USOO9388214B2

(12) United States Patent (10) Patent No.: US 9,388,214 B2 Wisniewski et al. (45) Date of Patent: *Jul. 12, 2016

(54) V1A RECEPTORAGONISTS Howl et al., “Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor.” Eur: J. (71) Applicant: FERRING B.V., Hoofddorp (NL) Biochem., 1993, 213, 711-19. Huguenin, "213. Synthesis of Phe2-Orn8-vasopressin and Phe2 (72) Inventors: Kazimierz Wisniewski, San Diego, CA Orn&-oxytocin, two vasopressin analogues endowed with selective (US); Geoffrey S. Harris, Cardiff, CA pressor activity.” Helv. Chim. Acta, 1964, 1934-41. (US); Robert Galyean, Escondido, CA Lenz et al., “Beneficial effect of 8-ornithin vasopressin on renal (US) dysfunction in decompensated cirrhosis.” Gut, 1989, 30(1), 90-96. Lenz et al., “Enhancement of renal function with ornipressin in a (73) Assignee: Ferring B.V., Hoofddorp (NL) patient with decompensated cirrhosis.” Gut, 1985, 26(12), 1385-86. (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. This patent is Subject to a terminal dis Primary Examiner — James H Alstrum Acevedo claimer. Assistant Examiner — Kaipeen Yang (74) Attorney, Agent, or Firm — Fish & Richardson P.C. (21) Appl. No.: 14/534,007 (22) Filed: Nov. 5, 2014 (57) ABSTRACT (65) Prior Publication Data Compounds of formula (I), salts thereof, and compositions US 2016/O12238.6 A1 May 5, 2016 and uses thereof are described. The compounds are useful as V1a vasopressin agonists, for the treatment of e.g., compli (51) Int. Cl. cations of cirrhosis, including bacterial peritonitis, HRS2 and AOIN37/18 (2006.01) refractory ascites. A6 IK38/04 (2006.01)

A6 IK38/2 (2006.01) C07K 7/06 (2006.01) (52) U.S. Cl. CPC ...... C07K 7/06 (2013.01) (58) Field of Classification Search CPC ..... A61K 38/10; A61K 38/00; A61K 38/177: A61 K38/085; A61K 38/17 See application file for complete search history. (56) References Cited U.S. PATENT DOCUMENTS 8,148,319 B2 * 4/2012 Wisniewski ...... CO7K 7/16 514/1.1 2015, 0126432 A1 5, 2015 Wisniewski et al.

FOREIGN PATENT DOCUMENTS

EP 1188.443 3, 2002 WO WO 2006/020491 2, 2006 WO WO 2009/020934 2, 2009 OTHER PUBLICATIONS Bisello et al., “Parathyroid hormone-receptor interactions identified directly by photocross-linking and molecular modeling studies. J. Biol. Chem., 1998, 273, 22498-505. Cardenas et al., “Tolvaptan, an oral vasopressin antagonist, in the treatment of hyponatremia in cirrhosis,” J. Hepatol., 2012, 56(3), 571-78. Durroux et al., “Fluorescent Pseudo-Peptide Linear Vasopressin Antagonists: Design, Synthesis, and Applications. J. Med. Chem.. 1999, 42, 1312-19. Fimiani et al., “The use of terlipressin in cirrhotic patients with refractory ascites and normal renal function: a multicentric study.” Eur: J. Intern. Med., 2011, 22(6), 587-90. Gulberg et al., "Long-term therapy and retreatment of hepatorenal syndrome type 1 with ornipressin and dopamine.” Hepatol., 1999, 30(4), 870-75. 39 Claims, No Drawings US 9,388,214 B2 Page 2

