RU2800369C1 - Dimeric dipeptide mimetics of neurotrofin-3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the new biologically active molecules containing dipeptides, and can be used in medicine. The invention discloses the possibility to obtain compounds characterized by the general formula (R-CO-L-AK-X-Asn-NH-)₂(CH₂)₆, where R = HOOC-(CH₂)₂-, or HOOC-(CH₂)₃-, or HO-(CH₂)₃-, AK = Glu or Asn, X = L- or D-configuration of the corresponding amino acid residue. Such compounds are dimeric dipeptide mimetics of the 4th loop of neurotrophin-3 (NT3).

EFFECT: invention can be used in medical practice in the treatment of depression and various neurodegenerative diseases.

7 cl, 13 ex, 14 tbl

Description

Field of invention

The invention relates to chemistry and medicine, namely to new biologically active dipeptides of the formula:

where R = HOOS-(CH ₂ ) ₂ - or HOOS-(CH ₂ ) ₃ - or HO-(CH ₂ ) ₃ -;

AK=Glu or Asn;

X = L or D configuration; Y = L or D configuration;

n = 6 or 7.

The new compounds are dimeric dipeptide mimetics of the 4th loop of neurotrophin-3 (NT3) and have neuroprotective, antidepressant-like, anxiolytic, antidiabetic, and antiaddictive activities. State of the art

Neurotrophin-3 (NT3), along with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), is a member of the family of neurotrophins - endogenous structurally homologous proteins responsible for the regulation of proliferation, differentiation, myelination of neurons, maintaining their viability and phenotypic stability. Neurotrophins and their tyrosine kinase receptors TrkA, TrkB, and TrkC are attracting much attention as potential therapeutic targets for the treatment of a wide range of diseases, in particular, neurodegenerative and psychiatric diseases. NT3, unlike other neurotrophins that are selective for different types of Trk receptors, interacts with all of these receptors, binding predominantly to TrkC, but also to TrkB and TrkA with lower affinity [Bothwell M. NGF, BDNF, NT3, and NT4. Handb Exp Pharmacol. 2014;220:3-15]. The involvement of NT3 in hippocampal neurogenesis and neuroplasticity has been shown. In particular, in mutant mice in which NT3 is not expressed in the brain, the differentiation of neuronal stem cells in the dentate gyrus of the hippocampus is impaired, as well as long-term potentiation (long-term potentiation), which underlies the cellular mechanisms of memory and learning [Shimazu K, Zhao M, Sakata K, et al. NT-3 facilitates hippocampal plasticity and learning and memory by regulating neurogenesis. LearnMem. 2006;13(3):307-315]. In the adult brain, NT3 and TrkC receptors are most expressed in the hippocampus, cortex, and cerebellum [Ueyama T, Kawai Y, Nemoto K, Sekimoto M, Tone S, Senba E. Immobilization stress reduced the expression of neurotrophins and their receptors in the rat brain. Neurosci Res. 1997;28(2):103-110; Zhang HT, Li LY, Zou XL, et al. Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains. J Histochem Cytochem. 2007;55(1):1-19; Dwivedi Y et al. Neurotrophin receptor activation and expression in human postmortem brain: effect of suicide. Biol Psychiatry. 2009;65(4):319-328; Memo J.-P. et al. Molecular cloning of rat TrkC and distribution of cells expression RNAs for members of the Trk family in the rat central nervous system. 1992;51(3):513-532]. NT3 and TrkC receptors are also expressed in the blue spot (locus coeruleus), which plays an important role in the behavioral and physiological response to stressful stimuli and is involved in the pathophysiology of mood disorders (mood disorders) [Smith M. A. et al. Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus. Neurobiology, v. 92, p. 8788-8792, 1995]. Interestingly, TrkC are the only Trk receptors found in locus coeruleus by in situ hybridization [Memo J.-P. et al. Molecular cloning of rat TrkC and distribution of cells expression RNAs for members of the Trk family in the rat central nervous system. 1992;51(3):513-532].

In vivo experiments revealed neuroprotective and neuroregenerative properties of NT3 in models of cerebral ischemia [Galvin K.A, Oorschot DE, Continuous low-dose treatment with brain-derived neurotrophic factor or neurotrophin-3 protects striatal medium spiny neurons from mild neonatal hypoxia/ischemia: a stereological study. Neuroscience.2003;118(4): 1023-1032; Zhang J et al. Neuroprotection of neurotrophin-3 against focal cerebral ischemia/reperfusion injury is regulated by hypoxia-responsive element in rats. neuroscience. 2012;222:1-9], spinal cord injury [Cong Y et al. NT-3 Promotes Oligodendrocyte Proliferation and Nerve Function Recovery After Spinal Cord Injury by Inhibiting Autophagy Pathway. J Surg Res. 2020;247:128-135] and peripheral nerves [Wang H et al. The Promotion of Neural Regeneration in A Rat Facial Nerve Crush Injury Model Using Collagen-Binding NT-3. Ann Clinic Lab Sc. 2016;46(6):578-585]; analgesic [Wilson-Gerwing TD et al. Neurotrophin-3 suppresses thermal hyperalgesia associated with neuropathic pain and attenuates transient receptor potential vanilloid receptor-1 expression in adult sensory neurons. J Neurosci. 2005;25(3):758-767; Gandhi R. et al. Neurotrophin-3 reverses chronic mechanical hyperalgesia induced by intramuscular acid injection. J Neurosci. 2004;24(42):9405-9413] and cognitotropic activity [Liu DB, Yang JS, Lu QB, Zhu ZF, Fang Q. Effect of NT-3 on infection-induced memory impairment of neonatal rats. Eur Rev Med Pharmacol Sci. 2019;23(5):2182-2187]. NT3, along with BDNF, is being considered as a potential target for the development of new therapeutic strategies for the treatment of depression. In addition to being involved in hippocampal neuroplasticity, the antidepressant-like potential of NT3 is due to its ability to modulate monoamine neurotransmission as well as stimulate BDNF expression [de Miranda AS et al. Is neurotrophin-3 (NT-3): a potential therapeutic target for depression and anxiety?. Expert Opin Ther Targets. 2020;24(12): 1225-1238]. The content of NT3 is reduced in the blood of people suffering from depression, and the stronger, the greater the severity of depression [ EA et al. Melatonin and neurotrophins NT-3, BDNF, NGF in patients with varying levels of depression severity. Pharmacol Rep. 2016;68(5):945-951]. A significant decrease in protein and mRNA expression of NT3 was found in the hippocampus and cerebral cortex of rats subjected to chronic unpredictable stress [Mao QQ. et al. Herbal formula SYJN increases neurotrophin-3 and nerve growth factor expression in brain regions of rats exposed to chronic unpredictable stress. J Ethnopharmacol. 2010;131(1):182-186]. Neurotrophin NT3, when injected into the dentate gyrus of the rat hippocampus, had an antidepressant-like effect on the model of learned helplessness and in the forced swimming test [Shirayama Y et al. Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci. 2002;22(8):3251-3261].

