NZ716342B2 - Vasopressin-2 receptor agonists - Google Patents

Abstract

Vasopressin-2 receptor agonists based on Formula (I), pharmaceutical compositions thereof and methods for using the foregoing for treating diabetes insipidus, primary nocturnal enuresis, and nocturia.

Description

VASD‘E‘RESSlN—Z RECEPTOR riiGONlSTS Related Applications This ation claims the benefit ofU.S. Provisional Applications 61/859,024 filed July 26, '20l3 and til/952,073 tiled March 12, 20M, both of which are hereby incorporated by reference in their entirety, Field The present invention relates to novel compounds with agonist activity at the vasopressin-Q (V3) receptor, ceutical compositions sing these, and use of the compounds for the manufacture of medicaments for treatment of diseases, Background There are three known subtypes of vasopressin receptors, V13, V11, and V3. The V yo receptor is also known as the V3 receptor and the Via receptor is also known as the V; receptor. Each subtype has a distinct pattern of expression in tissues, With V2 found primarily in the kidney, where it es the antidiuretic activity of the endogenous ligand vasopressin (E'avory et al, 2009), V711, is Widely distributed in the brain (l—lernando et at, 200.1) V1 is found in a variety of tissues, including smooth muscle, liver, kidney, platelets, spleen and brain (Zinggg l996; 0strowski et al, 1994).

Agonists of the V2 receptor are clinically useful. Desmopressin is a V2 or agonist that is ed in some territories for treatment of diabetes dns, primary nocturnal enuresis, noctnria, and coagulation disorders including haernophilia A and von Willebrand’s disease. Desmopressin binds and activates both the V: and Vb receptors, with weaker activity on the Via. ressin has been shown to be partly excreted via the s (cg, cstad— Panlsen et at, l993), and the half~life of desmopressih is increased in patients with renal impairment (anicka, et at. 2003; Agersoe et al. 2004). Agersoe et al. t that the increased halti-life might lead to prolonged antidittretic effects and increase the risk of hyponatremia, a drop in serum sodium levels that can lead to adverse events such as seizures or coma. They further state that “although desniopressin appears to he safe and well—tolerated by patients with impaired renal function, great caution should be exercised when titrating towards an ent dosing regimen, if ts with moderately or severe renal function are to he treated with desrnopressin at all.” Therefme, there is a need for onal Vg recepmr agonistg with reduced activity at the V125, receptor. Additit‘snallyfi 'Vg receptor agonisis that do not 1‘st as haavily on the kidneys for eliminatim may aise be desirable, Summarv In one embodiment, a eompmmd is provided according to formula 1 or a phamtaeeuticaliy acceptable salt thereof, wherein R’- is H C1—C4 aakyt, haiogen, —oH or —e—CJ-C4 alkyl; R3 is H 01" 2-OH or NR5R6; H4 is H or —CK=NH)-NH2; R5 and R5 are independently H, C1—C6 alkyl, -CE—I2—cyclopmpyl, —eyciopropy1 or arylaikyl with the proviso that R5 and RG are not both E; X and Y are independently »»-CHZ~~ or MS with the proviso that ifX is ~CHg-u, Y is not g----; Z is ~CHR7— or S and R7 is H or C1—C4 aikyi, halogen, 43H 01‘ —O—C 1(1); alkyl; R8 is H or -'CE{3; and Ar is iteteroaryl or phenyi optionaily substituted with one {fig-C44, aikyl, halegcn, —OH or ~-»O---—C1--C4 alkyi.

In some embodiments, R5 and R6 are independently H, C1436 alkyl, or arylailtyl. in some embodiments, RS and R6 are independently H, C1-C6 ailtyl or arylaikyl.

In some embodiments, R and R6 are not both H.

In some embodiments, only one ofX and Y is —S-. in some ments, X is --~CH2---. in some embodiments, X and Y are both --S--. in some embodiments, Air is thiophene, in some embodiments, R8 is {TH} in some ments, R5; is —C(_O}NR.5R5. In certain of these embodiments, R.5 is H and R6 is C1—C4 nikyi. in certain of these embodiments, both ofR and R6 is mCligCI-ig. in some ments, R2 is a halogen. in n of these embodiments, RZ is —Ci. in certain of these embodiments, \1' is —l€".

Also provided , according to an embodiment, is a method of treating one of diabetes insipidus, primary nocturnal enuresis, and nocturia comprising administering a therapeutically effective amount of a compound ing to formula 1 to a patient in need thereof. The invention also includes use of the compounds described herein in treating the conditions described herein, along with use of the compounds described herein in the manufacture of a medicament for treating the conditions described herein.

According to an embodiment, the compound of formula 1 is used in a medicament for the treatment of diabetes insipidus, y nocturnal enuresis, or nocturia.

Betailed lieseri tion Uniess otherwise stated, the following terms used in this ation, including the specification and claims, have the definitions given below. it must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plnrai referents unless the context clearly dictates otherwise. iBefinition of standard chemistry terms may be found in reference works, including Carey and Snndberg (200.7) Advanced Organic Chemistry 5th Ed. Vols. A and B, Plenum Press, New York. The practice of the present invention will employ, unless otherwise indicated, tional methods of synthetic organic chemistry, mass oscopy, preparative and analytics} methods of chromatography, protein chemistry, biochemistry and pharmacology, within the skill of the aft .

“All-:yl” is a €1-12 straight, or branched chain alkyl. Branched alkyi e ism, sec—, and tern-configurations, “Aryl” is inono— or iii—cyclic aromatic carbocyclic ring system of S—l’l’ carbon atoms ally substituted with C1—C4 alkyl, halogen, ~OH or ~O~C1~C4 alkyl. Exemplary mono— and bi-cyciic aromatic carbocyclic ring s include optionally substituted plienyl and ally substituted naphthyl.

