EA045183B1 - COMPOUNDS (IMMUNORELINS) STIMULATING GnRH RECEPTORS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a series of new peptides capable of stimulating the immune system, called immunorelins (immunorhelins). The invention also relates to new peptides capable of stimulating GnRH receptors on leukocytes. The present invention relates to novel compounds as such and compounds for use in medicine, especially in the treatment of viral diseases such as HIV and immunotherapeutic treatment of cancer. Immunorelins can also be used as immunomodulatory adjuvants in vaccination. New GnRH receptor-stimulating immunorelins maximize the modulatory effects of the immune system while minimizing therapeutically undesirable endocrine effects. The present invention also relates to methods for producing immunorelins according to the invention, which have improved properties for use in medicine.

Prerequisites for creating the invention

CD4+ T cells are key mediators of the immune response and primarily target HIV infection. Current antiretroviral therapy for HIV is compromised by patient compliance, drug toxicity, and drug resistance. Thus, there is a great need in the art for methods and means of increasing the immune competence of CD4+ T cells in HIV and certain immunologically related viral diseases, as well as in cancer.

GnRH I (also known as gonadotropin-releasing hormone or LHRH) is a decapeptide with the structure pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. It is produced as a 92 amino acid peptide that is modified post-translationally to form a final peptide with pyroglutamic acid at the amino terminus and a carboxamide at the carboxyl terminus. It has long been known to be responsible for the release of FSH and LH from the anterior pituitary gland, and is typically released from the hypothalamus in a pulsatile manner. Supraphysiological levels of GnRH I cause an immediate increase in FSH and LH secretion, which is soon followed by inhibition of FSH and LH secretion. This is due to the fact that high levels of GnRH I have an inhibitory effect on the GnRH I receptors of the anterior pituitary gland. Thus, continuous administration of GnRH I at high nonphysiological levels causes pharmacological castration (1). A large number of GnRH I agonists and antagonists have been synthesized for use in therapeutic areas such as the treatment of hormone-sensitive cancer. First, GnRH I salts (such as gonadorelin hydrochloride and gonadorelin diacetate tetarhydrate) were used therapeutically. The discovery and development of other drugs has led to the clinical use of a wide range of agents, including buserilin, triptorelin, nafarelin, histrelin, and leuprorelin, all of which have improvements over gonadorelin, such as increased half-life and superagonism at the GnRH I receptor.

It has been reported that GnRH I not only exhibits hormonal effects, but can also stimulate the immune system (2). McClean and McCluggage (3) observed massive infiltration of small mature lymphocytes in uterine leiomyomas after preoperative treatment with a GnRH I receptor agonist. Bardsley et al. (4) made the same observation, indicating a stimulatory effect on the migration of GnRH I on immune cells. Chronic plasma cell endometritis has been reported in hysterectomy specimens from HIV-infected women in retrospective analyzes (5) and elevated FSH and LH (hypergonadotropic) levels in HIV-infected men (6–7). By administering GnRH I to diabetic BB rats, displaying an AIDS-like lymphocyte profile, the number of CD4 T lymphocytes was increased (8).

The essence of the invention

In humans, there are two variants of the GnRH peptide, GnRH I and GnRH II, encoded by different genes. The structure of GnRH II is pyro-Glu-His-Trp-Ser-His-Gl·y-Trp-Tyr-Pro-Gl·y-NH ₂ (differences from GnRH I are underlined). GnRH II is a nonhypothalamic form produced initially outside the brain and is thought to be involved in nonendocrine aspects of the GnRH system ( 9 ). We unexpectedly discovered an effect of GnRH II stimulation on MHC class I expression on T cells, indicating that GnRH II directly activates these cells (Fig. 1).

Unlike other mammals, only one defined human GnRH receptor has been described, the GnRH receptor type I. In humans, a homologue of the GnRH receptor type II is present on the gene on chromosome 1q12, but contains a frameshift and a stop codon and is not believed to be functionally expressed (10) . Surprisingly, the authors' data suggest that the GnRH receptor is in fact expressed on T cells, as they respond to GnRH stimulation with increased expression of MHC class I (Fig. 1).

These functional conclusions are supported by qPCR analysis, where the authors were able to show the expression of GnRH type II receptor mRNA. In addition, the relative expression level of GnRH receptor type II is higher compared to the expression levels of GnRH receptor type I on naive and memory T cells (Fig. 4). Thus, we determined that GnRH receptor type II expression is dominant on T cells functionally responsive to GnRH stimulus.

The inventors also discovered that activating T cells leading to MHC expression

- 1 045183 class I, can GnRH I analogues. In recent clinical trials using the GnRH I analogue buserelin as a treatment for HIV, sex hormone replacement was provided to HIV-infected men to minimize the endocrine effects of GnRH I. This effect is mediated by the binding of GnRH I to GnRH receptors Type I pituitary gland, causing decreased testosterone production and subsequently impotence. It is very likely that GnRH I, in addition to its endocrine effects, cross-signals and stimulates the immune system by binding to the GnRH type II receptor on T cells when high castrating levels of GnRH analogues are used. It is interesting that GnRH I binding to receptors expressed in breast cancer cells displays low binding affinity (Kd, 1.6-3.0 x 10(-6) M), while central pituitary GnRH I binding displays 1000 times higher affinity (Kd, 4.8 x 10(-9) M) (11).

It is likely that the difference in binding affinities of GnRH I and GnRH II peptides reflects the expression of GnRH receptor type I, adapted to bind GnRH I on pituitary cells, while peripheral cells may have dominant expression of the GnRH receptor type II, and therefore low affinity and target abrogation action of GnRH I binding. Thus, the authors' unexpected finding that GnRH type II receptor is the dominant receptor on T cells is novel and may explain the physiology of GnRH I and GnRH II receptors. Therefore, by using GnRH II-like peptides in the treatment of HIV, the endocrine effect can be minimized, and the immunostimulatory effect can be isolated and enhanced.

Based on such discoveries, the inventors have prepared GnRH II-like peptides, called immunorelins, in order to optimize the immunostimulatory effect and minimize the effect on the hormonal system. These immunorelins have applications in stimulating MHC class I, which can lead to immune clearance responses against infectious agents such as HIV, and in the treatment of cancer. Therefore, several GnRH II-like peptides have been disclosed that have strong binding to GnRH II receptors but preferentially weaker binding to GnRH I receptors, leading to a comparable or stronger MHC class I response but weaker target stimulatory or inhibitory withdrawal effects hormones.

Description of the invention

In one aspect, the present invention relates to peptide analogues of human GnRH II. Thus, in one aspect the invention relates to a peptide of formula (I)

or a pharmaceutically acceptable salt thereof, and wherein

R5 = Me, Et, CH2CF3, iso-Pr, n-Pr, n-Bu, iso-Bu, sec-Bu, tert-Bu, cyclopropyl, CH2CONH2 or NHCONH2.

