United States Patent [191
`Tomiyoshi et al.
`
`llllllllllllllllllllllllllllllllllllllllllllllllllllIIIIHIIIIHIIIIIHIIII
`5,137,917
`Aug. 11, 1992
`
`US005137917A
`[11] Patent Number:
`[45] Date of Patent:
`
`[54] SPERGUALIN-RELATED COMPOUND AND
`USE THEREOF
`[75] Inventors: Tsugio Tomiyoshi; Takako Mae, both
`of Tokyo; Tetsushi Saino, Yono;
`Yoshihisa Umeda, Otsu, all of Japan
`[73] Assignees: Nippon Kayaku Kabushiki Kaisha,
`Tokyo; Takara Shuzo Co., Ltd.,
`Kyoto, both of Japan
`[21] Appl. No.: 731,805
`[22] Filed:
`Jul. 17, 1991
`[30]
`Foreign Application Priority Data
`Jul. 20, 1990 [JP]
`Japan ................................ .. 2492443
`
`[51] Int. Cl.5 . . . .
`
`. . . . . . . . . . .. A61K 31/195
`
`[52] US. Cl. .................................. .. 514/563; 514/558;
`562/439; 562/560; 554/53
`[58] Field of Search .............. .. 562/560, 439; 514/563,
`514/558; 260/404.5
`References Cited
`U.S. PATENT DOCUMENTS
`
`[56]
`
`4,430,346 2/1984 Umezawa et a1. ................ .. 424/311
`4,518,532 5/1985 Umezawa et a1. .
`260/4045
`4,529,549 7/ 1985 Umezawa et a1. .
`260/4045
`4,556,735 12/1985 Umezawa et a1.
`.... .. 564/157
`4,851,446 7/1989 Urnezawa et a1, ................ .. 514/620
`
`FOREIGN PATENT DOCUMENTS
`
`0105193 4/1984 European Pat. Off. .
`0241797 3/1987 European Pat. Off. .
`0213526 10/1987 European Pat. Off. .
`0309971 4/1989 European Pat. Off. .
`
`0347820 12/1989 European Pat. Off. .
`OTHER PUBLICATIONS
`The Journal of Antibiotics, vol. XL, No. 9, pp.
`1303-1315 and 1316-1324.
`The Journal of Antibiotics, vol. XLI, No. 11, pp.
`1629-1643.
`Patent Abstracs of Japan, vol. 10, No. 31 (C-327)
`(2088), Feb. 6, 1986; & JP—A—60185758 (Biseibutsu
`Kagaku Kenkyukai).
`Primary Examiner—Michael L. Shippen
`Attorney, Agent, or Firm—Nields & Lemack
`
`ABSTRACT
`'
`[57]
`Novel spergualin-related compounds represented by
`the general formula (I):
`
`wherein X represents —~(CH2)1_5-— or a phenylene
`group which may be substituted; in represents 0, 1 or 2;
`n represents 1 or 2; and R1 represents —(CH2)1.
`3—COOH, and pharmacologically acceptable salts
`thereof, possess an immunopotentiating activity, and are
`expected to be useful as immunopotentiators applicable
`to warm blooded animals.
`
`9 Claims, N0 Drawings
`
`NOVARTIS EXHIBIT 2028
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`Page 1 of 11
`
`

`
`5,137,917
`2
`pharmaceutically acceptable salts thereof, together
`with a pharmaceutically carrier.
`A third aspect of the present invention relates to a -
`method for immunopotentiation which comprises ad
`ministering an effective amount of the novel spergualin
`derivative represented by the general formula (I) or the
`pharmaceutically acceptable salt thereof to a warm
`blooded animal having a reduced immunity.
`A fourth aspect of the present invention relates to use
`of the novel spergualin derivative represented by the
`general formula (I) or the pharmaceutically acceptable
`salt thereof for the production of a pharmaceutical com
`position for immunopotentiation.
`
`‘1
`
`SPERGUALIN-RELATED COMPOUND AND USE
`THEREOF
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The present invention relates to a novel spergualin
`related compound which is useful for an immunopoten- 1O
`tiator and which has a high speci?city, and to medical
`use of the compound.
`2. Related Art Statement
`Spergualin is a compound having an anti-tumor activ
`ity and immunosuppressive activity which is obtained
`from the culture broth of Bacillus laterosporus (U .5. Pat.
`No. 4,416,899) and many derivatives of spergualin have
`been synthesized (cf., US. Pat. No. 4,430,346, US. Pat.
`No. 4,518,532, US. Pat. No. 4,529,549, US. Pat. No.
`4,556,735, US. Pat. No. 4,851,446, EP-A-213526, EP
`A-241,797). These compounds are expected to be drugs
`as carcinostatic agents or immunosuppresants.
`Currently, some immunopotentiators have been de
`veloped but new immunopotentiators having a higher
`speci?city have still been desired.
`
`20
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`The novel spergualin derivatives are represented by
`the general formula (I):
`
`SUMMARY OF THE INVENTION
`
`30
`
`Therefore, an object of the present invention is to
`provide a novel compound useful as an immunopoten
`tiator.