The ability of NT3 to stimulate monoaminergic transmission, participate in the regulation of synaptic plasticity and neurogenesis, and stimulate BDNF expression [de Miranda AS et al. Is neurotrophin-3 (NT-3): a potential therapeutic target for depression and anxiety?. Expert Opin Ther Targets. 2020;24(12): 1225-1238; Shimazu K. et al. NT-3 facilitates hippocampal plasticity and learning and memory by regulating neurogenesis. LearnMem. 2006;13(3):307-315], testifies in favor of the involvement of neurotrophin in the pathogenesis of anxiety disorders. This is supported by experimental and clinical data. Thus, TgNTRK3 transgenic mice with increased expression in the brain of the full-length TrkC receptor (TgNTRK.3) are used as a model of panic disorders [Dierssen M. et al. Transgenic mice overexpressing the full-length neurotrophin receptor TrkC exhibit increased catecholaminergic neuron density in specific brain areas and increased anxiety-like behavior and panic reaction. Neurobiol Dis. 2006;24(2):403-418]. In mice of the TgNTRK3 line, the extinction of conditioned reflex fear is impaired and there is a deficit of long-term potentiation in the infralimbic cortex, while the introduction of NT3 into the infralimbic cortex of these mice restores the process of fear extinction [D'Amico D. et al. Infralimbic Neurotrophin-3 Infusion Rescues Fear Extinction Impairment in a Mouse Model of Pathological Fear. neuropsychopharmacology. 2017;42(2):462-472]. Genetic studies in humans confirm the involvement of NT3/TrkC signaling in the development of panic and obsessive-compulsive disorders [ et al. Allele variants in functional MicroRNA target sites of the neurotrophin-3 receptor gene (NTRK3) as susceptibility factors for anxiety disorders. Hum Mutat. 2009;30(7):1062-1071].

There is evidence to support the involvement of NT3 in the pathogenesis of diabetes mellitus. In a streptozotocin diabetes model, NT3 has been shown to correct sciatic nerve conduction deficits in rats [Mizisin AP. et al. Neurotrophin-3 reverses nerve conduction velocity deficits in streptozotocin-diabetic rats. J Peripher Nerv Syst. 1999;4(3-4):211-221], prevents axonal atrophy, improves impaired calcium homeostasis, prevents mitochondrial dysfunction in rat sensory neurons with subcutaneous chronic administration [Huang TJ. et al. Neurotrophin-3 prevents mitochondrial dysfunction in sensory neurons of streptozotocin-diabetic rats. Exp Neurol. 2005;194(1):279-283]. It has been established that under conditions of experimental diabetes, there is a decrease in the production of NT3 in Schwann cells [Dey I. et al. Diabetic Schwann cells suffer from nerve growth factor and neurotrophin-3 underproduction and poor associability with axons. Glia. 2013;61(12): 1990-1999].

The nociceptive effects of NT3 are well known. Thus, intracerebral administration of NT3 increased the pain threshold of rats in the tail flick test [Siuciak JA. et al. Antinociceptive effect of brain-derived neurotrophic factor and neurotrophin-3. Brain Res. 1994;633(1-2):326-330]. Acute intraperitoneal administration of NT3 to rats led to the development of mechanical hypoalgesia associated with a decrease in the release of substance P in the spinal cord [Malcangio M. et al. Nerve growth factor- and neurotrophin-3-induced changes in nociceptive threshold and the release of substance P from the rat isolated spinal cord. J Neurosci. 1997;17(21):8459-8467]. Recombinant NT3 with a single subcutaneous injection abolished inflammatory hyperalgesia in rats [Watanabe M et al. Inhibition of adjuvant-induced inflammatory hyperalgesia in rats by local injection of neurotrophin-3. Neurosci Lett. 2000;282(1-2):61-64]. In a model of peripheral neuropathy, NT3, when administered intrathecally, reduced the overexpression of TRPV1 capsaicin receptors in the dorsal roots of the spinal cord and prevented the development of thermal hypersensitivity [Wilson-Gerwing TD et al. Neurotrophin-3 suppresses thermal hyperalgesia associated with neuropathic pain and attenuates transient receptor potential vanilloid receptor-1 expression in adult sensory neurons. J Neurosci. 2005;25(3):758-767].

Studies by Eric Nestler and his colleagues have revealed the role of neurotrophic factors in the mechanism of action of addictive psychoactive substances (surfactants) [Nestler EJ, Aghajanian GK. Molecular and cellular basis of addiction. Science. 1997;278(5335):58-63; Nestler EJ. Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci. 2001;2(2):119-128; Nestler EJ. From neurobiology to treatment: progress against addiction. Nat Neurosci. 2002;5 Suppl: 1076-1079]. The activity of dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (mesolimbic pathway) is considered central to the formation of reinforcing effects that determine the development of substance dependence. Chronic use of opiates and/or opioids induces lasting changes in synaptic function and signaling in dopaminergic neurons of the mesolimbic pathway, as well as noradrenergic neurons in the coeruleus [Nestler EJ, Aghajanian GK. Molecular and cellular basis of addiction. Science. 1997;278(5335):58-63; Williams J.T. et al. Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev. 2001;81(l):299-343]. It has been found that NT3 and TrkC are expressed in locus coeruleus [Smith MA. et al. Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus. Proc Natl Acad Sci USA. 1995; 92(19):8788-8792] and the ventral tegmental region [Numan S, Seroogy KB. Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study. J Comp Neurol. 1999; 403(3):295-308]. In in vitro experiments, NT3 exerted a trophic effect on noradrenergic neurons in the locus coeruleus [Friedman WJ et al. Differential actions of neurotrophins in the locus coeruleus and basal forebrain. Exp Neurol. 1993;119(1):72-78]. In vivo experiments have established the involvement of NT3 in the development of addiction states. Thus, long-term administration of morphine to rats caused an increase in the levels of BDNF and NT3 mRNA in the noradrenergic neurons of the coeruleus, and the withdrawal of morphine led to a significant increase in the expression of BDNF mRNA after 2, 6, 20, and 70 h, but a decrease in the levels of NT3 mRNA 20 and 70 h after the last opiate presentation, while the levels of TrkC mRNA were also below the control values after 20 and 70 h. 70 hours after morphine withdrawal [Numan S. et al. Differential regulation of neurotrophin and trk receptor mRNAs in catecholaminergic nuclei during chronic opiate treatment and withdrawal. J Neurosci. 1998; 18(24):10700-10708]. Treatment of locus coeruleus neurons with morphine led to a decrease in the uptake of norepinephrine by 20% and a decrease in the number of tyrosine hydroxylase-immunoreactive (TH+) cells by 12%, and when NT3 was introduced into the cell culture, an increase in the uptake of norepinephrine and the number of TH+ cells was noted, although a weakening of the effects of neurotrophin on the above indicators was recorded when morphine and NT3 neurons were co-introduced into the LC culture [Sklair- Tavron L, Nestler EJ. Opposing effects of morphine and the neurotrophins, NT-3, NT-4, and BDNF, on locus coeruleus neurons in vitro. Brain Res. 1995;702(1-2):117-125]. Infusion of NT3 directly into the ventral tegmental region of rats prevented biochemical changes in the mesolimbic dopaminergic system that occur with prolonged exposure to morphine [Berhow MT. et al. Influence of neurotrophic factors on morphine- and cocaine-induced biochemical changes in the mesolimbic dopamine system. neuroscience. 1995;68(4):969-979].

Due to the difficulties in delivering neurotrophins to the CNS due to poor penetration through the blood-brain barrier, short half-life, and rapid degradation, their low molecular weight mimetics with acceptable pharmacokinetic properties are being developed worldwide. In contrast to the small molecular weight NGF and BDNF mimetics, which have been the subject of a large amount of literature since the early 1990s, there are only a small number of publications that describe NT3 mimetics.