Formula I, relating to the compounds of the invention, can also be expressed as pGlu-Hi s-T gr-Ser-AAi - AA2-AAs - A Ad-Pro-X,

- 2 045183 where AA ₁ is selected from His and Tyr;

AA ₂ is selected from D-Ser(OtBu), D-Trp, D-Nal, D-Bhi, and D-Leu;

AA3 is selected from Leu and Trp; AA4 is selected from Arg and Tyr;

X is selected from -NHMe, -NHEt, -NHCH ₂ CF ₃ , -NH-iso-Pr, -NH-h-Pr, -NH-h-Bu, -NH-u30-Bu, -NHsec-Bu, -NH -tert-Bu, -NH-cyclopropyl and -NHCH ₂ CONH ₂ .

The invention does not include the following compounds of formula (I).

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Excluded are compounds of formula (1), expressed as pGlu-His-Trp-Ser-AA ₁ -AA2-AA ₃ -AA ₄ Pro-X, where AA1 is His, AA ₃ is Trp, AA4 is Tyr, and AA2 is selected from D-Leu, D-tert-Bu-Ser and D-Trp, and X is -NHEt or NH-NH-CO2.

Compounds P1 to P15 excluded from the invention may also be expressed as follows.

Pl. pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Trp-Tyr-Pro-Gly-NH2

P2. pGlu-His-Trp-Ser-His-D-Trp-Leu-Arg-Pro-Gly-NH2

RZ. pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Arg-Pro-Gly-NH2

P4. pGlu-His-Trp-Ser-His-D-Trp-Trp-Arg-Pro-Gly-NH2

P5. pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-Gly-NH2

P6, pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-Gly-NH2

P7. pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-NHEt

P8. pGlu-Hi s-Trp-Ser-Hi s-D-Nal-Trp-Tу r-Pro-NHEt

P9. pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-Gly-NH2

PIO. pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2

Pl 1. pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-Gly-NH2

P12. pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt

P13. pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Arg-Pro-NHEt

P14. pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

P15. pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Leu-Arg-Pro-NHNHCONH2

An interesting choice of compounds of the invention are the compounds of formula (I) or pharmaceutically acceptable salts thereof, in which

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Group Type I Type II Ri ABOUT N^/ R ₃ I r ₄ ^ΝΗ T 0 4

and wherein at least one of R1, R3 and R4 is selected from type II, and those of R1, R3 and R4 not selected from type II are selected from type I,

where ^R2

and wherein R5 = Me, Et, CH2CF3, iso-Pr, n-Pr, n-Bu, iso-Bu, sec-Bu, tert-Bu, cyclopropyl or CH2CONH2.

Compounds of this choice can also be expressed as pGlu-His-Trp-Ser-AAi-AA2-AA3-AA4-Pro-X, wherein AA1, AA2, AA3, AA ₄ and X are as defined above, and wherein at least one AA1, AA3 and AA4 are selected from His, Trp and Tyr; the remaining AA1, AA3 and AA4 are selected from Tyr, Leu, Arg, and with the exception of compounds P1-P15 described above, and those other compounds excluded in paragraph 1.

In one embodiment, two of the three R1, R3 and R4 are selected from Type II according to the list above, and the remaining R1, R3 and R4 are selected from Type I.

In one embodiment, one of the three R1, R3 and R4 is selected from Type I according to the list above, and two of R1, R3 and R4 are selected from Type II according to the list above.

Specific compounds of the invention include those listed below.

Connect singing no. R.I. R2 R3 R4 R5 1 NH N^/ MALAN h ₂ n^nh CH2CONH2 pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Leu-Arg-Pro-Gly-aMHfl 2 Oh....0 10 Η ₂ ΝγΝΗ .NH CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Trp-Arg-Pro-Gly-aMHfl 0 ,....0 hAALn 3^ T0 4 CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Leu-Tyr-Pro-Gly-aMHfl

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4 NH N^/ Η ₂ Ν^ΝΗ ^γ ^ΝΗ CH2CONH2 pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Trp-Arg-Pro-Gly-aMHfl 5 NH N^/ ΑΛΑ- Τ! Ο I CH2CONH2 pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Leu-Tyr-Pro-Gly-aiwi 6 NH N^/ AAA- η ₂ ν^νη J^ ^NH Et pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Leu-Arg-Pro-NHEt 7 O .0 ΑΑΑ- ο ο I CH2CONH2 pO1i-H13-Tgr-8er-Tyr-O-8er(tert-Bi)-Tgr-Tyr-Pro-O1y-amide 8 o ,.0 ΑΑΑ- η ₂ ν^νη J^ ^NH Et pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Trp-Arg-Pro-NHEt 9 Oh,.0 ΑΑΑ- τ ο 4 Et pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Leu-Tyr-Pro-NHEt 10 ^NH AAA η ₂ ν^νη J^ ^NH Et pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Trp-Arg-Pro-NHEt

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And NH N^/ LAan τ ο ‘L Et pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Leu-Tyr-Pro-NHEt 12 0' ABOUT LAan ΟI Et pGlu-His-Trp-Ser-Tyr-D-Ser(TpeT-Bu)-Trp-Tyr-Pro-NHEt 13 ^Т%Н N^/ AAAn τ ο Et pGlu-His-Trp-Ser-His-D-Ser(TpeT-Bu)-Trp-Tyr-Pro-NHEt 14 ABOUT Η Kommersant O I CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Tyr-Pro-Gly-amide 15 O Η Η ₂ ΝγΝΗ J^ ^NH Et pGlu-Hi s-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt 16 NH N^/ Η ΓζΚ 0 o I CH2CONH2 pGlu-His-Trp-Ser-His-D-Trp-Leu-Tyr-Pro-Gly-amide 17 NH N^/ Η H2N^NH J^ ^NH Et pGlu-Hi s-Trp-Ser-Hi s-D-Trp-Leu-Arg-Pro-NHEt

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18 Oh,.0 Η £ Η ₂ Ν^ΝΗ ^γ ^ΝΗ Et pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Arg-Pro-NHEt 19 O .0 Η γγ τ ο Et pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Tyr-Pro-NHEt 20 N.H. Νΐ/ Η /Ъ £ η ₂ ν^νη J^ ^NH Et pGlu-Hi s-Trp-Ser-Hi s-D-Trp-Trp-Arg -Pro-NHEt 21 V NH Νΐ/ Η £,κ γγ τ ο 4 Et pGlu-His-Trp-Ser-His-D-Trp-Leu-Tyr-Pro-NHEt 22 O .0 Η £j^ £ ο I Et pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-NHEt 23 NH N^/ <0 ΧΓ γγ η ₂ ν^νη J^ ^NH CH2CONH2 pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-Gly-amide 24 O .0 <0 JU η ₂ ν^νη ^γ ^ΝΗ CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-Gly-amide

25 ^VOH ηJU t o h CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-Gly-amide 26 O jQ JU γγ η ₂ ν^νη ^γ ^ΝΗ Et pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-NHEt 27 nh N^/ jQ JU And η ₂ ν^νη ^γ ^ΝΗ CH2CONH2 pGlu-His-Trp-Ser-His-D-Nal-Trp-Arg-Pro-Gly-amide 28 NH N^/ <0 JU γ^ τ ο 4 CH2CONH2 pGlu-His-Trp-Ser-His-D-Nal-Leu-Tyr-Pro-Gly-amide 29 NH N^/ jo JU γγ Η2Ν^ΝΗ J^ ^NH Et pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-NHEt thirty ABOUT jo JU And ί ο I CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Tyr-Pro-Gly-amide 31 O jo JU And Η ₂ ΝγΝΗ J^ ^NH Et