(56) References Cited Enkhbaatar et al., Arginine vasopressin Vla agonist attenuates methicillin-resistant Staphylococcus aureus-induced vascular leak OTHER PUBLICATIONS age by inhibiting bradykinin, Critical Care, 2013, 17(suppl. 4), Abstract No. P98 (Meeting Abstract of Sepsis 2013, Rio de Janeiro, Brazil, Nov. 5-6, 2013, p. 54). Russell et al., "Vasopressin versus norepinephrine infusion in Fernández-Varo et al., Vasopressin la receptor partial agonism patients with septic shock.” N. Engl. J. Med., 2008, 358(9), 877-87. increases sodium excretion and reduces portal hypertension and Sanyal et al., “A randomized, prospective, double-blind, placebo ascites in cirrhotic rats, Hepatology, 2016. 63(1), 207-16 (abstract). controlled trial of terlipressin for type 1 hepatorenal syndrome.” Huang et al., Terlipressin Resolves Ascites of Cirrhotic Rats through Gastroenterol, 2008, 134(5), 1360-68. Downregulation of Aquaporin2.J. Int. Med. Res., 2012, 40, 1735-44. Schmidt et al., “A radioiodinated linear vasopressin antagonist: A Koshimizu et al., Vasopressin Vla and V1b Receptors: From Mol ligand with high affinity and specificity for Vla receptors.” FEBS ecules to Physiological Systems, Physiol. Rev., 2012.92, 1813-64. Krag et al., Efficacy and safety of terlipressin in cirrhotic patients Lett., 1991, 282(1), 77-81. with variceal bleeding or hepatorenal syndrome, Adv. Ther. 2008, Terrillon et al., “Synthesis and Characterization of Fluorescent 25(11), 1105-40 (abstract). Antagonists and Agonists for Human Oxytocin and Vasopressin Vla Maybauer et al., The SelectiveVlaReceptor Agonist Selepressin (FE Receptors.” J. Med. Chem., 2002, 45,2579-88. 202158) Blocks Vascular Leak in Ovine Severe Sepsis, Crit. Care. Wisniewski et al., “New, Potent, Selective, and Short-Acting Peptidic Med., 2014, 42(7), e525-33. Vila Receptor Agonists,” J. Med Chem, 2011, 54,4388-98. Triantos et al., Terlipressin Therapy for Renal Failure in Cirrhosis, Yefet et al., “Extensive Epidermal Necrosis due to Terlipressin.” Eur, J. Gastroenterol. Hepatol., 2010, 22(4), 481-86 (abstract). IMA.J. 2011, 13, 180-81. Bolognesi et al., Splanchnic vasodilation and hyperdynamic circu Zhu, “Mechanistic explanation for the unique pharmacologic prop latory syndrome in cirrhosis, World J. Gastroenterol., 2014, 20010). erties of receptor partial agonists.” Biomed Pharmacother. 2005, 59. Fernández-Varo et al., Vasopressin la receptor partial agonism T6-89. increases sodium excretion and reduces portal hypertension and Zhu, “Rational Design of Receptor Partial Agonists and Possible ascites in cirrhotic rats, Hepatology, 2016. 63(1), 207-16. Mechanisms of Receptor Partial Activation: A Theory.” J. Theor: Biol., 1996, 181,273-91. * cited by examiner US 9,388,214 B2 1. 2 V1A RECEPTORAGONSTS and further contributes to the increase in HVPG diagnosed as portal hypertension once it exceeds 12 mm Hg. This shift of TECHNICAL FIELD the total blood volume towards the splanchnic circulation leads to a decrease in the effective blood volume (i.e., blood This disclosure relates to peptide compounds, and more 5 Volume in the central portion of the cardiovascular system), particularly to compounds which have partial V1a receptor which triggers reflex mechanisms aiming at increasing blood agonist activity, compositions containing Such compounds Volume, essentially sodium and water retention mechanisms and uses of Such compounds. and vasoconstrictor mechanisms, further increasing the intensity of blood volume shift towards the splanchnic circu BACKGROUND 10 lation which increases portal blood flow. Eventually, worsen ing vasoconstriction at the kidney starts reducing renal blood flow leading to either chronic (type II hepatorenal syndrome, The vasopressin-vasopressin receptor system is involved in HRS2) or acute renal failure (type I hepatorenal syndrome: two key homeostatic functions. A principal function of vaso HRS1) depending on the speed of deterioration. Both types of pressin is to regulate osmolality of the blood through the V2 renal failure are very difficult to manage clinically (i.e., receptor (V2R) found in the kidney. A second function of 15 reversing excessive renal vasoconstriction) without worsen vasopressin is as a pressor agent which is mediated by the Vla ing splanchnic vasodilation and portal hypertension. receptor (V1aR) found on blood vessels. Current medical management of the most severe cardio Experimentation has been performed with a number of vascular complications of cirrhosis primarily relies on either vasopressin receptor agonists and antagonists for use in the vasoconstrictor therapy or albumin administration. Vasocon treatment of a variety of diseases. Lenz, et al., Gut, 1985, strictor therapy targeted to specifically reduce splanchnic 26(12), 1385-1386; Lenz, et al., Gut, 1989, 30(1), 90-96: vasodilation without further deteriorating renal blood flow is Russell, et al., N. Engl. J. Med., 2008, 358(9), 877-887: Fimi the therapeutic intervention of choice. However, there is no ani, et al., Eur: J. Intern. Med., 2011, 22(6), 587-590; Carde current “gold standard of care, as available vasoconstrictive nas, et al., J. Hepatoll., 2012, 56(3), 571-578; Sanyal, et al., agents tend to have significant liabilities, such as an ineffec Gastroenterol., 2008, 134(5), 1360-1368. tive degree of splanchnic vasoconstriction and/or excessive Of particular clinical interest is the use of vasopressin 25 degree of extra-splanchnic vasoconstriction, too short a dura agonists for their pressor activity in patients with hypov tion of action, or too narrow a therapeutic window. In Euro olemia or hypotension in order to elevate arterial pressure. A pean countries, the emerging standard of care for the treat significant drawback of existing full vasopressin agonists for ment of HRS1 is administration of terlipressin. Other earlier, this use is the potential to induce severe vasoconstriction and less severe complications of cirrhosis are often managed with tissue hypoperfusion when used at pharmacological doses. 30 albumin as a Volume expander in the absence of a safe vaso Gulberg, et al., Hepatol., 1999, 30(4), 870-875; Yefet, et al., constrictor. Isr. Med. Assoc. J., 2011, 13(3), 180-181; Sanyal, et al., Gas Terlipressin has been shown to be effective in treating troenterol., 2008, 134(5), 1360-1368. The narrow therapeutic HRS1 in a large-scale, randomized, placebo-controlled, index of these compounds has restricted their use to patients blinded clinical trial (Orphan Therapeutics), providing proof where the risk of tissue hypoperfusion is acceptable due to the of concept that vasoconstriction can be effective in treating severity of the underlying condition being treated. renal failure in the context of cirrhosis (HRS1). Although the The V2 receptor (V2R) is primarily found in the kidneys, in trial did not achieve its primary endpoint (survival with a particular on the principal cells of the collecting ducts, where reversal of HRS), terlipressin will likely become the thera it is responsible for concentrating urine by reabsorbing water peutic of choice for HRS1 in the regions of the world where it from the glomerular ultrafiltrate. This water retention can is approved. While terlipressin is considered better than fluid/ lead to hyponatremia if fluid intake is not restricted propor 40 albumin therapy alone, it is only able to reverse renal failure tionately. The V2R is also found at extra-renal locations such in 30-40% of patients, leaving room for improvement. Terli as on endothelial cells where it appears to be responsible for pressin has demonstrated clinical efficacy in bleeding esoph a variety of effects, including release of von Willebrand factor ageal varices (BEV) and HRS1, but it has drawbacks such as and nitric oxide. a relatively short duration of action when used at lower, and The V1 a receptor (V1aR) is primarily found on smooth 45 hence safer, doses, and too much extra-splanchnic vasocon muscle cells throughout the vasculature where it acts as a key striction at higher doses. It is not practical to use terlipressin regulator of vascular tone. The vasopressin analog, terlipres outside of a monitored, inpatient setting due to its need for sin, has been approved in Some countries for the treatment of frequent dosing (every 4-6 h or via IV infusion) and the several cirrhotic complications (bleeding esophageal varices potential for severe adverse events. While these severe and type 1 hepatorenal syndrome) and has been used to dem adverse events are uncommon, they are potentially life threat onstrate the utility of using vasoconstriction to treat other 50 ening and must be managed accordingly. cirrhotic complications (spontaneous bacterial peritonitis, type-2 hepatorenal syndrome and post paracentesis circula tory dysfunction). SUMMARY Cirrhosis of the liver is a common end stage of excessive The present disclosure provides a compound according to alcohol consumption or of hepatitis. In about a third of cir 55 rhosis patients, fluid builds up in the peritoneal cavity, and this formula (I): is controlled by paracentesis. Complications of paracentesis (I) include hypovolaemia and an undesirable fall in arterial blood pressure. These have traditionally been checked by infusion of human albumin, and more recently, terlipressin. The development of portal hypertension as a consequence of cirrhosis is the key factor in the cardiovascular complica tions associated with end-stage liver disease. The liver has a normal hepatic venous pressure gradient (HVPG) of 1-5 mm Hg. An increase in HVPG is caused by active and passive increases in intrahepatic vascular resistance associated with the development of cirrhosis. This triggers a reflexsplanchnic arteriolar vasodilation leading to increase in portal blood flow US 9,388,214 B2 3 4 -continued ascites; vasodepressor Syncope; Vasovagal Syncope; toxic shock syndrome; and idiopathic systemic capillary leak Syn drome. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. Although methods and materials similar or equivalent O Q to those described herein can be used in the practice or testing b of the present invention, Suitable methods and materials are H 10 described below. All publications, patent applications, pat O ( Cs ents and other references mentioned herein are incorporated O NH2 HN by reference in their entirety. In case of conflict, the present O NH N 7 8 specification, including definitions, will control. In addition, ( p 5 HN O 15 the materials, methods and examples are illustrative only and 4. N i O HN O not intended to be limiting. O NH O R 9 The details of one or more embodiments are set forth in the O Rs. Ns NH2 accompanying drawings and the description below. Other 3 features, objects and advantages will be apparent from the R7 N 2 O description and drawings, and from the claims. H O DETAILED DESCRIPTION Air While not being limited by any theory, it is believed that a or a salt thereof, wherein the variables R. R. R. R. R. 25 Vlavasopressinagonist with a ceiling on its vasoconstrictive R. R. R', Ar. X, Q, m and p, are as described below. potential can be used for treatment using a bolus dosing Various embodiments of the disclosed compounds, includ paradigm that can provide long duration of action with a ing exemplary compounds are described. consistent level of vasoconstriction, thereby dramatically Also described is a pharmaceutical composition that improving safety, convenience and clinical efficacy in the includes a compound of formula (I), or any of the embodi 30 reversal of renal failure associated with cirrhotic complica ments thereof, or a pharmaceutically acceptable salt thereof, tions. It is believed that Such compounds can provide and a pharmaceutically acceptable carrier. improved safety allowing the use of Such a vasoconstrictor in Methods of treatment using the compounds of formula (I) low intensity clinical settings or even as an outpatient prod are also described. The methods include administering an uct, thereby enabling the treatment of conditions such as effective amount of the compound of formula (I) or any of the 35 spontaneous bacterial peritonitis, HRS2 and refractory embodiments thereof, or a pharmaceutically acceptable salt ascites. thereof, to an individual in need of the treatment. The disclosure describes selective, partial V1a agonists The compounds are also useful for treating complications that can substantially deliver the clinical benefits ofterlipres arising from cirrhosis, e.g., when increasing blood pressure is 40 sin, while providing improved safety and convenience therapeutically desirable. Complications of cirrhosis that can through longer duration of action than terlipressin. Undesired be treated with the claimed compounds include bacterial peri fluctuations in vasoconstrictive effect are also thereby tonitis, type II heptoarenal syndrome or refractory ascites. reduced. Such compounds could become the therapeutic The compounds are also useful, e.g., for increasing blood agents of choice in the treatment of cardiovascular complica pressure. The compounds are also useful for treating hypoV 45 tions where reduction of portal hypertension is clinically olemic shock; vasodilatory shock, bleeding esophageal efficacious. These advantages can enable the practical treat Varices; hepatorenal syndrome; type I hepatorenal syndrome; ment of cirrhotic complications generally, where a full ago type II hepatorenal syndrome; anesthesia-induced hypoten nist compound would not be suitable. sion; paracentesis-induced circulatory dysfunction; intra-op Existing full agonist compounds such as terlipressin have a erative blood loss; acute hemorrhage; blood loss associated 50 narrow therapeutic index. As the concentration of a full ago with burn debridement; blood loss associated with epistaxis; nist drug increases, it is possible to exceed the therapeutic spontaneous bacterial peritonitis; refractory ascites; hyper level of vasoconstriction and cause excessive vasoconstric tensive gastropathy bleeding; sepsis; severe sepsis; septic tion. This can result in severe tissue hypoxia and ischemia. shock; hypotension, including orthostatic hypotension and Reduced maximal efficacy or “partial efficacy” compounds intradialytic hypotension; cardiac arrest; trauma-related 55 will be able to be used at much higher concentrations than blood loss; vasodilatory shock induced by cardio-pulmonary would be possible with existing full agonist compounds while bypass; milrinone-induced vasodilatory shock in congestive not causing undesired additional vasoconstriction. The maxi heart failure; anaphylactic shock; cardiovascular instability mal vasoconstrictive effect that is attainable with such com induced by brain death; acute respiratory distress syndrome; pounds is reduced due to Submaximal agonist activation of acute lung injury; shock induced by metformin intoxication; 60 the V1 a receptor. shock induced by mitochondrial disease; shock induced by In the present disclosure, it is appreciated that certain fea cyanide poisoning; shock induced by vascular leak syndrome tures described herein, which are, for clarity, described in the induced by interleukin-2, another cytokine, denileukin difti context of separate embodiments, can also be provided in toX or another immunotoxin, or by ovarian hyperstimulation combination in a single embodiment. Conversely, various syndrome; hypotension induced by end-stage renal disease; 65 features described herein which are, for brevity, described in inflammatory bowel disease; reperfusion injury; infant respi the context of a single embodiment, can also be provided ratory distress syndrome; severe acute respiratory syndrome; separately or in any Suitable Subcombination. US 9,388,214 B2 5 6 I. Definitions remainder of the compound. The term “(C-C)alkynyl” Unless defined otherwise, all technical and scientific terms (wherein X and y are integers) denotes a radical containing X used herein have the same meaning as is commonly under toy carbons, wherein at least one carbon-carbon triple bondis stood by one of ordinary skill in the art to which this disclo present (therefore x must be at least 2). Some embodiments Sure belongs. are 2 to 4 carbons, some embodiments are 2 to 3 carbons and For the terms "e.g. and “such as and grammatical equiva Some embodiments have 2 carbons. Examples of an alkynyl lents thereof, the phrase “and without limitation' is under include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-buty stood to follow unless explicitly stated otherwise. nyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-penty As used herein, the singular forms “a” “an and “the nyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl include plural referents unless the context clearly dictates 10 and the like. The term “alkynyl' includes di- and tri-ynes. otherwise. As used herein, “halo' or “halogen' refers to —F. —Cl, As used herein, the term “about” means “approximately —Brand —I. (e.g., plus or minus approximately 10% of the indicated The term “haloalkyl as used herein refers to an alkyl group value). in which one or more of the hydrogen atoms has been As used herein, a “partial Vla agonist' is a compound 15 replaced by a halogen atom. The term “(C-C)haloalkyl” which provides agonism (as determined by the FLIPR assay (wherein X and y are integers) by itself or as part of another described herein) at the human V1 a receptor of between about Substituent means, unless otherwise Stated, an alkyl group 15% and about 70% of that provided by arginine vasopressin containing from X to y carbon atoms. The alkyl may be Sub (AVP), which is considered a full V1a agonist. stituted with one halogen up to fully Substituted, e.g., as As used herein, “alkyl refers to a saturated hydrocarbon represented by the formula CF; when more than one chain that may be a straight chain or a branched chain. An halogen is present they may be the same or different and alkyl group formally corresponds to analkane with one C–H selected from F, Cl, Br or I. Some embodiments are 1 to 3 bond replaced by the point of attachment of the alkyl group to carbons. Haloalkyl groups may be straight-chained or the remainder of the compound. The term “(C-C)alkyl branched. Examples include fluoromethyl, difluoromethyl, (wherein X and y are integers) by itself or as part of another trifluoromethyl, chlorodifluoromethyl, 2.2.2-trifluoroethyl, Substituent means, unless otherwise Stated, an alkyl group 25 pentafluoroethyl and the like. The term “perfluoroalkyl containing from X to y carbon atoms. For example, a (C-C) denotes the group of the formula —CF; stated differ alkyl group may have from one to six (inclusive) carbon ently, a perfluoroalkyl is an alkyl as defined herein wherein atoms in it. Examples of (C-C)alkyl groups include, but are the alkyl is fully substituted with fluorine atoms and is there not limited to, methyl, ethyl, n-propyl. n-butyl, n-pentyl, fore considered a subset of haloalkyl. Examples of perfluo n-hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 30 roalkyls include CF, CFCF, CFCFCF, CF(CF). neopentylandisohexyl. The (C-C)alkyl groups include (C- CFCFCFCF, CFCF (CF), CF(CF)CFCF and the Co)alkyl, (C-C)alkyl, (C-C)alkyl and (C-C)alkyl. like. The term “(C-C)alkylene" (whereinx and y are integers) As used herein, "cycloalkyl refers to a non-aromatic, Satu refers to an alkylene group containing from X to y carbon rated, monocyclic, bicyclic or polycyclic hydrocarbon ring atoms. An alkylene group formally corresponds to an alkane 35 system. The term “(C-C) cycloalkyl” (wherein x and y are with two C-H bonds replaced by points of attachment of the integers) denotes a cycloalkyl group containing from X to y alkylene group to the remainder of the compound. Examples carbonatoms in the ring. Cycloalkyl groups include (C-C) are divalent straight hydrocarbon groups consisting of meth cycloalkyl, (C-C7)cycloalkyl and (C)cycloalkyl. Represen ylene groups, such as, —CH2—, —CH2CH2— and tative examples of a (C-C)cycloalkyl include, but are not —CH2CHCH-. The (C-C)alkylene groups include (C- limited to, cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, C.)alkylene and (C-C)alkylene. 