“Arylalhyl” is an alltyl group which has as a. substituent an aryl or heteroaryl group, “Heteroaiyl” is an aromatic heterocyclic five— or six—incinbered ring system aptionally substituted with C1—C4 alkyl, n, «OH or ~~~O~~~C3~C4 alkyl. A five—menihered heteroaromatic ring system is a nionocyclic aromatic ring system having live ring atoms, wherein l, 2 3 or 4 ring atoms are ndently selected from N, 0 and S. Exemplary five- red heteroaromatic ring s include optionally substituted imidazolyl, thiazolyl, thienyl, luryl, pyrazolyl, and triazolyl. A six-niemhered heteroaroniatic ring system is a monocyclic aromatic ring system having six ring atoms, wherein l, 2’), 3 or 41mg atoms are ndently selected from N, O and S. Exemplary six—membered heteroaromatic ring systems include optionally substituted pyridyl, pyrimidyl and pyrazinyl.

One embodiment of the invention provides a ceutical composition comprising compounds of the invention. in a first embodiment, the pharmaceutical composition further comprises one or more pharrnaceuticaliy acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients. Such excipients are known to those of skill in the art. The compounds of the present invention include, without limitation, basic compounds such as free bases. A thorough discussion maceutically acceptable excipients and salts is available in Remington's ceutical Sciences, l8th n (Eastern Pennsylvania: Mack Publishing Company, 1990).

Examples ofpharniaceutically acceptable salts include acid addition salts, eg. a salt formed by reaction with alogen acids such as hydrochloric acid and mineral acids, such as sulphuric acid, phosphoric acid and nitric acid, as well as aliphatic, alicyclic, aromatic or heterocyclic sulpltonic or carhcxylic acids such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, nialic acid, tartaric acid, citric acid, henzoic acid, ascorbic acid, inaleic acid, hydroxynialeic acid, pyruvic acid, p—hydroxybenzoic acid, embcnic acid, methanesulphonic acid, ethan esulphonic acid, yethanesulphonic acid, halohenzenesulphonic acid, trill uoroacetic acid, trifluoromethanesulphonic acid, esulphonic acid and naphthalenesulphonic acid. (see, egx, Berge et al, J. Pizarro. Sci. 66:1 19, 1977 and Wernruth, CG. and RH. Stahl, eds. Pharmaceutical Salts: Properties, Selection and Use. Ziirich: Var/lug Helvetz'co (tut/mm Add, 2002).

Depending on the intended mode of administration, the pharmaceutical compositions may be in the form ol‘solid, semi-solid or liquid dosage forms, such as, for example, tablets, itories, pills, capsules, powders, s, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage.

The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical. agents, adiuyants, diluents, 61‘3, etc.

The ion includes a pharmaceutical composition comprising a compound of the present invention including s, c or non—racemic mixtures of isomers, or pharmaceutically acceptable salts or es thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients.

For solid compositions, conventional ic solid carriers include, for example, ceutical grades ofniannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like.

For oral administration, the composition will generally take the form of a tablet, capsule, a softgel capsule nonaqueous solution, suspension or syrup, 'l'ahlets and capsules are preferred oral administration forms. ln some embodiments, the tablet is a wafer, eg, a fast“ melt water. in some ments, the wafer is administered via a gual route of administration. Tablets and capsules for oral use will generally include one or more commonly used carriers such as lactose and corn starch. lubricating agents, such as ium stearate, are also typically added, When liquid suspensions are used, the active agent may be combined with emulsifying and suspending agents. if desired, flavoring, coloring and/or sweetening agents may be added as well. Qtlier optional components for incorporation into an oral fonnulation herein include, but are not limited to, preservatives \. suspending agents, thickening agents and the like.

The dosages for therapy will depend on tion, distribution, lism and excretion rates of the components of the combination therapy as well as other factors known to one of skill in the art. Dosage values will also vary with the severity ol‘the condition to be alleviated. It is to be further understoed that far any particniar subject, specific dosage ‘egimens and schedules may be ed ever time according to the dual's need and the professionai judgment 0f the persnn administering 01‘ supervising the administration 0f the therapy. in some embodiments, an intravenous dose is awund Kiting. In some embodiments, an era} dese is from 1 tag to 1mg. In same embediments, a nasal {Ease is fmm 3 mg to 6 mg.

Abbreviations used are: Abbreviatien firefinitinn Ac Aeety] ACOH Acetic acid AV? Arginine Vasopressin r—Phe—Gin—Asn~Cys—Pm—Arg-Giy—NHZ with disulfide bridge between Cys O NH H«N W A A WAE/ ; :V: NH; 00 Hiive Q :/ NHz _¢‘L‘N\ 3 o 3‘ i \______ H c; i"2N NH L 011% ° NH H \.;:_-.. N\ W“,.2 O ’5) ie. unbranched) i (isn), 5 (see) and E (tertiary) BZI Benzyl CHECN Acetonn‘rile DCE 1,2-—dichlor0eihane .\‘4 Abbreviatian im DCM Dichlommcthanc {EDAVP Desmopressin, {i-dcamino, 8arginine}--Va.sopressin U z * O NH. 0 Hz\ : H. H i HN N N ' I Y Mug ‘3 .