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pGlu-His-Trp-Ser-His-D-Nal-Trp-Arg-Pro-NHEt 32 ABOUT T about 4 Et pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-NHEt 33 n O I CH2CONH2 pGlu-His-Trp-Ser-His-D-Nal-Trp-Tyr-Pro-Gly-amide 34 JW-r NH N^/ J.U. h ₂ n^nh NH hLALx Et pGlu-His-Trp-Ser-His-D-Nal-Trp-Arg-Pro-NHEt 35 VnH N^/ <0 T about 4 Et pGlu-His-Trp-Ser-His-D-Nal-Leu-Tyr-Pro-NHEt 36 About „.P <0 JU O O I Et pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Tyr-Pro-NHEt 37 ^NH Nib/ h ₂ n^nh J^ ^NH CH2CONH2 pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-Gly-amide

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38 ABOUT & H ₂ N^NH J^ ^NH CH ₂ CONH ₂ pGlu-His-Trp-Ser-Try-D-Leu-Trp-Arg-Pro-Gly-amide 39 O O YY o I ch ₂ conh ₂ pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-Gly-amide 40 NH N^/ Y^ h ₂ n^nh J^ ^NH ch ₂ conh ₂ pGlu-His-Trp-Ser-His-D-Leu-Trp-Arg-Pro-Gly-amide 41 Nt/ γγ γγ o I ch ₂ conh ₂ pGlu-His-Trp-Ser-His-D-Leu-Leu-Tyr-Pro-Gly-amide 42 nh N^/ γ^ γγ h ₂ n^nh J^ ^NH Et pGlu-Hi s-Trp- Ser-Hi s-D-Leu-Leu-Arg-Pro-NHEt 43 ABOUT γ^ T o CH ₂ CONH ₂ pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Tyr-Pro-Gly-amide 44 A' ABOUT γ^ h ₂ n^nh ^γ ^ΝΗ Et pGlu-Hi s-Trp- S er-T y r-D -Leu-Trp - Arg-Pro-NHEt

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45 I O o I Et pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-NHEt 46 N.H. N^/ T about 4 CH2CONH2 pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-Gly-amide 47 NH N^/ h ₂ n^nh NH chaAa/ Et pGlu-Hi s-Trp- Ser-Hi s-D-Leu-Trp-Arg-Pro-NHEt 48 NH N^/ T about 4 Et pGlu-His-Trp-Ser-His-D-Leu-Leu-Tyr-Pro-NHEt 49 ABOUT - T about 4 Et pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Tyr-Pro-NHEt 50 Vnh N^/ X ¹¹ : O I Et pGlu-Hi s-Trp-Ser-Hi s-D-Leu-Trp-Tyr-Pro-NHEt 51 NH N^/ I'm A h ₂ n^nh .NH CH2CONH2 pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-Gly-amide

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57 O „...0 q r-N A ΗςΝ^ΝΗ J^ ^NH CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-amide 58 oh I A h ₂ n^nh J^ ^NH CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-amide 59 O q A T o Et pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-NHEt 60 NH N^/ q r-N A H ₂ N^NH J^ ^NH Et pGlu-His-Trp-Ser-His-D-Bhi-Trp-Arg-Pro-NHEt 61 N.H. I'm r-N oh oh T Et pGlu-His-Trp-Ser-His-D-Bhi-Leu-Tyr-Pro-NHEt

62 ABOUT q r-N I Oh oh I Et pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Tyr-Pro-NHEt 63 NH N^/ r-N A I 'b o I Et pGlu-Hi s-Trp-Ser-Hi s-D-Bhi-Trp-Tyr-Pro-NHEt 64 Ta H I J.U. CH2CONH2 pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-Gly-amide 65 G ⁷⁴ NH N^/ Hr-N I CH2CONH2 pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-Gly-amide

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pGlu-His-T rp-Ser-Tyr-D-Bhi-T rp-Arg-Pro-N HEt pGlu-His-Trp-Ser-His-D-Trp-Leu-Arg-Pro-Gly-amide amide - amide .

Of practical interest are compounds that are analogues of GnRH II, having predominantly i) a stimulating effect on or ii) affinity for the GnRH II receptor. Thus, compounds that do not bind or activate the GnRH I receptor resulting from an undesired therapeutic response are preferred. GnRH II analogues, which bind to or activate the GnRH I receptor, thereby stimulating endocrine signaling, are thought to be administered with sex hormones to counteract the endocrine effects.

In a preferred aspect the invention relates to the use of a compound of formula (I)

where the connection is selected from

- 15 045183 or a pharmaceutically acceptable salt thereof for treating cancer by stimulating the immune system by binding to the GnRH receptor type II on T cells, and wherein the cancer is selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, tumors brain/central nervous system, colon/rectal cancer, esophageal cancer, eye cancer, gallbladder cancer, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma , salivary cancer glands, basal cell and squamous cell skin cancer, Merkel cell skin cancer, thymus cancer, uterine sarcoma, Waldenström macroglobulinemia, or Wilms tumor.

General chemical methods.

Those skilled in the art will appreciate that the compounds of the invention can be prepared in a variety of ways known in the art. The routes described below are only illustrative of some of the methods that can be used to synthesize compounds of formula (I).

In general, synthetic methods for preparing the compounds of the invention can be divided into two methods: liquid-phase synthesis and solid-phase synthesis. Liquid-phase peptide synthesis involves reagents reacting in the solution phase. Disadvantages of this method include difficulty in separating and purifying the products. Solid-phase peptide synthesis is more common and has several advantages, including convenient extraction and purification, and the possibility of automation (Bodanszky et al., In Peptide Synthesis, John Wiley & Sons, 1976). A number of peptide synthesis resins have been developed that allow the synthesis of various peptides. These include chloromethyl and 2-chlorotrityl polystyrene resins. Examples of patents covering methods for synthesizing short peptides include US5602231, EP 0518655 and US6879289.

When a compound of the invention with a C-terminal amide, such as buseralin, is prepared, then one method of preparing the compounds is as follows, shown in Scheme II below. The peptide can be assembled on a solid support, typically 2-chlorotrityl polystyrene resin is used, but others will be apparent to one skilled in the art. The first amino acid is loaded and then the protecting group is removed, revealing a reactive amino group, which is then used to couple with the next amino acid. In turn, its protecting group can be removed and reattached. After several rounds of extension, the desired peptide sequence is obtained. The peptide is then cleaved from the resin by exposure to TPA or similar reagents. It is noted that when a tert-butyl side chain is required in the final compound, it is important to keep the reaction time short enough so that it is not completely cleaved off. Some tert-butyl groups will be cleaved, but this can be removed during purification. Finally, a secondary amide is prepared by connecting the peptide at the unprotected C-terminus with a selected primary amine. Coupling reactions typically use HBTU and DIPEA, although one skilled in the art can identify other activators and bases that can be used in combination to form the amide bond.