40 decahydronaphthalen-1-yl, octahydro-1H-inden-2-yl, As used herein, “alkoxy' refers to the group R-O- decahydro-1H-benzo.7 annulen-2-yl and dodecahydros-in where R is an alkyl group, as defined above. The term “(C- dacen-4-yl. Representative examples of a (C-C)cycloalkyl C.)alkoxy” (wherein X and y are integers) by itself or as part include, but are not limited to, cyclopropyl, cyclobutyl, cyclo of another Substituent means, unless otherwise Stated, an pentyl, cyclohexyl, cycloheptyl, cyclooctyl, decahydronaph alkyl group containing from X to y carbon atoms. (C-C) 45 thalen-1-yl and octahydro-1H-inden-2-yl. Representative alkoxy groups include, but are not limited to, methoxy, examples of a (C-C)cycloalkyl include, but are not limited ethoxy, n-propoxy, isopropoxy, n-butoxy and t-butoxy. The to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclo (C-C)alkoxy groups include (C-C)alkoxy and (C-C) heptyl, cyclooctyl and octahydropentalen-2-yl. alkoxy. A cycloalkyl can be unsubstituted or substituted. A substi As used herein, “alkenyl refers to an unsaturated hydro 50 tuted cycloalkyl can be substituted with one or more groups, carbon chain that includes a C=C double bond. An alkenyl e.g., 1, 2 or 3 groups, including: (C-C)alkyl, (C-C)alk group formally corresponds to an alkene with one C-H bond enyl, (C-C)alkynyl, halogen, (C-C)haloalkyl, —CN. replaced by the point of attachment of the alkenyl group to the - NO, —C(=O)R, —C(=O)CR, —C(=O)NR, remainder of the compound. The term “(C-C)alkenyl” C(=NR)NR, NR NRC(=O)R, NRC(=O)C) (wherein X and y are integers) denotes a radical containing X (C-C)alkyl, - NRC(=O)NR - NRC(=NR)NR, toy carbons, wherein at least one carbon-carbon double bond 55 —NRSOR, OR, —O(C-C)haloalkyl, —OC(=O)R. is present (thereforex must be at least 2). Some embodiments –OC(=O)C(C-C)alkyl, - OC(=O)NR –SR, S(O) are 2 to 4 carbons, some embodiments are 2 to 3 carbons and R. —SOR, —OSO(C-C)alkyl, —SONR, —(C-C) Some embodiments have 2 carbons. Alkenyl groups may alkylene-CN. —(C-C)alkylene-C(=O)CR, —(C-C) include both E and Z stereoisomers. An alkenyl group can alkylene-C(=O)NR —(C-C)alkylene-OR, —(C-C) include more than one double bond. Examples of alkenyl 60 alkylene-OC(=O)R, —(C-C)alkylene-NR —(C-C) groups include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, alkylene-NRC(=O)R, NR(C-C)alkylene-C(=O)CR, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, —NR(C-C)alkylene-C(=O)NR - NR(C-C)alkylene 5-hexanyl. 2,4-hexadienyl, and the like. OR, NR(C-C)alkylene-OC(=O)R. —NR(C-C)alky As used herein, “alkynyl refers to an unsaturated hydro lene-NR - NR(C-C)alkylene-NRC(=O)R. —O(C-C) carbon chain that includes a C=C triple bond. An alkynyl 65 alkylene-C(=O)CR, —O(C-C)alkylene-C(=O)NR, group formally corresponds to an alkyne with one C-H bond —O(C-C)alkylene-OR, O(C-C)alkylene-OC(=O)R, replaced by the point of attachment of the alkyl group to the —O(C-C)alkylene-NR and —O(C-C)alkylene-NRC US 9,388,214 B2 7 8 (=O)R. Each R can be, independently, hydrogen or (C-C) Zolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4- alkyl. Additionally, each of any two hydrogen atoms on the thiadiazolyl and 1.3,4-oxadiazolyl. same carbon atom of the carbocyclic ring can be replaced by Examples of polycyclic heterocycles include: indolyl, par an oxygen atom to form an oxo (=O) Substituent. ticularly 3-, 4-, 5-, 6- and 7-indolyl, indolinyl, quinolyl, tet The term “aromatic' refers to a carbocycle or heterocycle rahydroquinolyl, isoquinolyl, particularly 1- and 5-iso having one or more polyunsaturated rings having aromatic quinolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, character (i.e., having (4n+2) delocalized at (pi) electrons quinoxalinyl, particularly 1- and 5-quinoxalinyl, quinazoli where n is an integer). nyl, phthalazinyl, 1.5-naphthyridinyl, 1.8-naphthyridinyl, As used herein, “aryl employed alone or in combination 1,4-benzodioxanyl, coumarin, dihydrocoumarin, benzofuryl, with other terms, refers to an aromatic hydrocarbon group. 10 particularly 3-, 4-, 5-, 6- and 7-benzofuryl, 2,3-dihydroben The aryl group may be composed of, e.g., monocyclic or Zofuryl, 1,2-benzisoxazolyl, benzothienyl, particularly 3-, 4-, bicyclic rings and may contain, e.g., from 6 to 12 carbons in 5-, 6- and 7-benzothienyl, benzoxazolyl, benzthiazolyl, par the ring, such as phenyl, biphenyl and naphthyl. The term ticularly 2-benzothiazolyl and 5-benzothiazolyl, purinyl, “(C-C)aryl” (wherein X and y are integers) denotes an aryl benzimidazolyl, particularly 2-benzimidazolyl and benztria group containing from X toy ring carbonatoms. Examples of 15 Zolyl. a (C-C)aryl group include, but are not limited to, phenyl, A heteroaryl group can be unsubstituted or substituted. A C.-naphthyl, B-naphthyl, biphenyl, anthryl, tetrahydronaph substituted heteroaryl group can be substituted with one or more groups, e.g., 1, 2 or 3 groups, including: (C-C)alkyl, thyl, fluorenyl, indanyl, biphenylenyl and acenanaphthyl. (C-C)alkenyl, (C-C)alkynyl, halogen, (C-C)haloalkyl, Examples of a Co-Co aryl group include, but are not limited - CN, NO, C(=O)R, C(=O)CR, —C(=O)NR, to, phenyl, C.-naphthyl, B-naphthyl, biphenyl and tetrahy C(=NR)NR = NR, NRC(=O)R, NRC(=O)C) dronaphthyl. (C-C)alkyl, - NRC(=O)NR - NRC(=NR)NR, An aryl group can be unsubstituted or Substituted. A Sub —NRSOR, —OR, —O(C-C)haloalkyl, —OC(=O)R, stituted aryl group can be substituted with one or more –OC(=O)C(C-C)alkyl, - OC(=O)NR, SR, S(O) groups, e.g., 1, 2 or 3 groups, including: (C-C)alkyl, (C2 R. —SOR, —OSO(C-C)alkyl, -SONR, —(C-C) C.)alkenyl, (C-C)alkynyl, halogen, (C-C)haloalkyl, 25 alkylene-CN. —(C-C)alkylene-C(=O)CR, —(C-C) —CN, NO, C(=O)R, —C(=O)CR, C(=O)NR, alkylene-C(=O)NR —(C-C)alkylene-OR, —(C-C) C(=NR)NR, NR NRC(=O)R, NRC(=O)C) alkylene-OC(=O)R. —(C-C)alkylene-NR —(C-C) (C-C)alkyl, - NRC(=O)NR - NRC(=NR)NR, alkylene-NRC(=O)R, NR(C-C)alkylene-C(=O)CR, —NRSOR, OR, —O(C-C)haloalkyl, —OC(=O)R. —NR(C-C)alkylene-C(=O)NR - NR(C-C)alkylene –OC(=O)C(C-C)alkyl, OC(=O)NR –SR, -S(O) 30 OR, NR(C-C)alkylene-OC(=O)R, NR(C-C)alky R. —SOR, —OSO(C-C)alkyl, —SONR, —(C-C) lene-NR —NR(C-C)alkylene-NRC(=O)R. —O(C-C) alkylene-CN. —(C-C)alkylene-C(=O)CR, —(C-C) alkylene-C(=O)CR, —O(C-C)alkylene-C(=O)NR, alkylene-C(=O)NR —(C-C)alkylene-OR, —(C-C) —O(C-C)alkylene-OR, —O(C-C)alkylene-OC(=O)R. alkylene-OC(=O)R, —(C-C)alkylene-NR —(C-C) —O(C-C)alkylene-NR and —O(C-C)alkylene-NRC alkylene-NRC(=O)R, NR(C-C)alkylene-C(=O)CR, (=O)R. Each R can be, independently, hydrogen or (C-C) —NR(C-C)alkylene-C(=O)NR - NR(C-C)alkylene 35 alkyl. OR, NR(C-C)alkylene-OC(=O)R. —NR(C-C)alky The aforementioned listing of heterocyclyl and heteroaryl lene-NR - NR(C-C)alkylene-NRC(=O)R. —O(C-C) moieties is intended to be representative and not limiting. alkylene-C(=O)CR, —O(C-C)alkylene-C(=O)NR, The term “substituted” means that an atom or group of —O(C-C)alkylene-OR, —O(C-C)alkylene-OC(=O)R, atoms formally replaces hydrogen as a “substituent attached —O(C-C)alkylene-NR and —O(C-C)alkylene-NRC 40 to another group. The term “substituted, unless otherwise (=O)R. Each R can be, independently, hydrogen or (C-C) indicated, refers to any level of Substitution, namely mono-, alkyl. di-, tri-, tetra- or penta-Substitution, where Such Substitution is The term "heteroaryl' or "heteroaromatic” as used herein permitted. The substituents are independently selected, and refers to an aromatic ring system having at least one heteroa Substitution may be at any chemically accessible position. tom in at least one ring, and from 2 to 9 carbon atoms in the 45 In the description herein and in the claims, the nomencla ring system. The heteroaryl group has 1 or 2 oxygenatoms, 1 ture common in the art of peptide, and more specifically, or 2 sulfur atoms, and/or 1 to 4 nitrogenatoms in the ring, and vasopressin chemistry is used. The amino acids in the Sub may be bonded to the remainder of the molecule through a stances can be either L- or D-amino acids. When no configu carbon or heteroatom. Exemplary heteroaryls include furyl, ration is noted, the amino acid is in the L, or naturally occur thienyl, pyridyl, oxazolyl pyrrolyl, indolyl, quinolinyl or 50 ring form. The thio members of the B-mercaptopropionic acid isoquinolinyl, and the like. The heteroatoms of the heteroaryl (1) and cysteine (6) units are added for clarity in certain ring system can include heteroatoms selected from one or structural formulas. Substances described herein also include more of nitrogen, oxygen and Sulfur. peptides with sequences having reversed peptide bonds. Examples of non-aromatic heterocycles include monocy These sequences are preferably inverted sequences, more clic groups such as: aziridine, oxirane, thirane, aZetidine, preferably comprising D-amino acids. oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyra 55 The term "salt' includes any ionic form of a compound and Zolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihy one or more counter-ionic species (cations and/or anions). drofuran, tetrahydrofuran, thiophane, piperidine, 1.2.3,6-tet Salts also include Zwitterionic compounds (i.e., a molecule rahydropyridine, 1,4-dihydropyridine, piperazine, containing one more cationic and anionic species, e.g., Zwit morpholine, thiomorpholine, pyran, 2.3-dihydropyran, tet terionic amino acids). Counter ions present in a salt can rahydropyran, 1.4-dioxane, 1,3-dioxane, homopiperazine, 60 include any cationic, anionic, or Zwitterionic species. Exem homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin plary anions include, but are not limited to, chloride, bromide, and hexamethyleneoxide. iodide, nitrate, sulfate, bisulfate, sulfite, bisulfate, phosphate, Examples of heteroaryl groups include: pyridyl, pyrazinyl, acid phosphate, perchlorate, chlorate, chlorite, hypochlorite, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, periodate, iodate, iodite, hypoiodite, carbonate, bicarbonate, thienyl, furyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, 65 isonicotinate, acetate, trichloroacetate, trifluoroacetate, lac thiazolyl, oxazolyl pyrazolyl, particularly 3- and 5-pyra tate, Salicylate, citrate, tartrate, pantothenate, bitartrate, Zolyl, isothiazolyl, 1.2.3-triazolyl, 1,2,4-triazolyl, 1,3,4-tria ascorbate. Succinate, maleate, gentisinate, fumarate, glucon US 9,388,214 B2 10 ate, glucaronate, saccharate, formate, benzoate, glutamate, of symptoms, postponing or preventing the further develop methanesulfonate, trifluorimethan sulfonate, ethanesulfonate, ment of a disorder, and/or reducing the severity of symptoms benzenesulfonate, p-toluenesulfonate, p-trifluoromethylben that will or are expected to develop. The following abbreviations may also be found herein: Zenesulfonate, hydroxide, aluminates and borates. Exem AcOH (acetic acid); Boc (t-butoxycarbonyl); DCM (dichlo plary cations include, but are not limited, to monovalent alkali 5 romethane); DIAD (N,N'-diisopropyl azidodicarboxylate); metal cations, such as lithium, Sodium, potassium and DIC (N,N'-diisopropylcarbodiimide); DIPEA (N,N-diiso cesium, and divalent alkaline earth metals, such as beryllium, propylethylamine; DME (1,2-dimethoxyethane); DMF magnesium, calcium, strontium and barium. Also included (N,N-dimethylformamide); Et (ethyl); Fmoc (9-fluorenylm are transition metal cations, such as gold, silver, copper and ethylmethoxycarbonyl); h (hour(s)); iv.Dde (1-(4,4-dimethyl 10 2,6-dioxocyclohex-1-ylidene)3-methylbutyl; HIPF (1,1,1,3, Zinc, as well as non-metal cations, such as ammonium salts. 3.3-hexafluoro-2-propanol: HOBt References to the compounds described and disclosed (N-hydroxybenzotriazole); HPLC (high-performance liquid herein are considered to include both the free base and all chromatography): LC (liquid chromatography); MeOH addition salts. The addition salts may be either salts with (methanol); MS (mass spectrometry); Mitt (4-methyltrityl); pharmaceutically acceptable cations such as Na", Ca", K" or NMM (4-methylmorpholine); Pbf (2,2,4,6,7-pentameth Na" at a terminal acid group, such as when the C-terminal 15 yldihydrobenzofuran-5-sulfonyl); t-Bu (tert-butyl); TEAP amino acid is Gly or OH is present, or with a pharmaceutically (triethylammonium phosphate); TFA (trifluroracetic acid); acceptable acid addition salt at a basic center of the peptide, TFE (2.2.2-trifluoroethanol); TIS (triisopropylsilane); TPP Such as in an Arg unit. The acetate Salt forms are useful, and (triphenylphosphine); and Trt (trityl triphenylmethyl, hydrochloride, hydrobromide and salts with other strong (CHS)-C-I). acids are also useful. In the isolation procedures outlined in Other abbreviations used herein are as follows: 3-Pal the Examples, the peptide product is often isolated and puri (3-pyridylalanine); 5-Ava (5-amino Valeric acid); chexcarbo fied as an acetate salt. The compounds may also form inner nyl or chxCO (cyclohexylcarbonyl); Orn (ornithine); Tyr salts or Zwitterions when a free terminal carboxy group is (Me) (methoxy analog of tyrosine); Cit (citruline); Dab (2,4- present. The term “pharmaceutically-acceptable salt” refers diaminobutyric acid); Hmp (2-hydroxy-3- to salts which possess toxicity profiles within a range that mercaptopropionic acid); Hgn (homoglutamine); iBuCO affords utility in pharmaceutical applications. Pharmaceuti 25 (isovaleroyl), beta-Ala (beta alanine; 3-amino propionic cally unacceptable salts may nonetheless possess properties acid); and isohArg (isohomoarginine). Such as high crystallinity, which may render them useful, e.g., II. Compounds in processes of synthesis, purification or formulation of com Provided herein are peptidic partial V1 a receptor agonists pounds described herein. In general the useful properties of having a particular generic structural formula represented by the compounds described herein do not depend on whether 30 the compound is or is not in a salt form, so unless clearly formula (I) below: indicated otherwise (such as specifying that the compound (I) should be in “free base' or “free acid form), reference in the specification to a compound should be understood as includ ing salt forms of the compound, whether or not this is explic itly stated. Preparation and selection of suitable salt forms is 35 described in Stahl, et al., Handbook of Pharmaceutical Salts. Properties, Selection, and Use, Wiley-VCH 2002. When in the solid state, the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of Solvates, including hydrates. In general, the useful properties of the compounds described herein do not depend on whether the compound or salt thereof is or is in a particular Solid state form, Such as a polymorph or Solvate, so unless clearly indicated otherwise reference in the specifica tion to compounds and salts should be understood as includ ing any Solid State form of the compound, whether or not this 45 is explicitly stated. Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydro 50 gen include tritium and deuterium. The phrase “pharmaceutically acceptable' is employed herein to refer to those compounds, materials, compositions and/or dosage forms which are, within the scope of Sound medical judgment, Suitable for use in contact with the tissues 55 of human beings and animals without excessive toxicity, irri tation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The expression “therapeutically effective amount,” when used to describe an amount of compound administered in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, e.g., an amount that inhibits the abnormal growth or proliferation, or induces apoptosis of cancer cells, resulting in a useful effect. The terms “treating and “treatment’ mean causing a wherein: therapeutically beneficial effect, such as ameliorating exist 65 R" is selected from (C-Clio)alkyl, (C-Co)alkoxy, (C- ing symptoms, preventing or reducing additional symptoms, Co)alkylNH, Ar"-L- and unsubstituted or substituted ameliorating or preventing the underlying metabolic causes cycloalkyl;