NH2 0 DIC N;N"—I.)iEsopmpyicarbvdiimide DIPEA WEN—{iiisepmpyiathyiaminf: DIX/[F 1\.",i'\7~dimelhylfommmide dVP 1deammo—vasopressin 4 N7 \n/xfiAg:AVAVI/ 0 0 HR! 0 O O Abbreviatian 119mm“ EEt filthy} Pmoc 9‘-fluorenylmethoxycarbcnyl HBTU O—Benzotriazolc—N,N,N’,N’-tet1~a1net‘hjy"i—monimmhcxafluomphesphaie HF}? 1,1 1,3,3,3—hexafiuoro—Z—propanal HOB: Nuhydmxybenzotri322016 HPLC high mzmcc Hq‘uid (thrmnaiography i811 I'm-butyi CPI cyclopmpyi IIPr IZSO—pmpy‘; LC iiquid chroxnaiography Me methyl MeQH mathanai MS mass ometry Nvm’cthylmorphoiinc iert—buiyl e---------------------------------------------------------+----------------------------------------------------------------------------------------------------------------------------------------------- g tel/‘z—butylalcohol .................................................. trifluoroacetic acid triisopmpylsilam "E‘MO'F Trimethy‘; orthofmmatc, trimmhoxymethane Tr: trityl [triphengvrlmethy], (C5H5)3C—] Unless oéiiem'ise spsciiied, o acids were used and cmwenfiunal amino acid Ecrminoiogy is; used. Examples: ofamino acids other than the twsnty convmtional amino acids include: Abbreviation (Tonventimaai Name Thi fi—(Z—thifinylfilanine """"""" {pa{)(4chlomphen‘vi)ihnme Fpa {H4mfluorophenyi)alanine Hyp 4—Hydi‘0xypmiine 3,3-thiazoli(iin5-4—cai‘b13xyiicacid, i Th2 i i ihiopmiine Abu ‘Z-aminobutfy'ric acid Agm e, (4—amino‘butyi)guanidine PERM—MS) {i—(4-methy1phmyl)alanine {iiiigfiiiEiimm“WWW-E"ii:{El-5EEigfiéfifijfiéfiifié""""""""""""""""" {foniiggc'ands The campmzmds of tbs: invention have a structure of fomxula I: R2 and pharmaceuucafly acceptable‘. salts f, wherein: R7" is EL C1-C4 alkyL halogen "OH or --~C---C1--C4 aikyl; R3 is H M 4JH2~OH or — T(())—NR5R(’; R4 is H or -——C(=NI—i)--NHZ; R and R6 are indepemfiently PL {Tl-C5 , ~CH3-cyclupropy1, ~0yci0pmpyi or ziryiztikyl with the provise that R5 and R6 are not both H; X and Y are independentiy —CH2— or S with the proviso that if X is —CH2«, Y is net —CH2«; Z is --CBR7-- 0r S and R7 is i-i or C1-C4 alkyi, halegen, "SH or ----O---~C1--C4 alkyi; R8 is H or -CH;; Ar :13 heteroaryi or phenyi alfy substituted with one C3—C4 3&er halogen? —GH or —O—C1—C4 alkyi.

Tabie 1. Example Cempaunds {tithe lnventian.

CH;0H (B) C(16‘EH)—-NH2 ' thienyl i C(=C)—NH1-B(B) (1: wVElthH; (=0)..NBBBB (R) C(= Tum“; C(-—-0)—NH!:}3HR) C(=\'=H)-NB; 2 ~111ien'y} CHZOH (R) C(s— 3H3-NH2 2 ~thisnyl C(L'NHBNHZ fluorophc‘nyi ECHEH C(=\'='B)NB; s; a fluorophcnyl 9 0H H H 2—thienyi C(=NH)—NH; 2 ~thisnyl C(=C)}-NI-IcPr (R) C(=NE-D-NE—Iz Z-thienyl N {'I-CHz-CPI' (R) 2 mthie tiyi C(—O)M{B(R) 2 mienf1 C(==»O‘;—NHBu (R) C(\H)\TH C(=C)~NH::PB (R) C‘(=,\7 Eta-NH; i C(=0)-.NBB5BB (R) C:(= Bin-NH; CH(<:}H) awn (:4) CH C(-0)NHB_1 (R) C(:»-- H)NH2- CH, CH; C(T:())—NH2’B:_\(S) C(r=‘\?H)—NH2 C(=O}-NHMB (R) I)—NIIZ nyi -- CH;CH; ......... .

C(=0).Nm; (B) C(=NBI)--NB; 2 mthie'tiyi CH; CB;0H (S) C(BNHBNH; CH); CH;0H (S) C(r==\‘H)-NH1 CH; C: C.;H0H(B) C(‘NE—D-1\H2 CH; CH;0H (B) C(,-::\JH)*1\H3 CH; C(=())~NHI?BB (R) H)-1\ Hi CH; C(=0)--NBB5BB (R) _ Y“) -1\ H2 (3)3311) C(20)—NHB:+. (B) C(“VH)-1\ Hz 2—thienyl CB; C(T-GyNHEt (R) C(s— FIB—BB; phenyl S s CH; C(=0)-NHB:a (R) C(=NH)_NB2 CH; CH; C(=<)).N HE: (R) --NH~ ; :t- ; CBBOH) ----%_________ ' “Eiii‘(6'i§)""“: EC(=O)NHB: (B) C(==O)NHEE (R) C(r=»-‘EH)—1\Hz I5 Caj=0)-NI—1Ea (R) :3 wVECI-k Hg 2-ahienyl S S ram-Mag C(=0)~'\IH~CH2—2~thienyi ‘H)-‘a\'H2 77-—ahiaanyl {,f(={))-NH-CI-Ig--Euthifislyi : =\aa{).Ma2 EaR> fluomphcny‘a C(=(Z})NHBZE (R) <::a= ‘H)-I\H3 Za-ahienyl i.:(=0)—NHBZE (3a) ca: 7H)-‘a\H2 1y} =H)~N’Hz 2 ~t11ien'y} C(==— 1171}an fluoropheny‘a WO 13690 HO O HN~ o k HZN _ o m \n/firu: = *‘ HR: ,0 WO 13690 -)\\\NH := ,0 HN "’1 \RNi-i WO 13690 m o HN~ o k WO 13690 .2 o HN ' 0 kH7! .. O J H O H 53Y0 E S 1:} g *@s ' $0 HN 0 b?“ o HZN 0 kid : /U\/J\\ i : H Jr 0. \$ m o ._ "’IVU WO 13690 WO 13690 N N i H ..—-—' 0 MN 0 s T 8 HM "Wfl H \ NAfl .

WO 13690 $93N 0 W 0 /U\\ HZN o N AN 0 HM ,0 HQ 3 HN "Iii—O . Q Hgix.

WO 13690 N 3 N Y\N _ l )KCO H 5-: O HN O HN ’Ufl H \ WO 13690 H WO 13690 WO 13690 WO 13690 WO 13690 Mfr)?IN{IXHunk 21mg:I O WO 13690 HN 0 HN 0 H2::k WO 13690 \ HN o HZN “ o / H N U\ \H/\N _ H a 0 HM c- 8 32 H .u,,_.

