Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
  • C07K5/126—Tetrapeptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/08—Antiseborrheics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
  • C07D487/14—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

• Immunosuppressive drugs are commonly used in transplantation and in treatment of autoimmune diseases. Production of these drugs is expensive, and the most frequently used of these drugs, namely cyclosporine A, tacrolimus and rapamycin, exhibit undesirable side- effects.
• Cyclolinopeptide A (CLA), a very hydrophobic cyclic nonapeptide, was first isolated from linen seeds in 1959. CLA is strongly immunosuppressive, with a potency comparable to that of cyclosporine A (CsA). The mechanism of action of CLA was shown to be similar to that of CsA, i.e. CLA formed a complex with cyclophilin A causing inactivation of calcineurin, albeit at much lower affinity (Gaymes et al., Febs Lett, 1997, 418, 224-227).
• CLA inhibited both humoral and cellular immune response and graft- versus-host reaction; prolonged survival of allogeneic skin grafts; tempered post-adjuvant polyarthritis in rats and hemolytic anemia of New Zealand Black mice; and, similarly to CsA, inhibited IL1 and IL-2 production.
• k, m, n and p are each independently 0, 1 or 2;
• R and R' are each independently selected from H and C 1-3 alkyl, or, when taken together, R and R' are -CR ⁇ 1 -X-CH 2 -, wherein CR ⁇ 1 is attached to the backbone nitrogen, R 1 and R 1' are each independently selected from H and C 1-3 alkyl, and X is selected from -CH 2 - , -CH 2 CH 2 -, -CH(OH)-, -0-, -S- and -NII-;
• R" and R" ' are each independently selected from H and C 1-3 alkyl, or, when taken together, R" and R'" are -CR 2 R 2 -X'-CH 2 -, wherein CR 2 R 2' is attached to the backbone nitrogen, R 2 and R 2' are each independently selected from H and C 1-3 alkyl, and X' is selected from -CH 2 -, -CH 2 CH 2 -, -CH(OH)-, -0-, -S- and -NII-; and [0011] R 3 and R 4 are each independently selected from aryl, substituted aryl, heteroaryl and substituted heteroaryl;
• At least one of R 3 and R 4 is phenyl. In some embodiments, at least one of R 3 and R 4 is 4-hydroxyphenyl. In some embodiments, at least one of R 3 and R 4 is 4-i-butoxyphenyl. In some embodiments, at least one of R 3 and R 4 is 2-indolyl. In some embodiments, R 3 and R 4 are both phenyl. In some embodiments, one of R 3 and R 4 is phenyl and the other of R 3 and R 4 is 4-hydroxyphenyl. In some embodiments, one of R 3 and R 4 is phenyl and the other of R 3 and R 4 is 4-i-butoxyphenyl.
• one of R 3 J and R 4 is phenyl and the other of R 3 and R 4 is 2-indolyl.
• the carbon to which -CH 2 -R is attached has absolute (R)-stereochemistry.
• the carbon to which -CH 2 -R is attached has absolute ( ⁇ -stereochemistry.
• the carbon to which -CH 2 -R 4 is attached has absolute (R)-stereochemistry.
• the carbon to which -CH 2 -R 4 is attached has absolute ( ⁇ -stereochemistry.
• one of k, m, n and p is 1, and the remainder of k, m, n and p are 0.
• two of k, m, n and p are 1, and the remainder of k, m, n and p are 0.
• at least one of k and m is not 0.
• at least one of n and p is not 0.
• at least one of k and m is not 0 and at least one of n and p is not 0.
• both k and n are 0.
• both k and n are 0, one of m and p is 0, and the other of m and p is 1.
• both k and n are 0 and both m and p are 1.
• all four amino acids are L-amino acids.
• three of the amino acids are L-amino acids and one of the amino acids is a D-amino acid. In some embodiments, two of the amino acids are L-amino acids and two of the amino acids are D-amino acids. In some embodiments, one of the amino acids is an L-amino acid and three of the amino acids are D-amino acids. In some embodiments, all four amino acids are D-amino acids.
• R and R' are taken together to form -(CH 2 ) 3 -, i.e. R and R' are taken together to form -CR ⁇ 1 -X-CH 2 - wherein R 1 and R 1 are both H and X is CH 2 .
• R" and R' " are taken together to form -(CH 2 ) -, i.e. R" and R' " are taken together to form -CR 2 R 2 -X'-CH 2 - wherein R 2 and R 2' are both H and X' is CH 2 .
• R and R' are taken together to form -(CH 2 ) 4 -, i.e.
• R and R' are taken together to form -CR 1 R 1' -X-CH 2 - wherein R 1 and R 1' are both H and X is (CH 2 ) 2 .
• R" and R" ' are taken together to form -(CH 2 ) 4 -, i.e. R" and R" ' are taken together to form -
• R and R' are taken together to form -CH 2 -CH(OH)-CH 2 -, i.e. R and R' are taken together to form - CR 1 R 1' -X-CH 2 - wherein R 1 and R 1' are both H and X is CH(OH).
• R" and R'" are taken together to form -CH 2 -CH(OH)-CH 2 -, i.e. R" and R'" are taken together to form -CR 2 R 2' -X'-CH 2 - wherein R 2 and R 2' are both H and X' is CH(OH).
• the carbon at which R' is attached has absolute ( ⁇ -stereochemistry. In some embodiments in which R and R' are taken together, the carbon at which R' is attached has absolute (R)- stereochemistry. In some embodiments in which R" and R' " are taken together, the carbon at which R" ' is attached has absolute (S)- stereochemistry. In some embodiments in which R" and R" ' are taken together, the carbon at which R" ' is attached has absolute (R)-stereochemistry.
• the compound is selected from the group consisting of:
• the compound is a compound of formula 1-1. In some embodiments, the compound is a compound of formula 1-2. In some embodiments, the compound is a compound of formula 1-3. In some embodiments, the compound is a compound of formula 1-4. In some embodiments, the compound is a compound of formula 1-5. In some embodiments, the compound is a compound of formula 1-6. In some embodiments, the compound is a compound of formula 1-7. In some embodiments, the compound is a compound of formula 1-8.
• the compound is selected from the group consisting of:
• the compound is a compound of foraiula I-A. In some embodiments, the compound is a compound of foraiula I-B. In some embodiments, the compound is a compound of formula I-C. In some embodiments, the compound is a compound of formula I-D. In some embodiments, the compound is a compound of formula I-E. In some embodiments, the compound is a compound of formula I-F. In some embodiments, the compound is a compound of formula I-G. In some embodiments, the compound is a compound of formula I-H. In some embodiments, the compound is a compound of formula I-L In some embodiments, the compound is a compound of formula I-J. In some embodiments, the compound is a compound of formula I-K.