`Another object of the present invention is to provide
`a pharmaceutical composition comprising the novel
`compound as an active ingredient, which is particularly
`useful for an immunopotentiator.
`A further object of the present invention is to provide
`a method for immunopotentiation which comprises
`administering the novel compound to a warm-blooded
`animal.
`A still further object of the present invention is to
`provide use of the novel compound as an immunopoten
`
`tiator.
`>
`A ?rst aspect of the present invention relates to a
`novel spergualin derivative represented by the general
`formula (I):
`
`45
`
`55
`
`wherein X represents -—(CH2)1-s—- or a phenylene
`group which may be substituted; m represents 0, l or 2;
`n represents 1 or 2; and R1 represents —(CI-I2)1.
`3-COOI-I; and a pharmacologically acceptable salt
`thereof.
`'
`A second aspect of the present invention relates to a
`pharmaceutical composition for immunopotentiation
`comprising as an active ingredient the novel spergualin
`derivative represented by the general formula (I) or the
`
`65
`
`wherein X represents —(CH2)1-5— or a phenylene
`group which may be substituted; m represents 0, l or 2;
`n represents 1 or 2; and R1 represents —(CH2)1
`3-COOH.
`The phenylene group of X may be substituted with a
`halogen atom such as chlorine, ?uorine and bromine
`atom; a lower alkyl group such as methyl, ethyl, propyl,
`t-butyl and pentyl group; or a lower alkoxy group such
`as methoxy, ethoxy, propoxy, t-butoxy and pentoxy
`group. X is preferably —(CH2)3 or —(CH2)5—, more
`preferably ——(CH2)3—-. m is preferably 0 or 1. n is pref
`erably 1. R1 is preferably —(CH1)2-COOH or —(CH;.
`)3——COOH.
`The compounds represented by the general formula
`(I) may form salts with acids. As the acids for forming
`the salts, any of inorganic acids and organic acids may
`be used as long as they are non-toxic. As the inorganic
`acids, there are no particular limitation but hydrochlo
`ric acid, sulfuric acid, nitric acid and phosphoric acid
`are preferred. As the organic acids, there are no particu
`lar limitation but preferred are acetic acid, propionic
`acid, succinic acid, fumaric acid, maleic acid, malic
`acid, tartaric acid, glutaric acid, citric acid, benzenesul
`fonic acid, toluenesulfonic acid, methanesulfonic acid,
`ethanesulfonic acid, propanesulfonic acid, aspartic acid,
`and glutamic acid.
`I
`In the spergualin derivative of the general formula (I)
`of the present invention, steric con?guration of the
`carbon atom to which the hydroxy group is bound
`indicates S, R or R8 form. In particular, S or RS form
`are preferable. The representative compounds in the
`present invention are listed in the following Table l.
`
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`Page 2 of 11
`
`

`
`5,137,917
`
`(I)
`
`TABLE 1
`
`S1eric
`
`1
`
`S, R or R5
`
`—CH2—COOH
`
`111111111111111
`
`Compound
`N0
`
`1.234567009012345
`
`1111.11
`
`18
`
`1
`
`S,RorRS
`
`--(CHz)3-COOH
`
`
`
`901234567890 122222222223
`
`31
`
`Q1217’.
`max
`HHH
`I.
`
`000000000000
`
`0
`
`RRRRRRRRRRRR
`
`000000000000
`rrrvl-lrrrrrrr
`
`RRRRRRRRRRRR
`
`SSSSSSSSSSSS
`
`S, R or R5
`
`wooHoo?omnow
`OCCOCCOCCOCC C C C C
`
`HH HH HH HH
`
`0000000 00
`
`"CH3-COOH
`
`_ _ _ _ _ _ _ _
`
`0
`
`S, R or RS
`
`'—(CH1)z-COOH
`
`0
`
`S, R or RS
`
`-—(CHZ)3-COOH
`
`333
`456
`
`SR or RS
`5, Ror RS
`
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`
`

`
`5
`TABLE l-cbntinued
`
`5,137,917
`
`6
`
`NH;
`
`Compound
`No.
`
`X
`
`38
`
`39
`
`OH
`
`Steric
`Con?guration
`m of C‘
`
`R1
`
`0
`
`s, R or RS
`
`—(Cl-l2)2—COOl-l
`
`n
`
`2,
`
`2
`
`0
`
`s, R or Rs
`
`—-(CH2)3—-COOH
`
`Among the compounds listed in Table l, the follow-
`ing compound is the most preferable compound. The
`compound No. 2
`
`wherein X is as de?ned above. Thus, the compounds
`represented by the formula (II) are obtained.
`The condensation reaction as described above may be
`
`The steric con?guration of the asymmetric carbon
`atom in Compound No. 2 is preferably 5 form.