H2'\E WO 13690 WO 13690 WO 13690 I ‘ 33 :4ka a.) N WO 13690 40 H Hgfi/WMQ HN O AK Table 2: i’hysimchemical pmmrties (if campmxnds 143 (SEQ H} 141) M+H L‘s/HE HPES SEQ ID (calcuiatad) (abserved) purity ; 956.5 3 {017.4 .................................................................................................................. 4 $31.4 _______________________________________________________________________________________________________________ WO 13690 a:g:1*.) xc; 17 @9705 18 @10455 19 $10455 2a étoo35 l“) (X? E19354 i\) a E10294 m3§10174 31% K334 32 $19275 b) .1}. e 3: in vitm assay data far campflunds L43 ‘iépEfficacy 15559 E‘Mxiflfficacy Eavzuiz hVibuR thwa SEQ H) 42 (dDAVP) 43 ([‘a 4diAVP) 44 (AV?) Amim; Pasitian Ciaim Namenciamm Amati' ' PERM-Evie) 2 RA: "CH3 I—‘th—Et) 2 16: 3-(11-13 ....................................................................................................................................................................................................................

Amine Fesitisn Claim Nemenelature_ ----------------------------------------------------------------------------------------------------------------------------------------------------------------- ””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””””” Fpa3 """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" «has Hyp 7 The 7 Agm 8 = H and R" = =Ni—i)--Ni—i2 Examgies Amine acid tives were purchased item eennnerciai providers (Aapptecfi EMD ere and Peptides fienai). Resins were purchased from commercial suppiiers (PCAS tix inc. and Eli/l1) Millipore). All additional reagents, chemicals and seivents were purchased from Sigma~Aldrioh and VWR.

The compounds described herein were synthesized by standard methods in selid phase e ehemistiy utilising FH‘lOC methodolegy. The peptides were assembled either manually, automatically using a Tribute Peptide Synthesizer {Protein Technologies Line, 'l‘uesen, Arizena) or by combination 0f manual and antem atie syntheses.

Preparative HPLC was performed on a Waters Prep LC Sysie‘m using a Pi‘epi’ack cartridge Delta-Pack C l 8 300K 35 pin, 47 X 300 mm at a flow rate {if l 00 mL/lniii and/m on a l‘henomenex Luna Cl 8 column, lQOA, 5 pm, 30 x ll’lll mm at a flow rate of 40 mL/min.

Analytical reverse phase HPLC was pei‘fermed en an Agilent Teeimelogies 120011“ Series iiquid ehmmatogmph using an Agiient Zerbax Cl8 column, 1.8 pm, 4.6 x llO mm at a flow ext-1 -v I rate of 1.5 rnL/n'iin. Final compound analyses were performed on an Agilent logies 1200 Series chroniatograph by reverse phase HPLC on a Phenomenex Gemini 1101‘; C18 , 3 am, ‘2 x lSO mm at a flow rate of 0.3. mL/niin. Mass spectra were recorded on a MA'l.‘ Finningan LCQ electrospray mass spectrometer. Unless stated ise, all ons were performed at room temperature. The following rd reference literature provides further guidance on general experimental set up, as well as on the availability of required starting material and reagents: Kates, S.A.,, Aiherieio, F Eds, Solid Phase Synthesis: ,A Practical Guide, Marcel Dekker, New York, Basel, 2000; Greene, T.W., Writs, P.G.M., tive Groups in Organic Synthesis, John Wiley Sons inc, 2nd Edition, l991; Stewart, lit/L, Young, ill, Solid Phase Synthesis Pierce Chemical Company, 1984; Bisello, et al, J.

Biol. Chem. l998, 273, 22498—22505; Merrifield, .5. Am. Chem. Soc. 1963, 85, 2149—254; and Chang and White 3D,, ‘Frnoc Solid Phase Peptide Synthesis: a Practical ch: Oxford sity Press, Oxford, 2000, The following protecting groups were ed to protect the given amino acid side chain hmctional groups: be(2,2,4,6,'7--pentamethy1dihydrobenzoitu'ann5--sulfonyl) for Arg; tBu (t—hotyl) for 'l‘yr and lit (trityi) for Cys, Gin and Asn.

Couplings oi‘Finoc-proteeted amino acids on the Tribute synthesizer were mediated with HBTU/NMM in DMF except for cysteine derivatives that were coupled with DiC/HGBt in EMF, Single cycles of 30-60 minutes with a 5-fold excess of activated Frnocvproteoted amino acids were used during the synthesis. Removal oi” the Fmoe protecting group was monitored by UV. Multiple (up to 10 times, as needed) two-minute washes of the peptide resin with 20% piperidine in DMF were performed DIC/llOBt mediated couplings in DMF were employed for all amino acids in manual mode. Single cycles of at least 2 hours with a 3-fold excess of activated EniOC--pl‘0i€Cl€d amino acids were used during the synthesis. The completeness of ngs was assessed with nihidrine (Kaiser) test. Removal of the Pines protecting group was achieved with a single 30 min. wash of the peptide resin with 20% piperidine in DMF.

Upon completion of the peptide synthesis, the peptide resins were washed with DCM and dried in tracer). The resins were treated with TFA/HzO/TIS 96:2:2 (v/v/v) for 2 h to remove the side-chain protecting groups with concomitant cleavage ot‘the peptide from the resin, The peptides were d, precipitated with diethyl ether and ed, To obtain peptides with disulfide bridges, the precipitate was dissolved in neat TFA and the solution was subsequently poured into it) % acetonitrile in water. ln some eases an additional amount of acetonitrile was added to solubilize the substrate. The linear peptide was oxidized with 0.1M lg/MeOH . The oxidizer solution was added dropwise until yellow color persisted The excess of iodine was reduced. with solid ascorbic acid. The pH was then adjusted to about 4 with trated ammonia. The obtained solution was loaded directly onto an HPLC prep column and eluted with a gradient of component B i see table .