• the compound is a compound of formula I-L. In some embodiments, the compound is a compound of formula I-M. In some embodiments, the compound is a compound of formula I-N. In some embodiments, the compound is a compound of formula I-O. In some embodiments, the compound is a compound of formula I-P. [0017] In some embodiments, one or more amino groups in the compound of Formula I are in protected form.
• a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier, excipient or diluent therefor.
• the compound is a compound of formula 1-1.
• the compound is a compound of formula 1-2.
• the compound is a compound of formula 1-3.
• the compound is a compound of formula 1-4.
• the compound is a compound of formula 1-5.
• the compound is a compound of formula 1-6.
• the compound is a compound of formula 1-7.
• the compound is a compound of formula 1-8.
• the compound is a compound of formula I-A. In some embodiments, the compound is a compound of formula I-B. In some embodiments, the compound is a compound of formula I-C. In some embodiments, the compound is a compound of formula I-D. In some embodiments, the compound is a compound of formula I- E. In some embodiments, the compound is a compound of formula I-F. In some embodiments, the compound is a compound of formula I-G. In some embodiments, the compound is a compound of formula I-H. In some embodiments, the compound is a compound of formula I- I. In some embodiments, the compound is a compound of formula I-J. In some embodiments, the compound is a compound of formula I-K.
• the compound is a compound of formula I-L. In some embodiments, the compound is a compound of formula I-M. In some embodiments, the compound is a compound of formula I-N. In some embodiments, the compound is a compound of formula I-O. In some embodiments, the compound is a compound of formula I-P.
• a method of suppressing immune response in a patient comprising administering to a patient in need thereof an efficacious amount of a compound of formula I.
• the immune response which is suppressed is inflammation.
• the immune response which is suppressed is transplant rejection.
• the compound is a compound of formula 1-1.
• the compound is a compound of formula 1-2.
• the compound is a compound of formula 1-3.
• the compound is a compound of formula 1-4.
• the compound is a compound of formula 1-5.
• the compound is a compound of formula 1-6.
• the compound is a compound of formula 1-7. In some embodiments, the compound is a compound of formula 1-8. In some embodiments, the compound is a compound of formula I-A. In some embodiments, the compound is a compound of formula I-B. In some embodiments, the compound is a compound of formula I-C. In some embodiments, the compound is a compound of formula I-D. In some embodiments, the compound is a compound of formula I-E. In some embodiments, the compound is a compound of formula I-F. In some embodiments, the compound is a compound of formula I-G. In some embodiments, the compound is a compound of formula I-H. In some embodiments, the compound is a compound of formula I-I.
• the compound is a compound of formula I-J. In some embodiments, the compound is a compound of formula I-K. In some embodiments, the compound is a compound of formula I-L. In some embodiments, the compound is a compound of formula I-M. In some embodiments, the compound is a compound of formula I-N. In some embodiments, the compound is a compound of formula I-O. In some embodiments, the compound is a compound of formula I-P.
• a method of treating or preventing an immune-mediated disease or condition in a patient comprising administering to a patient in need thereof an efficacious amount of a compound of formula I.
• a method for lowering the toxicity profile of a second drug comprising administering a compound of formula I in conjunction with the second drug.
• the immune-mediated disease or condition is selected from the group consisting of auto-immune diseases, inflammation processes, transplant rejection, and allergic reactions.
• the immune-mediated disease or condition is selected from Psoriasis, lichen planus and other papulosquamous disorders.
• the immune-mediated disease or condition is selected from eczema and dermatitis.
• the eczemea or dermatitis is selected from eczema, atopic eczema, seborrheic dermatitis, and pompholyx.
• the immune-mediated disease or condition is a skin reaction to sunlight.
• the immune-mediated disease or condition selected from non-specific skin irritation and insect bite.
• the immune-mediated disease or condition is a urticaria.
• the immune-mediated disease or condition is selected from the group consisting of a primary skin tumor (e.g.
• the compound of formula I is administered in conjunction with a chemotherapeutic drug in order to reduce the toxic effects of the chemotherapeutic drug.
• the compound is a compound of formula 1-1.
• the compound is a compound of formula 1-2.
• the compound is a compound of formula 1-3.
• the compound is a compound of formula 1-4. In some embodiments, the compound is a compound of formula 1-5. In some embodiments, the compound is a compound of formula 1-6. In some embodiments, the compound is a compound of formula 1-7. In some embodiments, the compound is a compound of formula 1-8. In some embodiments, the compound is a compound of formula I-A. In some embodiments, the compound is a compound of formula I-B. In some embodiments, the compound is a compound of formula I-C. In some embodiments, the compound is a compound of formula I-D. In some embodiments, the compound is a compound of formula I-E. In some embodiments, the compound is a compound of formula I-F.
• the compound is a compound of formula I-G. In some embodiments, the compound is a compound of formula I-H. In some embodiments, the compound is a compound of formula I-I. In some embodiments, the compound is a compound of formula I-J. In some embodiments, the compound is a compound of formula I-K. In some embodiments, the compound is a compound of formula I-L. In some embodiments, the compound is a compound of formula I-M. In some embodiments, the compound is a compound of formula I-N. In some embodiments, the compound is a compound of formula I-O. In some embodiments, the compound is a compound of formula I-P.
• kits comprising a compound of formula I and instructions for using the compound to (a) suppress an immune response in a patient, (b) treat or prevent an immune-mediated disease or condition in a patient, or (c) lower the toxicity profile of a second drug.
• the immune response is inflammation.
• the immune response is transplant rejection.
• the immune-mediated disease or condition is selected from the group consisting of auto-immune diseases, inflammation processes, transplant rejection, allergic reactions.
• the immune-mediated disease or condition is selected from Psoriasis, lichen planus and other papulosquamous disorders.
• the immune-mediated disease or condition is selected from eczema and dermatitis.
• the eczemea or dermatitis is selected from eczema, atopic eczema, seborrheic dermatitis, and pompholyx.
• the immune-mediated disease or condition is a skin reaction to sunlight.
• the immune-mediated disease or condition selected from non-specific skin irritation and insect bite.
• the immune-mediated disease or condition is a urticaria.