`'
`The compounds listed above are all novel and may be
`prepared by the following process. That is, the com-
`pounds are obtained by removing a protective group
`from compounds represented by the formula (II):
`
`carried out by methods generally used in peptide chem
`30 istry. Examples of these methods include a carbodiim
`ide method using dicyclohexylcarbodiimide, l-ethyl-3
`(3'-dimethylaminopropyl)-carbodiimide or the like; a
`mixed acid anhydride method using ethyl chlorocar
`bonate, isobutyl chlorocarbonate or the like; an acti
`
`H2N—R2—P1
`
`-
`(III)
`
`wherein R2 and P1 are as de?ned above; are reacted
`with reactive derivatives of protected amino acids rep-
`resented by the formula (IV):
`,
`
`wherein X, m and n are as de?ned above; R2 represents 40 vated ester method using a cyanomethyl ester’ 3 viriyl
`-—(CH2)1-3—COO—; and P1 represents a protective
`ester, a SUbStIIUtEdIOI' unsubstituted phenyl ester, a thro
`group of the carboxy group
`phenyl ester or a hydroxysuccinimide ester or the like;
`The protected compounds of the general formula (II)
`an Q'acylhydroxylamme dFnvanve method “5mg ace‘
`which are the starting compounds of the present inven-
`tomme, cyclohexanon? oxlme or thf’j llkei an N'acyl
`tion may be synthesized by the following method.
`45 compomfd m§th°d_uS1Pg carbonyldnfmdazole; find F1
`Protected amino acids represented by the formula
`c'fuboxyllc acld acnvfmon mcthod “5mg 13' thmzoh'
`(In),
`d1ne-2-th1one or the like.
`_
`D
`As a solvent for the condensation reaction, any sol
`vent applied to conventional peptide bond-forming
`50 reactions may be used. Examples of such a solvent in
`clude ethers such as diethyl ether, tetrahydrofuran and
`diOXaIl; esters Such as ethyl acemte; ketones Such as
`acetone and methyl ethyl ketone; halogenated hydro
`carbons such as methylene chloride and chloroform;
`55 amides such as dimethylformamide and dimethylacet
`amide; nitriles such as acetonitrile. These solvents may
`be used singly or in combination thereof. Where the
`solvent is miscible with water, the solvent may be used
`as an admixture with water.
`As the protective group which may be used in the
`present invention, there are a lower alkyl group, t-butyl
`group, benzyl group, a substituted benzyl group, and
`the like.
`_
`The protective group in the compounds shown by the
`(v) 65 general formula (II) may be split off by reactions such as
`reduction, hydrolysis and acid decomposition. Such
`reactions are carried out generally in a solvent at —60°
`C. to the boiling point of a solvent, preferably at —50°
`
`0H
`
`wherein P2 represents a protective group of the amino
`group which is different from P1; and n and m are as 60
`de?ned above. Then the amino protective group P2 is
`removed and reacted with reactive derivatives of (0-
`guanidino-fatty acids represented by the formula (V):
`
`NH:
`
`NOVARTIS EXHIBIT 2028
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`
`

`
`5,137,917
`
`7
`to 100° C. Examples of the solvent used include water
`and hydrophilic organic solvents, for example, a lower
`alcohol such as methanol and ethanol; a ketone such as
`acetone and methyl ethyl ketone; an amide such as
`dimethylformamide and dimethylacetamide; a cyclic
`ether such as tetrahydrofuran and dioxane; a lower fatty
`acid such as acetic acid and trifluoroacetic acid; liquid
`ammonia; and liquid hydrogen ?uoride. These solvents
`may be appropriately used.
`The compounds of general formula (I) may be iso
`lated from the reaction solution of the compounds from
`which the protective group has been split off, by con
`ventional methods for puri?cation, for example, where
`the protective group is removed by catalytic reduction
`using palladium black, the ?ltrate obtained by ?ltering
`the catalyst off is concentrated under reduced pressure
`and the residue is puri?ed by known method using
`chromatography using CM-Sephadex® (Na-t) or Se
`phadex® LH-20. Where the protective group is re
`moved wi'th trifluoroacetic acid, the reaction solution is
`also concentrated under reduced pressure and the resi
`due is puri?ed by the method described above. Thus,
`the desired compounds may be puri?ed.
`By the puri?cation method described above, the com
`pounds of the general formula (I) may be obtained in the
`form of hydrochloride. The salt may also be converted
`into other salts. For example, the hydrochloride is dis~
`solved in water and the resulting aqueous solution is
`passed through a strongly basic ion exchange resin. The
`non-adsorbed fraction containing the compounds of
`general formula (I) is collected and a desired acid is
`added thereto for neutralization. The mixture is then
`evaporated to dryness under reduced pressure. In this
`case, water, or, if necessary, a hydrophilic organic sol
`vent such as methanol, ethanol, acetone, tetrahydrofu
`ran, dioxan, or the like is added. When the organic
`solvent is contained, the solvent is distilled. off under
`reduced pressure and freeze-dried to give the desired
`salts. The desired salts may also be obtained by adding
`an aqueous solution of silver hydroxide to the hydro
`chloride of the compounds of the general formula (I) to
`neutralize hydrochloric acid, ?ltering insoluble silver
`chloride, adding a desired acid to the ?ltrate to form the
`salts, and freeze-drying.