The first small molecule NT3 mimetic (compound 2b), created under the leadership of Burgess K. and Saragovi U.H. based on the beta rotary scaffold, was described in 2002. Compound 2b was a macrocyclic beta-turn peptidomimetic containing a Lys-Ser NT3 dipeptide fragment in its structure. It was found that 2b at concentrations of 0.4-50 μM potentiates the survival of TrkC-expressing NIH-3T3 cells upon incubation in a serum-free medium in the presence of NT3 at suboptimal concentrations (0.1 nM). Fluorescence-activated cell sorting (FACScan assay) showed direct binding of compound 2b to TrkC receptors at a concentration of 20 μM, and Western blot analysis showed that 2b at a concentration of 50 μM causes phosphorylation of TrkC tyrosine residues (antibody mAb 4G10). Subsequently, the same scientific group created other low-molecular-weight NT3 mimetics based on nitrogen-, sulfur-, and oxygen-containing beta-turn templates with an inserted dipeptide sequence of neurotrophin β-bends. Of these, a number of compounds bind to TrkC receptors, as shown by the FACScan method. The most interesting compound was the NT3 mimetic, which activated the neurotrophin-specific TrkC receptor without activating TrkA (1Aa). This peptidomimetic 1Aa was shown to be active in an in vitro model of sensorineural hearing loss [Kempfle JS. et al. A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In vitro. Front Cell Neurosci. 2021;15:666706], characteristic of full-length NT3 [Wan G. et al. Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma. elife. 2014;3:e03564]. At a concentration of 400 nM, 1Aa enhanced the growth of neurites in the culture of spiral ganglion neurons, and also in the culture of explants of the organ of Corti protected the ribbon synapses of the inner hair cells and promoted their regeneration under conditions of damage by cyanic acid [Kempfle JS. et al. A novel small molecule neurotrophin-3 analogue promotes inner ear neurite outgrowth and synaptogenesis in vitro. Front Cell Neurosci. 2021;15:666706].

In addition, a number of symmetrical and mixed bivalent NT3 mimetics were constructed, the structure of which contained two p-fold dipeptide fragments linked by a linker. The p-bend conformation is reproduced using a triazole template [Chen D. et al. Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals. ACS Chem Biol. 2009;4(9):769-781]. The FACScan method using biotin and fluoriscein labels showed direct binding of bivalent mimetics (at a concentration of 50 μM) to TrkA and TrkC receptors. Western blot analysis showed activation of TrkA and TrkC and their post-receptor signaling pathways PI3K/AKT and MAPK/ERK. In in vitro experiments, some mimetics had neuroprotective activity, while others only had differentiating activity. Thus, the symmetrical bivalent 6C mimetic, under conditions at a concentration of 20 μM, had its own neuroprotective activity, and also potentiated the neuroprotective effects of NT3 and NGF at their suboptimal concentrations. Mimetic 6C did not have differentiating activity, while its asymmetric analog 6F, in whose structure the side radicals aco Tyr, Trp, Leu, and Lys were reproduced, demonstrated a neuritogenic effect, but did not show a neuroprotective effect.

A scientific group from the University of Catania, (Catania, Italy) led by Enrico Rizzarelli synthesized a peptide containing 13 amino acid residues (1-13) of the N-terminal region of NT3 [Naletova I. et al. Copper complexes of synthetic peptides mimicking neurotrophin-3 enhance neurite outgrowth and CREB phosphorylation. metallomics. 2019;11(9):1567-1578]. In vitro experiments on SH-SY5Y human neuroblastoma cells showed that the peptide, like a full-sized neurotrophin, stimulates neuronal differentiation and neurite outgrowth, these effects being inhibited by blocking Trk receptors, and also causes CREB phosphorylation. In complex with copper ions, the peptide exhibited more pronounced activity, which can presumably be explained by improved internalization of the peptide.

Below are monovalent (2b, 1Aa) and bivalent (6C) NT3 mimetics containing its dipeptide sequences in the structure [Pattarawarapan M. et al. New templates for syntheses of ring-fused, C(10) beta-turn peptidomimetics leading to the first reported small-molecule mimic of neurotrophin-3. J Med Chem. 2002; 45(20):4387-4390; D. Chen et al. Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals. ACS Chem Biol. 2009; 4(9):769-781; Zaccaro MC et al. Selective small molecule peptidomimetic ligands of TrkC and TrkA receptors afford discrete or complete neurotrophic activities. Chem Biol. 2005; 12(9): 1015-1028].

The essence of the invention

The aim of our work was to obtain low molecular weight neurotrophin-3 (NT3) mimetics with neuroprotective, antidepressant-like, anxiolytic, antidiabetic, and antiaddictive activities. We have found that this goal can be achieved with compounds of the general formula:

where R = HOOS-(CH ₂ ) ₂ - or HOOS-(CH ₂ ) ₃ - or HO-(CH ₂ ) ₃ -;

AK=Glu or Asn;

X = L or D configuration; Y = L or D configuration; n = 6 or 7.

The following compounds can serve as examples of the invention:

I hexamethylenediamide bms-(N-monosuccinyl-L-asparaginyl-L-asparagine)

II bis-(N-monosuccinyl-D-asparaginyl-L-asparagine) hexamethylenediamide

III bis-(N-monosuccinyl-L-asparaginyl-D-asparagine) hexamethylenediamide

IV bis-(N-monosuccinyl-D-asparaginyl-D-asparagine) hexamethylenediamide

V hexamethylenediamide bms-(N-γ-hydroxybutyryl-L-glutamyl-L-asparagine)

VI hexamethylenediamide bms-(N-monoglutaryl-L-asparaginyl-L-asparagine)

VII heptamethylenediamide bms-(N-monosuccinyl-L-asparaginyl-L-asparagine)

The compounds according to the invention are further illustrated in Table 1.

The technical result of the invention is the expansion of the arsenal of drugs for the treatment of diseases, conditions and disorders of the nervous system, requiring the use of drugs with neuroprotective, antidepressant-like, anxiolytic and anti-addictive activities, as well as for the treatment of hyperglycemia, and not causing hyperalgesia, characteristic of full-sized neurotrophins.

Design of dimeric NT3 dipeptide mimetics

The design of the mimetics was based on the crystal structure of NT3 (pdb ID: lnt3), where 1, 2, 3, 4 are homodimer loops [Butte, M.J., Hwang, R.K., Mobley, W.C., Fletterick, R.J. // 1998. Biochemistry. 37. P. 16846-16852]:

Like other members of the neurotrophin family, NT3 is a symmetrical homodimer whose monomeric units contain 7 beta strands forming three antiparallel β-sheets. β-strands are connected by four outwardly exposed irregular regions called loops: 1st (residues 29-33), 2nd (42-47), 3rd (residues 58-75) and 4th (91-97). The 4th loop is the most exposed, the fragment of which Ser ⁹¹ -Glu ⁹² -Asn ⁹³ -Asn ⁹⁴ -Lys ⁹⁵ -Leu ⁹⁶ - containing turning regions in its structure presumably occupies the geometrically most favorable position for interaction with the receptor. When constructing, we retained the dipeptide fragment -Asn ⁹³ -Asn ⁹⁴ - as the central fragment of the beta-turn-like region, and the preceding a.o. Glu ⁹² was replaced by its bioisostere - succinic acid residue (GTS-301 and GTS-306) or glutaric acid residue (GTS-304). The dimeric structure of neurotrophin was reproduced using hexamethylenediamine or heptamethylenediamine spacers at the C-terminus. In addition, an NT3 mimetic was constructed based on the same fragment of the 4th loop, but with a shift by one a.a. to the left relative to Asn ⁹³ -Asn ⁹⁴ . At the same time, the dipeptide region -Glu ⁹² -Asn ⁹³ preceding the a.o. was retained. Ser ⁹¹ was replaced by its bioisostere, the residue of gamma-hydroxybutyric acid, dimerization was carried out using hexamethylenediamine. Received GTS-302.