US 9,388,214 B2 15 16 In some embodiments, R is methyl, isobutyl, -CH2OH. appropriate can be substituted, e.g., by 1, 2 or 3 substituents —(CH), NH, —(CH), NH, —(CH), NH, independently selected from (C-C)alkyl, (C-C)alkenyl, —(CH), C(=O)NH, -(CH), NHC(=NH)NH2, (C-C)alkynyl, halogen, (C-C)haloalkyl, -CN. —NO, —(CH) NHC(=O)NH2 or —CH2(1H-imidazol-4-yl). OR7, NR7, and NR7°C(=O)R’, wherein each R' In some embodiments, the amino acid having the carbon is independently selected from hydrogen and (C-C)alkyl, atom numbered 15 as its C-carbon atom (i.e., with R" as its e.g., methyl, ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, side-chain) can be, e.g., alanine, arginine, asparagine, citrul tert-butyl, neopentyl or n-hexyl. The aryl can be, e.g., mono line, 2,4-diaminobutyric acid, glutamine, histidine, isoleu Substituted, e.g., by any one of the aforementioned substitu cine, leucine, lysine, ornithine, phenylalanine, serine, tryp ent groups, e.g., in the para position of a phenyl group. The tophan or valine. 10 heteroaryl can be, e.g., monosubstituted, e.g., by any one of R is —(C-C)alkylene)-NR, or -((C-C)alkylene)- the aforementioned Substituent groups. NRC(=NR)NR'; wherein each R is independently In some embodiments, R7 can be unsubstituted or substi hydrogen or (C-C)alkyl, e.g., methyl. The (C-C)alkylene tuted cycloalkyl (Cy), e.g., unsubstituted or substituted (Cs chain can have 1, 2, 3, 4, 5 or 6 carbon atoms and can be C.)cycloalkyl or unsubstituted or substituted cycloalkyl((C- composed of methylenegroups. For example, Rican be of the 15 C.)alkylene) (Cy’-((C-C)alkylene)-), e.g., Cy-CH2- or formula—(CH), NR", or of the formula—(CH), Cy’-CH-CH ), e.g., unsubstituted or substituted (C-C) NRC(=NR)NR'. In some embodiments, R is cycloalkyl. When the cycloalkyl of the unsubstituted or sub -(CH), NR or -(CH), NRC(=NR) stituted cycloalkyl (Cy) or unsubstituted or substituted NR. In some embodiments, each R" is independently cycloalkyl((C-C)alkylene) group (Cy’-((C-C)alkylene)- hydrogen or methyl. In some embodiments, each R" is is Substituted, the cycloalkyl, e.g., C-C, cycloalkyl, e.g., hydrogen. In some embodiments, when Ris-((C-C)alky cyclopropyl, cyclobutyl, cylopentyl or cyclohexyl, can be lene)-NRC(=NR)NR', all of the R groups are hydro Substituted, e.g., by 1, 2 or 3 Substituents independently gen, or only one of the R" groups is (C-C)alkyl, e.g., selected from (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, methyl. halogen, (C-C)haloalkyl, -CN, NO, OR", NR', In some embodiments, Ris-(CH2). NH2-(CH2)4- 25 and - NR'C(=O)R’, wherein each R' is independently NH, -(CH), NH, or -(CH), NHC(=NH)NH2. In selected from hydrogen and (C-C)alkyl, e.g., methyl, ethyl, some embodiments, R is —(CH2). NHC(=NH)NH2. n-propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl In some embodiments, therefore, the amino acid having the or n-hexyl. The cycloalkyl can be, e.g., monosubstituted, e.g., carbon atom numbered 17 as its C-carbonatom (i.e., with R by any one of the aforementioned Substituent groups. as its side-chain) can be, e.g., alanine, arginine, 2,4-diami 30 In some embodiments, R is (C-C)alkyl In some embodi nobutyric acid, lysine or ornithine. ments, R is (C-C)alkyl. In some embodiments, R iss-bu In some embodiments, R' can be (C-C)alkyl, e.g., (C- tyl. C.)alkyl. R' can be, e.g., methyl, ethyl, n-propyl, isopropyl. In some embodiments, R7 is Ar’-. In some embodiments, isobutyl, sec-butyl, tert-butyl, neopenty1 or n-hexyl. R’ is Ar’-((C-C)alkylene)-. In some embodiments, R is In some embodiments, R7 can be unsubstituted or substi 35 Ar’-CH . In some embodiments, R7 is Ar"-CHCH-. In tuted cycloalkyl, e.g., (C-C2)cycloalkyl, e.g., (Cs-C7)cy some such embodiments, Ar" is phenyl. In some such cloalkyl or (C)cycloalkyl. R' can be, e.g., cyclopropyl, embodiments, Ar" is 4-hydroxyphenyl. In some embodi cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. In some ments, R is benzyl. In some embodiments, R is 4-hydroxy embodiments, the cycloalkyl can be unsubstituted. In some benzyl. embodiments, the cycloalkyl can be substituted. When R is 40 In some embodiments, R is Cy-. In some embodiments, Substituted cycloalkyl, the cycloalkyl can be substituted, e.g., R’ is Cy’-((C1-C4)alkylene)-. In some embodiments, R is by 1, 2 or 3 substituents independently selected from (C-C) Cy’-CH . In some embodiments, R7 is Cy-CHCH . In alkyl, (C-C)alkenyl, (C-C)alkynyl, halogen, (C-C)ha some such embodiments, Cy can be (C-C,)cycloalkyl or loalkyl, -OR" and oxo, wherein each R" is independently (C-C,)cycloalkyl. In some such embodiments, Cy can be selected from hydrogen and (C-C)alkyl, e.g., methyl, ethyl, 45 any one of cyclopropyl, cyclobutyl, cylopenty1 and cyclo n-propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl hexyl. In some such embodiments, R' can be any one of or n-hexyl. cyclopropyl, cyclobutyl, cylopenty1 and cyclohexyl. In some embodiments, R' can be unsubstituted or substi In some embodiments, therefore, the amino acid having the tuted aryl (Ar), e.g., unsubstituted or substituted phenyl. In carbonatom numbered 3 as its C-carbonatom (i.e., with R" as some embodiments, R' can be unsubstituted or substituted 50 its side-chain) can be, e.g., alanine, leucine, isoleucine, heteroaryl (Ar), e.g., unsubstituted or substituted 2-, 3-, or Valine, phenylalanine, or tyrosine. 4-pyridyl or unsubstituted or substituted 2- or 3-thienyl. In R can be NH or OH. In some embodiments, R is some embodiments, R' can be unsubstituted or substituted NH2. In some embodiments, Ris-OH. In some embodi aryl((C-C)alkylene) (Ar’-((C-C)alkylene)-, e.g., Ar ments, therefore, the acid having the carbon atom numbered CH or Ar-CHCH ), e.g., unsubstituted or substituted 55 1 as its C-carbonatom can be, e.g., cysteine or (R)-2-hydroxy benzyl. In some embodiments, R7 can be unsubstituted or 3-mercaptopropanoic acid. substituted heteroaryl((C-C)alkylene) (Ar’-((C-C)alky In some embodiments, R' is selected from —((C-C) lene)-, e.g., Ar’-CH2 or Ar-CH2CH2 ), e.g., unsubsti alkylene)-OR'' and -((C-C)alkylene)-C(=O)NR'. tuted or substituted 2-, 3- or 4-pyridylmethyl or unsubstituted The (C-C)alkylene chains can have 1, 2, 3, 4, 5 or 6 carbon or substituted 2- or 3-thienylmethyl. When the aryl of the 60 atoms and can be composed of methylene groups. For unsubstituted or substituted aryl (Ar) or unsubstituted or example, R' can be of the formula —(CH), C(=O) substituted aryl(C-C)alkylene) (Ar’-((C-C)alkylene)-) is NR', e.g., -(CH), C(=O)NR''. Each R' is substituted, or when the heteroaryl of the unsubstituted or independently selected from hydrogen and (C-C)alkyl, e.g., substituted heteroaryl (Ar) or unsubstituted or substituted methyl. In some embodiments, each R" is hydrogen. heteroaryl (C-C)alkylene) (Ar’-((C-C)alkylene)-) is sub 65 In some embodiments, R '' is Ar"-CH . In some stituted, the aryl, e.g., phenyl, or the heteroaryl, e.g., 2-, 3-, or embodiments, Ar' is unsubstituted heteroaryl. In some 4-pyridyl or unsubstituted or substituted 2- or 3-thienyl, as embodiments, Arm is substituted heteroaryl. In some