• the immune- mediated disease or condition is selected from the group consisting of a primary skin tumor (e.g.
• the instructions instruct administering the compound of formula I in conjunction with a chemotherapeutic drug in order to reduce the toxic effects of the chemotherapeutic drug.
• the compound is a compound of formula I- 1.
• the compound is a compound of formula 1-2.
• the compound is a compound of formula 1-3.
• the compound is a compound of formula 1-4. In some embodiments, the compound is a compound of formula 1-5. In some embodiments, the compound is a compound of formula 1-6. In some embodiments, the compound is a compound of formula 1-7. In some embodiments, the compound is a compound of formula 1-8. In some embodiments, the compound is a compound of formula I- A. In some embodiments, the compound is a compound of formula I-B. In some
• the compound is a compound of formula I-C. In some embodiments, the compound is a compound of formula I-D. In some embodiments, the compound is a compound of formula I-E. In some embodiments, the compound is a compound of formula I-F. In some embodiments, the compound is a compound of formula I-G. In some embodiments, the compound is a compound of formula I-H. In some embodiments, the compound is a compound of formula I-I. In some embodiments, the compound is a compound of formula I-J. In some embodiments, the compound is a compound of formula I-K. In some embodiments, the compound is a compound of formula I-L. In some embodiments, the compound is a compound of formula I-M. In some embodiments, the compound is a compound of formula I- N. In some embodiments, the compound is a compound of formula I-O. In some
• the compound is a compound of formula I-P.
• a method for making a compound of formula I comprising cyclizing a compound having the formula II- 1, ⁇ -2, II-3 or II-4, wherein R, R', R", R" ⁇ R 3 , R 4 , k, m, n and p are as defined in formula I (collectively referred to hereinafter as compounds of formula ⁇ ):
• the method further comprises synthesizing the compound of formula II- 1, ⁇ -2, II-3 or II-4.
• the compound of formula II- 1, ⁇ -2, II-3 or II-4 is formed by solid-phase synthesis.
• the compound of formula ⁇ is selected from the group consisting of:
• II-2-a H 2 N-Phe-Pro-Pro- ⁇ 3 hoPhe-OH
• II-3-a H 2 N- ⁇ 3 hoPhe-Phe-Pro-Pro-OH
• II-2-C H 2 N- ⁇ 3 hoPhe-Pro-Pro- ⁇ 3 hoPhe-OH
• II-3-C H 2 N- ⁇ 3 hoPhe-Pro-Pro- ⁇ 3 hoPhe-OH
• II-2-h H 2 N-Phe-Pro-Hyp- ⁇ 3 hoPhe-OH
• II-3-h H 2 N- ⁇ 3 hoPhe-Phe-Pro-Hyp-OH
• the compound is a compound of formula II-4-a. In some embodiments, the compound is a compound of formula II- 1 -a. In some embodiments, the compound is a compound of formula ⁇ -2-a. In some embodiments, the compound is is a compound of formula II-3-a. In some embodiments, the compound is a compound of formula ⁇ -4-b. In some embodiments, the compound is a compound of formula ⁇ -1-b. In some embodiments, the compound is a compound of formula II-2-b. In some embodiments, the compound is a compound of formula ⁇ -3-b. In some embodiments, the compound is a compound of formula ⁇ -4-c. In some embodiments, the compound is a compound of formula II-l-c.
• the compound is a compound of formula ⁇ -2-c. In some embodiments, the compound is a compound of formula ⁇ -3-c. In some embodiments, the compound is a compound of formula ⁇ -4-d. In some embodiments, the compound is a compound of formula ⁇ -1-d. In some embodiments, the compound is a compound of formula II-2-d. In some embodiments, the compound is a compound of formula ⁇ -3-d. In some embodiments, the compound is a compound of formula ⁇ -4-e. In some embodiments, the compound is a compound of formula ⁇ -1-e. In some embodiments, the compound is a compound of formula ⁇ -2-e. In some embodiments, the compound is a compound of formula II-3-e.
• the compound is a compound of formula ⁇ -4-f. In some embodiments, the compound is a compound of formula ⁇ -1-f. In some embodiments, the compound is a compound of formula ⁇ -2-f. In some embodiments, the compound is a compound of formula ⁇ -3-f. In some embodiments, the compound is a compound of formula ⁇ -4-g. In some embodiments, the compound is compound of formula II-1-g. In some embodiments, the compound is compound of formula II-2-g. In some embodiments, the compound is compound of formula II-3-g. In some embodiments, the compound is a compound of formula II-4-h. In some embodiments, the compound is compound of formula II-1-h. In some embodiments, the compound is compound of formula II-2-h. In some embodiments, the compound is compound of formula II- 3 -h.
• the compound of formula II is selected from the group consisting of
• the compound is a compound of foraiula ⁇ - ⁇ -l. In some embodiments, the compound is a compound of foraiula II-A-2. In some embodiments, the compound is a compound of foraiula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of foraiula ⁇ - ⁇ -4. In some embodiments, the compound is a compound of formula II- B-l. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of formula II-B-4. In some embodiments, the compound is a compound of formula II-C-1.
• the compound is a compound of formula II-C-2. In some embodiments, the compound is a compound of formula II-C-3. In some embodiments, the compound is a compound of formula II-C-4. In some embodiments, the compound is a compound of formula ⁇ -D-l. In some embodiments, the compound is a compound of formula II-D-2. In some embodiments, the compound is a compound of formula II-D-3. In some embodiments, the compound is a compound of formula II-D-4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -l. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3.
• the compound is a compound of formula ⁇ - ⁇ -4. In some embodiments, the compound is a compound of foraiula ⁇ -F-l. In some embodiments, the compound is a compound of foraiula II-F-2. In some embodiments, the compound is a compound of foraiula II-F-3. In some embodiments, the compound is a compound of foraiula II-F-4. In some embodiments, the compound is a compound of foraiula ⁇ -G-l. In some embodiments, the compound is a compound of foraiula II-G-2. In some embodiments, the compound is a compound of foraiula II-G-3.
• the compound is a compound of foraiula II-G-4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -1. In some embodiments, the compound is a compound of formula II-II-2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -4. In some embodiments, the compound is a compound of formula ⁇ -J-l. In some embodiments, the compound is a compound of formula II-J-2. In some embodiments, the compound is a compound of formula II-J-3. In some embodiments, the compound is a compound of formula II-J-4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -l.
