, 1450, 1425,
`1360, 1255, 1215, 1145,
`1025.
`TLC (ch1oroform:methano1:17%
`aqueous ammonia = 6:2.5:0.5 v/v)
`
`10-{N-(6-GuanidinohexanoyD-B-amino-a
`hydr0xypr0panoy1}-1,5,IO-triamdecane
`trihydrochloride
`NMR (D20, external TMS)
`s = 1.6~3.l (m, 1411), 3.3~
`4.1 (m, 1011), 3.9~4.3
`(11211.1 = s 111, 4.6~
`5.0 (1, 11, J = s 112).
`
`IR (KBr)
`v (cm-1) = 3260, 2930, 2770,
`2420,2180, 1615, 153s,
`1455, 1370, 1325, 1270,
`1235, 1160.
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 6 of 10
`
`

`
`11
`
`5,061,787
`
`12
`
`-continued
`l0{N-(6—GuanidinohexanoyD-B-amino-a
`hydroxypropanoyl}- l ,S-dibenzyloxy
`carbonyl-1,5,l0-triazadecane
`No. of
`compound hydrochloride
`
`hydroxypropanoyll-l,5,l0-triazadecane
`trihydrochloride
`
`Rf = 0.56
`
`10-{N-(7-GuanidinoheptanoyD-B-amino-a
`hydroxypropanoyl}-1,5-dibenzyloxy-
`carbonyl-1,5,10-triazadecane
`No. of
`compound hydrochloride
`
`22
`
`NMR (CD3OD)
`a = 0.9~2.0 (m, 14H), 2.0~
`2.5 (m, 2H), 2.8~3.8 (m.
`12H), 3.9~9.0 (b, 9H),
`4.0~4.4 (t, H, J = 5 Hz),
`5.03 (s, 2H), 5.07 (s, 2
`H), 7.3 (s, 1011),
`IR (KBr)
`v (cm-1) = 3310. 2930, 2860,
`i655, 1535, 1475, i425,
`1385, 1260, 1215, 1145,
`1120, 1025.
`TLC (chloroform:methanol:17%
`aqueous ammonia = 6:2.5:0.5 v/v)
`Rf = 0142.
`
`10{N-(7-Guanidinoheptanoyl)-B~amino
`a-hydroxypropanoyl}-1,5,10
`triazadecane lrihydrochloride
`NMR (D20, exlemal TMS)
`s = 1.4~3.1 (m, 16H), 3.3~
`4.0 (m, 1011), 3.9~4.2
`(d, 211, J = 5 111), 4.6~
`5.0 (1, 11, J = 5 Hz).
`
`IR (KBr)
`v (cm-1) = 3350, 2930, 1645,
`1530, 1460, 1165, 1110.
`
`No. of
`compound
`25
`
`lO—{N-(8-GuanidinooctanoyD-B-amino-a
`hydroxypropanoyU-1,5-dibenzyloxy
`carbonyl-1,5, l0~triazadecane
`hydrochloride
`
`10{N-(8-GuanidinooclanoyD-B-amino-a
`hydroxypropanoyl}-l,5, l0
`triazadecane trihydrochloride
`
`NMR (CD3OD)
`5 = 0.9~2.0 (m, 16H), 2.0~
`2.5 (m, 2H), 2.8~3.7 (m,
`12H), 3.9~4.3 (m, H),
`3.9~9.0 0), 9H), 5.03 (s,
`2H), 5.07 (s, 2H), 7.27
`(s, 10H).
`IR (KBr)
`V (cm*‘) = 3310, 2930, 2860,
`1650, 1530, 1420, 1360,
`1260, 1140.
`TLC (chloroformzmethanolzl7%
`aqueous ammonia = 612.505 v/v)
`Rf = 0.63.
`
`NMR (D20, external TMS)
`a = 1.5~3.0 (m, 18H), 3.3~
`4.0 (m, 1011), 3.s~4.2
`
`5.0 (t, H, J = 5 Hz).
`
`IR (K131)
`v (cm-1) = 3310, 2930, 1650,
`1530, 1460, 1360, 1260,
`1165, 1110.
`
`No. of
`compound
`28
`
`l0‘{N-[4-(4-Guanidinophenyl)butyryl]-B
`amin0-a-hydroxypropanoyl}-1,5-di
`benzyloxycarbonyl- 1, 5, lo‘triazadecane
`hydrochloride
`NMR (CD30D)
`6 = 0.9~3.7 (m, 22H) 3.5»
`9.0 (b, 9H), 3.9~4.3 (m,
`H), 5.03 (s, 2H), 5.07
`(s, 2H), 7.0~7.5 (m, 4H),
`7.3 (s, 10H)
`IR (KBr)
`v (cm-1) = 3300, 2930, 1660,
`1510, 1425, 1255, 1140,
`1020.
`TLC (chloroformzmethqnolzl7%
`aqueous ammonia = 6:l.5:0.25 v/v)
`Rf = 0.2
`
`10-{N-[4-(4-Guanidinophenyl)butyryl]
`B~amino-a-hydroxypropanyl}- 1,5, 10—
`triazadecane trihydrochloride
`
`NMR (D20, external TMS)
`a = 1.7~3.3 (m, 12H), 3.3~
`4.0 (m, 8H), 3.s~4.1
`
`4.9 (r, H, J = 5 Hz), 7.5~
`8.0 (m, 41-1).
`IR (KBr)
`v (cm-1) = 3320, 2950, 1640,
`1535, 1440, 1250, 1110.
`
`We claim:
`l. A novel spergualin-related compound represented
`by the general formula [I]:
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 7 of 10
`
`