To cyclize peptides via arnide bond formation the crude linear peptides were dissolved in DMF and a solution ofl-lBTU in DME was also prepared. The peptide solution and the activator solution were added interchangeably to a volume of vigorously stirred Dix/{F containing DIPEA. The pltl was maintained at 9—“) with the addition of neat l’JlPlfiA. The reaction was monitored by l-TPLC and ttmoieally no substrate peak t ’as detected after the last ns of the activator and peptide solutions have been added. The reaction e was diluted with 0.1% AcOll and the obtained solution was loaded. directly onto an lil’lLC prep column and eluted with a gradient of component B.

Each crude peptide was purified with buffer system T. The ons with a purity exceeding 93%, determined by reverse-phase ical ltll'JLC, were pooled and reloaded into the column and eluted with hotter T to provide triflttorottoetate salts. in some eases an additional ation with butler system C was performed. To obtain acetate salts the fractions front runs with buffer T or C were reloaded onto the eolnntn and the column was washed with 5 volumes oflll M um acetate. The final product was eluted with buffer A The li‘aetions were pooled and lyophilized.

Table, Buffer Compositions Buffer Component A Component 8 C 0.25 M 'l‘riethylamrnoniuni 60% acetonitrile, 40% Component orate, pH 2.3 A T (l.l% Trilluoroacetic aeid {TEA} 60% acetonitrile, 0.l‘l/.€.a TFA A 2% Acetic. acid (ACOH) 60% itrile, 2% AcOli, The compounds prepared were typically found to he at least about 95% pure.

Exam ,le l SE 13:21 The l—7 fragment was assembled manually starting from 7.8 g (6.9 mmel) of H—Pre—CZ- chloretrityl AM resin (Eli‘s/ED Millipore, catalog number 856057. 0.88 inrnol/g). DEC/HOE mediated couplings in DMF were employed. Single cycles of at least 2. hours with a 3-feld excess ot‘aetivated Fmee-protected amino acids were used during the synthesis. The completeness of couplings was assessed with ninhydrine test. Removal of the Frnoc protecting group was achieved with a single 30 min. wash of the peptide resin with 20% piperidine in DMF. The follnwing amino acid derivatives were used to assemble residues 1-7 of the resin.~hnund peptide: FrnoewCysfiCHfigC(0)OtBu)—OH. lE'nioc~Asn(”l‘rt)—Oiiy E'nioc— Val—OH, Fmoc—Thi~0l:l and Bec-Cpa-DH. After the l—7 peptide fragment was assembled the resin was washed thoroughly with DCM and treated with the T? 7:3 (v/v) cocktail ('2 x l h. 30 mi, each). The selv ents were their evaporated and the e was precipitated with ethyl ether. filtered and dried in vaeue. 5.79 g (4.63 inniol, 67 %) of the crude ted linear peptide was obtained. (The remainder of this product was used in the synthesis of other commands as bed herein.) H~T3~Arg~NEtz x ETFA. 2.81 g (5.4 ) of lfititt—D—Ai*g(l‘hfl—Oli (Client , eat it 05282), L95 mL (1 l2 mnroi) ot‘DlPEA and 2.13 g (5.6 rnnroi) ot‘l-IBTU were dissnlved in ll) hiL DMF. (3.62 n1L(6 inniol) ot‘diethylaniine was subsequently added tn the solution. No substrate was detected by analytical EFT/L", after 5 min, The reaction mixture was poured into 500 ml; of water and the precipitate was separated by fugation and dried in vacuo. The e was treated with 2C; lllL TFA/TlS/HZO 2. v/v/v) for l h and the solvents were evaporated. The residue was treated with ethyl ether and decanted. 1.65 g (3.6 nintol, 67%) of lid derivative was obtained which was used in the subsequent step without purification.

Coupling with H—l}—Arg~NEtg. 2.3 3 (ca. 1.86 mntol) of the linear ted peptide and 0.713 g t2 inrnol) of HBTU were dissolved in it) nil... EMF centaining 0.73 mL (4.2 innioi) DIPEA. 0.93 g (2.05 rental) of i’l—D—ArgtPhfl-Oll x ZTFA in l mL EMF was subsequently added to the reaction mixture. Ne substrate was detected after 5 min by HPLC.

The product was precipitated with l l ofwaten filtered off and dried in vacuo. 2.6 g {l .78 mmol, 96%) nt‘erude pretee'ted linear peptide was a itained. The fully protected e was treated with 20 mL TE; I’T‘lS/l-lgt) (96/2/2, v/v/v) for l h and the solvent was evaporated. The unprotected linear peptide was precipitated with ethyl ether and lyophilized. Yield 1.82 g (l .55 nimol, 83%). 2014/048317 The entire amount oi‘the linear peptide was dissolved in 50 rnL ol‘DMF. A solution of 0.59 g (ca. 1.55 nnnol) HBTU in 10 mi. ot‘DMF was also prepared. The peptide solution and the activator solution were added interchangeably to 50 mid of vigorously stirred DMF ning 200 nil. of lJ'il‘li'A in if) portions of 5 ml. and l mL, respectively. The pl-l was maintained at 9— it} with the addition of neat Dll’EA. No substrate peak was detected by HPLC after the last portions ofthe activator and peptide solutions have been added. The reaction mixture was diluted with 0.1% AcOH to l 1.. The obtained solution was loaded directly onto an HPLC prep colutnn and d with buffer system T eluted with a gradient of component B (see table above), The fractions with a purity exceeding 93%., determined by reverse—phase analytical HPLC, were pooled and reloaded onto the column. The column was washed with 5 volumes of 0. li‘vl AcONlh. and the compound was subsequently eluted with buffer C to provide acetate salt. The ons were pooled and lyophilized. 703.l mg (0.60 nitriol: 2 % overall based on 89. % peptide t) of white peptide powder was obtained.