• the compound is a compound of formula ⁇ - ⁇ -2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of formula II-K-4. In some embodiments, the compound is a compound of formula ⁇ -L-l. In some embodiments, the compound is a compound of formula II-L-2. In some embodiments, the compound is a compound of formula II-L-3. In some embodiments, the compound is a compound of formula II-L-4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -l. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3.
• the compound is a compound of formula ⁇ - ⁇ -4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -1. In some embodiments, the compound is a compound of formula II-N-2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -l. In some embodiments, the compound is a compound of formula ⁇ -0-2. In some embodiments, the compound is a compound of formula II-0-3. In some embodiments, the compound is a compound of formula ⁇ -0-4. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -l.
• the compound is a compound of formula ⁇ - ⁇ -2. In some embodiments, the compound is a compound of formula ⁇ - ⁇ -3. In some embodiments, the compound is a compound of formula II-P-4. DETAILED DESCRIPTION
• compounds of formula I exhibit immunosuppressive and/or antiinflammatory activity, while at the same time exhibiting less toxicity, than some known compounds.
• compounds of formula I may be useful as immunosuppressive and/or antiinflammatory agents.
• immunosuppressive and/or antiinflammatory agents As used herein, the term “immune-mediated” refers to a disease or condition in which the body' s immune system overreacts and/or attacks the body.
• Alkyl is intended to include linear, branched, or cyclic saturated hydrocarbon structures and combinations thereof.
• Lower alkyl refers to alkyl groups of from 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t- butyl and the like. Preferred alkyl groups are those of C 2 o or below.
• Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl and the like.
• Ci to C 2 o hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, camphoryl and naphthylethyl.
• the term "carbocycle” is intended to include ring systems consisting entirely of carbon but of any oxidation state. Thus (C3-Q0) carbocycle refers to such systems as cyclopropane, benzene and cyclohexene; (C 8 -C 12 ) carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene.
• Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen atom. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower- alkoxy refers to groups containing one to four carbons.
• Oxaalkyl refers to alkyl residues in which one or more carbons has been replaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the like.
• Acyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality.
• One or more carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t- butoxycarbonyl, benzyloxycarbonyl and the like.
• Lower-acyl refers to groups containing one to four carbons.
• Aryl means 6-membered aromatic ring; a bicyclic 9- or 10-membered aromatic ring system; or a tricyclic 13- or 14-membered aromatic ring system.
• the aromatic 6- to 14- membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene.
• Heteroaryl mean a 5- or 6-membered heteroaromatic ring containing 1-3 heteroatoms selected from O, N, or S; a bicyclic 9- or 10-membered heteroaromatic ring system containing 1-3 heteroatoms selected from O, N, or S; or a tricyclic 13- or 14-membered heteroaromatic ring system containing 1-3 heteroatoms selected from O, N, or S.
• the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene,
• Arylalkyl refers to a substituent in which an aryl residue is attached to the parent structure through alkyl. Examples are benzyl, phenethyl and the like. Heteroarylalkyl refers to a substituent in which a heteroaryl residue is attached to the parent structure through alkyl. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl and the like.
• Heterocycle means a cycloalkyl or aryl residue in which from one to three carbons is replaced by a heteroatom selected from the group consisting of N, O and S.
• the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
• heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like.
• heteroaryl is a subset of heterocycle in which the heterocycle is aromatic.
• heterocyclyl residues additionally include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxo-pyrrolidinyl, 2- oxoazepinyl, azepinyl, 4-piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl,
• Substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl, aryl, cycloalkyl, or heterocyclyl wherein up to three H atoms in each residue are replaced with alkyl, halogen, haloalkyl, hydroxy, loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phen benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryloxy.
• halogen means fluorine, chlorine, bromine or iodine.
• Boc t-butyloxy carbonyl
• DIEA ⁇ , ⁇ -diisopropylethyl amine
• DIPEA diisopropylethylamine
• HATU 0-(7-Azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium
• HBTU 0-(benzotriazol-l-yl)-l,l,3,3-tetramethyluronium
• HOBt 1-hydroxybenzotriazole
• TBTU 0-(benzotriazol-l-yl)-l,l,3,3-tetramethyluronium
• the notation " ⁇ 3 - ⁇ -" (or “beta 3-homo-") is used herein to refer to an amino acid having an extra methylene (-CH 2 -) in the backbone between the side-chain bearing carbon atom and the terminal nitrogen atom; the notation “ ⁇ 2 - ⁇ -” (or “beta 2-homo-”) is used herein to refer to an amino acid having an extra methylene in the backbone between the side-chain bearing carbon atom and the terminal carbon atom.
• Embodiments of the present invention include compounds of formula I in the form of salts, in particular acid addition salts.
• Suitable salts include those formed with both organic and inorganic acids.
• Such acid addition salts will normally be pharmaceutically acceptable, although salts of non-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question.
• preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p- toluenesulphonic, benzenesulphonic and isethionic acids.
• Salts of the compounds of formula I can be made by reacting the appropriate compound in the form of the free base with the appropriate acid.
• the compounds of formula I in accordance with the embodiments of the invention are cyclic tetrapeptides.
• the synthesis of these peptides may be accomplished by cyclizing corresponding linear peptides that are themselves synthesized using methodologies known in the art; see, for example, Merrifield, J. Am. Chem. Soc, 85:2149 (1964); Houghten, Proc. Natl. Acad. Sci. USA, 82:5132 (1985); Kelley & Winkler in Genetic Engineering Principles and Methods, Setlow, J. K, ed., Plenum Press, N.Y., vol. 12, pp 1-19 (1990); Stewart & Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, M. (1984); Mergler et al. (1988) Tetrahedron Letters 29:4005-4008; Mergler et al. (1988) Tetrahedron Letters
• Such synthesis may be accomplish via liquid-phase or solid-phase synthesis, or by a combination of both, as is known in the art.
• Liquid phase methods (often referred to as solution phase methods) of synthesis carry out all reactions in a homogeneous phase. Successive amino acids are coupled in solution until the desired peptide material is formed. During synthesis, successive intermediate peptides are purified by precipitation and/or washes.
• SPPS solid phase peptide synthesis
• a first amino acid or peptide group is bound to an insoluble support, such as a resin.
• Successive amino acids or peptide groups are added to the first amino acid or peptide group until the peptide material of interest is formed.
• the product of solid phase synthesis is thus a peptide bound to an insoluble support.
• Peptides synthesized via SPPS techniques are then cleaved from the resin, and the cleaved peptide is isolated.
