Somatostatin peptide analogue, chelate and application of somatostatin peptide analogue and chelate

Abstract

The invention discloses a somatostatin peptide analogue, the somatostatin peptide analogue has a cyclic or non-cyclic hexapeptide unit, and the residue sequence at the position numbered 2 to 5 in the hexapeptide unit is-X1-(D / L-) Trp-X2-X3-, and the residue sequence at the position numbered 2 to 5 in the hexapeptide unit is-X1-(D / L-) Trp-X2-X3-, or, the somatostatin peptide analogue has a ring-forming or non-ring-forming octapeptide unit, and the residue sequence at the position numbered 2 to 7 in the octapeptide unit is-Cys-X1-(D / L-) Trp-X2-X3-Cys-, and the residue sequence at the position numbered 2 to 7 in the octapeptide unit is-Cys-X1-(D / L-) Trp-X2-X3-Cys-; wherein X1 is an alpha-amino acid residue with an aryl group on a C alpha side chain, X3 is an alpha-amino acid residue, and the compound is characterized in that X2 is a Lys-variation residue: # imgabs0 #. The invention breaks through the'taboo 'that the Lys9 residue cannot be replaced, otherwise, the biological activity is greatly reduced. According to the present invention, the Lys9 residue in the somatostatin peptide analogue is changed, and the Lys9 residue is subjected to change variation, such that the somatostatin peptide analogue of the present invention has high (at least similar) binding affinity with SSTR2 due to the change variation;

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor compound synthesis, and specifically relates to a somatostatin peptide analog, a chelate and applications thereof. Background Art

[0002] Somatostatin (SST) is a 14-residue peptide (SST-14) or 28-residue peptide (SST-28) that regulates key aspects of animal physiology by inhibiting metabolic and growth-related neuroendocrine signaling systems, including insulin or glucagon, thyroid-stimulating hormone, prolactin, growth hormone, and digestion-related processes. These effects of somatostatin are signaled by somatostatin receptors (SSTRs). SSTRs are G protein-coupled receptors (GPCRs) that signal by inhibiting the Gi / o family of G proteins to prevent the release of secretory hormone vesicles. Neuroendocrine tumors (NETs) often overexpress SSTRs, and somatostatin agonists are therefore useful as diagnostic imaging tracers and therapeutic agents for the treatment of hormone excess disorders caused by NETs, ​​including carcinoid syndrome, gigantism, acromegaly, hyperthyroidism, and Cushing's disease.

[0003] The SSTR family includes five different subtypes, SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5. These receptors have different tissue expression profiles, including differential expression in different NETs. Among them, SSTR2 is overexpressed in most NETs, ​​so this subtype is the main target of clinical agonists. All US FDA-approved somatostatin agonists currently in clinical use are peptide analogs of SST-14, including SSTR2-selective drugs octreotide and lanreotide, and SSTR1, SSTR2, SSTR3, and SSTR5-selective pasireotide. A large amount of evidence shows that different SSTR subtypes also play an important role in a variety of tumor types other than NETs, ​​so more selective pharmacological molecules are needed to detect subtype-specific functions.

[0004] In this patent application, natural somatostatin is referred to as a tetradecapeptide (Formula V) having the following structure:

[0005]

[0006] The somatostatin peptide analogs used in this patent application refer to linear or cyclic peptide derivatives of naturally occurring SST-14, i.e., one or more residues of the above-mentioned sequence of formula V are deleted or replaced by one or more other amino acids, or one or more functional groups are deleted or replaced by one or more other functional groups. In general, the term covers all modified derivatives of natural SST-14 containing the above-mentioned sequence of formula V, which have high binding affinity to at least one somatostatin receptor subtype and reach nmol level.

[0007] The understanding of the role of key residues came from the synthesis of a large number of somatostatin analogs starting as early as the 1970s. The approach used by the Sandoz team was to start with the hexapeptide lead structure Cys-Phe-DTrp-Lys-Thr-Cys and by systematically extending the N- and C-termini, in 1980 they succeeded in synthesizing and characterizing a very stable and active analog octreotide with the following structure: H-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-Ol. This compound was more effective than two natural forms of somatostatin in inhibiting growth hormone release in vivo and showed sufficient stability in vivo, so it was selected for clinical studies. Octreotide was first registered as a drug by the FDA in 1988 for the treatment of acromegaly and carcinoid tumors. In addition, oxyoctreotide (an octreotide analog) can be used to detect tumor cells expressing somatostatin receptors by tumor focused scanning imaging by coupling with positron-emitting isotopic metal ions to form a coupled complex. Oxyoctreotide can also be used to kill tumor cells expressing somatostatin receptors by coupling with β-ray-emitting isotope metal ions to form a coupled complex.

[0008] Other somatostatin peptide analogs are also on the market, including lanreotide, vapreotide and pasireotide. A large number of studies have shown that the core of SST-14 binding to its receptor is composed of two residues, Trp8-Lys9. These two key binding residues are essential and indispensable for its biological activity. They were later proved to be the core pharmacophores of somatostatin peptide analogs binding to somatostatin receptors. In fact, a large number of Lys9 residue substitution studies have also failed to improve its receptor binding affinity and biological activity. Most of the substituted analogs have lost their receptor binding affinity or biological activity. These studies have led to a "taboo" conclusion: "Lys9 cannot be replaced, not even by its closest residues Arg or Orn. ​​Once replaced, its biological activity will be greatly reduced." Summary of the invention

[0009] According to a series of studies, the SSTR2 protein structure shows that the SSTR2 pocket bound to Lys9 still has a lot of space to accommodate other atoms. By changing the Lys9 group, the spatial tension can be appropriately increased and the number of possible conformations of the side chain can be limited, which is conducive to more effective binding between drug molecules and SSTR2.

[0010] Therefore, the object of the present invention is to provide a somatostatin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable complex thereof, or a pharmaceutically acceptable chelate thereof,

[0011] The somatostatin peptide analogs have a cyclic or non-cyclic hexapeptide unit, and the residue sequence at positions numbered 2 to 5 in the hexapeptide unit is: -X1-(D / L-)Trp-X2-X3-;

[0012] Alternatively, the somatostatin peptide analogue has a cyclic or non-cyclic octapeptide unit, and the residue sequence at positions numbered 2 to 7 in the octapeptide unit is: -Cys-X1-(D / L-)Trp-X2-X3-Cys-;

[0013] Wherein X1 is an α-amino acid residue having an aromatic group on the Cα side chain, X3 is an α-amino acid residue, characterized in that X2 is a Lys-variant residue:

[0014]

[0015] n is 0 or 1;

[0016] A1, A2, A3 are independently selected from C(R)2, O, S, NR, C=O, C=S, C=NR and C=C(R)2;

[0017] R is independently selected from R1, -R'OR1, -R'SR1, -R'N(R1)2, -R'C(O)R1, -R'S(O)R1, -R'S(O)2R1, -R'C(O)N(R1)2, -R'N(R1)C(O)R1, -R'C(O)OR1 and -R'OC(O)R1;

[0018] or independently, the R in C(R)2 at position A2 is connected to the R in C(R)2 at position A1 or position A3 to form a double bond or a triple bond;

[0019] or independently, R in C(R)2 forms a 3-8 membered carbocyclic ring with R in its own C(R)2, or with R in an adjacent C(R)2, or with R in a C(R)2 separated by one C(R)2, or forms a 3-8 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S;

[0020] R' is a linking bond, a C1-C6 alkylene group, a C2-C6 alkenylene group, a C2-C6 alkynylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkenylene group, a halogenated C1-C6 alkylene group, a halogenated C2-C6 alkenylene group, a halogenated C2-C6 alkynylene group, a halogenated C3-C8 cycloalkylene group, a halogenated C3-C8 cycloalkenylene group, a (C6-C8)arylene group, a 3-8 membered heterocyclylene group having 1 to 3 heteroatoms selected from N, O, and S, and a 3-8 membered heteroarylene group having 1 to 3 heteroatoms selected from N, O, and S;

[0021] R1 is independently selected from H, halogen, CN, NO2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C3-C8 cycloalkyl, halogenated C3-C8 cycloalkenyl, (C6-C8)aryl, 3-8 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-8 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S;

[0022] When n is 0, R in C(R)2 at the A1 position, A2 position and A3 position are not H at the same time.