US 9,388,214 B2 23 24 -continued Compound No. 28 (S)

Compound No. 29 (S)

Compound No. 30

Compound No. 31

Compound No. 32

PhAc-D-Tyr(Me)-Phe-Gln-Asn-Lys-Pro-Arg-Glu-NH2 Compound No. 33 (S) iBuCO-D-Tyr(Me)-Phe-Gln-Asn-Lys-Pro-Arg-Glu-NH2 Compound No. 35 (S)

PhAc-D-Tyr(Me)-Phe-Gln-Asn-Lys-Pro-Arg-Glu-NH2 Compound No. 36 (S)

Compound No. 37 (S)

PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Glu-NH2 Compound No. 38 (S)

Compound No. 39 (R)

Compound No. 41 (S) (S)

Compound No. (S) (S)

US 9,388,214 B2 30 The following are molecular structures of particular exem plified compounds.

Compound No. 18 (below) O

HN (EI O HN '', O HN n (S) O H (S) (R) N 2. S N1 (S) ... -NH H (S) O (S) S NH2 O N O HN s H NH O (S) O NH2 ON O (R): (S)-N (S) N H HN HN O O N NH2 ..(S) O N C s s O (S) O NH O NH O (S) Š N- () HN 1N1

1s GE) NH2 HN NH2 O

Compound No. 39 (below)

US 9,388,214 B2 31 32 -continued Compound No. 41 (below)

Compound No. 42 (below) US 9,388,214 B2 33 34 -continued Compound No. 47 (below)

HN H N

NH -

NH2

Compound No. 48 (below)

HN

US 9,388,214 B2

-continued Compound No. 54 (below) O

HN

O O y - HN O O H 2. . NH S N N NH H O O 2 HN N O O O NH2 O-N : H NH O N HN Sr. NH O N HN O s O t O NH2 w N s' H w w Nul NH2 O O NH O NH O s N ~ O sX-( N NH wa O HN 1Y1 HN

1s GE) NH2 HN NH2 O O Compound No. 55 (below) O

HN

O HN O i-tsO O H 10.1. NH S N N NH H O O 2 HN N O HN O NH O ONH, ON H O N HN N^ HN O N HN O w * N O ( H O NH2 w Null NH, 9 O NH O NH O S 1 O sX-( N NH w O HN 1n 1 HN