• solid phase synthesis begins at the carboxy terminus of the putative peptide by coupling a protected amino acid to an inert solid support.
• the inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid.
• the macromolecular support is a cross-linked polymeric resin (e.g. a polyamide or polystyrene resin) as shown in FIGS. 1-1 and 1-2, on pages 2 and 4 of Stewart & Young, supra.
• the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester.
• a macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the a-amino group of the blocked amino acids in peptide synthesis. If a base-labile a-protecting group is used, then it is desirable to use an acid- labile link between the peptide and the solid support.
• an acid-labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis as described on page 16 of Stewart & Young, supra.
• a peptide anchor link and a-protecting group that are differentially labile to acidolysis can be used.
• an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis as described on pages 11-12 of Stewart & Young, supra. Guiller et al., Chem Rev. 2000, 100, 2091-2157, reviewed linkers and cleavage strategies in solid-phase organic synthesis and combinatorial chemistry, including peptide synthesis.
• the a-amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, triethylamine (TEA).
• TFA trifluoroacetic acid
• TAA triethylamine
• the next a-amino and sidechain protected amino acid in the synthesis is added.
• the remaining a-amino and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support.
• some amino acids may be coupled to one another to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain
• the condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the axide method, mixed acid anhydride method, DCC ( ⁇ , ⁇ '-dicyclohexylcarbodiimide) or DIC ( ⁇ , ⁇ '-diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-l-yl-oxy-tris [dimethylamino] phosphonium hexafluorophosphate) method, N-hydroxysuccinic acid imido ester method, etc, and Woodward reagent K method.
• the axide method mixed acid anhydride method
• DCC ⁇ , ⁇ '-dicyclohexylcarbodiimide
• DIC ⁇ , ⁇ '-diisopropylcarbodiimide
• active ester method p-nitropheny
• a- and ⁇ -amino side chains can be protected, for example, with benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNoc), o-chlorobenzyloxycarbonyl [Z(2C1) or 2-C1- Z], p-nitrobenzyloxycarbonyl [Z(N0 2 )], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxy- carbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxy-carbonyl (Adoc), 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl (Pht), formy
• side chain protecting groups include acetyl (Ac), benzoyl (Bz), tert butyl (t-Bu), triphenylmethyl (trityl, Trt), tetrahydropyranyl, benzyl (Bzl), 2,6-dichlorobenzyl, nitro, p-toluenesulfonyl (Tos), xanthyl (Xan), benzyl, methyl, ethyl, and t- butyl ester, and aromatic or aliphatic urethan-type protecting groups, photolabile groups such as nitro veratryl oxycarbonyl (Nvoc), and fluoride labile groups such as
• amino terminal protecting groups include: (1) acyl-type protecting groups, such as formyl, acrylyl (Acr), benzoyl (Bz) and acetyl (Ac); (2) aromatic urethan-type protecting groups, such as benzyloxy- carbonyl (Z) and substituted Z, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycar- bonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups
• Preferred protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4- biphenylyl)-propyl(2)oxycarbonyl (Bpoc), 2-phyenlpropyl(2)-oxycarbonyl (Poc), and t- butyloxycarbonyl (Boc).
• Protective groups for the carboxy functional group are exemplified by benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmeth- yl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group.
• the guanidino group of arginine may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-meth- oxybenzesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysul- fonyl (Mts), and the like.
• the thiol group of cysteine can be protected with p-methoxybenzyl, trityl, and the like.
• a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients.
• the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
• the most suitable route may depend upon the condition and disorder of the recipient.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods for making such formulations include the step of bringing into association a compound of formula I or a pharmaceutically acceptable salt or solvate thereof ("active ingredient”) with the carrier which constitutes one or more accessory ingredients.
• active ingredient a pharmaceutically acceptable salt or solvate thereof
• the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
• Formulations in accordance with embodiments of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
• the active ingredient may also be presented as a bolus, electuary or paste.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
• Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
• Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
• Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
• Formulations for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.
• Preferred unit dosage formulations are those containing an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of the active ingredient.
• formulations in accordance with embodiments of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
• compounds in accordance with embodiments of the invention may be used for the treatment or prevention of certain diseases or conditions.
• preventing refers to administering a medicament beforehand to forestall or obtund an attack.
• the person of ordinary skill in the medical art recognizes that the term “prevent” is not an absolute term. In the medical art it is understood to refer to the
• embodiments of the invention may be used to suppress an immune response, "suppress” will be understood to include reducing the degree of the response, and not necessarily absolutely preventing the response.
• Fig. 1 shows antigen- specific increase of the ear thickness derived from the experiment described in Example 2;
• Fig. 2 shows total numbers of cells in the parotid lymph nodes observed in the experiment described in Example 2;
• Fig. 3 shows the content of viable and dead cells in the parotid lymph nodes described in Example 2;
• Fig. 4 presents number and participation of cell types in the draining lymph nodes registered in the experiment described in Example 2;
• Fig. 5 presents toxicity of the compound of formula IA against mononuclear cells from human blood in as described in Example 3;
• Fig. 6 presents effects of the intraperitoneal administration of the compound of formula IA on the humoral immune response of mice to sheep erythrocytes as described in Example 4;
• FIG. 7 shows effects of the intraperitoneal administration of the compound of formula IA on the cellular immune response to ovalbumin as described in Example 5;
• FIGs. 8 A and 8B show the effects of peptides in accordance with embodiments of the invention on PMBC survival in vitro;
• FIGs. 9 A and 9B show the effects of peptides in accordance with embodiments of the invention on PHA-induced PBMC proliferation in vitro;
• FIGs. 10A and 10B show the effects of several compounds on changes in ear thickness in response to antigenic challenge, as described in Example 7;
• FIG. 11 shows the permeability of capillary blood vessels in the Evans blue test, as described in Example 7;
• Fig. 12 shows the total number of cells in the draining lymph nodes, as described in Example 7;
• Fig. 13 shows the effects of the compounds on the numbers of circulating leukocytes, as described in Example 7;
• Fig. 14 which shows the types of leukocytes present in different cases, as described in Example 7.
• Fig. 15 provides morphometric data on the number and composition of cells in mouse auricles, as described in Example 7.
• Cyclic tetrapeptides according to embodiments of the invention can be synthesized by the use of known peptide synthesis methods, such as solution-phase or solid-phase methods. In general the synthesis involves two consecutive steps: (1) synthesis of a linear tetrapeptide and (2) cyclization to yield the cyclic tetrapeptide.