[0023] In the present invention, "R in C(R)2 forms a 3-8 membered carbocyclic ring with R in its own C(R)2, or with R in an adjacent C(R)2, or with R in a C(R)2 separated by one C(R)2, or forms a 3-8 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S", which includes the following 4 forms:

[0024] Independently, the two Rs in C(R)2 at the A1 position, A2 position or A3 position together form a 3-8 membered carbocyclic ring, or a 3-8 membered heterocyclic ring having 1-3 heteroatoms selected from N, O and S;

[0025] or independently, R in C(R)2 at position A1 and R in C(R)2 at position A3 are connected together to form a ring so that A1, A2, and A3 form a 3-8 membered carbocyclic ring or a 3-8 membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, and S;

[0026] Or independently, R in C(R)2 at position A1 and R in C(R)2 at position A2 are connected together to form a ring so that A1 and A2 form a 3-8 membered carbocyclic ring or a 3-8 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S;

[0027] Alternatively, independently, the R in C(R)2 at position A2 and the R in C(R)2 at position A3 are connected together to form a ring so that A2 and A3 form a 3-8 membered carbocyclic ring or a 3-8 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S.

[0028] In a preferred example,

[0029] The somatostatin peptide analogs have a cyclic or non-cyclic hexapeptide unit, and the residue sequence at positions numbered 1 to 6 in the hexapeptide unit is: Xa-X1-(D / L-)Trp-X2-X3-Xb;

[0030] The somatostatin peptide analogs have a cyclic or non-cyclic octapeptide unit, and the residue sequence at positions numbered 1 to 8 in the octapeptide unit is: X0-Cys-X1-(D / L-)Trp-X2-X3-Cys-Xb;

[0031] Wherein Xa and Xb are amino acid residues; X0 is an α-amino acid residue having an aromatic group on the Cα side chain.

[0032] In a preferred example,

[0033] X1 is a Phe, Tyr, Trp or Phg (phenylglycine) residue; X3 is a Thr, Ser, Cys or tyrosylbenzyl residue.

[0034] In a preferred example, the somatostatin peptide analog has a cyclic octapeptide unit structure as shown in the following general formula (I):

[0035]

[0036] n is 0 or 1;

[0037] A1, A2, A3 are independently selected from C(R)2, O, S, NR, C=O, C=S, C=NR and C=C(R)2;

[0038] R is independently selected from R1, -R'OR1, -R'SR1, -R'N(R1)2, -R'C(O)R1, -R'S(O)R1, -R'S(O)2R1, -R'C(O)N(R1)2, -R'N(R1)C(O)R1, -R'C(O)OR1 and -R'OC(O)R1;

[0039] or independently, the R in C(R)2 at position A2 is connected to the R in C(R)2 at position A1 or position A3 to form a double bond or a triple bond;

[0040] or independently, R in C(R)2 forms a 3-8 membered carbocyclic ring with R in its own C(R)2, or with R in an adjacent C(R)2, or with R in a C(R)2 separated by one C(R)2, or forms a 3-8 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S;

[0041] R' is a linking bond, a C1-C6 alkylene group, a C2-C6 alkenylene group, a C2-C6 alkynylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkenylene group, a halogenated C1-C6 alkylene group, a halogenated C2-C6 alkenylene group, a halogenated C2-C6 alkynylene group, a halogenated C3-C8 cycloalkylene group, a halogenated C3-C8 cycloalkenylene group, a (C6-C8)arylene group, a 3-8 membered heterocyclylene group having 1 to 3 heteroatoms selected from N, O, and S, and a 3-8 membered heteroarylene group having 1 to 3 heteroatoms selected from N, O, and S;

[0042] R1 is independently selected from H, halogen, CN, NO2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C3-C8 cycloalkyl, halogenated C3-C8 cycloalkenyl, (C6-C8)aryl, 3-8 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-8 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S;

[0043] When n is 0, R in C(R)2 at the A1 position, A2 position and A3 position are not H at the same time.

[0044] In a preferred example,

[0045] The R in C(R)2 at position A1 and the R in C(R)2 at position A3 are connected together to form a ring so that A1, A2, and A3 form a 3- to 8-membered carbocyclic ring or a 3- to 8-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, and S;

[0046] Preferably,

[0047] The R in C(R)2 at position A1 and the R in C(R)2 at position A3 are connected together to form a ring so that A1, A2, and A3 form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring having 1-2 heteroatoms selected from N, O, and S.

[0048] In a preferred example,

[0049] The R in C(R)2 at the A2 position is connected to the R in C(R)2 at the A1 position or the A3 position to form a double bond.

[0050] In a preferred example,

[0051] R' is a connecting bond, C1-C4 alkylene, C2-C4 alkenylene, C2-C4 alkynylene, C3-C7 cycloalkylene, C3-C7 cycloalkenylene, halogenated C1-C4 alkylene, halogenated C2-C4 alkenylene, halogenated C2-C4 alkynylene, halogenated C3-C7 cycloalkylene, halogenated C3-C7 cycloalkenylene, (C6-C8)arylene, 3-7 membered heterocyclylene having 1 to 3 heteroatoms selected from N, O, and S, and 3-7 membered heteroarylene having 1 to 3 heteroatoms selected from N, O, and S;

[0052] R1 is independently selected from H, halogen, CN, NO2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C7 cycloalkyl, halogenated C3-C7 cycloalkenyl, (C6-C8)aryl, 3-7 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-7 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S;

[0053] Preferably,

[0054] R' is a linking bond, C1-C3 alkylene, C2-C3 alkenylene, C2-C3 alkynylene, C5-C6 cycloalkylene, C5-C6 cycloalkenylene, halogenated C1-C3 alkylene, halogenated C2-C3 alkenylene, halogenated C2-C3 alkynylene, halogenated C5-C6 cycloalkylene, halogenated C5-C6 cycloalkenylene, (C6-C8)arylene, 5-6 membered heterocyclylene having 1-2 heteroatoms selected from N, O, and S, and 5-6 membered heteroarylene having 1-2 heteroatoms selected from N, O, and S;

[0055] R1 is independently selected from H, halogen, CN, NO2, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, halogenated C1-C3 alkyl, halogenated C2-C3 alkenyl, halogenated C2-C3 alkynyl, halogenated C5-C6 cycloalkyl, halogenated C5-C6 cycloalkenyl, (C6-C8)aryl, 5-6 membered heterocyclic group having 1 to 2 heteroatoms selected from N, O, and S, and 5-6 membered heteroaryl having 1 to 2 heteroatoms selected from N, O, and S.