`The physiological activity of the compounds of the
`present invention is demonstrated by the following
`experiments wherein the effects of potentiating anti
`body production were determined.
`
`5
`
`15
`
`25
`
`35
`
`45
`
`8
`from the next day after sensitization. Mice were sacri
`?ced 4 days after the sensitization. The count of anti
`SRBC antibody-producing cells (plaque forming cell,
`PFC) in the spleen cells was determined and the PFC
`count was calculated per 106 of the spleen cells. As is
`shown in the following equation, the effect of the com
`pound of the present invention is expressed by a potenti
`ation rate (%) of the PFC count in the group adminis
`tered with the compound of the present invention, as
`compared to the PFC count in the control group.
`
`Potentiation rate (%) =
`
`PFC count in the
`Law
`X 100
`PFC count in the
`control group
`
`2. Results
`The effects of representative examples of the com
`pounds of the present invention on potentiating anti
`body production is shown in Table 2.
`TABLE 2
`Effects of Compounds of This Invention on
`Potentiating Antibody Production
`Effect of Potentiating
`Antibody Production
`(control: 100%)
`Dose
`
`Compound
`No.
`
`2'
`3'
`5'
`14'
`21'
`24‘
`Control:
`Physiological
`saline
`
`‘RS form
`
`3 mg/kg
`
`3 mg/ltg
`
`I27
`128
`93
`84
`152
`169
`
`100
`
`151
`128
`127
`113
`173
`166
`
`100
`
`EXPERIMENT 2
`
`1. Method
`Methylprednisolone was intraperitoneally adminis
`tered to BALE/C mice (female, age of 8 weeks, Japan
`Kurea) in a dose of 400 mg/kg for 9 days to prepare
`immunosuppressed mice. On Day 6, the mice was
`boosted by intravenous injection of sheep red blood
`cells (SRBC, Japan Biological Material Center) in a
`dose of 1 X l06 cells. On Day 6 after the sensitization the
`count of antibody-producing cells (plaque forming cell,
`PFC) in the spleen cells was determined. A test com‘
`pound was intravenously administered in each dose
`shown in Table 3 only on the next day after the sensiti
`zation.
`In the same way as in Experiment 1, a potentiation
`rate of PFC count was determined.
`The tested compounds were as follows:
`
`EXPERIMENT l
`
`50
`
`1. Method
`Sheep red blood cells (SRBC) were intravenously
`injected to CDF1-SLC mice (5 mice in each group) in a
`dose of l X 108/02 ml for booster. The compound of the
`present invention was diluted with physiological saline
`in various concentrations. Each diluted solution was
`administered once in a daily dose of 0.1 ml per 10 g of
`body weight (0.1 ml/lO g/day) for consecutive 3 days
`
`55
`
`Compound No. 2 (RS form)
`
`Compound No. 2 (S form)
`
`(RS)
`
`OH
`
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`Page 5 of 11
`
`

`
`5,137,917
`
`-continued
`
`10
`
`The effects of the tested compounds on potentiating
`antibody production are shown in Table 3.
`TABLE 3
`Potentiation Rate of PFC Count
`vs. Methyl
`prednisolone (%)
`
`vs. Normal (%)
`
`Treatment
`
`Normal
`Methylprednisolone
`Compound No. 2
`(RS form!
`10 rug/kg
`30 mg/kg
`m
`Normal
`Methylprednisolone
`Compound No. 2
`(S form)
`30 mg/kg
`
`100
`37.5
`
`65.6
`96.0
`
`100
`77.2
`
`163.5
`
`100
`
`15
`
`174.8
`255.8
`
`100
`
`211.8
`
`20
`
`25
`
`the compound of the general formula (I) may be admin
`istered to the warm blooded animal, whereby antibody
`production is potentiated and immunity is activated.
`Where the compound of the general formula (I) is
`orally administered, the compound is used in the form
`of a tablet, a capsule, a powder, a granule, a solution or
`a dry syrup, generally together with solid carriers or
`liquid carriers as described above. The capsule, granule
`and powder contain generally 5 to 100 wt %, preferably
`25 to 98 wt % of the active ingredient.
`Dose may be determined depending upon age, body
`weight and condition of patient to be administered and
`therapeutic purpose but is generally 1-500 mg/kg/day,
`that is, 1-100 mg/kg/day in parenteral administration
`and 5 to 500 mg/kg/day in oral administration.
`
`PREPARATION EXAMPLE 1
`Distilled water for injection is added to 30 parts by
`weight of the hydrochloride of Compound No. 2 shown
`in Table 1 to make the whole volume 2000 parts. After
`dissolving the hydrochloride of Compound 2, the solu
`tion is subjected to cell-free ?ltration using Millipore
`Filter GS type. The ?ltrate (2 g) is taken up in a vial
`bottle of 10 ml volume and freeze-dried to give a freeze
`dried injection containing 30 mg of the hydrochloride
`of Compound No. 2 per vial.