To study the stereospecificity of the pharmacological effect on the example of GTS-301, its diastereomers ITC-301LD, TTC-301DL, TTC-301DD were obtained.

Thus, dimeric dipeptide mimetics of the 4th loop of NT3 were constructed:

bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) hexamethylenediamide (GTS-301)

bis-(N-monosuccinyl-L-asparaginyl-D-asparagine) hexamethylenediamide (GTS-301LD)

bms-(N-monosuccinyl-D-asparaginyl-L-asparagine) hexamethylenediamide (GTS-301DL)

bms-(N-monosuccinyl-D-asparaginyl-D-asparagine) hexamethylenediamide (GTS-301DD)

bms-(N-γ-hydroxybutyryl-L-glutamyl-L-asparagine) hexamethylenediamide (GTS-302)

bms-(N-monoglutaryl-L-asparaginyl-L-asparagine) hexamethylenediamide (GTS-304)

heptamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-306)

Synthesis of NT3 mimetics

NT3 mimetics are compounds of a peptide nature and can be obtained by methods of classical peptide synthesis in a homogeneous phase.

Condensation in a homogeneous phase can be done as follows:

a) condensing an amino acid having a free carboxyl group and a protected other reactive group with an amino acid having a free amino group and protected other reactive groups in the presence of a condensing agent such as a carbodiimide;

b) condensation of an amino acid having an activated carboxyl group and a protected other reactive group with an amino acid having a free amino group and protected other reactive groups;

c) condensation of an amino acid having a free carboxyl group and a protected other reactive group with an amino acid having an activated amino group and protected other reactive groups.

The carboxyl group can be activated by converting it to an azide, anhydride group or an activated ester such as N-hydroxysuccinimide, N-oxybenzotriazole, pentachlorophenyl, pentafluorophenyl or p-nitrophenyl ethers. The amino group can be activated by converting it to a phosphitamide or by the "phosphorase" method.

The most common for the above condensation reactions are: carbodiimide method; azide method; mixed anhydride method; the activated ester method described in "The Peptides". Vol.1. 1965 (Academic Press), E. Schroeder, K. Lubke, or in "The Peptides", Vol. 1, 1979 (Academic Press) E. Cross, L. Meinhofen.

The preferred condensation methods for the preparation of peptides of formula (1) is the method of activation of the carboxyl group, which is carried out mainly using pentafluorophenyl ethers of amino-protected amino acids. The best solvent for obtaining an activated ester of a protected amino acid is ethyl acetate, and for the condensation of a.k.o. - dimethylformamide.

Reactive groups that should not participate in the condensation can be protected with groups that are easily removed, for example, by hydrolysis or reduction. Thus, the carboxyl group can be protected by esterification with methanol, ethanol, tert-butanol, benzyl alcohol.

The amino group is usually effectively protected with acidic groups, for example aliphatic, aromatic, heterocyclic carboxylic acid residues such as acetyl, benzoyl, pyridinecarboxylic acid groups, carbonic acid derivatives such as ethoxycarbonyl, benzyloxycarbonyl, tert-butyloxycarbonyl; or acid groups derived from a sulfonic acid such as para-toluenesulfonyl.

Protecting groups are removed according to the nature of these groups. The carbobenzoxy group is removed by catalytic hydrogenolysis in a stream of hydrogen in methanol with the addition of 10% palladium on carbon; the tert-butyloxycarbonyl group is removed in the presence of anhydrous trifluoroacetic acid in dichloromethane.

In the synthesis of the claimed compounds, N-acyl derivatives were obtained using anhydrides of succinic and glutaric acids, butyrolactone. The dimeric structure of the dipeptides according to formula (1) was reproduced by the interaction of the activated ester of protected asparagine with hexa- or heptamethylenediamine.

Below are examples of the invention.

Abbreviations used:

Boc - tert-butyloxycarbonyl

DIEA - diisopropylethylamine

DCC - Dicyclohexylcarbodiimide

DCM - dichloromethane

DCU - Dicyclohexylurea

DMF - dimethylformamide

DMSO - dimethyl sulfoxide

EtOAc - ethyl acetate

OPfp - pentafluorophenyl

Pd/C - palladium nanoparticles on the surface of activated carbon

TFA - trifluoroacetic acid

Z - benzyloxycarbonyl

DCHA - Dicyclohexylamine

TLC - thin layer chromatography

NMR - nuclear magnetic resonance

Starting substances and auxiliary reagents:

Reagents: L-asparagine (Diaem, China), Z-L-Glu(OtBu)-OH*DCHA (China), pentafluorophenol and trifluoroacetic acid (AlfaAesar, USA), dicyclohexylcarbodiimide (Sigm-Aldrih, Germany), di-tert-butylpyrocarbonate (Chemical Line, Russia), Pd/C catalyst (Acros organics, Germany).

Solvents: ethyl acetate, dimethylformamide (DMF), diethyl ether, dichloromethane, hexane, methanol were obtained from OOO TD Himmed (Russia). DMF was purified by vacuum distillation over ninhydrin. Diethyl ether was kept over NaOH, then filtered through a paper filter. Dichloromethane was passed through a column of Al ₂ O ₃ . Ethyl acetate and alcohols were used without further purification.

To determine the physicochemical characteristics of the obtained substances, the following equipment was used:

melting points were determined on an Optimelt MPA100 instrument (Stanford Research Systems, USA) in open capillaries without correction;

The specific optical rotation was recorded on an automatic polarimeter ADP 440 Polarimeter (Bellingham + Stanley Ltd., UK).

The structure and diastereomeric purity of the target peptides and intermediates were determined by one-dimensional ¹ H-, ¹³ C- and two-dimensional (COSY - homonuclear correlation, HSQC - heteronuclear single-quantum correlation, HMBC - heteronuclear multiply connected correlation) NMR spectroscopy. ^1H- and ^3C -NMR spectra were recorded in δ scale, ppm on a Bruker FOURIER 300 HD spectrometer (Bruker Corporation, Leipzig, Germany, 300 and 75 MHz for ^1H- and ^13C nuclei, respectively) in DMSO-d ₆ and CDCl ₃ solutions, internal standard tetramethylsilane (0 ppm). Spin-spin interaction constant J, Hz. The following abbreviations were used to designate resonant signals: s - singlet, d - doublet, t - triplet, m - multiplet.