[0056] Preferably, X2 is a Lys-variant residue:

[0057] Selected from:

[0058]

[0059]

[0060] Among them, Lys(c), Lys(d), Lys(f), Lys(h), Lys(j), Lys(l), Lys(m) can also be any one of the side chain stereoisomers. For example, Lys(d) can be any one of the following two stereoisomers, namely:

[0061]

[0062] Lys(j) can be any of the following four stereoisomers, namely:

[0063]

[0064] In a preferred embodiment of the present invention, the somatostatin peptide analogs are specifically: Core amino acid sequence Phe-DTrp-Lys(a)-Thr, The core amino acid sequence is Phe-DTrp-Lys(b)-Thr, Core amino acid sequence Phe-DTrp-Lys(d)-Thr, The core amino acid sequence is Phe-DTrp-Lys(j)-Thr,

[0065] The core amino acid sequence is Phe-DTrp-Lys(n)-Thr.

[0066] Another object of the present invention is to provide a somatostatin chelate, which is a chelate formed by removing the H atom from the N-terminal NH2 substituent of the somatostatin peptide analog of the invention general formula (I) and directly connecting it to the chelate group or indirectly connecting it through a spacer. The structural formula of the chelate of the present invention is: chelate group-(spacer)-dehydrogenation general formula (I).

[0067] The chelating group includes any feasible chelating group mentioned in the prior art. Preferred chelating groups are DTPA, DOTA, TETA or substituted EDTA or DTPA chelating groups. More preferred chelating groups are DTPA or DOTA.

[0068] Suitable spacers include those disclosed in German Patent GB-A-2,225,579, for example a divalent residue of an aminocarboxylic acid, such as β-Ala or a divalent residue derived from 6-aminocaprioic acid.

[0069] The structural formulas of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) and EDTA (ethylenediaminetetraacetic acid) are as follows:

[0070]

[0071] The structural formulas of β-Ala and 6-aminocaprioic acid (6-aminohexanoic acid) are as follows:

[0072]

[0073] One structure of the somatostatin chelate of the present invention is, for example:

[0074]

[0075] The chelating group may be bound to a detectable element. Suitable detectable elements include, for example, 19F, heavy metal elements or rare earth ions, such as paramagnetic ions used in CAT scanning (computed axial tomography), such as Gd 3+ , Fe 3+ , Mn 2+ and Cr 2+ , fluorescent metal ions, such as Eu 3+ and radionuclides, such as the radioactive lanthanides, in particular gamma-emitting radionuclides, beta-emitting radionuclides, alpha-emitting radionuclides, Auger-e - Emitting radionuclides, positron-emitting radionuclides e.g. 68 Ga.

[0076] Suitable gamma-emitting radionuclides include those useful in diagnostic techniques, such as those derived from gallium, indium, technetium, ytterbium, rhenium, terbium, thallium and samarium, e.g. 67 Ga, 111 In, 99m Tc, 161 Tb, 169 Yb and 186 Re.

[0077] Suitable beta-emitting radionuclides include those useful for therapeutic applications, e.g. 90 Y. 67 Cu, 177 Lu, 186 Re, 188 Re, 169 2. 121 Sn, 127 Te, 143 Pr, 198 Au, 109 Pd, 165 Dy, 32 P. 142 Pr and 156 Sm.

[0078] Suitable alpha-emitting radionuclides are those used in therapy, e.g. 211 At 212 Bi, 225 Ac, or 201 Tl.

[0079] The compounds of the present invention may exist in free or salt form.

[0080] Another object of the present invention is to provide the use of somatostatin peptide analogs of the general formula (I) of the present invention in the preparation of anti-tumor drugs, preferably in neuroendocrine tumors, more preferably acromegaly and carcinoid tumors. For example, the anti-tumor drug is used to treat, prevent and / or diagnose those diseases, diseases and / or pathologies expressing SSTR1, SSTR2, SSTR3, SSTR4 and / or SSTR5 somatostatin receptors, the causes of which include or are related to excessive secretion of growth hormone, gastrointestinal diseases, malignant cell proliferative diseases, angiogenesis-related diseases, or to prevent or combat transplant vascular diseases, restenosis and vascular occlusion after vascular injury. The method comprises administering a therapeutically effective amount of a somatostatin analog or a chelate thereof, or a pharmaceutically acceptable salt or a complex with a detectable element to a subject in need of such treatment.

[0081] The positive improvement effect of the present invention is that the reference compound of the present invention is octreotide, and its molecular structure is as follows (VI):

[0082]

[0083] The core amino acid sequence of octreotide is Phe-DTrp-Lys-Thr.

[0084] The present invention breaks the taboo that Lys9 residue cannot be replaced, otherwise the biological activity will be greatly reduced. The present invention changes and mutates the Lys9 residue in the somatostatin peptide analogs, and this change and mutation makes the somatostatin peptide analogs of the present invention show a higher (at least similar) binding affinity with SSTR2. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 The graph is a result of measuring the effect of the concentration of the reference compound on the binding affinity of SSTR 2;

[0086] Figure 2 The graph is a result of measuring the effect of the concentration of target compound 1 on the binding affinity of SSTR 2;

[0087] Figure 3 The graph is a result of measuring the effect of the concentration of target compound 2 on the binding affinity of SSTR 2;

[0088] Figure 4 The graph is a result of measuring the effect of the concentration of target compound 3 on the binding affinity of SSTR 2;

[0089] Figure 5 The graph is a result of measuring the effect of the concentration of target compound 4 on the binding affinity of SSTR 2;

[0090] Figure 6 The graph is a result of measuring the effect of the concentration of target compound 5 on the binding affinity of SSTR 2;

[0091] Figure 7 This is a graph showing the effect of the concentration of target compound 6 on the binding affinity of SSTR 2. DETAILED DESCRIPTION

[0092] Some abbreviations used in the present invention are as follows:

[0093] RT is room temperature; MS is mass spectrometry; Min is minute; HPLC is high performance liquid chromatography; UPLC is ultra-high performance liquid chromatography; LC-MS is liquid chromatography-mass spectrometry; MW is molecular weight; N2 is nitrogen; I2 is iodine; VC is vitamin; EC50 is half maximal effect concentration;

[0094] DMF is dimethylformamide; TFA is trifluoroacetic acid; BOC is tert-butyloxycarbonyl; Fmoc = 9-fluorenylmethoxycarbonyl; HOBT = hydroxybenzotriazole; DODT = 3,6-dioxa-1,8-octanedithiol; AcOH = Acetate acid; DCM = dichloromethane; ACN = acetonitrile; DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TETA = 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid acid (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid); EDTA = ethylenediaminetetraacetic acid; DTPA = diethylenetriaminepentaacetic acid; EA = ethyl acetate;

[0095] Cys is cysteine; Trp is tryptophan; Lys is lysine; Tyr is tyrosine; Ser is serine; Ala is alanine; Asp is aspartic acid; Glu is glutamic acid; Gly is glycine; His is histidine; Ile is isoleucine; Leu is leucine; Met is methionine; Asn is asparagine; Pro is phenylalanine; Pro is proline; Gln is glutamine; Arg is arginine; Thr is threonine; Val is valine; Phg is phenylglycine; Orn is ornithine; DTrp = (D-)Trp; DPhe = (D-)Phe. Unless otherwise specified; amino acid residues are usually L-type.