`-
`
`PREPARATION EXAMPLE 2
`50 parts by weight of the hydrochloride of Com
`pound No. 21 shown in Table l, 600 parts of lactose, 330
`parts of crystalline cellulose and 20 parts of hydroxy
`propyl cellulose are thoroughly kneaded with each
`other and the mixture is compressed using a roller com
`pacter. By grinding and sieving through a mesh of 16 to
`60 mesh, granules are obtained.
`'
`Next, the present invention is described more speci?
`cally with reference to the examples below but is not
`deemed to be limited thereto.
`
`EXAMPLE 1
`Synthesis of
`16-guanidino-7-(RS)-hydroxy-5, 10-dioxo-4,9-diazahex
`adecanoic acid (Compound No. 2)
`a) Synthesis of benzyl
`9-t-butyloxycarbonyl-7-(RS)-hydroxy-5-oxo-4,9
`diazanonanoate
`In 60 ml of dichloromethane were dissolved 2.5 g
`(11.40 mmols) of y-N-t-butyloxycarbonyl-B-(RS)4
`hydroxybutanoic acid and 2.6 g (17.10 mmols) of N
`hydroxybenzotriazole. The solution was cooled with
`ice and 3.5 g (17.10 mmols) of N,N’-dicyclohexylcar
`bodiirnide was added to the solution. The mixture was
`reacted for 15 minutes under ice cooling, Then, a solu
`tion of 4.0 g (11.40 mmols) of 8-a1anine benzyl ester
`p-toluenesulfonic acid salt and 1.3 g (12.54 mmols) of
`triethylamine in dichloromethane was added to the
`solution under ice cooling. The temperature was then
`reverted to room temperature and the reaction was
`carried out for several hours. Insoluble matters were
`?ltered off and the ?ltrate was concentrated under re
`
`As is'evident from the foregoing experimental results,
`the compounds of the present invention possess an ex
`cellent immunopotentiating activity and are expected as
`immunopotentiators and other drugs.
`Where the compounds of the present invention are
`used as drugs, the compounds are formulated into phar
`maceutical preparations in a conventional manner, if
`necessary, together with additives used for pharmaceu
`tical preparations. The preparations may be adminis
`tered orally or parenterally. The additives such as ex
`cipients or carriers may be chosen from those pharma
`cologically acceptable. The kind and composition of
`excipients or carriers to be chosen may vary depending
`on route or method for administration. As liquid carri
`ers, there may be used, for example, water, an alcohol,
`animal and vegetable oil such as soybean oil, olive oil
`and mineral oil; or synthetic oil. As solid carriers, there
`may be used sugars such as maltose and sucrose; amino
`acids; cellulose derivatives such as hydroxypropyl cel
`lulose; or organic acid salts such as magnesium stearate.
`In the case of injection, it is desired to use, as a disso
`lution liquid, physiological saline, various buffer solu
`tions, a solution of sugar such as glucose, inositol, man
`nitol and lactose, or a glycol such as ethylene glycol and
`polyethylene glycol. Alternatively, a sugar such as ino
`sitol, mannitol and lactose, or an amino acid such as
`phenylalanine may be used as the carriers to prepare a
`freeze-dried preparation. Upon administration, such a
`preparation may be dissolved in a solvent suitable for
`injection such as liquid for intravenous administration,
`e.g., sterile water, physiological saline, a glucose solu
`tion, an electrolyte solution or an amino acid solution,
`and the resulting solution may be administered. I
`A content of the compound of the present invention
`in the pharmaceutical preparation may vary depending
`upon the mode of preparation but is generally in a range
`of 0.1 to 100 wt %, preferably 1 to 98 wt %. In the case
`of, e.g., injection, it is desired to contain the active
`ingredient generally in a range of 0.1 to 30 wt %, prefer
`ably 1 to 10 wt %.
`65
`Where it is desired to immunopotentiate a warm
`blooded animal (including human being) using the com
`pound of the general formula (I), an effective dose of
`
`45
`
`60
`
`NOVARTIS EXHIBIT 2028
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`Page 6 of 11
`
`

`
`5,137,917
`
`12
`
`.
`NMR (CD3OD)
`5=0.9-2.1 (m, 8H), 2.1-3.0 (m, 6H), 3.0-3.9 (m, 6H),
`3.9-4.4 (m, H), 5.23 (s, 2H), 7.46 (s, 5H)
`IR (KBr)
`v (cm‘1)=3300, 2940, 2600, 2500, 1730, 1650, 1540,
`1170
`TLC (chloroformzmethanol: l 7%
`ter=6:2.5:0.5, v/v)
`Rf: 0.34
`
`ammonia wa
`
`11
`duced pressure. The oily residue was dissolved in 150
`ml of ethyl acetate followed by washing sequentially
`with distilled water, 5% phosphoric acid, 5% sodium
`carbonate aqueous solution and then saturated sodium
`chloride aqueous solution. The organic layer was dried
`over anhydrous sodium sulfate. The supernatant was
`concentrated under reduced pressure to give 5.11 g of
`light yellow oily product. The oily product was sub
`jected to column chromatography using Silica Gel 60
`(manufactured by Merck Inc.) and development was
`performed with a solvent mixture of chloroform
`methanol (30:1, v/v) to give 3.5 g (yield, 80.83%) of the
`oily product.