`
`13
`
`NH
`
`OH
`
`5,061,787
`14
`or a pharmacologically acceptable salt thereof.
`5. A novel spergualin-related compound represented
`by the formula:
`>
`
`wherein X is -(CH2)15- or
`
`NH
`
`OH
`
`15
`
`Y is a hydrogen atom or a residue obtained by removing
`a hydroxyl group from the carboxyl group of an amino
`acid di or tripeptide; m and n are both 1, or a pharmaco
`logically acceptable salt thereof.
`2. A novel spergualin-related compound or a pharma
`cologically acceptable salt thereof ‘as set forth in claim
`1, wherein Y is a hydrogen atom.
`3. A novel spergulain-related compound or a pharma
`cologically acceptable salt thereof as set forth in claim
`1, wherein X is -(CH2)35-, Y is a hydrogen atom and m
`and n are each 1.
`_
`25
`4. A novel spergualin-related compound represented
`by the formula:
`
`or a pharrnacologically acceptable salt thereof.
`6. An immunosuppressant containing a novel sper
`gualin-related compound represented by the general
`formula [I]:
`
`NH
`
`OH
`
`wherein X is -(CH2)15- or
`
`Y is a hydrogen atom or a residue obtained by removing
`a hydroxyl group from the carboxyl group of an amino
`acid or di- or tri-peptide; m and n are both 1, or a phar
`macologically acceptable salt thereof as an active ingre
`dient.
`
`* t i
`
`t #
`
`35
`
`45
`
`50
`
`55
`
`65
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 8 of 10
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`5,061 , 787
`Page 1 of 2
`
`PATENT NO. :
`
`DATED
`
`; October 29, 1991
`
`‘
`
`INVENTUR(S) ; Tetsushi Saino , Tsugio Tomiyoshi , KYuiChi NeIHOtO and YOSIIJIihiSa
`meda
`it is certified that error appears in the above-identified patent and that said Letters Patent is hereby
`corrected as shown below:
`
`On the title page:
`Attorney, Agent , or Firm — "Nields & Lamack" should read ——Nields &
`
`Lemack——
`
`Column 8 line 40 "(111, 15H) , 14. 2" should read ——(m, 15H) ,
`
`1 .42-
`
`vColumn 13 line 7 "wherein X is —(CH
`
`or" should read ——wherein X
`
`Column 13 line 23 "X is —(CH2)35—," should read -—X is —(CH2)3_ 5—,-
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 9 of 10
`
`

`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENT NO. I
`
`5,061 , 787
`
`_
`
`Page 2 of 2
`
`DATED
`
`: October 29, 1991
`
`|NVENTUR(S) I
`
`Tetsushi Saino, Tsugio Tomiyoshi, Kyuichi Nemoto and
`
`_
`
`‘
`
`_
`
`Yoshihisa Umeda
`
`It rs certlfred that error appears m the above-ldentlfled patent and that sad Letters Patent 1s hereby
`corrected as shown below:
`Column 14 line 21 "X is —(CHZ) 15- or" should read ——X is ——(CI-I )
`..
`or"
`2 1-5
`
`_
`
`. ‘
`
`,_
`
`_.
`
`Signed and Sealed this
`
`Second Day of February, 1993
`
`Arrest:
`
`Arresting Oj?cer
`
`Acting Commissioner of Patents and Trademarks
`
`STEPHEN G. KUNIN
`
`NOVARTIS EXHIBIT 2027
`Par v Novartis, IPR 2016-00084
`Page 10 of 10