[0096] The synthesis process of the somatostatin peptide analogs of the general formula (I) of the present invention is as follows:

[0097] Step S1: Swelling of resin

[0098] Chloro-2-chloro-trityl resin (1 g, degree of substitution: 0.7 mmol per gram) was added with DCM (dichloromethane, 20 ml), and swelled under nitrogen for half an hour. Then, the DCM was removed and washed three times with DMF (N,N-dimethylformamide solution).

[0099] Step S2 Removal of Fmoc protecting group

[0100] To the product of step S1, Fmoc-Thr(OtBu)-OH (2.1 mmol) and diisopropylethylamine (DIPEA, 8.4 mmol) were added, reacted for two hours, filtered, and washed with DMF six times to obtain Fmoc-Thr(OtBu)-O-2-chloro-trityl resin.

[0101] Step S3: Condensation reaction of amino acids

[0102] To the product of step S2, add Fmoc-protected amino acid (2.1 mmol); HOBt (2.1 mmol); HBTU (2.1 mmol); diisopropylethylamine (DIPEA, 4.2 mmol) and N, N-dimethylformamide (15 ml), react for 1 hour, and filter. The obtained Fmoc-protected amino acid-Thr(OtBu)-O-2-chloro-trityl resin is washed with N, N-dimethylformamide 3 times (2 minutes each time).

[0103] Step S4

[0104] Repeat steps S2 and S3 until all designed polypeptide units are assembled.

[0105] Step S5: Shrinkage of resin

[0106] In the product obtained in step S4, the Fmoc protecting group of the last amino acid is removed according to the method of step S2, and then the deprotection liquid is extracted, and finally washed three times with DMF, DCM and methanol respectively, and dried to obtain a resin polypeptide.

[0107] Step S5: Formation of disulfide bonds by iodine methanol method

[0108] The resin polypeptide was dissolved in H2O / acetonitrile, the concentration was controlled at about 0.001 mol / L, AcOH (acetic acid) was added to adjust the pH to 4-5, and iodine solution (0.04 mol / L) (about 1g of iodine plus 100ml of methanol) was slowly added dropwise until the solution no longer turned colorless, and the reaction was continued for 30 minutes. After the reaction was completed, VC (ascorbic acid) was added to remove excess iodine, and the reaction was continued for 30 seconds until the solution turned colorless. The obtained white solid was separated by high performance liquid chromatography (prep-HPLC) and freeze-dried to obtain the product.

[0109] Example 1 Synthesis of Compound 1

[0110] Lys (a) is a commercially available raw material. The synthesis of compound 1 was carried out according to the general synthesis process of somatostatin peptide analogs described above, and the specific steps are:

[0111]

[0112] Compound 1 is a white powder, molecular weight (average): calculated value 1033.3 g / mol; LC-MS measured value (M+H)+: 1034.4; (M+2H)2+: 517.3

[0113] Purity determined by UPLC (214 nm): >99%

[0114] Example 2 Compound 2

[0115] The synthesis steps of Lys(b) variant residue in compound 2 are as follows:

[0116]

[0117] Synthesis of compound 2b

[0118]

[0119] Isobutyronitrile 2a (2.0 g) was dissolved in THF (20 mL), the temperature was lowered to -78 °C, LDA (15.9 mL, 2M in THF) was added dropwise, the mixture was kept at -78 °C and stirred for 45 min, 3-bromopropylene (7.1 g) was added dropwise, the temperature of the reaction solution was slowly raised to room temperature, and the reaction was carried out at room temperature for 3.5 h. The reaction solution was quenched with water (50 mL), the aqueous phase was extracted with n-hexane (3×70 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1 (3.12 g) as a yellow oil.

[0120] 1 H NMR (400MHz, DMSO) δ5.91–5.75(m,1H),5.25–5.12(m,2H),2.30-2.26(m,2H),1.29(s,6H).

[0121] Synthesis of compound 2c

[0122]

[0123] Compound 2b (2.55 g) was dissolved in MTBE (30 mL), and LAH (3.56 g) was added in batches at 0°C. The mixture was slowly warmed to room temperature and reacted overnight at room temperature. The reaction solution was quenched with 6M NaOH (20 mL, aq.) under an ice bath, the aqueous phase was extracted with MTBE (3×40 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 2c (2.64 g) as a light yellow viscous oil.

[0124] 1H NMR (400MHz, DMSO) δ5.88–5.71(m,1H),5.07–4.95(m,2H),2.28(s,2H),1.94-1.89(m,2H),0.77(s,6H).

[0125] Synthesis of compound 2d

[0126]

[0127] Compound 2c (2.64 g) was dissolved in MTBE (20 mL), (Boc)2O (7.01 g) and TEA (6 mL) were added, and the mixture was reacted at room temperature for 4 h. The reaction solution was diluted with water (20 mL), the aqueous phase was extracted with EA (3×40 mL), the organic phase was washed with saturated aqueous sodium chloride solution (1×50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=5 / 1) to obtain compound 2d (2.08 g).

[0128] 1 H NMR(400MHz,DMSO)δ6.79(t,J=6.1Hz,1H),5.87-5.74(m,1H),5.06–4.93(m, 2H), 2.76 (d, J = 6.4Hz, 2H), 1.88 (d, J = 7.5Hz, 2H), 1.38 (s, 9H), 0.76 (s, 6H).

[0129] Synthesis of compound 2e

[0130]

[0131] Compound 2d (1.0 g) was dissolved in a mixed solution of MeOH (20 mL) and DCM (20 mL), and after the temperature was lowered to -78°C, O3 was continuously introduced until the reaction solution turned blue. The reaction solution was concentrated and dried by spin drying and used directly in the next reaction.

[0132] 1 H NMR (400MHz, DMSO) δ11.51 (s, 1H), 6.67 (t, J = 6.2Hz, 1H), 2.79-2.75 (m, 2H), 1.46-1.41 (m, 2H), 1.38 (s, 9H), 0.82 (s, 6H).

[0133] Synthesis of compound 2f

[0134]

[0135] Compound 2e (921.1 mg) was dissolved in DCM (10 mL), compound 2w (1.463 g) and DBU (758 mg) were added, and the mixture was reacted at room temperature overnight. The reaction solution was diluted with water (20 mL), the aqueous phase was extracted with DCM (3×40 mL), the organic phase was washed with water (1×50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by column chromatography to obtain compound 2f (550 mg).

[0136] 1 H NMR(400MHz,DMSO)δ8.32(br,1H),6.80(t,J=6.1Hz,1H),6.35(br,1H),3.66(s ,3H),2.77(d,J=6.4Hz,2H),2.01(d,J=7.8Hz,2H),1.38(s,18H),0.78(s,6H).

[0137] Synthesis of compound 2g

[0138]

[0139] Compound 2f (550 mg) was dissolved in MeOH (20 mL), (S,S)-Et-DuPHOS-Rh (13 mg) was added, and the mixture was reacted at room temperature for 3 h under a hydrogen environment (400 psi). The solid was filtered off and the filtrate was concentrated to obtain compound 2g (630 mg, crude).