The product purity was determined by analytical HPLC as 99.7% and the observed M+lil was l045.6 (calc. i‘s/Hl—l = lt’lilftfi). 2.32 g (about 1.3 rnrnol) of the protected linear peptide prepared in the synthesis of SEQ ll) NO: 21 was ved in 7 inL ofDMF and 0.63 niL (3.6 t 2 ed) NMM was added followed by 0.76 g {2. inmol. 1.1 cu) HBTU. in a separate vial, 0.64 g {2.8 inntolfl l5: ed) of agmatine sulfate was suspended in 7 tnL DMF containing 0.49 mL (2.8 tnnrol) of DIPEA. NtOuBis(triinethylsilyl)acetzaniide (ETA, Sigma-Aldrich, cat # ) was added to the oceasionally vottexed/sonicated sion. A clear solution was obtained after 4 eq of BTA were added to the suspension. The two solutions were combined and no substrate peptide was detected by HPLC after 5 min. The product was precipitated with l L ol‘water, filtered off and dried in vacuo. The resulting powder was treated with 50 mi of the 'l‘FA/TlS/HZO 3 (v/v/v) il for l.5 hrs. The solvent was ated and the linear peptide was precipitated with ethyl ether, tituted in water/aeetonitrile and l yophilized.

The entire amount ofpeptide (2.13 g, ca. 2 nnnol) obtained in the preceding step was dissolved in 50 ml}. of DMF. A solution of 0.76 g ('2 inniol) HBTU in 10 int; ofDMF was also prepared. The peptide solution and the activator solution were added interchangeably to 50 niL of vigorously stirred DMF containing 400 uL ofDlPEA in 10 portions of2.5 inL and 9.5 mil, respectively. The pH was maintained. at 9~l O with the addition of neat. l3ll?EA.. No substrate peak was detected after the last portions of the activator and peptide solutions have been added. The reaction mixture was diluted with 0.1% AcOl-i to l L and the obtained solution was loaded directly onto an l—lPLC prep column and purified with buffer system T eluted with a gradient of component B (see table above). The fractions with a purity exceeding 93%, determined. by reverse-phase analytical l-lll‘lfl, were pooled and reloaded onto the column. The column was washed with 5 volumes of (l. ll‘vl AcONH4 and the nd was subsequently eluted with buffer C to provide acetate salt. The fractions were pooled and lyophilized. 656.7 mg (0‘62 inniol, 23% overall yield based on 895% peptide content) of white peptide powder was obtained. The product purity was determined by analytical HPLC as l00.0% and the observed Mill was 946.6 (calc. M-l-H was .

Exam )le 3 — SE ll) N05 l g (era l mmol) ofFMPB AM resin (EMD Millipore, cat #1 855028) was swollen in l5 nil of DCE/"l‘lv’lOF l :1 mixture. To the resin suspension isobutyl amine ( l .5 ml; l5 mmol) was added followed by 3.2 g solid sodium triaeetoxyborohydride. The suspension was shaken overnight. The resin was washed with MeOH, DMF and DCM and was subsequently ed with Fmoovl}—Arg(Pbl)~Ol¥l/llllC (4 en) in DCJyl, The resin was washed with DMF and tested for acylati on completeness with the chloranil test (negative). The resin was split into three equal portions and the sis was continued at 0.33 mmol scale on the Tribute Synthesizer. Single couplings mediated with HBTU/NMM in [>th or with DlC/llOBt (for Cys") with a 5—fold excess of Fmoe—proteeted amino acids were used. The Fmoc protecting group was removed with several consecutive 2 min. washes with 20% piperidine in DMF.

The following amino acid tives were used in the automatic synthesis: Fmoc-Pro-Dl-l, Ftnoo—Cys((Cl-lg)3C(O)OtBu)—OH, ane—Asnfl‘tt}0ll, Fmoc—Vai—Ofl lE'nioc—Thi—OH and Boc—Cpa—Oll. After the entire peptide sequence has been assembled the e was cleaved from the resin with 20 mL of TFA.I’T-igO/TTS 96:2:2 (v/v/v) for 2 h. The linear peptide was dissolved in 40 mL of DMF containing 260 uL ot‘DlPEA. A on of ii? mg (ca. 0.4 niniol) HB'l'U in 5 ml; of9th was also prepared The e solution and the activator solution were added interchangeably to 40 ml; of vigorously stirred Dr‘s/ll“ in it} portions of 4 inL and 0.5 mL, respectively. The pH was ined at 9—10 with the on of neat DIPEA. No ate peak was detected by HPLC after the last portion of the activator solution has been added. The reaction mixture was diluted with 0. l % AeOll to l L. "the obtained solution was loaded directly onto an HPLC prep column. The compound was purified by three consecutive runs in buffer T.

WO 13690 2014/048317 The fractions exceeding 9794:; purity were pooled and lyophilized. 49.0 mg (0.042 mmol, 129/5 overall. assuming 90% peptide content) of white peptide powder was obtained.

The product purity was determined by analytical HPLC as 99.5% and the observed M+H was 1045.6 (calc. M+H = 1045.5).

EsaniplezlSEQlDNO9 0.37 g (ca. 0.3 rnnrol) of l,4—diaminohtitane—Z—chlorotrityl resin (Eh/ll) Millipore, cat # ) was swollen in 10 iii ofDMF and the resin placed in an automatic synthesis reaction vessel. The peptide assembly was carried out on the Tribute Synthesizer. Single couplings ed with HBTU/Nl‘v’lh’l in DMF or with DEC/HOBt {for Cys) with a 5—fold excess of Force-protected amino acids were used. The Finoc protecting group was removed with several consecutive '2. min. washes with 20% piperidine in DMF. The following amino acid derivatives were used in the automatic synthesis: Fmoc-Pro—OH, Fmoc- Cys((Cltlg)3C(G)OrBu)-Ol~i, Frnoc—Asnfirtl—Oi-i, Fmoc-Val-Gl-l, Finoc-Thi-Oi-I and Boc- TyrttBn)—-Dl—i. After the entire peptide sequence has been assembled the peptide was cleaved from the resin with 30 ml. ot‘HFlP/DCM 3:7 (ii/V) for 2 h, The resin was filtered and the solvents were evaporated. The linear protected peptide was precipitated with anhydrous ethyl ether. The precipitate was decanted and suspended in 20 rnL acetonitrile. ill mg ((3.4 inniol) oi‘ZtZ-Cl)--OSu and 0.136 niL (0.8mnicl) DIPEA were subsequently added to the suspension.