• the linear tetrapeptide may be prepared in protected form and then deprotected prior to cyclization.
• the resin was swelled in dimethylformamide (DMF) (0.25 mmol, 10 ml/g resin) for 15 min.
• DMF dimethylformamide
• Amino acid or peptide amino groups on the resin were acylated by shaking with a mixture of Fmoc-protected amino acid (4 eq), HBTU or TBTU (4 eq) and DIPEA (4 eq) dissolved in anhydrous DMF (4 ml/mmol), for 20 hours.
• the Kaiser test (for all amino acids except proline) was used to estimate if all amino groups were acylated.
• the resin was washed according to points 3-5 of the protocol.
• the Fmoc group was removed from the peptide as described in point 2, and the resin was washed again with DMF, MeOH and DCM as in points 3-5.
• the resin was dried overnight in a desiccator over KOH pellets under reduced pressure at room temperature.
• Peptides were cleaved from the dried, Wang-type resin with a mixture of trifluoroacetic acid /water /triisopropylsilane 95:2.5:2.5 (v/v/v; 10 ml/lg of peptidyl-resin).
• the solution obtained was partially evaporated at room temperature under reduced pressure and peptide was precipitated with 10 volumes of ether. After being filtered off, the crude peptide was dissolved in 0.05 M aqueous HCl and evaporated to dryness. The residue was dissolved in water and lyophilized.
• Peptides were cleaved from 2-Cl-trityl resin by treatment with a 1:3: 1 mixture of acetic acid / dichloromethane / trifluorethanol (v/v/v; 10 ml/lg of peptidyl-resin).
• the resulting cleavage solution plus collected washings were filtered, evaporated to dryness at ambient temperature and reduced pressure, and the residue dissolved in a minimal volume of DCM, diluted with 20 volumes of hexane and re-evaporated (twice). Crude detached peptide was dissolved in water and lyophilized.
• Example 2 Therapeutic efficacy of the peptide as a 0.1% ointment in the model of contact sensitivity to oxazolone in BALB/c mice
• the aim of this experiment was to verify the therapeutic action of compound I-A and its toxicity in a generally accepted animal model.
• the compound of formula I-A was applied as a therapeutic preparation in the form of 0.1% wt/wt ointment based on a commonly used pharmaceutical vehicle, namely an ointment composed of 50% vaseline and 50% lanoline.
• the usefulness of the preparation in reduction of the effector phase of the contact sensitivity to oxazolone in mice, in comparison with reference
• tacrolimus Protopic ®
• pimecrolimus Elidel ®
• mice BALB/c female mice, 8-10 week-old, delivered by the Institute of Laboratory Medicine, Lodz, Tru, were used for the study. The mice were fed a commercial, pelleted food and water ad libitum. The local ethics committee approved the study.
• Hydrocortisonum ® (hydrocortisone) was purchased from Aflofarm Farmacja Polska, Poland. DMSO was obtained from Fluka; oxazolone, acetone, Evans blue, Giemsa, May-Grunwald, and formalin were from Sigma.
• lymph node cell numbers Superficial parotid, mandibular and accessory mandibular lymph nodes were isolated, homogenized by pressing against a stainless screen into phosphate buffered solution (PBS), washed twice and re-suspended in PBS containing 0.2% Trypan blue. The total and nonviable cell numbers were determined using a light microscope and Biirker's hemocytometer. Mice treated only with the eliciting dose of antigen served as a background control.
• PBS phosphate buffered solution
• Fig.1 The data included in Fig.1 show the therapeutic efficacy of compound of formula I- A (labeled “4B8M” in Fig. 1 and subsequent figures) and the reference preparations in mice with fully developed contact sensitivity reaction to oxazolone. The preparations were applied topically as described in the Methods.
• Fig. 1 presents only antigen- specific increases of the ear thickness (as a result of subtracting background values measured in non- sensitized mice which were given only the eliciting dose of antigen).
• Compound I-A caused about 80% inhibition of the ear edema; Protopic ® and Elidel ® respectively caused about 30% and 50% inhibition.
• Fig. 2 shows that both the compound of formula I-A as well as Elidel ® decreased the numbers of lymphocytes in the draining lymph nodes to the level registered in non-sensitized mice. However, in mice treated with Protopic ® the number of lymph node cells was similar to that in untreated mice.
• Fig. 3 shows the proportions of viable and dead cells in the draining lymph nodes, expressed in percentages.
• the compound of formula I-A exhibits a negligible toxic effect as compared to the control, non-treated mice. A higher toxic effect is caused by Elidel ® , and Protopic ® is exceptionally toxic with regard to lymph node cells.
• Fig. 4 depicts the numbers and participation of basic cell types involved in the local inflammatory process.
• Mast mastocytes
• L lymphocytes
• M0 macrophages
• Ne neutrophils.
• the auricles from untreated mice (K +) are characterized by a high infiltration of neutrophils.
• Application of the compound of formula I-A almost entirely reversed the changes observed in control mice (normalization of neutrophil number with some increase in the macrophage content).
• Protopic ® caused some changes in the proportion of respective cell types with no reduction of the total cell infiltrate. Elidel ® caused a moderate diminution of the total cell number.
• Example 3 The toxicity of compound I-A versus that of cyclolinopeptide against human blood mononuclear cells
• PBMC peripheral mononuclear blood cells
• CLA cyclolinopeptide
• the cytotoxic test Venous blood from a single donor was taken into heparinized syringes, diluted twice with phosphate buffered saline (PBS) and applied onto Lymphoprep ® (Polfa, Kutno, Tru) (density of 1.077 g/ml). After centrifugation at 1200 x g for 20 min, the mononuclear cells from the interphase were harvested and washed 3 times with PBS.
• PBS phosphate buffered saline
• the cells were re- suspended in a standard culture medium consisting of RPMI-1640 medium, L- glutamine, sodium pyruvate, 2-mercaptoethanol, 100 ⁇ g/ml each of streptomycin and penicillin, and 10% fetal calf serum.
• the cells were distributed to in 96- well flat-bottom culture plates at density of 2 x 10 5 /100 ⁇ .
• the compounds (formula I-A and CLA) were initially dissolved in DMSO (5 mg/500 ⁇ ) and subsequently in the culture medium. DMSO, appropriately diluted with the culture medium, was used as a control. After 24 h incubation in a cell culture incubator, cell viability was determined by a colorimetric method (Hansen et al., J Immunol Methods, 1989, 119, 203-210).