[0140] 1 H NMR (400MHz, DMSO) δ7.11(d,J=7.5Hz,1H),6.78(t,J=6.2Hz,1H),3.87-3.79(m,1H),3.61(s,3H),2.77- 2.68(m,2H),1.64–1.44(m,2H),1.41–1.34(m,18H),1.24-1.19(m,1H),1.12-1.02(m,1H),0.74(s,6H).

[0141] Synthesis of compound 2h

[0142]

[0143] Compound 2g (630 mg, crude) was dissolved in a mixed solution of THF (10 mL) and H2O (10 mL), LiOH.H2O (138 mg) was added, and the mixture was reacted at room temperature overnight. After the reaction solution was concentrated, the pH value of the solution was adjusted to about 3, the aqueous phase was extracted with EA (3×30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound 8 (450 mg).

[0144] 1 H NMR (400MHz, DMSO) δ12.36(br,1H),6.92(d,J=7.6Hz,1H),6.77(t,J=6.3Hz,1H),3.81–3.67(m,1H),2.80 –2.64(m,2H),1.66–1.45(m,2H),1.40–1.35(m,18H),1.29–1.21(m,1H),1.12-1.02(m,1H),0.75(s,6H).

[0145] Synthesis of compound 2i

[0146]

[0147] Compound 2h (210 mg) was dissolved in 2M NaHCO3 (2.4 mL, 420 mg) and CuSO4.5H20 (300 mg) H20 solution (2.40 mL) was added. NaHCO3 (100 mg) was then added followed by (Boc)2O (414 mg) dissolved in 3 mL acetone. The mixture was stirred for 24 hours. Methanol (1 mL) was added to the solution and stirring continued for 14 hours. H2O (2 mL) was added to the mixture and the product was filtered, washed with H2O (3 x 2 mL) and dried to give a light blue solid. To remove copper, the light blue Cu-chelate was suspended in H2O (27 mL). Afterwards, 8-quinolinol (234 mg) was added and stirred for 18 hours (a light salad green precipitate was formed). The suspension was filtered, the precipitate was washed with H2O (3 x 7 mL) and the filtrate was washed with ethyl acetate (3 x 15 mL). The aqueous layer was lyophilized to obtain 120 mg of white solid. LC-MS: [M+H] + =275.2

[0148] Synthesis of compound 2j

[0149]

[0150] Compound 2i (120 mg) was dissolved in a mixed solution of H2O (5 ml), and a solution of Fmoc-OSu (168 mg) dissolved in ACN (5 mL) was added, and stirred at room temperature for 3 hours. The mixture was extracted with EA (3×6 mL), and the organic phase was concentrated to obtain a residue which was purified by flash (C18 column) to obtain compound 2j (150 mg) as a white solid.

[0151] LC-MS: [M+H] + =497.4

[0152] Synthesis of compound 2

[0153] According to the general synthesis process of somatostatin peptide analogs described above, the specific steps are:

[0154]

[0155] Compound 2 is a white powder, molecular weight (average): calculated value 1047.3 g / mol; LC-MS measured value (M+2H)2+: 524.3. UPLC measured purity (214 nm): >99%

[0156] Example 3 Compound 3

[0157] The synthesis steps of the Lys(d) variant residue in compound 3 are as follows:

[0158]

[0159] Synthesis of compound 3b

[0160]

[0161] Compound 3a (7.98 g) was dissolved in THF (100 mL), TEA (11.35 g) and (Boc)2O (24.50 g) were added, and the reaction mixture was stirred at room temperature overnight. The reaction solution was diluted with water (50 mL), and the aqueous phase was extracted with EA (3×50 mL). After the organic phases were combined, they were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1) to obtain compound 3b (13.4 g).

[0162] Synthesis of compound 3d

[0163]

[0164] Compound 3c (13.4 g) was dissolved in NaHCO3 aqueous solution (29.36 g, 30 mL), CbzCl (29.82 g) was added dropwise at room temperature, and the mixture was reacted at room temperature overnight. The reaction solution was adjusted to pH 2-3 with 1M HCl, the aqueous phase was extracted with EA (3×100 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain an amide intermediate (40.5 g, crude product), the amide intermediate and benzyl alcohol (12.58 g) were dissolved in DCM (100 mL), DCC (26.40 g) and DMAP (1.42 g) were added at 0°C, the reaction mixture was stirred at room temperature for 5 h, the white solid was filtered off, the filtrate was concentrated, and the residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 3) to obtain compound 3d (6.7 g).

[0165] Synthesis of compound 3e

[0166]

[0167] Compound 3d (5.64 g) and compound 3 (8.53 g) were dissolved in DCM (50 mL), 2nd Grubbs catalyst (833 mg) was added, and the mixture was stirred at 60°C overnight. After the reaction solution was cooled, water (50 mL) was added to dilute it, and the aqueous phase was extracted with DCM (3×50 mL). After the organic phases were combined, they were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / dichloromethane = 1 / 1) to obtain compound 3e (4.44 g).

[0168] Synthesis of compound 3g

[0169]

[0170] Compound 3e (4.0 g, 8.30 mmol) was dissolved in methanol (30 mL), and after replacing nitrogen, 10% Pd / C (500 mg) was added to replace hydrogen. The mixture was reacted at room temperature overnight under a hydrogen atmosphere. The solid was filtered off, and the filtrate was dried by rotary evaporation. The obtained residue (compound 3f) was directly used in the next step.

[0171] Compound 3f (1.7 g) and NaCO3 (1.3 g) were dissolved in dixoane (20 mL) and H2O (6 mL), and Fmoc-Cl (1.68 g) was added at 0°C. The mixture was stirred at room temperature for 30 min, then quenched with water (20 mL). The aqueous phase was extracted with EA (3×40 mL). The organic phases were combined, washed with saturated aqueous sodium chloride solution (1×60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by Prep-HPLC (acetonitrile / water (containing 0.5% formic acid)) to obtain compound 3g (300 mg).

[0172] Molecular weight (calculated): 482.6; LCMS: [MH] - =481.2, Purity:97.39%, Rt=1.814min;

[0173] 1H NMR (400MHz, DMSO) δ7.897-7.878(m,2H),7.678-7.661(m,2H),7.429-7.307(m,4H),6.741-6.726(m,1H),6.420-6.408(m,1H),4.256- 4.212(m,3H),3.515-3.499(m,1H),2.822-2.626(m,2H),1.598-1.459(m,3H),1.409(s,9H),1.235-1.193(m,2H),0.786-0.744(m,3H).

[0174] Synthesis of compound 3

[0175] According to the general synthesis process of somatostatin peptide analogs described above, the specific steps are:

[0176]

[0177] Compound 3 is a white powder, molecular weight (average): calculated value 1033.3 g / mol; LC-MS measured value (M+2H)2+: 517.4

[0178] Purity determined by UPLC (214nm): >99%

[0179] Example 4 Compound 4

[0180] The synthesis steps of Lys(j) variant residue in compound 4 are as follows:

[0181]

[0182] Synthesis of compound 4b

[0183]

[0184] Compound 4a (10 g) was dissolved in DCM (60 mL), O-Allyl-N-[(9-anthracenyl)methyl]cinchonidium bromide (2.05 g) and CsOH.H2O (57 g) were added, and the mixture was cooled to -78°C, and a solution of 2-cyclohexene-1-one (10.4 g) in DCM (20 ml) was slowly added dropwise, and the reaction mixture was kept at -78°C and stirred for 6 h. The reaction solution was poured into water (50 mL) for quenching, the aqueous phase was extracted with EA (3×50 mL), the organic phase was washed with saturated sodium chloride aqueous solution (1×70 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1) to obtain compound 4b (7.2 g) as a white solid.