Mass spectrometry: high-resolution spectra were recorded on a Bruker microTOF II instrument by electrospray ionization (ESI-MS). Measurements were made on positive (capillary voltage 4500 V) or negative (capillary voltage 3200 V) ions. Mass scanning range (m/z) 50-3000 Yes, calibration - external or internal (Electrospray Solution, Fluka). Syringe injection of the substance was used. Flow rate 3 µl/min. Spray gas - nitrogen (4 l / min); interface temperature 180°C.

Thin layer chromatography (TLC) was performed on glass silica gel plates DC Kieselgel 60 G/F ₂₅₄ (Merck, Germany) in solvent systems: chloroform - MeOH - water - acetic acid, 15:10:2:3 (A);

n-butanol - acetic acid - water, 3:1:1 (B);

EtOAc (C);

hexane - EtOAc, 1:5 (D).

chloroform - MeOH - water - acetic acid, 8:10:2:3 (E);

chloroform - MeOH - acetic acid, 8:1:0.1 (F).

Amine-containing compounds were detected with ninhydrin, compounds with amide groups - using a chloro-tolidine test, compounds with an open carboxyl group - with bromcresol green.

Preparation of a solution for the chlor-tolidine test: a sample of 160 mg of o-tolidine, 1 g of KI was placed in a dark glass container with a volume of 1 l, and 30 ml of ice was added. acetic acid and 500 ml of distilled water, stirred. Chromatogram development: the plate was placed in an atmosphere of chlorine (freshly prepared from 1.5% KMnO ₄ solution and 10% HCl solution) for 2-3 min. Then the plate was left for 3–5 min under air flow to remove excess chlorine, then it was immersed in a tolidine solution for 5 s, and the plate was washed with water.

Analytical HPLC of the dipeptide GTS-301 was performed using a Wellchrom 2001 chromatographic system (KNAUER, Germany) on a LUNA-C18 column (4.6 × 250 mm, 5 µm) in a concentration gradient of acetonitrile in 0.1% TFA (5–50%, 20 min). Flow rate 1 ml/min, detection at 214 nm.

EXAMPLE 1. Obtaining bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) hexamethylenediamide (GTS-301)

a) N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OH

To a solution of 56.75 g (0.378 mol) of HL-Asn-OH monohydrate in 380 ml of 1 M NaOH was successively added with stirring a solution of 18.9 g (0.225 mol) of NaHCO ₃ in 190 ml of water and 380 ml of i-PrOH. Then, at room temperature, 113 ml (0.491 mol) of di-tert-butyl pyrocarbonate were added in portions to the reaction mixture over 40 min and stirred for 20 h. The reaction mixture was evaporated to 1/3 of the volume and diluted with 400 ml of water; The aqueous solution was cooled to +10°C and acidified with 1 M HCl to pH 2.5. The precipitate formed was filtered off, washed with 100 ml of cold water (+10°C), i-PrOH (100 ml), hexane (50 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Product yield Boc-L-Asn-OH 79.5 g (91%) as a white powder. R _ƒ 0.84 (E), R _ƒ 0.71 (B); m.p. 176-176.4°С (with decomposition); [α] _D ²¹⁵ - 11.2° (с = 1, DMF), [α] _D ²¹ - 9.6° (с = 2, DMF). ¹ H-NMR (DMSO-d ₆ ): 1.38 (s, 9H, -OC(CH ₃ ) ₃ Boc), 2.37-2.53 (m, 2H, C ^β H ₂ Asn), 4.27 (m, 1H, C ^α H Asn), 6.60 (d, minor conformer NH Asn), 6.88 (d, 1H, major conformer ep, ³ J=8.5 Hz, NH Asn), 6.91 and 7.32 (two s, 2H, -C(O)NH ₂ Asn), 12.52 (s, 1H, COOH), and 4.14 (m, 1H, C ^α H Asn). Lit. [Keller O, Keller WE, Look G van, Wersin G. tert -Butoxycarbonylation of Amino Acids and their Derivatives: N-tert -Butoxycarbonyl-l-phenylalanine. In: Organic Syntheses. Coll; Vol.7 (1990), 70]: T pl. 176°C (decomp); [α] _D ²⁰ - 7.2° (c = 2, DMF). Lit. [T. Munegumi, K. Akao, Y. Kawatu, T. Yamada and K. Harada //Asian Journal of Chemistry; Vol.26, No. 19 (2014), 6541-6548 Heating Reactions of Nt-Butyloxycarbonyl-Asparagine and Related Compounds]: M.p. 177-179°C (EtOH); [α] _D ²⁰ - 7.8° (c=1, DMF).

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OPfp

A solution of 12.01 g (52 mmol) PfpOH in 60 ml EtOAc was added to a suspension of 10.0 g (43 mmol) Boc-L-Asn-OH in 150 ml EtOAc at room temperature with stirring. The solution was cooled to 10°С and a solution of 10.73 g (52 mmol) DCC in 50 ml EtOAc was added in portions. The reaction mass was stirred for 7 hours at 10–12°C and left at this temperature for 12 hours without stirring. The precipitated DCU was filtered off (filtered at 10°C), washed with 60 ml of EtOAc (10°C). The combined filtrates were evaporated, the resulting solid was triturated under petroleum ether (75 ml) and left in the refrigerator for 12 hours. The precipitate was filtered off, washed with petroleum ether (50 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Product yield Boc-L-Asn-OPfp 16.53 g (96%) as a white powder. R _ƒ 0.73 (C), R _ƒ 0.66 (D), R _ƒ 0.63 (F); m.p. 120-123°C; [α] _D ²³ - 17.9° (с = 1, EtOAc). ¹ H-NMR (CDCl ₃ ): 1.47 (s, 9H, -OC(CH ₃ ) ₃ Boc), 2.90 (dd, 1H, ² J=16.5 Hz; ³ J=4.1 Hz, C ^β H ₂ Asn), 3.15 (dd, 1H, ² J=16.5 Hz; ³ J=4.7 Hz C ^β H ₂ Asn), 4. 91 (m, 1H, C ^α H Asn), 5.92 (d, ³ J=6.15 Hz, 1H, NH Asn), 5.94 and 6.03 (two br s, 2H, -C(O)NH ₂ Asn).

Lit. [Kisfaludy L, Low M, Nyeki O, Szirtes T, Schon I. Die Verwendung von Pentafluorphenylestern bei Peptidsynthesen. Justus Liebigs Ann Chem. 1973; 1973(9): 1421-1429]: T. pl. 124-126°C (diethyl ether/hexane); [α] _D ²⁵ - 16.7° (c=1, EtOAc).