`-
`NMR (CD3OD)
`5:1.47 (s, 911), 2.1-2.5 (d, 2H, J=6 Hz), 2.4-2.8 (t,
`2H, J=6 Hz), 2.9-3.3 (d, 2H, J=6 Hz), 3.2-3.7 (t, 2H,
`J=6 Hz), 3.8-4.3 (q, H, J=6 Hz), 5.15 (s, 2H), 7.37 (s,
`5H)
`TLC (chloroformzmethanol:10:1, v/v)
`Rf :04
`
`10
`
`20
`
`25
`
`30
`
`35
`
`d) Synthesis of
`16-guanidino-7-(RS)-hydroxy-5, 10-dioxo-4,9-diazahex
`adecanoic acid
`After 4.1 g (8.43 mmols)_of benzyl 16-guanidino-7
`(RS)-hydroxy-5,10-dioxo-4.9-diazahexadecanoate was
`dissolved in 40 ml of methanol, 10 ml of acetic acid and
`0.4 g of palladium black were added to the solution
`followed by catalytic reduction at 50° C. for 4 hours
`under normal pressure. The catalyst was ?ltered off and
`the ?ltrate was concentrated under reduced pressure to
`give 5.0 g of the oily product. This oily product was
`dissolved in 60 ml of distilled water and the solution was
`passed through a column packed with 500 ml of CM
`Sephadex® C-25 (Na-1'). After eluting with distilled
`water, the fraction containing the desired product was
`collected and evaporated to dryness under reduced
`pressure to give 3.5 g of the dry solid. In order to re
`move a small amount of impurities, the resulting dry
`solid was dissolved in 40 ml of distilled water and the
`solution was passed through a column packed with 300
`ml of I-IP-20® manufactured by Mitsubishi Chemical
`Industry Co., Ltd. By eluting with distilled water, the
`fraction containing the desired product was collected
`and concentrated under reduced pressure. The resulting
`oily product was dissolved in 20 ml of distilled water.
`Insoluble matters were ?ltered off and the ?ltrate was
`freeze-dried to give 1.34 g (yield, 44.42%) of the desired
`product.
`NMR (D20, external TMS) .
`8:1.5-25 (m, 8H), 2.5-3.2 (m, 6H), 3.4-4.1 (m, 6H),
`4.3-4.8 (m, H)
`IR (KBr)
`v(cm*1)=3320, 2940, 1635, 1550, 1410, 1185, 1095
`As is described in the followings, the various desired
`compounds represented by the general formula (I) were
`obtained in a manner similar to the procedures in d) in
`the example described above, using the corresponding
`compounds represented by the general formula (II).
`The compounds represented by the general formula (II)
`can be obtained by forming peptide bond, in sequence,
`in a conventional manner, in a manner similar to the
`procedures of a) to c) in the example described above,
`using the corresponding amino acids.
`SYNTHESIS OF COMPOUND NO. 1
`i) Benzyl
`l5-guanidino-6-(RS)-hydroxy-4.9-dioxo-3, 8-diazapen
`tadecanoate hydrochloride (Corresponding Compound
`of formula (11))
`NMR (CD3OD)
`8=0.9-2.0 (m, 8H), 2.0-2.7 (m, 4H), 2.8-3.5 (m, 4H),
`3.8-4.4 (m, H), 4.0 (s, 2H), 5.1 (s, 2H), 7.37 (s, 5H)
`IR (KBr)
`v (cm“1)=3330, 2940, 1745, 1650, 1545, U95, 1090,
`1030
`TLC (chloroformmethanol = 17%
`ter=6:2.5:0.5, v/v)
`
`ammonia wa
`
`b) Synthesis of benzyl
`8-amino-7-(RS)-hydroxy-5-oxo-4-azaoctanoate
`hydrochloride
`After 3.5 g (9.20 mmols) of benzyl 9-t-butyl-oxycar
`bonyl-7-(RS)-hydroxy-5-oxo-4,9-diazanonanoate was
`dissolved in 10 ml of dichloromethane, 10 ml of 4N
`hydrochloric acid-dioxan solution was added to the
`solution under ice cooling. The temperature was re
`verted to room temperature and the mixture was re
`acted for 3 hours. The reaction solution was concen
`trated under reduced pressure. The resulting white
`residue was subjected to decantation with n-hexane and
`ether. Concentration under reduced pressure gave 2.9 g
`(yield, 100%) of white crystals.