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Scheme I

Reaction - Reaction.

When a compound with a C-terminal primary amide is prepared, as in triptorelin, then the process for preparing the compounds is as follows and is shown in Scheme II below. The peptide can be assembled on a solid support, Ramage resin is typically used, but others will be apparent to one skilled in the art. The first amino acid is loaded and then the protecting group is removed, revealing a reactive amino group, which is then used to couple with the next amino acid. In turn, its protecting group can be removed and reattached. After several rounds of extension, the desired peptide sequence is obtained. The peptide is then cleaved from the resin by exposure to TPA or similar reagents. Coupling reactions typically use HBTU and DIPEA, although one skilled in the art can identify other activators and bases that can be used in combination to form the amide bond.

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Scheme II

Reaction - Reaction.

Compounds of formula (I) can be prepared by combining the methods described above and other methods known to those skilled in the art. General Use of the Compounds of the Invention

The compounds described herein may be used in medicine, medical research, or in the manufacture of a composition for such use. In addition, the invention also relates to compounds P1-P21 described herein for use in medicine, medical research or in the manufacture of a composition for such use. Accordingly, when the term compounds of the invention are further used in connection with a medical use or pharmaceutical composition, the term is intended to also include compounds P1-P21, provided that these compounds are not known for such use.

In addition, the compounds are believed to exhibit improved properties for the treatment of these and related diseases, including reduced binding to the GnRH receptor I compared to the GnRH receptor II.

The compounds of the invention disclosed herein can be used to treat diseases, disorders, conditions and symptoms where stimulation of the immune response is useful, for example in the treatment of patients infected with viral agents or with viral diseases such as HIV, alphavirus, arbovirus, Borna disease, bunyavirus, calicivirus, condyloma acuminata, coronavirus, coxsackievirus, cytomegalovirus, Dengue fever virus, ecthyma contagiosum, Epstein-Barr virus, erythema infectiosum, hantavirus, hemorrhagic fever virus, viral hepatitis, herpes simplex virus, herpes zoster virus, infectious mononucleosis, influenza, Lassa fever virus, measles, mumps, molluscum contagiosum, paramyxovirus, phlebotomy fever,

- 18 045183 polyomavirus, Rift Valley fever, rubella, slow virus, smallpox, subacute sclerosing leukoencephalitis, oncogenic viral infections, West Nile virus, yellow fever virus, rabies virus and respiratory syncytial virus.

In addition, the compounds are believed to be suitable for use in the treatment of cancer, in particular adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors, breast cancer, Castleman's disease, cervical cancer , colon/rectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal cancer and hypopharyngeal cancer, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodys plastic syndrome, cavity cancer nose and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, basal cell and squamous cell skin cancer, melanoma, Merkel cell skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, Wilms' tumor.

Thus, the beneficial properties of a compound of the invention may include one or more of the following properties:

improved binding to the GnRH II receptor compared to the GnRH I receptor;

improved MHC class I stimulation;

improved immunomodulation;

improved activation of antigen presenting cells;

improved anticancer activity.

Pharmaceutical compositions comprising a compound of the invention.

The present invention also relates to a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable diluents or carriers. This section applies primarily to composition with new GnRH analogues. In cases where new compounds have an effect on the GnRH I receptor that is undesirable and causes castration of such effects, compositions containing sex hormones known in the art can be co-administered.

The compounds of the invention or compositions thereof may be administered by any convenient route, for example, but not limited to, the drug may be administered parenterally, orally, topically, or mucosally (including buccal, sublingual, transdermal, vaginal, rectal, nasal, ocular). medical device (eg stent), by inhalation. Treatment may consist of a single administration or several administrations over a period of time.

Treatment may be administered once a day, three times a day, four times a day, etc., depending on the particular disease being treated and the weight and age of the patient being treated. Treatment can also be carried out by continuous administration, such as intravenous administration by drop infusion.

Although it is possible to administer a compound of the invention as such, it is preferable that it be present in a pharmaceutical preparation together with one or more suitable carriers. The carrier(s) must be acceptable in the sense of being compatible with the compound of the invention and not harmful to recipients thereof. Examples of suitable media are described in more detail below.

The preparations may be conveniently presented in a suitable dosage form, including unit dosage form, and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of mixing together the active ingredient (a compound of the invention) with a carrier that consists of one or more auxiliary ingredients. In general, preparations are prepared by uniformly and thoroughly mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compound of the invention will generally be administered by any convenient route of administration, typically the oral or any parenteral route, in the form of a pharmaceutical preparation comprising the active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending on the disorder and patient being treated, as well as the route of administration, the compositions may be administered at different doses and/or frequencies.

Pharmaceutical compositions must be stable under production and storage conditions; Thus, if necessary, they should be protected from the contaminating action of microorganisms, such as

- 19 045183 bacteria and fungi. In the case of liquid preparations such as solutions, dispersions, emulsions and suspensions, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerin, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof . For example, a compound of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, films, granules, elixirs, solutions, emulsions or suspensions, which may contain flavoring agents or dyes.

The preparations of the present invention suitable for oral administration may be presented in discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, in multi-dose units, for example, in the form of a tablet or capsule; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of 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.

Solutions or suspensions of a compound of the invention suitable for oral administration may also contain one or more solvents, including water, alcohol, polyol, etc., as well as one or more excipients such as pH adjuster, stabilizers, surfactants substances, solubilizers, dispersants, preservatives, fragrances, etc. Specific examples include, for example, K,K-dimethylacetamide, dispersants, e.g. polysorbate 80, surfactants and solubilizers, e.g. polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soy fatty acids, ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate). The preparations of the present invention may also be in the form of emulsions, wherein the compound of formula (I) may be present in an emulsion such as an oil-in-water emulsion or a water-in-oil emulsion. The oil may be a natural or synthetic oil or any oil-like substance such as, for example, soybean oil or safflower oil or combinations thereof.

Tablets may contain excipients such as microcrystalline cellulose, lactose (eg lactose monohydrate or lactose anhydrous), sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, butylated hydroxytoluene (E321), crospovidone, hypromellose, disintegrants such as starch (preferably corn, potato or cassava starch), sodium starch glycolate, croscarmellose sodium and some complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), macrogol 8000, sucrose, gelatin acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

A tablet may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient into a free-flowing form such as powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactant or dispersant. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may optionally be coated or scored, and may be formulated to provide slow or controlled release of the active ingredient used therein, for example, with hydroxypropyl methylcellulose in varying proportions to provide the desired release profile.