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparagine), (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₆

To a solution of 3.87 g (9.73 mmol) of Boc-L-Asn-OPfp in 20 ml of DMF, a solution of 0.51 g (4.40 mmol) of hexamethylenediamine in 15 ml of DMF was added with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (40°С), 200 ml of distilled water (43–45°С) was added to the residue and left at room temperature until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), petroleum ether (2×40 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . 1.90 g (79%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.91 (E), R _ƒ 0.75 (B); m.p. 206-211°C (with decomposition); [α] _D ²¹ - 2.0° (c=1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.20 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.37 (м, 22Н, 2 С ² Н ₂ спейсер, 2 -ОС(СН ₃ ) ₃ ), 2.36 (м, 4Н, 2 С ^β Н ₂ Asn), 3.02 (м, 4Н, 2 С ¹ Н ₂ спейсер), 4.16 (м, 2Н, C ^α H Asn), 6.82 (д, ³ J=8.1 Гц, 2Н, NH Asn) 6.88 и 7.26 (два с, 4Н, 2 -C(О)NH ₂ Asn), 7.67 (т, ³ J=5.3 Гц, 2Н, -NH(CH ₂ ) ₆ NH-).

d) Hexamethylenediamide ditrifluoroacetate bis-(L-asparagine), (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆

A solution of 1.20 g (46.45 mmol) (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of a mixture of DCM and TFA (1:1) was stirred at room temperature for 2 h, then evaporated, reevaporated with DCM (2 × 15 ml), the residue was triturated under dry diethyl ether with decantation (2 × 20 ml) and left under diethyl ether (20 ml) for 2 h for the formation of a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . Received 2.00 g (87%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.36 (E), R _ƒ 0.17 (B); m.p. 160-178°C (with decomposition); [α] _D ²⁵ +4.7° (c=1, MeOH), [α] _D ²⁵ +1.0° (c = 1, DMSO). ¹ H-NMR (DMSO-d ₆ ): 1.26 (m, 4H, 2 C ³ H ₂ spacer), 1.40 (m, 4H, 2 C ² H ₂ spacer), 2.62 (m, 4H, 2 C ^β H ₂ Asn), 3.09 (m, 4 H, 2 C ¹ H ₂ spacer), 3.98 (m, 2Н, 2 С ^α Н Asn), 7.23 and 7.66 (two s, 4Н, 2 -C(O)NH 2 Asn), 8.09 (br s, 6Н, 2 N ⁺ H ₃ Asn), 8.33 (t, ³ J _5.5 Hz, 2Н, -NH(CH ₂ ) ₆ NH-).

e) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparaginyl-L-asparagine), (Boc-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆

To a solution of 1.68 g (2.90 mmol) (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml of DMF was added 1.12 ml (6.45 mmol) of DIEA, and 2.57 g (6.45 mmol) of Boc-L-Asn-OPfp was sprinkled with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (53°С), reevaporated with water (3×20 ml), 50 ml of distilled water (45°С) was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), hexane (20 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.50 g (66%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.89 (E), R _ƒ 0.36 (B); m.p. 200-227°C (with decomposition); [α] _D ²⁴ - 9.2° (c=1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.19 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.38 (м, 22Н, 2 С ² Н ₂ спейсер, 2 -ОС(СН ₃ ) ₃ Вос), 2.39-2.55 (м, 8Н, 4 С ^β Н ₂ ¹ Asn и ² Asn), 3.00 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.17 (м, 2Н, С ^α Н ² Asn), 4.41 (м, 2Н, С ^α Н ¹ Asn), 6.88 и 6.96, 7.33 и 7.38 (четыре с, 8Н, 4 -C(O)NH ₂ ¹ Asn и ² Asn), 7.00 (д, 2Н, ³ J=7.3 Гц, 2 NH ² Asn), 7.61 (т, ³ J=4.8 Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 8.11 (д, 2Н, ³ J=7.9 Гц, 2 NH ¹ Asn).

f) Hexamethylenediamide ditrifluoroacetate bis-(2,-asparaginyl-2,-asparagine), (CF ₃ COOH*HL-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ (6)

A suspension of 1.047 g (1.35 mmol) (Boc-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of a mixture of DCM and TFA (1:1) was stirred for 2.5 h, then the reaction mass was evaporated, reevaporated with DCM (2 × 15 ml), the residue was triturated under dry diethyl ether with decantation (2 × 20 ml) and left under diethyl ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . Received 0.96 g (88%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.25 (A), R _ƒ 0.03 (B), R _ƒ 0.28 (E); m.p. 197-21 GS (with decomposition); [α] _D ²⁴ - 5.0° (c = 1, DMSO), [α] _D ²⁴ - 7.5° (c = 0.66, MeOH:H ₂ O = 2:1). ¹ Н-ЯМР (DMSO-d ₆ ): 1.20 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.36 (м, 4Н, -2 С ² Н ₂ спейсер), 2.41 и 2.52 (два дд, ² J=15.7 Гц, ³ J=8.0 Гц, ³ J=5.3 Гц, 4Н, 2 C ^β H ₂ ² Asn), 2.63 и 2.74 (два дд, ² J=17 Гц, ³ J=7.5 Гц, ³ J=5.0 Гц, 4Н, 2 С ^β Н ₂ ¹ Asn), 3.02 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.05 (м, 2Н, 2 C ^α H ² Asn), 4.53 (м, 2Н, 2 C ^α H ¹ Asn), 6.91 и 7.37; 7.29 and 7.75 (four s, 8Н, 4 -C(O)NH ₂ ¹ Asn and ² Asn), 7.84 (t, ³ J=5.6 Hz, 2Н, -NH(CH ₂ ) ₆ NH-), 8.15 (br s, 6Н, 2 N ⁺ H ₃ ² Asn), 8.61 (d, 2Н, ³ J=7.7 Hz, 2 NH ¹ Asn).

g) Hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine), (HOOC-(CH ₂ ) ₂ -CO-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ , GTS-301

To a solution of 0.90 g (1.12 mmol) (CF ₃ COOH*HL-Asn-L-Asn-NH-) ₂ (CH ₂ ) 6 in 20 ml DMF was added 0.46 ml (2.60 mmol) DIEA, then 0.30 g (2.60 mmol) succinic anhydride was sprinkled with stirring. The reaction mass was stirred at room temperature for 4 h, then left overnight without stirring. DMF was evaporated (53°C), reevaporated with water (3 × 20 ml), 40 ml of acetone was added to the residue and left for 2 h under stirring, the formed precipitate was filtered off, washed with hot MeOH (3 × 10 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.80 g (93%) of a chromatographically homogeneous product was obtained in the form of a white powder. R _ƒ 0.24 (A), R _ƒ 0.08 (B), R _ƒ 0.42 (E); t = 10.15 min; m.p. 214-229°C (from methanol, with decomposition); [α] _D ²² - 20.2° (с = 1, DMSO); Found, m/z: 71334 [M+H] ⁺ ; m/z: 771.33 [M-N] ^- . C ₃₀ H ₄₈ N ₁₀ O ₁₄ . Calculated, m/z: 772.77.

¹ H-NMR (DMSO-d ₆ ): 1.21 (s, 4H, 2 C ³ H ₂ spacer), 1.36 (m, 4H, 2 C ² H ₂ spacer), 2.37-2.60 (m, 16H, 4 C ^β H ₂ ¹ Asn and ² Asn; 2-CH ₂ CH ₂ -COOH Suc), 3.00 (m, 4 H, 2 C ¹ H ₂ spacer), 4.42 (m, 4H, 4 C ^α H ¹ Asn and ² Asn), 6.87 and 7.33; 6.99 and 7.44 (four s, 8Н, 4 -C(О)NH ₂ ¹ Asn and ² Asn), 7.61 (t, ³ J=5.3 Hz, 2Н, -NH(CH ₂ ) ₆ NH-), 8.15 (d, 2Н, ³ J=7.9 Hz, 2 NH ² Asn), 8.27 (d, 2Н, ³ J= 7.0 Hz, 2 NH ¹ Asn), 12.11 (br s, 2 H, 2 COOH Suc).