1. GE) NH2 HN NH2 O O

III. Synthesis available for further coupling reactions. Due to the well In general, the methods of peptide synthesis are applicable developed methods available for peptide synthesis, methods to the synthesis of the compounds of formula (I). Methods of that are suitable for synthesis of the compounds of formula (I) peptide synthesis are well-developed in the art and typically 60 will be apparent to one skilled in the art from the structure of use protected amino acids, typically protected with a carbam Such compounds. Syntheses of particular compounds are ate group (e.g., t-butyloxycarbonyl (“BOC) or fluorenyl described in the Examples, and the methods described can be methoxycarbonyl (Fmoc). In a typical process, the protected adapted to additional compounds within the scope of formula amino acid is coupled to a free amino group of a growing (I), e.g., by Substituting appropriate amino acid derivatives as peptide chain to give a peptide extended by an additional 65 necessary. amino acid unit. The amino group of the new terminal amino Due to the biological importance of peptides and peptide acid of the growing peptide chain is then deprotected, and is analogues, a wide variety of amino acids is commercially US 9,388,214 B2 39 40 available or known in the art. In addition, numerous methods or Suspending system. Bland fixed oils, including synthetic of making Such compounds are known in the art. Therefore, mono- or di-glycerides, and fatty acids, such as oleic acid, the amino acids (as well as other intermediates) required to may be used. make compounds of formula (I) are commercially available, The amount of compound or composition to be adminis known in the art, or may be made by methods known in the tered will be determined by the responsible physician, taking art. into consideration all the relevant factors. In a preferred Methods of synthesizing amino acids and peptides are embodiment, the amount of compound or composition described, e.g., by: Benoiton, Chemistry of Peptide Synthesis, administered in each injection will be between about 0.001 CRC Press, 2006; Hughes, et al., Amino Acids, Peptides and mg to about 2.5 mg per Kg of body weight per day, with about 10 0.2 mg/Kg/day usually being Sufficient. Proteins in Organic Chemistry, Vol. 1, Origins and Synthesis The compounds, and compositions containing the com of Amino Acids, Wiley-VCH 2009: Hughes, et al., Amino pounds, could be given intravenously or subcutaneously, e.g., Acids, Peptides and Proteins in Organic Chemistry, Vol. 2. once, or chronically, to increase systemic vascular resistance Modified Amino Acids, Organocatalysis and Enzymes; and/or reduce splanchnic blood flow to treat any of the indi Wiley-VCH 2010; Hughes, et al., Amino Acids, Peptides and 15 cations. In some embodiments, the compounds are adminis Proteins in Organic Chemistry Vol. 3: Building Blocks, tered by intravenous injection. A course of treatment may Catalysis and Coupling Chemistry, Wiley-VCH 2011; involve a single injection or repeated injections. Hughes, et al., Amino Acids, Peptides and Proteins in Organic In general, dosing frequency can range from as infre Chemistry, Vol. 4: Amino Acids, Peptides and Proteins in quently as several times a week, up to several times per day. In Organic Chemistry, Protection Reactions, Medicinal Chem general, duration of therapy can range from as short as about istry, Combinatorial Synthesis, Wiley-VCH 2011; Amino a few days or a week, up to continuously. When treatment of Acids, Peptides and Proteins in Organic Chemistry, Vol. 5: the patient includes paracentesis, the course of treatment can Amino Acids, Peptides and Proteins in Organic Chemistry, comprise an injection immediately before the start of para Analysis and Function of Amino Acids and Peptides, Wiley centesis and one or more (such as two or three) injections VCH 2011; Howl, et al., Peptide Synthesis and Applications 25 following the paracentesis and at least one. Such as two, (Methods in Molecular Biology Vol. 298), Humana Press, injections afterwards. The injections may be separated by a 2010; Jones, Amino Acid and Peptide Synthesis, 2nd edn. period of a few hours, such as a period of between 4 and 12 h. Oxford University Press, 2002; Jones, The Chemical Synthe more preferably between 6 and 10 h. A course of treatment sis of Peptides (International Series of Monographs on Chem can comprise an injection before the start of paracentesis and istry), Oxford University Press, 1994; Pennington, et al., Pep 30 follow-up injections 8 and 16 h later. tide Synthesis Protocols (Methods in Molecular Biology Vol. The compounds provided herein are often administered in 35), Humana Press, 1994: Sewald, et al., Peptides: Chemistry the form of pharmaceutically acceptable, nontoxic salts, such and Biology, Wiley-VCH, 2009; Williams, et al., Chemical as acid addition salts, or of metal complexes, e.g., with Zinc, Approaches to the synthesis of Peptides and Proteins (New barium, calcium, magnesium, aluminum, or the like (which Directions in Organic & Biological Chemistry), CRC Press 35 are considered as addition salts for purposes of this applica 1997. tion), or of combinations of the two. Illustrative of such acid IV. Formulation and Administration addition salts are hydrochloride, hydrobromide, sulfate, Compositions provided herein may comprise the partial phosphate, nitrate, oxalate, fumarate, gluconate, tannate, V1 a receptor agonists of formula (I) described herein, their pamoate, maleate, acetate, citrate, benzoate, Succinate, algi salts, or any of the embodiments thereof. The compounds 40 nate, malate, ascorbate, tartrate, and the like. provided herein are particularly soluble at physiological pHs V. Methods of Use (e.g., about 6.8 to about 7.4) and can be prepared as relatively Also provided herein are methods of treatment and meth concentrated Solutions for administration, particularly for ods of using the compounds and compositions described and Subcutaneous injection. These compounds are well-tolerated provided herein, e.g., for the manufacture of medicinal prod in the body and do not tend to gel when administered Subcu 45 ucts for therapeutic effect. The compounds, and compositions taneously at effective concentrations. containing the compounds, are useful for treatment of, e.g., Generally, pharmaceutical compositions including Such complications of cirrhosis, including bacterial peritonitis, compounds and a suitable pharmaceutically acceptable HRS2 and refractory ascites. excipient can be administered parenterally, e.g., intrave Provided herein are compounds that have a reduced maxi nously, intraperitoneally, intramuscularly, Subcutaneously, or 50 mal efficacy at the V1 a receptor such that the risk of excessive the like. The pharmaceutical compositions will usually con vasoconstriction is significantly reduced. The compounds are tain an effective amount of the compound in conjunction with also useful, e.g., for treatment to increase blood pressure. a conventional, pharmaceutically-acceptable carrier or dilu These compounds are especially useful in the treatment of ent. Usually, the dosage will be from about 1 micrograms to conditions where a modest increase in blood pressure is desir about 2.5 milligrams of the peptide per kilogram of the body 55 able. Such as shock of hypovolemic (e.g., hemorrhagic) or weight of the host when given intravenously. The nature of vasodilatory (e.g., septic) origin, bleeding esophageal varices these compounds may permit effective oral administration; (BEV), hepatorenal syndrome (HRS), including type I and however, oral dosages might be higher. type II heptoarenal syndrome, cardiopulmonary resuscitation For parenteral administration, the compound may be for and anesthesia-induced hypotension. These compounds are mulated, e.g., as a sterile solution or Suspension. The com 60 also especially useful in the treatment of complications aris pounds may be formulated, e.g., as a sterile aqueous prepa ing from cirrhosis, including spontaneous bacterial peritoni ration that may be isotonic with the blood of the recipient. An tis, type II heptoarenal syndrome (HRS2) and refractory aqueous preparation may be formulated, e.g., according to ascites. Refractory ascites refers to an inability to mobilize known methods using Suitable dispersing agents, wetting asciitic fluid and can be diagnosed by the following criteria: agents, and/or Suspending agents. Water, Ringer's solution, 65 lack of response to maximal doses of diuretic for at least one and isotonic sodium chloride Solution are examples of Suit week; diuretic-induced complications in the absence of other able diluents. Sterile, fixed oils may be employed as a solvent precipitating factors; early recurrence of ascites within 4 US 9,388,214 B2 41 42 weeks of fluid mobilization; persistent ascites despite sodium further guidance on general experimental setup, as well as on restriction; mean weight loss less than 0.8 kg over 4 days the availability of required starting material and reagents: despite maximal doses of diuretics; and urinary sodium Kates, et al., Solid Phase Synthesis. A Practical Guide, Mar excretion less than Sodium intake (Siqueira, et al., Gastroen cel Dekker, New York, Basel, 2000; Greene, et al., Protective terol. Hepatol., (N.Y.), 2009, 5(9), 647-656.) Groups in Organic Synthesis, John Wiley Sons Inc., 2" Edi The compounds described herein will also have clinical use tion, 1991; Stewart, et al., Solid Phase Synthesis, Pierce in the treatment of orthostatic hypotension, paracentesis-in Chemical Company, 1984; Bisello, et al., J. Biol. Chem., duced circulatory dysfunction, acute hemorrhage, intra-op 1998, 273, 22498-22505; Merrifield, J. Am. Chem. Soc., erative blood loss and blood loss associated with burn debri 1963, 85,2149-2154; and Chang, et al., Fmoc Solid Phase dement and blood loss associated with epistaxis. 10 Peptide Synthesis: a Practical Approach, Oxford University Other conditions that can be treated with the compounds Press, Oxford, 2000. H-Rink-ChemMatrix resin (PCAS described herein include: hypertensive gastropathy bleeding: BioMatrix Inc., St-Jean-sur-Richelieu, Canada) was used as sepsis; severe sepsis; septic shock; hypotension, including starting material for automatic synthesis and Fmoc-Rink-AM prolonged and severe hypotension, and orthostatic hypoten resin (EMD Biosciences, San Diego, Calif.) was used for sion and intradialytic hypotension; cardiac arrest; trauma 15 manual synthesis. related blood loss; vasodilatory shock induced by cardio The following protecting groups were utilized to protect pulmonary bypass; milrinone-induced vasodilatory shock in the given amino acid side chain functional groups: Pbf (2.2, congestive heart failure; type I hepatorenal syndrome; type II 4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) for Arg; hepatorenal syndrome; anaphylactic shock; cardiovascular tBu (t-butyl) for Glu, Asp, Ser. Thr and Tyr; Trt (trityl) for instability induced by brain death; acute respiratory distress Cys, His, Gln and Asn; Boc (t-butoxycarbonyl) group for syndrome; acute lung injury; shock induced by metformin Dab, Ornand Lys. The Mtt (4-methyltrityl) or iv.Dde (1-(4.4- intoxication; shock induced by mitochondrial disease; shock dimethyl-2,6-dioxocyclohex-1-ylidene)3-methylbutyl) pro induced by cyanide poisoning; shock induced by vascular tecting groups were used in the side chain of the diamino acid leak syndrome induced by interleukin-2, another cytokine, residue of alpha amino acid 8 to provide an additional level of denileukin diftitoX or another immunotoxin, or by ovarian 25 orthogonality for branching. hyperstimulation syndrome; hypotension induced by end Peptides were prepared on Solid Support starting with resi stage renal disease; inflammatory bowel disease; reperfusion dues 1-9 of the peptide shown in Formula (I), followed by the injury; infant respiratory distress syndrome; severe acute res removal of the position 8 side chain orthogonal protecting piratory syndrome; ascites; vasodepressor Syncope; vasova group and the addition of the alpha amino acid 10-18 con gal syncope, e.g., postural hypotension with Syncope, or neu 30 taining peptide fragment. The Solid phase peptide synthesis rocardiogenic syncope; toxic shock syndrome; and idiopathic was performed manually, automatically on the Tribute pep systemic capillary leak syndrome (Clarkson's disease). tide synthesizer (Protein Technologies Inc., Tucson, Ariz.) or These compounds also display an improved therapeutic by a combination of the manual and automatic methods. index over therapy involving, e.g., terlipressin. Couplings of Fmoc-protected amino acids on the Tribute 35 synthesizer were mediated with HBTU/NMM in DMF except EXAMPLES for cysteine derivatives that were coupled with DIC/HOBt in DMF. Single cycles of 30-60 min. with a 5-fold excess of 1. General Methods activated Fmoc-protected amino acids were used during the Amino acid derivatives were purchased from commercial synthesis. Removal of the Fmoc protecting group was moni providers (Bachem, EMD Biosciences and Peptides Interna 40 tored by UV. Multiple (up to 10 times, as needed) two-minute tional). Resins were purchased from commercial Suppliers washes of the peptide resin with 20% piperidine in DMF were (PCAS BioMatrix Inc. and EMDBiosciences). Alladditional performed. reagents, chemicals and solvents were purchased from DIC/HOBt mediated couplings in DMF were employed for Sigma-Aldrich and VWR. all amino acids in manual mode. Single cycles of at least 2h Most of the compounds herein were synthesized by stan 45 with a 3-fold excess of activated Fmoc-protected amino acids dard methods in Solid phase peptide chemistry utilizing Fmoc were used during the synthesis. The completeness of cou methodology. The peptides were assembled either manually, plings was assessed with the ninhydrin (Kaiser) test. Removal automatically using a Protein Technologies Tribute Peptide of the Fmoc protecting group was achieved with a single 30 Synthesizer or by combination of manual and automatic Syn min. wash of the peptide resin with 20% piperidine in DMF. theses. If more convenient the compounds were assembled 50 After the peptide fragment containing alpha carbons 1-9 manually using combination of Boc and Fmoc strategies was assembled the position 8 side chain protecting group was (e.g., compounds 26, 29). removed. Peptide resins protected with the Mtt group were Preparative HPLC was performed on a Waters Prep LC treated with the HIPF/TFE/TIS/DCM 4/2/1/13 (v/v/v/v) System using a PrepPack cartridge Delta-Pack C18, 300 A. cocktail (3 times for 1 h). To remove the ivode protecting 15um, 47x300mm at a flow rate of 100 mL/min and/or on a 55 group the peptide resins were treated with 2% hydrazine/ Phenomenex Luna C18 column, 100 A, 5um,30x100mm at DMF (3 times for 10 min.). After the orthogonal protecting a flow rate of 40 mL/min. Analytical reverse phase HPLC was group was removed the remaining part (residues 10-18) of the performed on an Agilent Technologies 1200rr series liquid peptide was assembled by adding each amino acid sequen chromatograph using an Agilent Zorbax C18 column, 1.8 um, tially. 4.6x110 mm at a flow rate of 1.5 mL/min. Final compound 60 Upon completion of the peptide synthesis, the peptide res analyses were performed on an Agilent Technologies 1200 ins were washed with DCM and dried in vacuo. The resins Series chromatograph by reverse phase HPLC on a Phenom were treated with TFA/HO/TIS 96:2:2 (v/v/v) for 2 h to enex Gemini 110 A C18 column, 3 um, 2x150 mm at a flow remove the side-chain protecting groups with concomitant rate of 0.3 mL/min. Mass spectra were recorded on a MAT cleavage of the peptide from the resin. The peptides were Finningan LCQ electrospray mass spectrometer. Unless 65 filtered, precipitated with diethyl ether and decanted. The stated otherwise, all reactions were performed at room tem precipitate was dissolved in 10 mL of neat TFA and the perature. The following standard reference literature provides solution was subsequently poured into 200 mL of 10% aceto US 9,388,214 B2 43 44 nitrile in water. The linear peptide was oxidized with 0.1 M DMF and DCM. The resin was wet-split at this point and the I/MeoH. The oxidizer solution was added dropwise until synthesis was continued at a 1 mmol scale. (The remainder of yellow color persisted. The excess of iodine was reduced with the split portion was used to synthesize other compounds, solidascorbic acid. The pH was then adjusted to about 4 with according to the description herein.) concentrated ammonia. The solution obtained was loaded The 2-nitrobenzenesulfonyl group was introduced with directly onto an HPLC prep column and eluted with a gradient 2-nitrobenzenesulfonyl chloride (1.11 g, 5 mmol) and 2.4.6- of component B (see table below). collidine (1 mL, 7.5 mmol) in DCM. After 2 h the ninhydrin Each crude peptide was purified with buffer system P. The test was negative. The resulting resin-bound Sulfonamide was fractions with a purity exceeding 93%, determined by washed with dry DME and suspended in 5 mL of dry DME. reverse-phase analytical HPLC, were pooled and reloaded 10 2.63 g (10 mmol) 5-Fmoc-amino-1-pentanol and 2.63 g (10 onto the column and eluted with buffer T to provide trifluo mmol) were Subsequently added to the Suspension followed roacetate salts. To obtain acetate salts the fractions from runs by a solution of 1.97 mL (10 mmol) of DIAD and the resin with buffer P were reloaded onto the column and the column was shaken overnight. An aliquot of the resin was cleaved to was washed with 5 volumes of 0.1 Mammonium acetate. The test the completeness of the alkylation. No substrate peak was final product was eluted with buffer A. The fractions were 15 detected by HPLC analysis of the cleaved peptide. The resin pooled and lyophilized. was split again and the synthesis was carried on at a 0.2 mmol scale. The resin was then placed in two automatic synthesis vessels each containing about 0 1 mmol resin-bound interme TABLE 1. diate peptide. The synthesis was continued in parallel fashion Buffer Compositions on the Tribute peptide synthesizer. Single couplings (with each amino acid 10-18 added, one-at-a-time) were mediated Buffer Component A Component B with HBTU/NMM in DMF with a 5-fold excess of Fmoc P 0.25 M Triethylammonium 60% acetonitrile, 40% protected amino acids were used. The Fmoc protecting group Phosphate (TEAP) (pH 5.2) Component A was removed with two consecutive 10 min. washes with 20% T 0.1% Trifluoroacetic acid (TFA) 60% acetonitrile, 0.1% TFA 25 piperidine in DMF. The following derivatives were used in the A. 2% Acetic acid (AcOH) 60% acetonitrile, 2% AcOH automatic synthesis: Fmoc-Glu-NH., Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Asn (Trt)-OH, Fmoc To prepare the alkyl-linked (alkyl as substituent “O'” in Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-D-Tyr(Me)-OH and Formula (I) hybrids, residues 1-9 were assembled with an PhAc-OH. orthogonal protecting group (Mtt or iv.Dde) in position 8 as 30 After the entire peptide has been assembled, the two resins described above. The orthogonal protecting group was then were pooled and the 2-nitrobenzenesulfonyl group was removed and the 2-nitrobenzenesulfonyl group was intro removed with 5% potassiumthiophenolate in DMF (2 washes duced with 2-nitrobenzenesulfonyl chloride/2.4.6-collidine of 30 min. each). Upon completion of the peptide synthesis, in DCM. The resulting resin-bound sulfonamide was alky the peptide resin was washed with DCM and dried in vacuo. lated with an appropriate primary alcohol (e.g. 5-Fmoc 35 The peptide was cleaved from the resin with 20 mL of TFA/ amino-1-pentanol) under the Mitsunobu reaction conditions HO/TIS 96:2:2 (v/v/v) for 2 h. The resin was filtered off and (10 equivalents of alcohol/TPP/DIAD in dry DME, over TFA was evaporated. The crude product was precipitated with night). The remaining residues 10-18 were Subsequently diethyl ether and decanted. The precipitate was dissolved in added one-by-one and the 2-nitrobenzenesulfonyl was 10 mL of neat TFA and the solution was subsequently poured removed with 5% potassium thiophenolate in DMF (3 times 40 into 200 mL of 10% acetonitrile in water. The linear peptide for 30 min.). The cleavage, cyclization and purifications were was oxidized with 0.1 MI/MeoH. The oxidizer solution was performed as described above. added dropwise until yellow color persisted. The excess of The compounds prepared were typically found to be at iodine was reduced with solidascorbic acid. The pH was then least about 90% pure, e.g., at least about 95% pure, or at least adjusted to about 4 with concentrated ammonia. The obtained about 97% pure, or at least about 98.5% pure. 45 solution was loaded directly onto an HPLC prep column and Illustrative syntheses of some of the compounds described purified with buffer system Peluted with a gradient of com herein are provided below. ponent B (see table below). The fractions with a purity 2. Synthesis of Compound No. 2 exceeding 93%, determined by reverse-phase analytical The 1-9 fragment was assembled manually starting from HPLC, were pooled and reloaded onto the column and eluted 15 g (10 mmol) of Rink Amide AM resin (EMD Biosciences, 50 with buffer T to provide trifluoroacetate salt. The fractions catalog number 855004, 0.68 mmol/g). DIC/HOBt mediated were pooled and lyophilized. 46.2 mg (0.018 mmol, 9% couplings in DCM/DMF (1:1 V/V, for Gly, Orn, Pro, Cys, Ile, assuming 85% peptide content) of white peptide powder was Phe and Cys) or in DMF (Asn., Gln) were employed. Single obtained. cycles of at least 2 h with a 1.5-3-fold excess of activated The product purity was determined by analytical HPLC as Fmoc-protected amino acids were used during the synthesis. 55 99.0% and the observed M+H as 2213.8 (calc. M+H was The completeness of couplings was assessed with the ninhy 2214.1). drintest. Removal of the Fmoc protecting group was achieved 3. Synthesis of Compound No. 42 with a single 30 min. wash of the peptide resin with 20% The fragment comprising residues 5-9 (referring to For piperidine in DMF. The following amino acid derivatives mula (I)) was assembled manually starting from 0.68 g (1 were used to assemble residues 1-9 of the resin-bound pep 60 mmol) of Rink Amide AM resin (EMD Biosciences, catalog tide: Fmoc-Gly-OH, Fmoc-Orn(Mtt)-OH, Fmoc-Pro-OH, number 855004, 0.68 mmol/g). DIC/HOBt mediated cou Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)- plings in DMF were employed. Single cycles of at least 2h OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Boc-Cys(Trt)-OH. with a 3-4-fold excess of activated Fmoc-protected amino After the residue 1-9 peptide fragment has been assembled acids were used during the synthesis. The completeness of the resin was washed thoroughly with DCM and treated with 65 couplings was assessed with the ninhydrin test. Removal of the DCM/HFIP/TFE/TIS 13:4:2:1 (v/v/v/v) cocktail (2x2 h, the Fmoc protecting group was achieved with a single 30 min. 200 mL each). The resin was then washed with DCM, MeOH, wash of the peptide resin with 20% piperidine in DMF. The US 9,388,214 B2 45 46 following amino acid derivatives were used to assemble the used in the automatic synthesis. The Fmoc protecting group residue numbers 5-9 resin-bound peptide: Fmoc-Gly-OH, was removed with several consecutive 2 min. washes with Fmoc-Dab(ivDde)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH 20% piperidine in DMF. The following derivatives were used and Fmoc-ASn(Trt)-OH. After the fragment comprising resi in the automatic synthesis: Boc-Lys(Fmoc)-OH, Fmoc-Glu dues numbered 5-9 was assembled, the resin was wet-split NH., Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cit-OH, and the synthesis was continued at a 0.3 mmol scale on the Fmoc-ASn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Tribute peptide synthesizer with UV monitoring. (The Fmoc-D-Tyr(Me)-OH and Phac-OH. Upon completion of remainder of this product was used in the synthesis of other the peptide synthesis, the peptide resin was washed with compounds as described herein.) DCM and dried in vacuo. The peptide was cleaved from the Single couplings mediated with HBTU/NMM in DMF 10 with a 5-fold excess of Fmoc-protected amino acids were resin with 20 mL of TFA/HO/TIS 96:2:2 (v/v/v) for 2 h. The used. The Fmoc protecting group was removed with several Subsequent steps were the same as in the synthesis of com consecutive 2 min. washes with 20% piperidine in DMF. The pound 2. The fractions were pooled and lyophilized. 174.3 following amino acid derivatives were used to assemble the mg (0.063 mmol. 21% assuming 85% peptide content) of fragment comprising residues numbered 1-4 (see Formula 15 white peptide powder was obtained. (I)) as a resin-bound peptide: Fmoc-Gln(Trt)-OH, Fmoc-Ile The product purity was determined by analytical HPLC as OH, Fmoc-Phe-OH and Boc-Cys(Trt)-OH. After the residue 96.9% and the observed M+H was 2343.2 (calc. number 1-4 fragment was assembled the iv.Dde group was MH=2343.1). removed with 2% hydrazine/DMF (20 mL, 3x10 min.) and 5. Synthesis of Compound No. 29 by Boc/Fmoc strategy. the synthesis was continued on Tribute to introduce the linker The fragment comprising residues 8-9 (referring to For (Q) and the residues 10-18 of the peptide sequence. The mula (I)) was assembled manually starting from 5.00 g (3.5 synthesizer settings were identical as those used in the assem mmol) of MBHA resin (EMD Biosciences, catalog number bly of the 1-4 fragment. The following derivatives were used 855.006, 0.70 mmol/g). DCC or DIC/HOBt mediated cou in this part of automatic synthesis: Boc-Lys(Fmoc)-OH, plings in DCM were employed. Single cycles of at least 2h Fmoc-Lys(Mtt)-OH, Fmoc-Glu-NH., Fmoc-Arg(Pbf)-OH, 25 with a 2-4-fold excess of activated Boc-protected amino acids Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Asn (Trt)-OH, Fmoc were used during the synthesis. The completeness of cou Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-D-Tyr(Me)-OH and plings was assessed with the ninhidrin test. Removal of the Phac-OH. After the entire peptide sequence has been Boc protecting group was achieved with two consecutive assembled the resin was washed thoroughly with DCM and washes (5 and 25 min.) of the peptide resin with 50% TFA/ treated with the DCM/HFIP/TFE/TIS 13:4:2:1 (v/v/v/v) 30 DCM containing 1% m-cresol. Neutralization of the peptide cocktail (3x1 h, 20 mL each). The resin was thenwashed with DCM and DMF and acetylated with acetic anhydride (0.28 resin was accomplished with two 5 min. washes with 5% mL, 3 mmol) in DMF. Finally, the resin was washed with TEA/DCM. The following amino acid derivatives were used DMF, MeOH and DCM and dried in vacuo. The peptide was to assemble the residue numbers 8-9 resin-bound peptide: cleaved from the resin with 20 mL of TFA/HO/TIS 96:2:2 35 Boc-Gly-OH and Boc-Dab(Fmoc)-OH. After the fragment (v/v/v) for 2 h. The subsequent steps were the same as in the comprising residues numbered 8-9 was assembled, the resin synthesis of compound 2. The fractions were pooled and was split and the synthesis was continued manually at a 0.5 lyophilized. 249.6 mg (0.088 mmol. 29% assuming 85% mmol scale. (The remainder of this product was used in the peptide content) of white peptide powder was obtained. synthesis of other compounds as described herein.) The frag The product purity was determined by analytical HPLC as 40 ment comprising residues numbered 2-7 (see Formula (I)) 95.3% and the observed M+H was 2413.0 (calc. was Subsequently assembled using synthetic methods M+H=2413.2). described for the 8-9 fragment. The following derivatives 4. Synthesis of Compound No. 47 were used in this segment: Boc-Pro-OH, Boc-Cys(Mob)-OH, The fragment comprising residues numbered 5-9 (see For Boc-Asn-OH, Boc-Gln-OH, Boc-Ile-OH and Boc-Phe-OH. mula (I)) was assembled manually starting from 0.6 g (1 45 The resin was split again and the synthesis was continued mmol) of Rink Amide ChemMatrix resin (PCAS BioMatrix manually at a 0.13 mmol scale. The position 1 amino acid was Inc, catalog number 7-600-1310, 0.6 mmol/g). DIC/HOBt introduced as Z(2-Cl)-Cys(Mob)-OH and the Fmoc group mediated couplings in DMF were employed. Single cycles of was removed with two consecutive washes with 25% piperi at least 2 h with a 3-4-fold excess of activated Fmoc-protected dine in DMF (5 and 20 min., respectively). Fmoc-Lys(Boc)- amino acids were used during the synthesis. The complete 50 OH was then coupled and the remaining fragment comprising ness of couplings was assessed with the ninhydrin test. residues numbered 10-18 (see Formula (I)) was subsequently Removal of the Fmoc protecting group was achieved with a assembled by Boc chemistry. The following amino acid single 30 min. wash of the peptide resin with 20% piperidine derivatives were used to synthesize this fragment: Boc-Glu in DMF. The following amino acid derivatives were used to NH, Boc-Arg(Tos)-OH, Boc-Pro-OH, Boc-Ala-OH, Boc assemble the 1-9 resin-bound peptide: Fmoc-Gly-OH, Fmoc 55 Asn-OH, Boc-Gln-OH, Boc-Phe-OH, Boc-D-Tyr(Me)-OH Dab(ivDde)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc and Phac-OH. After the entire peptide sequence has been ASn(Trt)-OH, Fmoc-Hgn(Trt)-OH, Fmoc-Ile-OH, Fmoc assembled the resin was washed thoroughly with DMF and Phe-OH and Boc-Cys(Trt)-OH. After the 1-9 peptide treated with 25%piperidine in DMF (5 and 20 min). The resin fragment was assembled, the resin was washed 3 times with was then washed with DMF, suspended in NMP/DMSO (1:1, 20 mL of 2% hydrazine/DMF and wet-split at this point and 60 V/v) and guanylated with 1H-Pyrazole-1-carboxamidine HCl the synthesis was continued at the 0.3 mmol scale on the (Aldrich #02729LB)/DIPEA. Finally, the resin was washed Tribute peptide synthesizer with UV monitoring to introduce with DMF, MeOH and DCM and dried in vacuo. The peptide the linker (Q) and the residue 10-18 peptide sequence. (The was cleaved from the resin with 20 mL of HF/anisole 20:1 remainder of the 1-9 residue resin product was used to syn (v/v) for 1.5 h at 0°C. The resin/crude peptide was washed thesis other compounds described herein) 65 with 100 mL of ethyl ether and the peptide was extracted with Single couplings mediated with HBTU/NMM in DMF 100 mL of acetic acid/water 3:1, (v/v). The subsequent steps with a 5-fold excess of Fmoc-protected amino acids were were the same as in the synthesis of compound 2. The frac