Solid compositions of this type can be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweeteners or flavoring agents, coloring agent or dyes, emulsifiers and/or suspending agents, and diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored mass, usually sucrose and gum arabic or tragacanth; lozenges comprising the active ingredient in an inert mass such as gelatin and glycerin, or sucrose and gum arabic; and rinses comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, powders and the like. Such compositions can be prepared by conventional methods involving the inclusion of an active agent. Thus, they may also include compatible conventional carriers and additives such as preservatives, solvents to aid drug penetration, emollient in creams or ointments, and ethanol or oleyl alcohol for lotions. Such carriers may be present in from about 1% to about 98% of the composition. More often they will make up up to about 80% of the composition. Just as an explanation, cream or

- 20 045183 ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing about 5-10% by weight of the compound, in sufficient quantities to obtain a cream or ointment having the desired consistency.

Pharmaceutical compositions adapted for transdermal administration may be presented in the form of discrete patches designed to maintain close contact with the recipient's epidermis over an extended period of time. For example, the active agent can be delivered from the patch by iontophoresis.

For applications on external tissues, such as the mouth and skin, the compositions are preferably applied in the form of a topical ointment or cream. When formulating an ointment, the active agent can be used with an ointment base that can be mixed with either paraffin or water.

Alternatively, the active agent may be formulated into a cream with an oil-in-water cream base or a water-in-oil cream base.

For parenteral administration, liquid unit dosage forms are prepared using the active ingredient and a sterile medium, such as, but not limited to, water, alcohols, polyols, glycerin and vegetable oils, with water being preferred. The active ingredient, depending on the medium and concentration used, may be in a colloidal state, suspended or dissolved in the medium. When preparing solutions, the active ingredient may be dissolved in water for injection and filtered before loading into a suitable vial or ampoule and sealing.

Advantageously, agents such as local anesthetics, preservatives and buffering agents may be dissolved in the medium. To enhance stability, the composition may be frozen after loading into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in a vial, and the vial can be accompanied by water for injection to rehydrate before use.

Pharmaceutical compositions of the present invention suitable for injection include sterile aqueous solutions or dispersions. In addition, the compositions may be in the form of sterile powders for immediate administration of such sterile solutions or dispersions for injection. In all cases, the final injection form must be sterile and must be truly fluid for ease of syringability.

Parenteral suspensions are prepared in essentially the same manner as solutions, except that the active ingredient is suspended in a medium instead of dissolved, and sterilization cannot be accomplished by filtration. The active ingredient can be sterilized by exposure to ethylene oxide before suspension in a sterile environment. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.

It should be understood that in addition to the ingredients specifically mentioned above, the preparations of the present invention may include other agents known in the art relevant to the type of preparation in question, for example, agents suitable for oral administration may include flavoring agents. One skilled in the art will know how to select an appropriate drug and how to prepare it (see, for example, Remington's Pharmaceutical Sciences, 18 Ed. or later). One skilled in the art will also know how to select the appropriate route of administration and dosage.

One skilled in the art will appreciate that the optimal amount and range of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that the physician in will ultimately determine the appropriate dosages to use. This dosage may be repeated as often as required. If side effects occur, the amount and/or frequency of dosage may be adjusted or reduced according to normal clinical practice.

All % values stated herein are % w/w unless the context otherwise requires.

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List of sequences

The sequence list is obtained according to WIPO standard ST.25. In the sequence listing, the unnatural amino acids of compounds 1-63 and P1-P21 are represented as the corresponding naturally occurring amino acids, as follows.

Unnatural amino acid Corresponding Natural Amino Acid pGlu, pyroglutamate L-glutamate, Glu , D-Ser(O-TpeT-Bu) L-serine, Ser , D-Trp L-tryptophan, Trp LL , D-Nal L-phenylalanine, Phe G , D-Bhi L-histidine, His , D-Leu L-leucine, Leu Pro-Et L-proline, Pro Pro-NHNHCONH ₂ L-proline, Pro Gly-NH ₂ Gly

In the sequence list, entries 1-63 correspond to connections 1-63, and entries 64-78 correspond to connections P1-P15. Entries 78 and 80 correspond to wild-type GnRH I and GnRH II. Inputs 81-84 correspond to primers. Inputs 85-89 correspond to connections 64-68. Inputs 90-95 correspond to connections P16-P21. However, the sequences SEQ ID NOs: 1-78 and 85-89 as they appear in the sequence listing, i.e., without the unnatural amino acids described above, are not claimed, but are included only to fulfill the requirements of EPC R. 30(1).

Excerpt from free text list of sequences.

To comply with paragraph 36 of WIPO Standard ST.25, the body of the description repeats the free text included under the sequence list numeric identifier <223>.

SEQ ID NO Free text included in <223> 1-78 Artificial analogue of GnRH II 79 GnRHI 80 GnRH II 81 GnRH receptor type I forward primer 82 GnRH receptor type I reverse primer 83 GnRH receptor type II forward primer 84 GnRH receptor type II reverse primer 85-95 Artificial analogues of GnRH II

Definitions.

The singular number as used herein refers to one or more than one (ie, at least one) of the grammatical entities. For example, analog means one analog or more than one analog.

As used herein, the terms immunorelins and compound(s) of the invention are used interchangeably and refer to compounds of formula (I).

Pharmaceutically acceptable salts of the compounds of the invention include conventional salts such as

- 22 045183 baths with pharmaceutically acceptable inorganic or organic acids or bases, as well as addition salts of quaternary ammonium acids. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic, naphthalene-2sulfonic, benzenesulfonic, hydroxynaphthoic, hydroiodic, malic, steroic, tannic and similar acids. Other acids, such as oxalic acid, which are not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of salts of suitable bases include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, Nmethylglucamine and procaine salts.

Brief description of the figures

Fig. 1. Expression of MHC class I after stimulation of T cells with GnRH II at increasing concentrations. PBMCs from a healthy donor are stimulated with GnRH II and IL-2 for 72 hours. These points represent the average fluorescence intensity of MHC class I expression on CD4+ T cells (blue triangles) or CD8+ T cells (black squares) measured by flow cytometry.

Fig. 2. MHC class I expression after stimulation of T cells with increasing concentrations of GnRH I (red line) and GnRH II (black line) analogues. PBMCs from a healthy donor are stimulated with GnRH analogue I or GnRH II and IL-2 for 72 hours. These points represent the average fluorescence intensity of MHC class I expression on CD4+ T cells (A) or CD8+ T cells (B) measured by flow cytometry.

Fig. 3. Expression of MHC class I after stimulation of CD4+CD14+ monocytes with GnRH analog I (red line) and GnRH II (black line) in increasing concentrations. CD14+ monocyte PBMCs from a healthy donor are stimulated with GnRH analog I or GnRH II and IL-2 for 72 hours. These points represent the average fluorescence intensity of MHC class I expression on CD4+CD14+ monocytes measured by flow cytometry.

Fig. 4. GnRH receptor expression in human T cells is analyzed by quantitative real-time PNR. Bars represent GnRHR I or GnRHR II mRNA ratios normalized to RNA polymerase II expression in sorted naïve T cells (open bars) or memory T cells (gray bars). The MCF-7 breast cancer cell line (black bars) was used as a positive control.

experimental part

General biological methods.

The preferential effect of the compounds of the invention on GnRH receptors can be tested using one or more of the methods described below.