¹³C-NMR (DMSO-d₆): 174.42 (s, 2 CO, COOH Sue), 172.31 (s, 4 CONH₂ ¹Asn and²Asn), 172.01 (s, 2 CONH Sue), 171.13 (s, 2 CONH²Asn), 170.92 (s, 2 CONH¹Asn), 50.52 (s, 4С^{α 1}Asn and²Asn), 39.19 (s, 2 С¹ spacer), 37.47 (s, 2 C^{β 2}Asn), 37.09 (s, 2 C^{β 1}Asn), 30.35 (s, 2 С² Sue), 29.56 (s, 2 С¹ Sue), 29.34 (s, 2 С² spacer), 26.41 (s, 2 C³ spacer).

EXAMPLE 2 Preparation of bis-(N-monosuccinyl-L-asparaginyl-D-asparagine) hexamethylenediamide (GTS-301LD)

a) N-tert-butyloxycarbonyl-D-asparagine, Boc-D-Asn-OH

Received similarly to example 1 point a.

A solution of 6.30 g (0.075 mol) of _NaHCO3 in 56.75 ml of water and 113.5 ml of i-PrOH was successively added to a solution of 17.04 g (0.1135 mol) of HD)-Asn-OH monohydrate in 130 ml of 1 M NaOH with stirring. Then, 35 ml (0.164 mol, 30% excess) of di-tert-butylpyrocarbonate was added in portions to the reaction mixture at room temperature over 40 min and stirred for 20 h. The reaction mixture was evaporated to 1/3 of the volume and diluted with 200 ml of water; The aqueous solution was cooled to +10°C and acidified with 1M HCl to pH=2.5.

The precipitate formed was filtered off, washed with 100 ml of cold water (+10°C), i-PrOH (100 ml), hexane (50 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . Product yield Boc-D-Asn-OH 22.79 g (87%) as a white crystalline product. R _ƒ 0.51 (A), R _ƒ 0.84 (E), R _ƒ 0.71 (B); m.p. 172.4-173.1°С; [α] _D ²² +7.5° (с = 1, DMF); [α] _D ²² +7.0° (c = 2, DMF). ¹ H-NMR (DMSO-d ₆ ): 1.37 (s, 9H, -OC(CH ₃ ) ₃ Boc), 2.42 and 2.52 (two dd, 2H, ³ J=5.59 Hz and ³ J=7.36 Hz, ² J=15.27 Hz C ^β H ₂ Asn), 4.23 (m, 1H, C ^α H Asn), 6.8 7 (d, 1H, ^3J 8.5 Hz, NH Asn), 6.91 and 7.32 (two s, 2H, -C(O)NH ₂ Asn), 12.51 (s, 1H, COOH).

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-D-asparagine, Boc-D-Asn-OPfp

Received similarly to example 1 point b.

A solution of 12.01 g (52 mmol) PfpOH in 60 ml EtOAc was added to a suspension of 20.0 g (0.0861 mol) Boc-D-Asn-OH in 150 ml EtOAc at room temperature with stirring. The solution was cooled to 10°С and a solution of 10.73 g (52 mmol) DCC in 50 ml EtOAc was added in portions. The reaction mass was stirred for 7 hours at 10–12°C and left at this temperature for 12 hours without stirring. The precipitated DCU was filtered off (filtered at 10°C), washed with 60 ml of EtOAc (10°C). The combined filtrates were evaporated, the resulting solid was triturated under petroleum ether (75 ml) and left in the refrigerator for 12 hours. The precipitate was filtered off, washed with petroleum ether (50 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 31.23 g (91%) as a white crystalline product. R _ƒ 0.74 (C), R _ƒ 0.67 (D), R _ƒ 0.63 (F); m.p. 116.5-119°С; [α] _D ²³ +17.7° (c = 1, EtOAc); ¹ H-NMR (CDCl ₃ ): 1.47 (s, 9H, -OC(CH ₃ ) ₃ Boc), 2.91 (dd, 1H, ² J=16.5 Hz, ³ J=4.0 Hz, C ^β H ₂ Asn), 3.15 (dd, 1H, ² J=16.5 Hz, ³ J=4.7 Hz C ^β H ₂ Asn), 4. 91 (m, 1H, C ^α H Asn), 5.90 (d, ³ J=8.7 Hz, 1H, NH Asn), 5.73 and 5.91 (two br s, 2H, -C(O)NH ₂ Asn).

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-2)-asparagine), (Boc-D-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 point in.

To a solution of 3.87 g (9.73 mmol) of Boc-D-Asn-OPfp in 20 ml of DMF, a solution of 0.51 g (4.40 mmol) of hexamethylenediamine in 15 ml of DMF was added with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (40°С), 200 ml of distilled water (43–45°С) was added to the residue and left at room temperature until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), petroleum ether (2×40 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.68 g (70%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.95 (E), R _ƒ 0.75 (B); m.p. 210-219°С (with decomposition); [α] ²⁷ _D +0.51° (с = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.21 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.37 (м, 22Н, 2 С ² Н ₂ спейсер, 2 -ОС(СН ₃ ) ₃ ), 2.36 (м, 4Н, 2 С ^β Н ₂ Asn), 3.02 (м, 4Н, 2 С ¹ Н ₂ спейсер), 4.16 (м, 2Н, С ^α Н Asn), 6.81 (д, ³ J=8.2 Гц, 2Н, NH Asn) 6.86 и 7.24 (два с, 4Н, 2 -C(О)NH ₂ Asn), 7.65 (т, ³ J=4.7 Гц, 2Н, -NH(CH ₂ ) ₆ NH-).

d) Hexamethylenediamide ditrifluoroacetate bis-(D-asparagine), (CF ₃ COOH*HD)-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item d.

A solution of 1.50 g (2.75 mmol) (Boc-D-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of a mixture of DCM and TFA (1:1) was stirred at room temperature for 2 h, then evaporated, reevaporated with DCM (2×15 ml), the residue was triturated under dry diethyl ether with decantation (2×20 ml) and left under diethyl ether (20 ml) for 2 h for the formation of a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . 1.43 g (91%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.17 (B), R _ƒ 0.33 (E); m.p. 171-180°C (with decomposition); [α] _D ²⁴ -3.7° (c = 1, MeOH), [α] _D ²³ -1.4° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.26 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.40 (м, 4Н, 2 С ² Н ₂ спейсер), 2.58 и 2.66 (два дд, 4Н ² J=17.0 Гц, ³ J=5.22, ³ J=7.82 Гц 2 С ^β Н ₂ Asn), 2.99-3.19 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 3.98 (уш с, 2Н, 2 C ^α H Asn), 7.24 и 7.66 (два с, 4Н, 2 -C(О)NH ₂ Asn), 8.09 (уш с, 6Н, 2 N ⁺ H ₃ Asn), 8.34 (т, ³ J=5.5 Гц, 2Н, -NH(CH ₂ ) ₆ NH-).

e) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparaginyl-D-asparagine), (Boc-L-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