[0185] LC-MS: [M+H] + =392.4

[0186] 1 H NMR (400MHz, CDCl3) δ7.70–7.68(m,2H),7.53–7.33(m,6H),7.19-7.14(m,2H),3.92(d,J=4.3Hz,1H),2.70( t,J=13.1Hz,1H),2.58–2.36(m,3H),2.33–2.21(m,1H),2.07–2.01(m,1H),1.75–1.55(m,3H),1.48(s,9H).

[0187] Synthesis of compound 4c

[0188]

[0189] Trimethyl phosphonoacetate (6.7 g) was dissolved in DMF (300 mL). NaH (1.8 g) was added in batches at 0°C under nitrogen protection. After the mixture was stirred at 0°C for 20 min, compound 4b (7.2 g) was added and the reaction mixture was stirred at room temperature for 1.5 h. The reaction solution was poured into water (500 mL) for quenching. The aqueous phase was extracted with EA (3×500 mL). The organic phase was washed with saturated sodium chloride aqueous solution (1×700 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1) to obtain compound 4c (7.2 g).

[0190] LC-MS: [M+H] + =448.4

[0191] Synthesis of compound 4d

[0192]

[0193] Compound 4c (5 g) was dissolved in MeOH (100 ml), Pd / C was added, and the mixture was stirred at room temperature for 3 h under a hydrogen atmosphere. The solid was filtered out and the filtrate was dried to obtain compound 4d, which was directly used in the next step reaction.

[0194] LC-MS: [M+H] + =286.3

[0195] Synthesis of compound 4e

[0196]

[0197] Compound 4d (3.19 g) was dissolved in a mixed solution of MeOH and H2O (100 ml), LiOH (1.4 g) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated. The residue obtained by concentration was purified by silica gel column chromatography (dichloromethane / methanol = 5 / 1) to obtain compound 4e (3 g) as a colorless oil.

[0198] LC-MS: [M+H] + =272.2

[0199] Synthesis of compound 4f

[0200]

[0201] Compound 4e (3 g) was dissolved in DMF (50 mL), FmocOSu (11.2 g) and DIEA (4.3 g) were added, and the mixture was stirred at room temperature for 5 h. The reaction solution was diluted with water (80 mL), the aqueous phase was extracted with EA (3×100 mL), the organic phase was washed with saturated sodium chloride aqueous solution (1×150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 1) to obtain compound 4f (3 g).

[0202] LC-MS: [M+H] + =494.3

[0203] 1 H NMR (400MHz, CDCl3) δ7.76(d,J=7.5Hz,2H),7.61(d,J=7.4Hz,2H),7.39(t,J=7.4Hz,2H),7.33-7.28(m,2H),5.39-5.33(m,1H),4 .42-4.37(m,2H),4.25-4.21(m,2H),2.36(br,1H),2.24-2.22(m,1H),1.98–1.41(m,16H),1.38-1.25(m,1H),1.12–0.78(m,2H).

[0204] Synthesis of compound 4g

[0205]

[0206] Compound 4f (2.9 g) was dissolved in DMF (20 mL), DPPA (1.94 g) and DIEA (2.3 g) were added, and the mixture was stirred at room temperature for 3 h. The reaction solution was diluted with water (50 mL), the aqueous phase was extracted with EA (3×70 mL), the organic phase was washed with saturated sodium chloride aqueous solution (1×100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=5 / 1) to obtain compound 4g (1.13 g) as a white solid.

[0207] LC-MS: [M+H] + =519.2

[0208] 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.5Hz,2H),7.60(d,J=7.3Hz,2H),7.40(t,J=7.4Hz,2H),7.32-7.29(m,2H),5.30-5.21(m, 1H),4.43-4.40(m,2H),4.23(t,J=6.8Hz,2H),2.47–2.13(m,2H),1.95–1.63(m,4H),1.63-1.48(m,13H),1.13–0.78(m,3H).

[0209] Synthesis of compound 4h

[0210]

[0211] Compound 4g (600 mg) was dissolved in t-BuOH (5 ml), and the mixture was reacted at 100° C. for 2 h under microwave. The reaction solution was concentrated, and the residue obtained by concentration was purified by silica gel column chromatography (petroleum ether / ethyl acetate=7 / 1) to obtain compound 4h (600 mg).

[0212] LC-MS: [M+H] + =565.4

[0213] 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.6Hz,2H),7.61(d,J=7.3Hz,2H),7.40(t,J=7.4Hz,2H),7.36–7.29(m,2H),5.32-5.29(m,1H),4.40- 4.38(m,2H),4.25-4.21(m,2H),3.17-2.88(m,2H),1.84-1.70(m,3H),1.65-1.52(m,4H),1.39(s,9H),1.36(s,9H),1.13–0.67(m,3H).

[0214] Synthesis of compound 4j

[0215]

[0216] Compound 4h (600 mg) was dissolved in a mixed solution of TFA (10 ml) and DCM (10 ml), and the mixture was reacted at room temperature for 30 min. The reaction solution was spin-dried to obtain compound 4i, which was directly used in the next step.

[0217] Compound 4i (600 mg) was dissolved in DCM (20 ml), (Boc)2O (267 mg) and DIEA (569 mg) were added, and the mixture was reacted at room temperature for 1 h. The reaction solution was concentrated, and the residue obtained by concentration was purified by silica gel column chromatography (dichloromethane / methanol=10 / 1) to obtain compound 4j (1 g).

[0218] LC-MS: [M+H] + =509.4

[0219] 1 H NMR (400MHz, DMSO) δ7.90(d,J=7.5Hz,2H),7.76-7.63(m,2H),7.50-7.45(m,1H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),6.81-6.7 7(m,1H),4.26–4.22(m,3H),3.96–3.80(m,1H),2.90–2.71(m,2H),1.72-1.54(m,4H),1.37(s,9H),1.36-1.30(m,2H),1.15–0.63(m,4H).

[0220] Synthesis of compound 4:

[0221] The synthesis of compound 4 was carried out according to the general synthesis process of somatostatin peptide analogs described above, and the specific steps are:

[0222]

[0223]

[0224] Compound 4 is a white powder, molecular weight (average): calculated value 1059.3 g / mol; LC-MS measured value (M+2H)2+: 530.4. UPLC measured purity (214 nm): 93%

[0225] Example 5 Compound 5

[0226] Lys(n) residue was a commercially available raw material. The synthesis of compound 5 was carried out according to the general synthesis process of somatostatin peptide analogs described above, and the specific steps were:

[0227]

[0228] Compound 5 is a white powder, molecular weight (average): calculated value 1067.3 g / mol; LC-MS measured value (M+H)+: 1068.4; (M+2H)2+: 534.4. UPLC measured purity (214 nm): 99%

[0229] Example 6 Compound 6

[0230] The specific steps for the synthesis of compound 6 are:

[0231]

[0232] Among them, compound 6a was prepared according to the general synthesis process of somatostatin peptide analogs mentioned above.