After the substrate has dissolved. the solvent was ated and the residue was treated with 2G rnL of the 'l‘FA/TlS/HZO 95,"2..5/'2..5 cocktail for l5 h. TFA was then evaporated and the residue was precipitated with diethyl, ether. The crude linear peptide was dissolved in 10.0 ml. of DMF ning 200 pl. of [)lPEA. A solution of lZO mg (0.3l minol) HBTU in 5 ml. of DMF was subsequently added to the vigorously stirred reaction e. After '36) min. the reaction e was diluted with l L 0.1% AcOH and the obtained solution was uploaded onto prep HPLC column. The cyclic peptide was eluted with fast (on. 3% MeCN/min.) in buffer system 'l". Fractions exceeding 97% purity by analytical HPM‘J were peeled and lyophilized. The liophilizate was treated with S inL of the r/thioanisole/TFA cocktail ( l/ 1/6, v/v/v) for l h at 0°C. TEA was evaporated and the peptide was precipitated with ethyl ether. The final product was ed by a single run in buffer T.

The fractions exceeding 97% purity were pooled and lyophilized. 77.5 mg (0.079 mnrcl, 26% overall. assuming 90% peptide t) of white peptide powder was ed.

The product purity was determined by analytical HPLC as 99.6% and the observed M+H was 886.4 (calc. Mal-l 886.4).

Exam ,le 5 SE ll) N02l7 0.43 g (ca. 0.3 nunol) of H-Arg(l>ht‘)—0—2—chlorotrityi resin {EMU Millipore, cat # 856067) was swollen in it) ml. ofDMF and the resin placed in an automatic synthesis reaction vessel. The peptide assembly was carried out on the Tribute Synthesizer. Single couplings mediated with l-lBTU/NMM in DMF or with DiC/HGBt (for Cys) with a 5-fold excess of Fmoc—protected amino acids were used. The Frnoc protecting group was removed with several consecutive 2 min. washes with 20% piperidine in DMF, The liillowing amino acid derivatives were used in the automatic synthesis: Einocnl’ro-OH, Pinoc- CysfriCllzliC(C)OtBu)—OH, Pinioc~Asn("i‘tt",t—(')l:l'y Enioc—VaLOH and Ftnocfl‘thH. After the 3—8 peptide sequence has been assembled ll‘rnoc—Phet’4—Et)-OH was coupled ly using DIC/HGBt tnethod with 2-fold excess of reagents. The Frnoc group was then replaced with the Boo group by treating the resin with 20% PIP/DMF for 30 min. and acyiating the N- terminal amino on, with BoczO in DMP. The linear peptide was cleaved front the resin with 3 0 mL of ll/DCl‘s/l 3:7 (v/V) for 2 h. The resin was filtered and the ts were ated. The linear protected peptide was itated with anhydrous ethyl ether. The precipitate was decanted and dried in ya.cuo.450 mg of the crude protected peptide was obtained. The entire amount of the peptide (ca. 0.3 nimul) was dissolved in it) nil. 1,2— dichloroethane containing 0.5 rnL DMF and (il pL (0.45 rnmol) NMM. The solution was cooled to GT on ice bath and 61 pl. (0.45 mmol) ot‘isohutyi chlorothrrnate was added. The on mixture was magnetically stirred for it) min. at 0°C. A solution of res mg (4.5 nnnol) sodium horohydride in 5 ml. water i 'as added in one portion. The on was diluted with 200 ml. water and the product was separated by centrifugation and dried in vacuo. The t was then was treated with 20 niL of the 'I‘FA/TIS/l-IZO 95/25/25 cocktail for 1.5 h. 'I‘FA was then evaporated and the e was precipitated with diethyl ether. The crude ioear peptide was dissolved in 80 ml... of DMF containing 200 at. of DIPEA. A solution offal mg (9.15 mmol) HB'l‘rU in 5 inL of DMF was subsequently added to the vigorously d reaction mixture.

After 30 min. the reaction mixture was diluted with l L 0.19/6 AcOl-l and the obtained solution was uploaded onto prep HPLC column. The cyclic peptide was purified by two consecutive runs in buffer T.

The fractions exceeding 97% purity were pooled and lyophilized. 41.7 mg (0.039 tnmol, 13% l. assuming 90% peptide content) of white peptide powder was obtained.

The product purity was determined by analytical lll’lifl as 95. l% and the observed M+ll was 970.6 (calc. Mil-l 970.5).

WO 13690 EX erlmental Biolo stinr in vitro rose tor assa‘ Agonist activity of compounds on the human V2 receptor (h VgR") was determined. in a transcriptional reporter gene assay by transiently ecting an h V2 receptor expression DNA into EEK-293 (human embryonic kidney 293 cell line) cells in concert with a reporter DNA containing intracellular calcium sive er elements regulating expression of firefly luciferase. See Boss, Vs ’l‘alpade, Di, Murphy, ll}. J; Biol, Chem. l996fl May 3; 2’7l(li§), 10429404332 for further guidance on this assay. Cells were exposed to serial dilutions ofcompounds diluted til-fold per dose for S h, ed by lysis ofcells, determination of luciferase activity, and determination of nd efficacies and Eng values through non-linear regression. Desmopressin (dl‘lA'Vl’) was used as an internal control in each experiment. Results for the tested nds are shown in Table 3 V”, Recenter Activitv To determine ivity, compounds were tested in lucilerase-based transcriptional reporter gene assays expressing the human V jb or (it‘s/Halt). Agonist activity of compounds on the liV 13R was determined in a transcriptional reporter gene assay in a Ftp— lnTM 293 cell line (llEK—tlpin) stably transfected to express the thR. These cells are transiently trartstected with an NFAT sive elements-luciferasc Luc) reporter.

Cells were exposed to serial dilutions of compounds diluted lit-fold per dose for 5 hours: followed by lysis of cells, determination of J.uciferase activity, and determination of compound efficacies and EC” values through non-linear regression. AV? was used as an internal control in each experiment. Results for the tested compounds are shown in Table 3.