• mice had free access to water and pelleted food.
• the local ethics committee approved the study.
• Sheep erythrocytes (SRBC) were delivered by Wroclaw
• mice were immunized with 0.2 ml of 5% SRBC suspension (0.5 ml of SRBC pellet re-suspended to a volume of 10 ml of 0.9% NaCl), intraperitonelly. After 4 days the number of antibody-forming cells (AFC) in the spleens was determined using an assay of local hemolysis in agar gel (per Mishell et al., J Exp Med, 1967, 126, 423-442). The results are presented in Fig. 6 as a mean value of 5 mice + standard error and expressed as AFC number per 10 6 of viable splenocytes.
• AFC antibody-forming cells
• mice were immunized with SRBC as described above and after 2 h were given 10 or 100 ⁇ g of the compound of formula I-A.
• Cyclosporin A (CsA) served as a reference drug.
• the number of antibody-forming cells to SRBC was measured after 4 days.
• the compound of formula IA was more inhibitory at both doses than CsA.
• mice 8-12 weeks old were delivered by The Institute of Laboratory Medicine, Lodz, Tru. The mice had free access to water and pelleted food. Ovoalbumin was from Sigma and the adjuvants from Difco.
• DTH test Mice were sensitized subcutaneously with 5 ⁇ g of ovalbumin (OVA) emulsified in Freund' s complete adjuvant in the tail base. After 4 days the mice were challenged with 50 ⁇ g of OVA in Freund' s incomplete adjuvant in the hind footpads. Following the next 24 hours, the footpad thickness was measured using a caliper. Controls (background response mice) were not sensitized but received the challenging dose of OVA. The compound of formula I-A and the reference compound were administered to mice in two 100 ⁇ g intraperitoneal doses, 2 h before and 24 h after the sensitizing dose of antigen. The results, presented in Fig.
• Cyclic tetrapeptides were tested in vitro for their effects on phytohemagglutinin A (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMC) and for lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNF-a) production by whole blood cell cultures at l-100 ⁇ g/ml concentration range. Compounds were also tested for cell toxicity at 1-100 ⁇ g/ml concentration range against human PBMC.
• PHA phytohemagglutinin A
• PBMC peripheral blood mononuclear cells
• LPS lipopolysaccharide
• TNF-a tumor necrosis factor alpha
• Reagents RPMI- 1640 medium (Cibi/Life Technologies, UK), fetal calf serum (FCS, Gibco), DMSO, phytohemagglutinin A (PHA), lipopolysaccharide (LPS) from E. coli strain 0111:B4 (Sigma), 93-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), SDS and DMF (Sigma).
• the culture medium consisted of RPMI- 1640, 10% addition of FCS, L-glutamine, sodium pyruvate, 2-mercaptoetanol and antibiotics (streptomycin and penicillin). Cyclic tetrapeptides were initially dissolved in DMSO (5 mg/ml), the dissolved in the culture medium to the desired concentration.
• PBMC peripheral blood mononuclear cells
• lymphocytes (20%) and monocytes (80%) were then washed three times with Hanks' medium and re-suspended in the culture medium at density of 2xl0 6 cells/ml.
• the proliferative response of PBMC to PHA The isolated PBMC were distributed into 96- well flat-bottom plates in 100 ⁇ aliquots (2xl0 5 cells/well). PHA was used at concentration of 5 ⁇ g/ml. The compounds were tested at concentrations of 1, 10 and 100 ⁇ g/ml. DMSO at appropriate dilutions served as control. After a four-day incubation in a cell culture incubator, the proliferative response of cells was determined by the colorimetric MTT method (Hansen et al., J. Immunol. Methods, 1989, pp. 203-210). The data are presented as a mean OD value from quadriplicate wells + standard error (SE). The cultures "Control (-)" contained no mitogen (PHA). The cultures "Control (PHA)” contained PHA but not cyclic tetrapeptides.
• Toxicity test PBMC, at a density of 2x10 5 /100 ⁇ 1/well, re-suspended in the culture medium, were cultured for 24 h in a cell culture incubator with the cyclic tetrapeptides at concentrations of 1, 10 and 100 ⁇ g/ml concentrations. Cell survival was determined by the MTT colorimetric method (Hansen et al., J. Immunol. Methods, 1989, pp. 203-210). The data are presented as a mean OD value from quadriplicate wells + standard error (SE). The cultures "Control (-)" contained only cells in the culture medium.
• TNF alpha activity (per Espevik et al., J. Immunol. Methods, 95 (1986):99-103): Human whole blood was diluted 10-fold with RPMI-1640 medium and distributed to 24- well culture plates in 1ml aliquots. LPS was added to the culture at a concentration of ⁇ g/ml. The studied peptides were used at concentrations of 1, 10 and 100 ⁇ g/ml. After overnight incubation, the supernatants were harvested and frozen at -20°C until cytokine determination. TNF-a activity was determined using a bioassay.
• WEHI 164.13 cells (ATCC CRL 1751) were seeded at a concentration of 2x10 4 cells/well in quadriplicate. Increasing dilutions of the assayed supernatant were mixed with the target cells in the presence of actinomycin D ( ⁇ g/ml). After 20 h of incubation, MTT was added into the wells, and the cultures were incubated for an additional 4 h. Next, a lysing buffer (20% SDS with 50% DMF, pH 4.7) was added and the optical density at 550 nm with the reference wavelength of 630 nm in a Dynatech 5000 spectrophotometer was measured after 24 h. The detection limit of the assay was about 2.5 pg/ml.
• TNF-a activity was defined as an inverse of supernatant dilution where 50% cell death took place.
• the cultures labeled "Control (-)” contained no LPS.
• the cultures labeled “Control (LPS)” contained LPS and none of the studied compounds. Statistical analysis was not applied since the data derive from single cultures (wells).
• Brown-Forsyth's test was used to determine the homogeneity of variance between groups. When the variance was homogenous, analysis of variance (one-way ANOVA) was applied, followed by post hoc comparisons with the Tukey's test to estimate the significance of the differences between groups. Significance was determined at P ⁇ 0.05. Statistical analysis was performed using STATISTICA 6.1 for Windows.
• P01 compound I-D
• P02 compound I-E
• P03 compound I-F
• P04 compound I-G
• P05 compound I-H
• P06 compound I-J
• P07 compound I-K
• P08 compound I-L
• PlOa compound I-M
• PlOb compound I-N
• PI 1 compound I-O.