[0233] Compound 6a is a white powder with the chemical formula: C 67 H 94 N 14 O 19 S2; molecular weight (average): calculated value 1463.69 g / mol; LC-MS measured value (M+H)+: 1463.6; (M+H)+: 1485.6; UPLC measured purity (214 nm): 99%.

[0234] Compound 6a (5 mg) and 0.1 M sodium acetate (1 mL, pH = 5.5) were mixed, LuCl3·6H2O (5.6 mg) was slowly added, and the mixture was stirred at room temperature for 12 hours, then separated by high performance liquid chromatography (prep-HPLC), and freeze-dried to obtain compound 6. Chemical formula: C 67 H 91 LqCy 14 O 19 S2; Molecular weight (average): calculated value 1635.6 g / mol; LC-MS measured value (M+H)+: 1635.7; HPLC measured purity (214 nm): 100%

[0235] Effect Example Determination of Competitive Affinity of Radioligands

[0236] SSTR2 overexpressing CHO-K1 cells (Hunter express SSTR2 CHO-K1: Discover X) were used to 125 I-TYR 11- Radioligand competition binding assay for Somatostatin-14 (Perkin Elmer).

[0237] Step S1: Extraction of cell membrane

[0238] SSTR2-overexpressing CHO-K1 cells (from Discover X) were cultured in complete medium (F12 basic medium with 10% FBS and 1% P / S (100U / ml penicillin and 100μg / ml streptomycin)) in a 37°C incubator with 5% CO2. When the cell confluency reached 80-90%, the cells were washed twice with pre-cooled PBS. The cells were then scraped off with a cell scraper and transferred to a 50mL centrifuge tube and centrifuged at 500×g and 4°C for 10 minutes. The supernatant was discarded and the cell pellet was retained. Cell membrane lysis buffer (containing 25mM HEPES, 1mM EDTA, 2mM MgCl2 and 1× protease inhibitor, pH=7.4) was added at a ratio of 4E+07 cells / 10mL and placed on ice for 10 minutes. The cells were then disrupted using an IKA disruptor. The operation procedure is 15000rpm, 10 seconds, twice. Centrifuge the broken cell suspension at 4℃, 650×g for 5 minutes. Transfer the supernatant to a new centrifuge tube and centrifuge at 4℃, 13000rpm for 30 minutes. Discard the supernatant and resuspend the cell membrane precipitate with 1.6mL of resuspension solution, which is cell membrane lysis solution and adds 10% sucrose. The cell membrane lysis solution is repeatedly aspirated 4 times with a 27-gauge needle to break the DNA. The protein concentration of the cell membrane extract is determined by the BCA method, and the cell membrane extract is frozen in a -80℃ refrigerator.

[0239] Step S2 Cell membrane binding

[0240] (1) Dilution of compounds: The buffer used for the assay contained 50 mM Hepes, 5 mM MgCl2, 1 mM CaCl2 and 0.5% BSA, pH = 7.4, and the buffer was filtered. The assay buffer was maintained at 4°C. The ligand solution was serially diluted using an Agilent Bravo. Each test compound was diluted 3×8 times in succession. The eight binding concentrations of typical compounds were: 50 nM, 12.5 nM, 3.13 nM, 0.78 nM, 0.20 nM, 0.05 nM, 12.2 pM and 3.05 pM. The affinity was determined twice for each concentration.

[0241] Transfer 1 μL of each of the diluted compounds to the corresponding wells of the 96-well V-bottom plate.

[0242] (2) 125 I-SST preparation:

[0243] The storage solution 125 I-SST was diluted to 3 nM (10×) with binding buffer (containing 50 mM Hepes, 5 mM MgCl2, 1 mM CaCl2 and 0.5% BSA, pH=7.4, stored at 4° C.) and 10 μL was transferred to the corresponding compound wells.

[0244] (3) Combined experiment:

[0245] The extracted cell membrane extract was diluted to 2.5 μg / 90 μL with binding buffer and added to the corresponding wells. Incubated at 25°C on a shaker at 500 rpm for 2 hours.

[0246] The membrane complexes were collected into 0.5% PEI pre-coated GF / B plates using Cell Harvester, and washed three times with 200 μL of 4°C pre-cooled buffer (the same buffer used to dilute the compounds) per well. Dry at 37°C for 2 hours. 50 μL of liquid scintillation fluid (MicroScint20, Perkin Elmer) was added to each well and allowed to stand for 30 minutes before reading (radioactivity) on a Topcount plate reader, 30 seconds per well.

[0247] Step S3 Curve fitting

[0248] For each compound, the 8-point reading data (2× of 8 readings) were imported into X Lfit software to fit the sigmoidal curve. The EC value of the compound on SSTR2 was 50 It is calculated based on the mathematically optimal Sigmoidal dose-response model equation:

[0249]

[0250] Where A is the bottom of the curve, B is the top of the curve, and C is the relative EC 50 , D is the Hill slope, y is the %Effect value of the curve fitting, and x is the molar concentration. Accordingly,

[0251]

[0253] Where A, B, C, and D have the same meaning, y is the % Effect reading, and x is the molar concentration calculated by curve fitting. When y is 50%, the calculated x is the absolute EC 50 Values, as shown in Table 1 and Figures 1 to 6 shown.

[0254] Table 1 Binding affinity of target compounds 1, 2, 3, 4, and 5 to SSTR2

[0255]

[0256] in conclusion:

[0257] By changing the Lys9 group, appropriately increasing the spatial tension and limiting the number of possible conformations of the side chain, it can be beneficial for the drug molecule and SSTR2 to produce more effective binding, thus breaking the "taboo" that the Lys9 residue cannot be replaced, otherwise the biological activity will be greatly reduced.

Claims

1. A somatostatin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable complex thereof, or a pharmaceutically acceptable chelate thereof, The somatostatin peptide analogs have a cyclic or non-cyclic hexapeptide unit, and the residue sequence at positions numbered 2 to 5 in the hexapeptide unit is: -X1-(D / L-)Trp-X2-X3-; Alternatively, the somatostatin peptide analogue has a cyclic or non-cyclic octapeptide unit, and the residue sequence at positions numbered 2 to 7 in the octapeptide unit is: -Cys-X1-(D / L-)Trp-X2-X3-Cys-; wherein X1 is an α-amino acid residue having an aromatic group on the Cα side chain, and X3 is an α-amino acid residue characterized in that X2 is a Lys-variant residue: n is 0 or 1; A1, A2, A3 are independently selected from C(R)2, O, S, NR, C=O, C=S, C=NR and C=C(R)2; R is independently selected from R1, -R'OR1, -R'SR1, -R'N(R1)2, -R'C(O)R1, -R'S(O)R1, -R'S(O)2R1, -R'C(O)N(R1)2, -R'N(R1)C(O)R1, -R'C(O)OR1 and -R'OC(O)R1; or independently, the R in C(R)2 at position A2 is connected to the R in C(R)2 at position A1 or position A3 to form a double bond or a triple bond; or independently, R in C(R)2 forms a 3-8 membered carbocyclic ring with R in its own C(R)2, or with R in an adjacent C(R)2, or with R in a C(R)2 separated by one C(R)2, or forms a 3-8 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S; R' is a linking bond, a C1-C6 alkylene group, a C2-C6 alkenylene group, a C2-C6 alkynylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkenylene group, a halogenated C1-C6 alkylene group, a halogenated C2-C6 alkenylene group, a halogenated C2-C6 alkynylene group, a halogenated C3-C8 cycloalkylene group, a halogenated C3-C8 cycloalkenylene group, a (C6-C8)arylene group, a 3-8 membered heterocyclylene group having 1 to 3 heteroatoms selected from N, O, and S, and a 3-8 membered heteroarylene group having 1 to 3 heteroatoms selected from N, O, and S; R1 is independently selected from H, halogen, CN, NO2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C3-C8 cycloalkyl, halogenated C3-C8 cycloalkenyl, (C6-C8)aryl, 3-8 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-8 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S; When n is 0, R in C(R)2 at the A1 position, A2 position and A3 position are not H at the same time.