`TLC (ch1oroform:methano1:17%
`ter=6:2.5:0.5, v/v)
`Rf=0.35
`
`ammonia wa
`
`c) Synthesis of benzyl
`1é-guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-diazahex
`adecanoate hydrochloride
`After 2.47 g (11.04 mmols) of 7-guanidinoheptanoic
`acid was dissolved in 40 ml of dimethylformamide, 1.53
`g (13.25 mmols) of N-hydroxysuccinimide and 2.73 g
`45
`(13.25 mmols) of N,N’-dicylohexylcarbodiimide were
`added to the solution under ice cooling. The mixture
`was reacted at room temperature overnight. The pre
`cipitates were ?ltered off and the ?ltrate was used for
`the subsequent reaction as it was.
`After 2.9 g (9.20 mmols) of white crystalline benzyl
`S-amino-7-(RS)-hydroxy-5-oxo-4-azaoctanoate hydro
`chloride was dissolved in 40 ml of dimethylformamide,
`1.02 g (10.12 mmols) of triethylamine was added to the
`solution under ice cooling. Then the aforesaid solution
`of 7-guanidinoheptanoate hydrochloride N-hydrox
`ysuccinimide ester in dimethylformamide was added to
`the mixture followed by reacting at room temperature
`overnight. The reaction solution was ?ltered and the
`?ltrate was concentrated under reduced pressure. After
`decantation of the resulting oily residue twice with 100
`ml of n-hexane, the system was concentrated under
`reduced pressure. The resulting oily-product was sub
`jected to column chromatography using Silica Gel 60
`(manufactured by Merck Inc.) and development was
`performed with a solvent mixture of chloroform
`methanoll7% ammonia water_(6:2.5 :0.5, v/v) to give
`4.1 g (yield, 91.72%) of the oily product.
`
`50
`
`55
`
`65
`
`NOVARTIS EXHIBIT 2028
`Par v Novartis, IPR 2016-00084
`Page 7 of 11
`
`

`
`5,137,917
`
`14
`SYNTHESIS OF COMPOUND NO. 8
`i) Benzyl
`1S-guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-diaza-pen
`tadecanoate hydrochloride (Corresponding Compound
`of formula (11))
`NMR (CD30D, external TMS)
`8: 1.4-2.1 (m, 6H), 2.1-3.0 (m, 6H), 3.2-3.9 (m, 6H),
`4.0-4.5 (m, H), 5.28 (s, 2H), 7.50 (s, 5H)
`IR (KBr)
`v (cm_1)=3310, 3180, 2940, 1730, 1645, 1540, 1400,
`1170
`TLC '(ch1oroform:methano1:17%
`ter=6:2.5:0.5, v/v)
`Rf =0.48
`
`ammonia wa
`
`Rf=0.28
`
`13
`
`ii)
`15-Guanidino-6-(RS)-hydroxy-4,9-dioxo-3,8-diazapen
`tadecanoic acid (Compound No. 1)
`NMR (D20, external TMS)
`8=1.5-2.5 (m, 8H), 2.5-3.3 (m, 4H), 3.4-4.0 (m, 4H),
`4.3-4.9 (m, H), 4.47 (s, 2H)
`IR (KBr)
`8 (cm-1)=3340, 2930, 1640, 1540, 1410, 1230
`
`5
`
`10
`
`SYNTHESIS OF COMPOUND NO. 3
`i) Benzyl
`17-guanidino-8-(RS)-hydroxy-6,11-dioxo-5,10
`diazaheptadecanoate hydrochloride (Corresponding
`Compound of formula (11))
`NMR (CD3OD)
`6=0.9-2.0 (m, 10H), 2.0-2.7 (m, 6H), 2.8-3.5 (m, 6H),
`3.8-4.2 (m, H), 5.07 (s, 2H), 7.28 (s, 5H)
`IR (KBr)
`v (cm-1)=3300, 2940, 2360, 1735, 1640, 1460, 1165,
`1090
`TLC (chloroform :methanol : 1 7%
`ter=6:2.5:0.5, v/v)
`Rf =0.63
`
`ammonia wa
`
`ii)
`15-Guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-diazapen
`tadecanoic acid (Compound No. 8)
`NMR (D20, external TMS)
`6: 1.6-2.5 (m, 6H), 2.6-3.5 (m, 6H), 3.6-4.4 (m, 6H),
`4.5-5.1 (m, H)
`IR (KBr)
`v (cm-1)=333O, 2940, 1645, 1540, 1415, 1360, 1180,
`1075
`
`SYNTHESIS OF COMPOUND NO. 14
`i) Benzyl
`18-guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-diazaoc
`tadecanoate hydrochloride (Corresponding Compound
`of formula (II))
`NMR (CD3OD, external TMS)
`6: 1.1-2.0 (m, 12H), 2.1-3.0 (m, 6H), 3.0-3.8 (m, 6H),