I. Expression of GnRH receptors on T cells.

Human naïve and memory T cells are fluorescently labeled with CD45RA, CD45RO, and CD4 antibodies and sorted by flow cytometry. Total RNA was extracted using the Rnaeasy kit (Qiagen) and reverse transcribed using the iScript select cDNA synthesis kit (Biorad). cDNA was amplified from the template using SYBR Green (Applied Biosystem) and performed on CFX96 PCR (Biorad). The ratios of GnRH receptor type I and GnRH receptor type II mRNA are normalized for RNA polymerase II expression in sorted naïve or memory T cells. The breast cancer cell line MCF-7 was used as a positive control.

Primer sequences.

Type I GnRH receptor fwd 5'-tgc etc ttc ate ate cct ct-3' rev 5'-gca aat gca ace gtc att tt-3'

Type II GnRH receptor fwd 5'-act gtt caa tgg etg get gt-3' rev 5'-gcc ccc aga agt ttc ett ac-3'

I. GnRH I versus GnRH II assay.

The compounds are tested on cells prepared to express type I or type II GnRH receptors by transfection. The cells are exposed to the labeled GnRH compound, washed and then assessed by measuring the label on the cells. The label is measured either directly (radioactive isotope label or fluorescent label) or indirectly (biotin-labeled peptide).

Signal transduction induced by GnRH compounds is measured in cell lines expressing GnRH type I and GnRH type II receptors, respectively. GnRH compounds are screened for their respective affinities for GnRH I and GnRH II receptors using competitive assays.

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Calcium influx is measured using cells labeled with Fluo-4-Direct either using a flow cytometer or using live cell imaging microscopy in order to estimate the EC50 values of their potency enhancement. Signal transduction is also examined by Western blotting using antibodies to pERK or p-JNK.

To evaluate the effect of cellular activation on LH and FSH production and compare it with stimulation of immunorelated functions, the effects of the compounds on pituitary cells and immune cells expressing either GnRH type II or GnRH type I receptors were examined.

II. Immune stimulation assay.

The potency of compounds to cause activation of immune cells can be assessed using an assay such as the following assay.

Human peripheral blood mononuclear cells (PBMCs) from healthy donors are purified by Ficoll-Hypaque density gradient centrifugation. Cells were cultured in RPMI1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 μg/ml ampicillin, 100 μg/ml streptomycin, and IL-2 (100 U/ml) for 72 hours at 37°C, 5% CO2. Cells are stimulated with GnRH II or GnRH I analogues at increasing concentrations and analyzed for expression of specific cell surface activation markers CD25, CD69 and MHC class I monoclonal antibodies from BD Pharmingen using flow cytometry.

Solubility and stability data (examples 2 and 3).

Each compound (0.2 mg) was added to PBS (pH 7.4, 0.2 mg/ml) and sonicated for 10 min and then shaken for 20 min. Take sample A T = 0 hour (80 µl) for LC/MS analysis. The solutions are then incubated (37°C with stirring) in a Techne Roller-Blot Hybridiser HB-3D. Samples are also taken at T = 4, 24, 96 h for LC/MS analysis.

LC/MS analysis is performed on an Aglent HP 1100 HPLC system equipped with a diode array detector (DAD), one Waters ZQ quadrupole mass analyzer and a Waters X select CSH C18 column, 2.1 mm x 50 mm, 3.5 μm. Collect LC/MS data for compound identity. Collect LC/UV area under the curve (AUC) data for each compound at 280 nm at each of four time points (T = 0, 4, 24, 96 hours). The trend in these data is interpreted to give each compound a solubility score from 1 (good) to 5 (poor) and a stability score (T _1/2 ).

Analysis of calcium in Cno-Κι cells (Genscript).

Sample solutions of the test compounds are dissolved in HBSS buffer (with 20 mM HEPES buffer, pH 7.4) to form 5x working solutions. Use the FLIPR Calcium 4 Assay Kit from Molecular Deviced (R8141) when required. Briefly, CHO-K1 cells expressing GnRHR (CHO-K1/GnRHR/Ga15, Genscript accession NM_000406) were respectively cultured in 10 cm dishes and maintained at 37°C/5% CO ₂ . CHO-K1 cells expressing GnRHR were respectively seeded in 384-well dark clear-bottom plates at a density of 15,000 cells per well in 20 μl of growth medium approximately 18 hours before the day of the experiment and maintained at 37°C/5% CO ₂ . 20 μl of dye solution is then added to the wells, and the plates are then placed in an incubator at 37°C for 60 minutes, followed by a 15-minute incubation at room temperature. Finally, 10 μL of compound or control agonist solutions are added to the appropriate wells of the assay plate during FLIPR reading. A plate containing 5x compound and control agonist solution is placed in FLIPR. Solutions are added to the wells of the plate automatically over 20 seconds, and the fluorescence signal is monitored for another 100 seconds (21 s - 120 s). Data are captured by ScreenWorks (version 3.1) as FMD files with FLIPR and stored on the GenScript network for offline analysis. Data are collected, analyzes performed using ScreenWorks (version 3.1) and loaded into Excel. Calculate the average reading over the first 20 seconds as the baseline, and calculate the values in relative fluorescence intensity units (ΔRFU) by subtracting the average of the baseline from the maximum fluorescence units (21 s - 120 s).

Calculate % stimulation from the following equation:

% stimulation = ((ARFU compound - ARFU 6a3bi)(ARFU control agonist - ARFU base) x 100.

Dose response curves are constructed using a four-parameter logistic equation using GraphPad Prism 6. Equation: Four-parameter logistic equation.

Y = bottom + (top - HH3)/(l+10((LogEC50 - X)xslope))

X represents the logarithm of the concentration. Y represents the reaction.

Materials.

Unless otherwise noted, all reagents used in the examples below are obtained from commercial sources.

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General synthesis method.

Method A.

Reaction - Reaction.

Peptides are prepared using standard solid phase synthesis with Fmoc as shown in the scheme above. Protected amino acids are used (Fmoc and tert-Bu or Trt, if necessary), and the synthesis is performed on 2-chlorotrityl polystyrene resin. The reactions are performed in the order A, B, C, followed by several repetitions of B and C to assemble the desired peptide. When the last amino acid (pyroglutamate - note: reaction C is used for this, although the amino acid is not protected by Fmoc), the last two reactions D and E are carried out in that order to generate the compound of the invention.

Reaction A: The resin is suspended in dichloromethane (10-20 volume equivalents of resin) and stirred at room temperature. Fmoc protected amino acid (2 equivalents) is added to 1 equivalent of resin in the presence of diisopropylethylamine (6 equivalents). The reaction mixture is stirred for 0.5 hour - 1 hour at room temperature. The resin is collected by filtration and washed 6 times with DMF and then used directly in the next step.

Reaction B: Remove the Fmoc protecting group by treatment with piperidine (20%) in dimethylformamide (5-10 volume equivalents of resin) at room temperature. The reaction mixture is stirred for up to 1 hour, the resin is collected by filtration, and then the resin is washed 6 times with DMF and used directly in the next step.