Renal Clearance Desnronressin is cleared from the body primarily by the kidneys (“renal clearance”).

Compounds of the invention. have a higher extent of clearance through. non~renal mechanisms. Pliarnracolrinetic experiments were performed in neplirectoniized and sham- operated rats. Non-renal clearance (CLnr) was determined in nephrec'tonnzed rats, and total nce was determined in sham-operated rats tCLsham). % Non-Renal Clearance was calculated by (Clint/Closharn) x, MK).

For the pharmacokinetic studies, adult male Sprague Dawley rats were terized via the jugular vein (for compound administration) and carotid artery (for blood collection), A solution containing multiple compounds ette dosing) was injected into the jugular vein catheter (0.1 mg l'r‘B/ml of each compound, 0.3 ml/animal; nominal dose of ill mg FBI’lig/compound). Blood samples were collected at 2, 6, l0, iii, 20, '30, 45, 60, 90, and l2ll minutes post-administration using an ted blood sampling system, the h Laboratories Automated Blood Sampling Unit 2nd generation (A882). Plasma was prepared from whole blood using KZEDTA as agulant. uent hioanalysis of samples included compound extraction and plasma, concentration determination using standard LCD/MS methods. Analyte concentration was calculated from peak areas and calibration curves. PK ters were obtained by best g of the nd concentration—time profile for each animal by means of a noncompartmental analysis method using WINNGNLINTM *3/63 software (Pharsight Corporation).

Antidiuresis nds were tested for antidiuretic activity in a rat. model. in brief, catlieterized euvolemic Sprague Dawley rats were placed in metabolic cages, Each metabolic cage 3 as set up for continuous measurement of spontaneous urine output via for re transducers placed above the urine collection vials to monitor and record the time course of urine output using NOTOCORDTM software. The rats received an intravenous infusion of test compound or vehicle for three hours using a syringe pump and swivel/tether method. Data for urine output was collected during the administration ol‘compound (ti-3 hours) and was collected for the 5. hours post~adininistration In some cases, urine osniolality was also determined Compounds of the invention showed uretic activity.

Pharmaceutical itions There is also provided the use of a compound of a (l), as define herein, as a pharmaceutical. Further provided a pharmaceutical composition comprising a compound of formula (l), as defined herein, as active ingredient in association with a, pharmaceutically acceptable adjuvant, diluent or carrier.

The pharmaceutical composition may he adapted for s modes of administration including for example, oral and nasal: The composition may thus for instance he in the form of tablets, capsules, powders, microparticles, granules, syrups, suspensions and solutions.

The pharmaceutical composition may ally comprise tag. at least one further additive selected from a disintegrating agent, hinder, lubricant, llavouring agent, preservative, colourant. and any mixture f, Examples of such and other additives are found in ‘iiz’andbook (5”,I3intzmtacettzz‘ca1' Ii'xczfpz’ems"; lid. All. Kibhe, 3rd Ed, American ceutical ation, USA and Pharmaceutical Press UK, 3000.

Methods of Treatment In a, further aspect the present invention provides the use of a compound as outlined above for the cture of a medicament for treatment of diabetes insipidus, primary nocturnal enuresis, and nocturia. Further, methods of treating diabetes insipidus, primary nocturnal enuresis, and nocturia are provided. As used herein ‘treatrnent’ means the ation of symptomsg postponement of the onset of the disease and/or the cure of the disease when a. compound of the invention is administered in a suitable dose.

The typical dosage ol‘the compounds according to the present invention varies within a Wide range and Will depend on various factors such as the individual needs of each patient and the route of administration. The dosage may be stered once daily or more frequently than once daily, eg. intermittently. A physician of ordinary skill in the art will be able to optimize the dosage to the situation at hand All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be orated by reference.

Although the ing invention has been described in some detail by way of illustration and example for purposes of y of understanding it will he readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and cations may he made thereto without departing from the spirit or scope of the appended claims.

Claims (15)

1. A compound of formula I: , or a pharmaceutically acceptable salt f, wherein: R2 is H, C1-C4 alkyl, halogen, –OH or –O–C1-C4 alkyl; R3 is H or –CH2-OH or –C(O)-NR5R6; R4 is H or –C(=NH)-NH2; R5 and R6 are independently H, C1-C6 alkyl, -CH2-cyclopropyl, -cyclopropyl, or arylalkyl, with the proviso that R5 and R6 are not both H; X and Y are ndently –CH2– or –S– with the proviso that if X is –CH2–, Y is not –CH2–; Z is -CHR7- or S and R7 is H or C1-C4 alkyl, halogen, –OH or –O–C1-C4 alkyl; R8 is H or -CH3; Ar is heteroaryl or phenyl optionally substituted with one C1-C4 alkyl, halogen, –OH or –O– C1-C4 alkyl.

2. The nd of claim 1 wherein R5 and R6 are independently H, C1-C6 alkyl or kyl.

3. The compound of claim 1 wherein only one of X and Y is –S–.

4. The nd of claim 1 wherein X is –CH2–.

5. The compound of claim 1 wherein X and Y are both –S–.

6. The compound according to any one of claims 1-5 wherein Ar is thiophene.

7. The compound according to any one of claims 1-6 wherein R8 is -CH3.

8. The compound according to any one of claims 1-7 wherein R3 is –C(O)-NR5R6.

9. The compound according to any one of claims 1-8 wherein R5 is H and R6 is C1-C4 alkyl.

10. The nd according to any one of claims 1-8 wherein both of R5 and R6 is –CH2CH3.

11. The compound according to any one of claims 1-10 wherein R2 is a halogen.

12. The compound of claim 11 wherein R2 is –Cl.

13. The compound of claim 11 wherein R2 is –F.

14. A pharmaceutical composition comprising a nd according to any of claims 1- 13 for use in the treatment of diabetes insipidus, primary nocturnal enuresis, or nocturia.

15. Use of a compound according to any of claims 1-13 in the manufacture of a medicament for the treatment of diabetes insipidus, primary nocturnal enuresis, or nocturia.