`3.8-4.4 (m, H), 5.20 (s, 2H), 7.38 (s, 5H)
`IR (KBr)
`v (cm-1)=3310, 2930, 2860, 2330, 1730, 1635, 1460,
`1170
`TLC (ch1oroform:methanol:17%
`ter=6:2.5:0.5, v/v)
`Rf=0.48
`
`ammonia wa
`
`11)
`18-Guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-d1azaoc
`tadecanoic acid (Compound No. 14)
`NMR (D20, 6x16111111 TMS)
`6=1.5-2.6(m, 12H), 2.7-3.6 (m, 6H), 3.7-4.5 (m, 611),
`4.6-5.2 _(m, H)
`IR (KBr)
`v (cm-1)=3300, 3160, 2920, 2850, 1720, 1640, 1555,
`1410, 1375, 1350, 1180, 1095
`>
`
`SYNTHESIS OF COMPOUND NO. 21
`i) Benzyl
`15-guanidino-7-(RS)-hydroxy-6,10-dioxo-5,9-diazapen
`tadecanoate hydrochloride (Corresponding Compound
`of formula (11))
`,
`NMR (CD3OD, external TMS)
`8=1.1-2.9 (m, 12H), 3.1-3.9 (m, 6H), 4.1-4.5 (t, H,
`J=6 Hz), 5.27 (s, 2H), 7.47 (s, 5H)
`IR (KBr)
`v (cm-'1)=3310, 2940, 1725, 1645, 1540, 1450, 1255,
`1165, 1110
`TLC (chloroform:methanol:17%
`ter=6:2.5:0.5, v/v)
`'
`
`ammonia wa
`
`ii)
`17-Guanidino-8-hydroxy-6,1 1-dioxo-5,10-diazahep
`tadecanoic acid (Compound No. 3)
`NMR (D20, external TMS)
`8: 1.5-2.5 (m, 10H), 2.5-3.1 (m, 6H), 3.3-4.0 (m, 6H),
`4.2-4.8 (m, H)
`IR (KBr)
`v (cm-1)'=3320, 3150, 2930, 2860, 1670, 1640, 1540,
`1400, 1305, 1110
`
`30
`
`40
`
`SYNTHESIS OF COMPOUND NO. 5
`i) Benzyl
`14-guanidino-7-(RS)-hydroxy-5,10-dioxo-4,9-diazatet
`radecanoate hydrochloride (Corresponding Compound
`45
`-
`of formula (II))
`NMR (CD3OD, external TMS)
`8=1.5-2.l (m, 4H), 2.2-3.6 (m, 6H), 3.1-3.9 (m, 6H),
`3.9-4.5 (m, H), 5.26 (s, 2H), 7.48 (s, 5H)
`IR (KBr)
`v(cn1-‘)=3300, 3180, 1730, 1650, 1540, 1435, 1400,
`1250, 1170, 1075
`TLC (ch1oroform:methanol:17%
`ter=6:2.5:0.5, v/v)
`Rf=0.48
`
`ammoma wa- 55
`
`50
`
`ii)
`14-Guanidino-7-(RS)-hydroxy-5, l0-dioxo-4,9-diazatet
`radecanoic acid (Compound No. 5)
`NMR (D20, external TMS)
`8: 1.7-2.5 (m, 4H), 2.6-3.4 (m, 611), 3.4-4.2 (m, 6H),
`4.3-1.9 (m, H)
`IR (KBr)
`v (cm-1)=3340, 3260, 1720, 1645, 1555, 1540, 1445,
`1410, 1250, 1185
`
`NOVARTIS EXHIBIT 2028
`Par v Novartis, IPR 2016-00084
`Page 8 of 11
`
`

`
`15
`
`5,137,917
`
`16
`EXAMPLE 2
`Synthesis of
`16-guanidino-7-(S)-hydroxy-S,10-dioxo-4,9-diazahex
`adecanoic acid (S form of Compound No. 2)
`a) Synthesis of benzyl
`9-t-butyloxycarbonyl-7-(S)-hydroxy-5-oxo-4,9
`diazanonanoate
`In 70 ml of dichloromethane were dissolved 1.76 g
`(8.02 mmols) of 'y-N-t-butyloxycarbony1-B~(S)-hydrox
`ybutanoic acid and 1.84 g (12.02 mmols) of N-hydrox
`ybenzotriazole. The solution was cooled with ice and
`2.48 g (12.02 mmols) of N,N’-dicyclohexylcarbodiimide
`was added to the solution. The mixture was reacted for
`15 minutes under ice cooling. Then, a solution of 2.82 g
`(8.02 mmols) of B-alanine benzyl ester p-toluenesulfonic
`acid salt and 0.89 g (8.79 mmols) of triethylamine in
`dichloromethane was added to the solution under ice
`cooling. The temperature was then reverted to room
`temperature and the reaction was carried out for several
`hours. Insoluble matters were filtered off and the ?ltrate
`was concentrated under reduced pressure. The oily
`residue was dissolved in 130 ml of ethyl acetate fol
`lowed by washing sequentially with distilled water, 5%
`phosphoric acid, 5% sodium carbonate aqueous solu
`tion and then saturated sodium chloride aqueous solu
`tion. The organic layer was drived over anhydrous
`sodium sulfate. The supernatant was concentrated
`under reduced pressure to give 4.5 g of light yel