2. The somatostatin peptide analogue according to claim 1, characterized in that The somatostatin peptide analogs have a cyclic or non-cyclic hexapeptide unit, and the residue sequence at positions numbered 1 to 6 in the hexapeptide unit is: Xa-X1-(D / L-)Trp-X2-X3-Xb; The somatostatin peptide analogs have a cyclic or non-cyclic octapeptide unit, and the residue sequence at positions numbered 1 to 8 in the octapeptide unit is: X0-Cys-X1-(D / L-)Trp-X2-X3-Cys-Xb; Wherein Xa and Xb are amino acid residues; X0 is an α-amino acid residue having an aromatic group on the Cα side chain.

3. The somatostatin peptide analogue according to claim 1 or 2, characterized in that X1 is a Phe, Tyr, Trp or Phg residue; X3 is a Thr, Ser, Cys or tyrosylbenzyl residue.

4. The somatostatin peptide analogue according to claim 1, characterized in that The somatostatin peptide analogs have a cyclic octapeptide unit structure as shown in the following general formula (I): n is 0 or 1; A1, A2, A3 are independently selected from C(R)2, O, S, NR, C=O, C=S, C=NR and C=C(R)2; R is independently selected from R1, -R'OR1, -R'SR1, -R'N(R1)2, -R'C(O)R1, -R'S(O)R1, -R'S(O)2R1, -R'C(O)N(R1)2, -R'N(R1)C(O)R1, -R'C(O)OR1 and -R'OC(O)R1; or independently, the R in C(R)2 at position A2 is connected to the R in C(R)2 at position A1 or position A3 to form a double bond or a triple bond; or independently, R in C(R)2 forms a 3-8 membered carbocyclic ring with R in its own C(R)2, or with R in an adjacent C(R)2, or with R in a C(R)2 separated by one C(R)2, or forms a 3-8 membered heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S; R' is a linking bond, a C1-C6 alkylene group, a C2-C6 alkenylene group, a C2-C6 alkynylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkenylene group, a halogenated C1-C6 alkylene group, a halogenated C2-C6 alkenylene group, a halogenated C2-C6 alkynylene group, a halogenated C3-C8 cycloalkylene group, a halogenated C3-C8 cycloalkenylene group, a (C6-C8)arylene group, a 3-8 membered heterocyclylene group having 1 to 3 heteroatoms selected from N, O, and S, and a 3-8 membered heteroarylene group having 1 to 3 heteroatoms selected from N, O, and S; R1 is independently selected from H, halogen, CN, NO2, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C3-C8 cycloalkyl, halogenated C3-C8 cycloalkenyl, (C6-C8)aryl, 3-8 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-8 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S; When n is 0, R in C(R)2 at the A1 position, A2 position and A3 position are not H at the same time.

5. The somatostatin peptide analogue according to claim 1 or 4, characterized in that The R in C(R)2 at position A1 and the R in C(R)2 at position A3 are connected together to form a ring so that A1, A2, and A3 form a 3- to 8-membered carbocyclic ring or a 3- to 8-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, and S; Preferably, The R in C(R)2 at position A1 and the R in C(R)2 at position A3 are connected together to form a ring so that A1, A2, and A3 form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring having 1-2 heteroatoms selected from N, O, and S.

6. The somatostatin peptide analogue according to claim 1 or 4, characterized in that The R in C(R)2 at the A2 position is connected to the R in C(R)2 at the A1 position or the A3 position to form a double bond.

7. The somatostatin peptide analogue according to claim 1 or 4, characterized in that R' is a connecting bond, C1-C4 alkylene, C2-C4 alkenylene, C2-C4 alkynylene, C3-C7 cycloalkylene, C3-C7 cycloalkenylene, halogenated C1-C4 alkylene, halogenated C2-C4 alkenylene, halogenated C2-C4 alkynylene, halogenated C3-C7 cycloalkylene, halogenated C3-C7 cycloalkenylene, (C6-C8)arylene, 3-7 membered heterocyclylene having 1 to 3 heteroatoms selected from N, O, and S, and 3-7 membered heteroarylene having 1 to 3 heteroatoms selected from N, O, and S; R1 is independently selected from H, halogen, CN, NO2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, halogenated C1-C4 alkyl, halogenated C2-C4 alkenyl, halogenated C2-C4 alkynyl, halogenated C3-C7 cycloalkyl, halogenated C3-C7 cycloalkenyl, (C6-C8)aryl, 3-7 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O, and S, and 3-7 membered heteroaryl having 1 to 3 heteroatoms selected from N, O, and S; Preferably, R' is a linking bond, C1-C3 alkylene, C2-C3 alkenylene, C2-C3 alkynylene, C5-C6 cycloalkylene, C5-C6 cycloalkenylene, halogenated C1-C3 alkylene, halogenated C2-C3 alkenylene, halogenated C2-C3 alkynylene, halogenated C5-C6 cycloalkylene, halogenated C5-C6 cycloalkenylene, (C6-C8)arylene, 5-6 membered heterocyclylene having 1-2 heteroatoms selected from N, O, and S, and 5-6 membered heteroarylene having 1-2 heteroatoms selected from N, O, and S; R1 is independently selected from H, halogen, CN, NO2, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, halogenated C1-C3 alkyl, halogenated C2-C3 alkenyl, halogenated C2-C3 alkynyl, halogenated C5-C6 cycloalkyl, halogenated C5-C6 cycloalkenyl, (C6-C8)aryl, 5-6 membered heterocyclic group having 1 to 2 heteroatoms selected from N, O, and S, and 5-6 membered heteroaryl having 1 to 2 heteroatoms selected from N, O, and S.

8. The somatostatin peptide analogue according to claim 1 or 4, characterized in that X2 is a Lys-variant residue: Selected from:

9. The somatostatin peptide analogue according to claim 1 or 4, characterized in that Somatostatin peptide analogs are specifically:

10. A somatostatin chelate, characterized in that The somatostatin chelate is a chelate formed by removing the H atom from the N-terminal NH2 substituent of the somatostatin peptide analogue described in claim 1 or 4 and then directly connecting it to the chelating group or indirectly connecting it through a spacer.

11. The somatostatin chelate according to claim 10, characterized in that The chelating group is a chelating group of DTPA, DOTA, TETA or substituted EDTA or DTPA, and the spacer is a divalent residue of an aminocarboxylic acid, such as a β-Ala residue or a divalent residue derived from 6-aminocaprioic acid.

12. Use of the somatostatin peptide analogs according to claim 1 or 4 in the preparation of anti-tumor drugs, preferably for neuroendocrine tumors, acromegaly and carcinoid tumors.

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