Reaction C: Fmoc protected amino acid (4 equivalents) is dissolved in DMF and DiPEA (2 equivalents) is added. After stirring at room temperature for one minute, the mixture was added to the amino acid resin (1 equivalent) from Reaction B, treated with HBTU (1 equivalent). The reaction mixture is stirred for up to 1 hour, the resin is collected by filtration and washed 6 times with DMF and used directly in the next step. The next step is either reaction B or reaction D, depending on the target sequence.

Reaction D: The protected peptide is cleaved from the resin by treatment with 3-5% trifluoroacetic acid in dichloromethane. The resin is removed by filtration and the peptide is obtained by precipitation with ice-cold diethyl ether and collection by centrifugation. The solid is also washed with diethyl ether and then dried in vacuo before being used in the next step.

Reaction E Prepare the C-terminal amide by dissolving the peptide from reaction D (1 equivalent) in DMF, add monoalkylamine (20-50 equivalents) and HBTU (2-3 equivalents), and stir the reaction mixture at room temperature for up to 3 hours. The reaction mixture is diluted with water and the crude peptide is then purified as detailed below.

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Method B.

Reaction - Reaction.

Peptides are prepared using standard solid phase synthesis with Fmoc as shown in the scheme above. Use protected amino acids (Fmoc and tert-Bu or Trt, if necessary), and perform synthesis on Ramage resin. The reactions are performed in the order F, G, H, I, followed by several repetitions of Y to assemble the desired peptide. When the last amino acid (pyroglutamate note: this uses reaction I, although the amino acid is not protected by Fmoc) is added, the last reaction Y is carried out in this order to generate the compound of the invention.

Reaction F: Fmoc-Ramage resin is suspended in DMF (5-10 resin volume equivalents) containing 20% piperidine. The reaction mixture is stirred for up to 1 hour at room temperature, and the resin is collected by filtration, washed 6 times with DMF and used directly in the next step.

Reaction G: Fmoc protected amino acid (5 equivalents) is dissolved in DMF and DiPEA (2 equivalents) is added. After stirring at room temperature for one minute, the mixture is added to the amino acid resin (1 equivalent) from reaction F, treated with the addition of HBTU (1 equivalent). The reaction mixture is stirred for up to one hour, the resin is collected by filtration and washed 6 times with DMF and used directly in the next step.

Reaction H: Remove the Fmoc protecting group by treatment with piperidine (20%) in dimethylformamide (5-10 volume equivalents of resin) at room temperature. The reaction mixture is stirred for up to 1 hour, the resin is collected by filtration, and then the resin is washed 6 times with DMF and used directly in the next step.

Reaction I: Fmoc protected amino acid (4 equivalents) is dissolved in DMF and DiPEA (2 equivalents) is added. After stirring at room temperature for one minute, the mixture was added to the amino acid resin (1 equivalent) from Reaction H, treated with HBTU (1 equivalent). The reaction mixture is stirred for up to one hour, the resin is collected by filtration and washed 6 times with DMF and used directly in the next step. The next stage

- 26 045183 is either reaction H or reaction J, depending on the target sequence.

Reaction J: The protected peptide is cleaved from the resin by treatment with 90% trifluoroacetic acid with 2.5% water, 2.5% triisopropylsilane and 5% dichloromethane. The resin is removed by filtration and the peptide is obtained by precipitation with ice-cold diethyl ether and collection by centrifugation. The crude peptide is then purified as detailed below.

Cleaning.

Crude peptides are dissolved individually in acetonitrile/H ₂ O (1:1, v/v) and purified by preparative HPLC with a C18 column using a water (0.1% TFA) - acetonitrile (0.1% TFA) gradient ). The final purity of the peptides was confirmed by analytical HPLC. Peptides are lyophilized before storage at -20°C.

Compound analysis - identity and purity.

Method of analysis A.

For analysis, compounds are dissolved in methanol:water (9:1, 0.1 mg/ml) and a 150 μl portion is placed in an HPLC microvial and centrifuged at 14,000 rpm for 3 minutes. The sample is then tested by high-performance liquid chromatography with diode array (HPLC-DAD) and mass spectrometry detection (HPLC-MS). HPLC-DAD-MS is performed using an Agilent 1100 HPLC system comprising a quaternary pump, autosampler, column oven, and diode array detector coupled with a single Waters ZQ quadrupole mass spectrometer. Also, for all connections, use a reverse phase column Waters Xselect CSH C18, 2.1 mm x 50 mm, particle size 3.5 μm, and connect to a safety column Waters VanGuard CSH C18, 2.1 mm x 5 mm, particle size 3.5 µm, and inline column filter, Waters Acquity, 0.2 µm. The column is used at a flow rate of 1 ml/min, maintained at 60°C. The solvents used are 0.17% formic acid in 95% acetonitrile, 5% water (solvent B) and 10 mM ammonium formate, 0.2% formic acid in water (solvent A), with the following gradient: 5% solvent B from 0 to 0.2 min, 5-50% solvent B from 0.2 to 9.3 min, 50-95% solvent B from 9.3 to 9.5 min, 95% solvent B from 9.5 to 11 min , 95-5% solvent B from 11 to 11.05 min and re-equilibration of 5% solvent B from 11.05 to 11.5 min. Nitrogen is used as an auxiliary and shielding gas. The voltage source is set to 3400 V, the cone voltage is set to 31 V with a gas flow of 50 l/h, the drying gas flow rate is 550 l/h and the drying gas temperature is 350°C.

Analysis of compounds - solubility and stability in solution.

Method of analysis B.

For solubility and stability assays, compounds are dissolved (0.2 mg/mL) in phosphate buffered saline (PBS, 10 mM, pH 7.4) and shaken at room temperature for 20 minutes. A T = 0 sample (80 μl) is taken and centrifuged at 14,000 rpm for 3 minutes and then analyzed using Assay Method A described above. The sample bulk is placed in a Techne RollerBlot HB-3D Rolling Hybridiser at 37°C and removed only when a sample (80 μl) is drawn at T = 4, 24 and 96 hours. Samples were centrifuged at 14,000 rpm for 3 min and then analyzed by HPLC-DAD-MS as described above. The UV area under the curve is recorded at 280 nm at each time point.

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Examples

Example 1. Synthesis of compounds.

The compounds of the invention are prepared according to the methods set out in the General Synthesis Method section.