US 9,388,214 B2 57 58 TABLE 2-continued Analytical Data for Example Compounds

Mass Spectrometric Data (M+H

Com- Calcu- Ob pound Structure lated served

60 (R) 22291 2229.2 H-Cys-Phe-Ile-Asn-Asn-Cys-Pro-Dab (CO-CH(NH2)-(CH2)4- It is N. PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Glu-NH2

61 23141 2314.4 (R) H-Cys-Phe-Ile-Hgn-Asn-Cys-Pro-Dab(CO-CH(NH-(CH2)4-NH)-Gly-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Gln-Pro-Arg-Glu-NH2

62 (R) 2257.1 2257.3 H-Cys-Phe-Ile-Hgn-Asn-Cys-Pro-Dab(CO-CH(NH-(CH2)4-NH)-Gly-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-D-Glu-NH2

63 (S) 22291 2229.2 H-Cys-Phe-Ile-Hgn-Asn-Cys-Pro-Dab(CO-CH(NH2) (CH2)2 -joo y-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Lys-NH2

64 (S) 2257.1 2257.3 H-Cys-Phe-Ile-Hgn-Asn-Cys-Pro-Dab(CO-CH(NH2)-(CH2)2 -joo y-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Lys-NH2

65 (R) 2257.1 2257.3 H-Cys-Phe-Ile-Hgn-Asn-Cys-Pro-Dab(CO-CH(NH2) (CH2)2-CO)-Gly-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Lys-NH2