• Figs. 8A and 8B show the effects of the tested peptides on the survival of PBMC.
• Example 7 Inhibitory effect of Compound LA on toluene diisocyanate-induced ear inflammation in mice
• mice BALB/c female mice, 8-10 weeks old, were delivered by the Institute of Laboratory Medicine, Lodz, Tru. The mice were fed a commercial, pelleted food and water ad libitum. The local ethics committee approved the study. [00128] Reagents. Compound I-A was synthesized as described above; Protopic
• tacrolimus was from Astellas, Ireland; Elidel ® (pimecrolimus) from Novartis; DMSO from Fluka; TDI, acetone, Evans blue, Trypan blue, Giemsa, May-Grunwald, haematoxylin, eosin, toluidine blue and formalin were from Sigma.
• Compound I-A was applied topically in the form of 0.1% ointment on both sides of the ears (total volume of 100 ⁇ - 50 ⁇ per ear), one hour after each challenge with TDI.
• the reference drugs were applied in a similar way.
• lymph node cell numbers were isolated, homogenized by pressing against a stainless screen into PBS, washed 2 xwith PBS and re-suspended in PBS containing 0.2% Trypan blue. The total and nonviable cell numbers were counted using a light microscope and Biirker's hemocytometer.
• Evans blue test Mice were given 1 mg of Evans blue in 0.2 ml of 0.9% NaCl, intravenously. After 30 min mice were sacrificed, the ears were cut off, weighed and immersed in 50 ⁇ of 1M KOH for 18 h at 37°C. The dye was extracted from the ears using 450 ⁇ of 0.2 M phosphate acid and acetone (5: 13 ratio). The samples were centrifuged at 3,000 rpm for 15 min. The optical densities (OD) of the supernatants were measured at 630 nm. The amount of Evans blue ⁇ g/ml) was determined based on a standard curve. The results were presented as the amount of Evans blue per 100 mg of wet tissue.
• mice treated only with the eliciting dose of antigen served as a background control.
• Histological analysis The auricles were fixed in 4% formalin solution for 48 h, washed for 24 h, dehydrated in an alcohol series and embedded in paraffin. The paraffin blocks were sliced in a Micron HM310 microtome into 6 ⁇ sections. The sections were stained with haematoxylin and eosin and with toluidine blue. Histological analysis was performed using a Nikon Eclipse 8011ight microscope. Morphometric estimations of neutrophils, macrophages, lymphocytes and mast cells in the perivascular and subepithelial connective tissue were performed on the histological slides containing cross-sections of auricles.
• tetrapeptide (formula I-A, labeled 4B8M in Figs. 10A and 10B) and the reference compounds one hour after each challenge with antigen.
• the effects of the treatments are presented in Figs. 10A and 10B, which show ear thickness measure 5 h (Fig. 10A) and 24 h (Fig. 10B) after administration on the day of the test indicated in the figure.
• Control responses to TDI gradually elevated after each antigen challenge (best seen in the 5 h measurement). The results showed differentiated efficacy of the compounds in reducing the ear swelling.
• FIG. 11 shows the permeability of capillary blood vessels in the Evans blue test. The procedure, as described aboved, was performed 24 h after the fifth challenge with TDI (on day 28). As shown in the Fig. 11, the rates of blood vessel permeability were strictly correlated with the effects of the compounds on ear thickness in the respective mouse groups.
• Fig. 12 shows the total number of cells in the draining lymph nodes. As shown in Fig. 12, the treatment of mice with compound I-A resulted in a reduction of the lymph node cell numbers almost to the background levels (non-sensitized mice).
• FIG. 13 shows the effects of the compounds on the numbers of circulating leukocytes; the application of the studied preparations lowered the numbers of circulating leukocytes to the levels observed in control, unsensitized mice.
• Fig. 15 provides morphometric data on the number and composition of cells in the auricles.
• the composition of cell types within the auricles differed among the studied mouse groups is presented in Fig. 15.
• the predominant, residing cell types in control non-sensitized mice are mastocytes and neutro- fils (10 and 5 cells per the analyzed area, respectively).
• the number of mastocytes increased twofold and neutrophils almost 5- fold (20 and 23 cells, respectively).
• Protopic ® and Compound I-A were effective in reducing the cell numbers to 14 and 14.8.
• Ne neutrophils
• BG vs Control P 0.0001;
• the cells were then distributed into 96-well flat-bottom tissue culture plates (Nunc) at a density of 2 x 10 5 cells/100 ⁇ l/well. Con A (2.5 ⁇ g/ml) was added to induce cell proliferation. The compounds were added to the cultures at doses of 1 - 100 ⁇ g/ml. After a three-day incubation, cell proliferation was determined using the colorimetric MTT method (Hansen MB, J Immunol Methods, 1989, 119, 203-210). The results were presented as the mean optical density (OD) at 550 nm + SE from quadriplicate determinations (wells).
• SRBC sheep erythrocytes
• the compounds were added to the cultures at the beginning of the four-day incubation period at concentration ranges of 1-100 ⁇ g/ml.
• the number of antibody-forming cells (AFC) in the cultures was determined using a method of local hemolysis in agar gel according to Mishell et al., J Exp Med, 1967, 126, 423-442.
• Toxicity test Splenocytes, at a density of 2 x 10 5 cells/100 ⁇ l/well, re- suspended in the culture medium, were cultured for 24 h in a cell culture incubator with the compounds (1- 100 ⁇ g/ml). Cell survival was determined by MTT colorimetric method. The results were presented as mean optical density (OD) at 550 nm from 4 wells. The viability of cells in respective compound concentrations was compared to appropriate DMSO control groups (100% survival), corresponding to respective compound concentrations.
• compound I-C showed a strong inhibitory effect on concanavalin A-induced mouse splenocyte proliferation. At 100 ⁇ g/m, this compound showed 70% toxicity to splenocytes. At 10 ⁇ g/m and 100 ⁇ g/m, compound I-C showed 33% and 80% suppression, respectively, in the model of in vitro humoral immune response to SRBC in mouse splenocyte cultures . In the model of delayed type hypersensitivity to ovalbumin, compound I-C showed 26.9% inhibition at the dose of 100 ⁇ g, compared to 72.7% inhibition by compound I-A.
• compound I-B demonstrated a weak antiproliferative effect on concanavalin A-induced splenocyte proliferation; no such effect was observed at lower concentrations. Compound I-B had 30% toxicity at this concentration.

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