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23 NW <0 1 J γ Η ₂ ΝγΝΗ j/ ^NH ch ₂ conh ₂ IN 24 I Z. γ X HaN^NH ^χ ^ΝΗ ch ₂ conh ₂ IN 25 Xl ii *' Ύ To ch ₂ conh ₂ IN — 27 T'nh Λ T nh /¼ w H ₂ N^NH J^ ^NH ch ₂ conh ₂ V 28 X^NH N^/ ? μΖ” X O X ch ₂ conh ₂ V thirty '^^'oh X ^nh ύ XT ch ₂ conh ₂ V 33 ^T^NH NW > ₍ W X I ch ₂ conh ₂ V 42 'X^NH NW G h ₂ n^nh z Et A 44 AND -W. XNH u h ₂ n^nh j/ ^nh Et A 45 Xx 1 z^z ^OH Et A 47 k^NH NW X w'M Η ₂ Ν^ΝΗ ^χ ^ΝΗ Et A 48 ^^/ ^NH 1 w^ To Et A

-29045183

49 ^ΌΙ- SI g To Et A 50 'k^NH «%/ ^χ^ CH 5 To Et A 56 ''Υ ^Α νη Ν«=/ I Λ’ ΗζΝ^ΝΗ Et A 59 ΥΊ I Λ' To Et A 60 τ\η No.=/ I? vVW Ο ΗζΝ^ΝΗ Et A 61 ΝΗ No.=/ I to Ο X Et A

- 30 045183

62 AND ) _Γ -Ν ί J^NH ί / X Ο X Et A 63 ' ^X F ^A NH ы=/ Μ X ? G-M J f ^Ν θ' X ο X Et A 64 XL ίΧ ] ΝΗ X. ' XI CH2CONH2 65 V Χι NX Η fL < ί Ό X .2k ,ιο X Ο X CH2CONH2 66 Χχ -Ν Λ ιί. - ΊΟ* ^У 1.x Η ₂ ΝγΝΗ j^ ^NH CH2CONH2 67 ΎΧ Η ο ) χ. y' 'Ν 'νη /M To η ₂ ν^νη jT^ ^NH Et 68 Η X- Γ ΝΗ : Λ ^ц NX *, η ₂ ν^νη J^ ^NH CH2CONH2

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Connection number Salt form Retention time (assay method A) m/z (analysis method A) 6 TFC 4.15 1213.8 8 TFC 5.5 1312.7 9 TFC 6.64 1246.8 10 TFC 4.27 1286.9 AND TFC 5.35 1220.9 12 TFC 6.75 1319.7 13 TFC 5.53 1293.7 14 TFC 6.13 1318.7 16 TFC 4.93 1292.8 20 TFC 4.02 1358.9 23 TFC 4.79 1296.7 24 TFC 5.84 1395.7 25 TFC 7.19 1329.6 27 TFC 4.91 1369.6 28 TFC 5.92 1303.8 thirty TFC 7.20 1402.7 33 TFC 6.00 1376.4 42 TFC 3.90 1183.8 44 TFC 5.23 1282.8 45 TFC 6.35 1216.8 47 TFC 4.00 1256.8 48 TFC 5.12 1190.8 49 TFC 6.56 1290.0 50 TFC 5.30 1263.7 56 TFC 3.42 1297.7 59 TFC 5.22 1330.8 60 TFC 3.81 1370.6 61 TFC 4.36 1304.7 62 TFC 5.60 1403.5 63 TFC 4.69 1377.7 64 TFC 6.19 1391.7 65 TFC 5.09 1366.0 66 TFC 4.92 1384.7 67 TFC 4.47 1396.9 68 TFC 3.83 1285.8

Example 2: Solubility Analysis.

The solubility of the compounds of the invention is tested as described in the General Methods. Solubility is then classified according to a scale of 1 to 5, where 1 means the most solubility and 5 means the least solubility.

Connection number Solubility classification Buserelin acetate 1 Triptorelin acetate 2 Napherelin acetate 2 Histrelin acetate 4

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Leuprorelin acetate 2 Buserelin-TFC 2 Triptorelin-TPA 2 Naferelin-TFA 1 G istrelin-T FC 2 Leuprorelin-T FC 1 6 1 8 2 9 1 10 1 AND 1 12 4 13 3 14 4 16 2 20 3 23 1 24 5 25 4 33 2 thirty 1 28 2 27 1 56 1 67 1 59 4 63 5 62 5 61 3 60 2 42 1 44 2 45 2 50 1 49 4 48 2 47 1 68 1 66 5 14 4 65 4 64 5 16 2 20 3

Example 3. Stability analysis.

The stability of the compounds of the invention in aqueous media (PBS, pH 7.4) is tested as described in the general methods. Resistance is then classified on a scale where t1/2>96 minutes is shown as +, and resistance less than this is shown as -.

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Connection number Stability classification 6 + 8 + 9 + 10 + AND + 12 + 13 + 14 + 16 + 20 + 23 + 24 - 25 - 33 - thirty = 28 + 27 + 56 + 67 - 59 - 63 - 62 - 61 + 60 + 42 + 44 + 45 + 50 + 49 + 48 + 47 + 68 + 66 + 14 + 65 + 64 16 + 20 +

Example 4. Stimulation of GnRH R.

The ability of the compounds of the invention to stimulate GnRHR was assessed using the Cho-Κι cell calcium assay (Genscript), see General Methods for details. Activity is then recorded as percent stimulation at 1 μM.

Connection number GnRHR stimulation at 1 µM Buserelin 94 Leuprorelin 107(n=2) G oserelin 99

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Gonadorelin 102 Nafarelin 100(n=2) 6 88 8 86 9 78 10 81 AND 76 12 80 13 87 14 78 16 76 20 75 23 106 24 87 25 82 33 67 thirty 12 28 95 27 108 56 96 67 117 59 85 63 77 62 77 61 112 60 102 42 102 44 111 45 105 50 105 49 77 48 118 47 119 68 86 66 83 14 78 65 72 64 50 16 76 20 75

Links

1. Gonadotropin secretion and its control. Fink G., The physiology of reproduction, 1998.

2. Immunomodulatory actions of gonadal steroids may be mediated by gonadotropinreleasing hormone. Jacobson J.D. and Ansari M.A., Endocrinology, 2004; 145(1): 330-6.

3. Unusual morphological features of uterine leiomyomas treated with gonadotropinreleasing hormoneagonists: massive lymphoid infiltration and vasculitis. McClean G. and

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Claims (9)

1. McCluggage W. G., Int. J. Surg. Pathol., 2003; 11(4): 339-44.
2. 4. Massive lymphocytic infiltration of uterine leyomyomas associated with GnRH agonist treatment. Bardsley V. et al., Histopathology, 1998; 33(1): 80-2.
3. 5. Chronic plasma cell endometritis in hysterectomy specimens of HIV-infected women: a retrospective analysis. Kerr-Layton J. A. et al., Infect. Dis. Obstet Gynecol., 1998; 6(4): 18690.
4. 6. Serum dihydrotestosterone and testosterone concentrations in human immunodeficiency virus-infected men with and without weight loss. Arver S. et al., J. Androl. 1999; 20(5): 611-8.
5. 7. Prevalence of endocrine dysfunction in HIV-infected men. Brockmeyer G. et al., Horm. Res., 2000; 54(5-6): 294-5.
6. 8. Gonadotropin-releasing hormone increases CD4-T-lymphocyte numbers in an animal model of immunodeficiency. Jacobson J. D. et al., J. Allergy Clin. Immunol., 1999; 104: 653-8.
7. 9. Second gene for gonadotropin-releasing hormone in humans. White et al., PNAS 1998; Jan 6; 95(1): 305-9.
8. 10. A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1 q. 12. Morgan et al., Endocrinology, 2003 Feb; 144(2): 423-36.
9. 11. Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists. Eidne et al., J. Clin. Endocrinol. Metab., 1987 Mar; 64(3): 425-32.

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