66 (R) 22291 2229.3 H-Cys-Phe-Ile-Asn-Asn-Cys-Pro-Dab (CO-CH(NH2) (CH2)2-CO)-Gly-NH2 PhAc-D-Tyr(Me)-Phe-Gln-Asn-Ala-Pro-Arg-Lys-NH2

7. Biological Data for Illustrative Compounds 45 single integrated Flp Recombination Target (FRT) site from a. Agonist Activity at Vasopressin V1a Receptors pFRT/lacZeo or pFRT/lacZeo2. To generate the HEK-flpin The method is designed to determine the agonist activity of cell line stably expressing the human V1aR, the cells were compounds at the vasopressin V1a receptors in a cell-based co-transfected with the Flp-InTM expression vector contain Fluorescence Imaging Plate Reader (FLIPR) assay and to 50 ing the gene of interest (pcDNA5/FRT-hV1aR) and the Flp evaluate their ECso values (the concentration of a compound recombinase expression plasmid pCG44. Flp recombinase that produces 50% of the maximum possible response). Effi mediates insertion of the Flp-InTM expression vector into the cacy (%MPE) is also determined as the percentage of maxi genome at the integrated FRT site through site-specific DNA mal possible effect. This agonist assay utilizes cells from a recombination. Stable cell lines expressing the hV1aRfrom stable cell line (HEK-flpin) expressing the vasopressin V1a 55 the Flp-InTM expression vector can be generated by selection receptor. Intracellular calcium increase in response to agonist using hygromycin B. See Life Technologies/Invitrogen is measured through real-time fluorescence of an intracellular manual, Growth and Maintenance of Flp-InTM Cell Lines, calcium-sensitive dye. Cells are exposed to varying concen Version E, published Feb. 12, 2003, for detailed information. trations of test agonist compounds whereupon release of Arginine vasopressin (AVP) was used as reference agonist intracellular calcium is measured to determine the agonist 60 in the assay. Efficacy of this compound was set to be 100%. A potency and efficacy. stock solution of 5 mM was made up in DMSO and stored at Materials -20° C. Human cells used were from the Flp-InTM 293 cell line Reagents used were the following: Dimethyl sulfoxide (HEK-flpin). The cell line stably expresses the lacZ-ZeocinTM (DMSO) (Sigma, D8779): Dulbecco's Modification of fusion gene and is designed for use with the Flp-InTM expres 65 Eagle's Medium (DMEM) with glucose and sodium pyruvate sion vector containing the gene of interest and the Flp recom without L-glutamine (Mediatech, 15-013-CV); Fetal Bovine binase expression plasmid, pCG44. The cell line contains a Serum-Heat inactivated (FBS-HI) (Invitrogen, 16140-071); US 9,388,214 B2 59 60 FLIPR Calcium 4 Assay Kit, bulk format (Molecular The remaining steps of the assay were carried forward by Devices, R8141); Hanks’ Balanced Salt Solution (HBSS) FLIPR Tetra. A baseline reading was taken at 1-second (s) (Invitrogen, 14025-092); Hepes Buffer, pH 7.2 (Mediatech, intervals for 5 s followed by the addition of 5 ul of 10x 25-060-CI); Hygromycin B 50 mg/mL (Mediatech, 30-240 compounds (test, reference, or Blank). The agonist-induced CR); GlutaMAXTM-I Supplement, 200 mM (Invitrogen, fluorescence signal was then measured for 180s with initial 35050-061); Phenol red-free DMEM: DMEM with 4.5 g/L 120 readings at 1-second intervals followed by 20 readings at glucose and sodium pyruvate without L-glutamine and phe 3-second intervals. Overall, each well in the FLIPRassay was nol red (Mediatech, 17-205-CV); Probenecid (Sigma-Ald composed of the following components in a total Volume of rich, P-8761); and Trypsin EDTA: 0.05% Trypsin/0.53 mM 50 ul: 20 ulcells; 20 ul Calcium 4 Loading Buffer; 4.4 ul 10x EDTA (Mediatech, 25-052-CI). 10 test or reference compound. Supplies used were the following: 384 well black clear Averaged ECso (in nM) and Efficacy Average (as compared bottom, poly-D-lysine-coated Assay Plate (Corning, 3712); to AVP) are presented in Table 3. 384 well V-bottom plate, Dilution Plate (Greiner, 781280); Polystyrene test tube (BD Biosciences, 352057); and T175 TABLE 3 Cell" Flask (Sarstedt, 83.1812.302). Equipment and software 15 used were the following: Fluorometric Imaging Plate Reader Assay Results (FLIPR Tetra) (Molecular Devices); and ActivityBase soft Com- ECso Efficacy ware (IDBS, UK). pound if (Avg) nM Avg. (%) HEK-flpin-hV1aR, cmcqV1aR, dV1aR and pV1aR cells AVP O.O7 1OO.O were maintained in DMEM containing 10% (v/v) FBS-HI, 4 1 1.O 30 mM GlutaMAXTM-I, 25 g/mL Hygromycin B at 37° C. 2 O.69 52 under 5% CO in a humidified atmosphere. Subculture was 3 2.9 39 4 1.5 24 achieved by splitting semi-confluent cultures 1:3 to 1:6. On 5 1.3 28 the day prior to the assay, cells were removed from culture 6 2.5 28 flasks using Trypsin EDTA and harvested in phenol-red free 25 7 >10,000 24 DMEM containing 10% FBS-HI, 4 mM GlutaMAXTM-I. 8 >10,000 50 Cells were seeded into 384-well, black clear bottom, poly-D- 9 >10,000 34 10 >10,000 26 lysine-treated plates (20 ul/well), at cell density of 20,000 11 2.2 51 cells/well and incubated overnight. 12 2.3 39 All compounds were made up in 100% DMSO at 10 mM 30 13 2.4 48 14 2.1 37 stock concentrations and stored at -20°C. The compounds 15 2.3 44 were allowed to thaw just before the assay. Compounds were 16 2.4 46 assayed in duplicate at descending concentrations in half-log 17 2.9 62 increments with the highest concentration of 1 or 10 um 18 3.9 49 depending on the potency of the compounds. Typical dilution 35 19 2.5 50 2O 2.7 42 procedure of 10x final assay concentration involved a 1:100 21 .5 47 top dilution (e.g., 2 ul Stock into 198 ul dilution medium) 22 O.95 29 followed by half-log serial dilutions (e.g., 25.3 ul into 54.7 ul 23 7 51 24 2.3 33 dilution medium supplemented with 1% DMSO (v/v)). (Dilu 25 .4 49 40 tion media consisted of phenol-red free DMEM containing 26 O.93 32 10% FBS-HI, 4 mM GlutaMAXTM-I). The reference (AVP) 27 O 70 was tested at 1 um highest concentration (e.g. 1 Jul stock into 28 .2 44 29 .2 39 999 ul dilution medium). The final DMSO concentration in 30 .1 50 the assay was 0.1%. The reference (AVP) and the Blank 31 O.83 63 consisting of dilution media supplemented with 0.1% DMSO 45 32 O 56 (v/v) were included in each study. 33 .1 50 35 .2 43 ECso Determination 36 .2 46 Loading Buffer was prepared by dissolving 1 vial of Cal 37 3 44 cium 4 Assay reagent in 100 mL of 1x HBSS-20 mM Hepes 38 .4 46 buffer. Probenecid was resuspended at 250 mM in 1 MNaOH 50 39 8 48 41 .2 37 followed by a 1:100 dilution in the Loading Buffer for a final 42 O 37 working concentration of 2.5 mM. The pH was adjusted to 43 .4 59 74. 44 2.0 40 The cells were loaded with Loading Buffer as follows. Cell 45 O.8O 72 55 46 .4 62 plates were removed from the incubator and 20 ul of Loading 47 8 45 Buffer containing probenecid (2.5 mM) was added to each 48 .4 67 well. The cells were incubated for 1 hr at 37° C. under 5% 49 O.76 55 CO in a humidified atmosphere. The calcium image was 50 O.90 40 51 .4 43 obtained as follows. FLIPR Tetra was setup with the follow 52 .5 47 ing default parameters and a read mode with an excitation 60 53 7 43 wavelength of 470-495 nm and an emission wavelength of S4 .4 51 515-575 nm as determined by filter selection: Gain of 20; 55 7 56 Excitation Intensity of 80% (Default); Exposure Time of 0.4 56 8 60 57 .4 63 seconds (Default). 58 2.6 26 The cell plates were transferred to FLIPR Tetra, along with 65 59 7 26 a 384 well V-bottom plate pre-loaded with half-log concen 60 2.3 42 trations of test compounds at 10x final test concentrations. US 9,388,214 B2 61 62 TABLE 3-continued R is selected from (C-C)alkyl, unsubstituted or substi tuted cycloalkyl and Cy-CH : ASSay Results Cy- is unsubstituted or substituted aryl or unsubstituted or Com- ECso Efficacy substituted cycloalkyl; pound if (Avg) nM Avg. (%) R" is selected from (C-C)alkyl, (C-C)alkenyl, (C-C) 61 2.5 38 alkynyl, (C-C)haloalkyl, -((C-C)alkylene)-OR", 62 2.5 31 —((C-C)alkylene)-NR", -((C-C)alkylene)-S 63 1.1 36 (C-C)alkyl, —((C-C)alkylene)-C(=O)CR', 64 2.9 42 —((C-C)alkylene)-C(=O)NR', -((C-C)alky 65 4.5 31 10 lene)-C(=NR)NR', —((C-C)alkylene)-OC 66 2.3 30 (—O)R*, -((C-C)alkylene)-OC(=O)CR", —((C-C)alkylene)-OC(=O)NR', -((C-C)alky OTHER EMBODIMENTS lene)-NR“C(=O)R', -((C-C)alkylene)-NR“C 15 (=O)CR', -((C-C)alkylene)-NR“C(=O)NR', It is to be understood that while the invention has been -((C-C)alkylene)-NR“C(=NR)NR'', Ar" and described in conjunction with the detailed description -((C-C)alkylene)-Ar; thereof, the foregoing description is intended to illustrate and each R" is independently selected from hydrogen and not limit the scope of the invention, which is defined by the (C-C)alkyl; Scope of the appended claims. Other aspects, advantages and Ar" is selected from unsubstituted or substituted aryland unsubstituted or substituted heteroaryl; modifications are within the scope of the following claims. R is selected from —((C-C)alkylene)-NR", and What is claimed is: -((C-C)alkylene)-NRC(=NR)NR; 1. A compound according to formula (I): each R is independently selected from hydrogen and 25 (C-C)alkyl; Q is selected from the groups Q', Q, Q and Q': (I) R2 Q 30 O

Y O O R10 pi

HN n R6 RI 1's--" N 35 O R3 O Q2 O O R4 O Y pi 4 N NHsul 18 N 16 NH ) 40 Q E NH2 O O Rs | R9 X a O Y )r b O O (N" 45 X Ny b. *

pi HN 8 HN a R6 O O HNst O 50 Q O 55

or a salt thereof, wherein: a and b denote the bonds attaching Q to the remainder of the R" is selected from (C-Co)alkyl, (C-Clio)alkoxy, (C- 60 molecule: Co.)alkylNH. Ar"-L- and unsubstituted or substituted R is selected from hydrogen, (C-C)alkyl and cycloalkyl; C(-NR)NR; Ar"-L"- is selected from Ar"-, Ar"-CHCH , Ar-O , each R is hydrogen or (C-C)alkyl: Ar"-CHO. , Ar-NH- and Ar"-CH-NH : R’ is selected from (C-C)alkyl, Ar-, Ar’-((C-C)alky Ar' is unsubstituted aryl or substituted aryl; 65 lene)-, Cy-, and Cy’-((C-C)alkylene)-, R is selected from hydrogen, (C-C)alkyl, hydroxy, (C- Ar" is unsubstituted or substituted aryl or unsubstituted or C.)alkoxy and halogen; substituted heteroaryl;