To a solution of 1.45 g (2.53 mmol) of (CF ₃ COOH*HD-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml of DMF was added 1.04 ml (6.00 mmol) of DIEA, and 2.42 g (6.07 mmol) of Boc-L-Asn-OPfp was sprinkled with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (53°С), reevaporated with water (3×20 ml), 50 ml of distilled water (45°С) was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), hexane (20 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . 1.16 g (59%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.82 (A), R _ƒ 0.58 (B); m.p. 201-218°C (with decomposition); [α] _D ²³ +8.1° (с = 1, DMSO). ¹ H-NMR (DMSO-d ₆ ): 1.23 (m, 4H, 2 C ³ H ₂ spacer), 1.38 (m, 22H, 2 C ² H ₂ spacer, 2-OC(CH ₃ ) ₃ Boc), 2.42-2.50 (m, 8H, 4 C ^β H ₂ ¹ Asn and ² Asn), 3.00 (m , 4 H, 2 C ¹ H ₂ spacer), 4.14 and 4.42 (two m, 4H, C ^α H ¹ Asn and ² Asn), 6.86 and 7.31; 6.94 and 7.34 (four s, 8Н, 4 -C(О)NH ₂ ¹ Asn and ² Asn), 7.04 and 8.19 (two d, 4Н, ³ J=7.0, ³ J=8.3 Hz, 4 NH ¹ Asn and ² Asn), 7.55 (t, ³ J=5.1 Hz, 2Н, -NH(CH ₂ ) ₆ NH-).

e) Hexamethylenediamide ditrifluoroacetate bis-(L-asparaginyl-D-asparagine), (CF ₃ COOH*HL-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

To a suspension of 1.03 g (2.62 mmol) (Boc-L-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ in 15 ml DCM at room temperature was added 15 ml TFA, stirred for 2.5 h, then the reaction mass was evaporated, reevaporated with DCM (2 × 15 ml), the residue was triturated under dry diethyl ether with decantation (2 × 20 ml) and left under diethyl ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.98 g (92%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.06 (A), R _ƒ 0.08 (V); m.p. 144-153°C (with decomposition); [α] _D ^23.5+ 12.9° (s,1; MeOH), [α] _D ^23.5 +29.2° (s = 1, _H2O ), [α] _D ^23.1 +6.0° (s = 1, DMSO). ¹ H-NMR (DMSO-d ₆ ): 1.23 (m, 4H, 2 C ³ H ₂ spacer), 1.36 (m, 4H, -2 C ² H ₂ spacer), 2.34-2.68 (m, 8H, 4 C ^β H ₂ , ¹ Asn and ² Asn), 3.01 (m, 4 H, 2 C ¹ H ₂ spacer), 4.12 (m, 2H, 2 C ^α H ¹ Asn), 4.52 (m, 2H, 2 C ^α H ² Asn), 6.87 and 7.36; 7.25 and 7.67 (four s, 8Н, 4 -C(О)NH ₂ ¹ Asn and ² Asn), 7.89 (t, ³ J=5.4 Hz, 2Н, -NH(CH ₂ ) ₆ NH-), 8.11 (br s, 6Н, 2 N ⁺ H ₃ ² Asn), 8.62 (d, 2Н, ³ J=1.9 Hz, ^2NH2Asn ).

g) Hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine), (HOOC-(CH ₂ ) ₂ -CO-L-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ , GTS-301LD

Received similarly to example 1 item g.

To a solution of 0.98 g (1.71 mmol) of (CF ₃ COOH*HL-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of DMF was added 0.66 ml (3.76 mmol) of DIEA, then 0.38 g (2.20 mmol) of succinic anhydride was sprinkled with stirring. The reaction mass was stirred at room temperature for 4 h, then left overnight without stirring. DMF was evaporated (53°C), reevaporated with water (3 × 20 ml), 40 ml of acetone was added to the residue and left for 2 h under stirring, the formed precipitate was filtered off, washed with hot MeOH (3 × 10 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.86 g (68%) of a chromatographically homogeneous product was obtained in the form of a white powder. R _ƒ 0.38 (A), R _ƒ 0.22 (B); m.p. 191-213°C (from methanol, with decomposition); [α] _D ^25.5 +1.56° (с = 1, DMSO). Found, m/z: 773.34 [M+H] ⁺ ; m/z: 771.33 [M-N] ^- . C ₃₀ H ₄₈ N ₁₀ O ₁₄ . Calculated, m/z: 772.77. ¹ Н-ЯМР (DMSO-d ₆ ): 1.20 (с, 4Н, 2 С ³ Н ₂ спейсер), 1.37 (м, 4Н, 2 С ² Н ₂ спейсер), 2.34-2.56 (м, 16Н, 4 C ^β H ₂ ¹ Asn и ² Asn; 2 -СН ₂ СН ₂ -СООН Suc), 3.01 (м, 4 H, 2 С ¹ Н ₂ спейсер), 4.36 (м, 4Н, 4 С ^α Н ¹ Asn и ² Asn), 6.88 и 7.31, 6.95 и 7.41 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.62 (т, ³ J=5.4 Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 8.16 (д, 2Н, ³ J=8.2 Гц, 2 NH ² Asn), 8.31 (д, 2Н, ³ J=7.0 Гц, 2 NH ¹ Asn), 12.23 (уш с, 2 Н, 2 СООН Suc).

¹³C-NMR (DMSO-d₆): 174.54 (s, 2 CO, COOH Suc), 172.46 (s, 2 CONH₂ ¹Asn), 172.44 (s, 2CONH₂ ²Asn), 172.14 (s, 2 CONH Suc), 171.29 (s, 2 CONH¹Asn), 170.85 (s, 2 CONH²Asn), 50.79 (s, 2C^{α 1}Asn), 50.53 (s, 2C^{α 2}Asn), 39.23 (s, 2 С¹ spacer), 37.09 (s, 2 C^{β 1}Asn), 36.94 (s, 2C^{β 2}Asn), 30.46 (s, 2 С² Sue), 29.66 (s, 2 С¹ Suc), 29.30 (s, 2 C² spacer), 26.43 (s, 2 C³spacer).

EXAMPLE 3 Preparation of bis-(N-monosuccinyl-D-asparaginyl-L-asparagine) hexamethylenediamide (TTC-301DL)

a) N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OH

See example 1 point a.

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OPfp

See example 1 point b.

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparagine), (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₆

See example 1 point c.

d) Hexamethylenediamide ditrifluoroacetate bis-(L-asparagine), (CF ₃ COOH * HL-Asn-NH-) 2 (CH ₂ ) ₆

See example 1 paragraph d.

e) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-D-asparagnyl-L-asparagine), (Boc-D-Asn-Z,-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

To a solution of 1.50 g (2.62 mmol) (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml DMF was added 1.0 ml (5.74 mmol) DIEA and 2.50 g (6.30 mmol) Boc-D-Asn-OPfp was sprinkled with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (53°С), reevaporated with water (3×20 ml), 50 ml of distilled water (45°С) was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), hexane (20 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.60 g (79%) chromatographically homogeneous product in the form of a white powder with a slight creamy tint. R _ƒ 0.71 (A), R _ƒ 0.46 (B); m.p. 196-215°C (with decomposition); [α] _D ²² - 4.8° (с = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.23 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.38 (м, 22Н, 2 С ² Н ₂ спейсер, 2 -ОС(СН ₃ ) ₃ Boc), 2.42-2.50 (м, 8Н, 4 С ^β Н ₂ ¹ Asn и ² Asn), 3.00 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.11-4.18 и 4.39-4.45 (два м, 4Н, C ^α H ¹ Asn и ² Asn), 6.87 и 7.32, 6.94 и 7.36 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.06 (д, 2Н, ³ J=7.2 Гц, 2 NH ² Asn), 7.56 (т, ³ J=5.2 Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 8.20 (д, 2Н, ⁵ J=8.1 Гц, 2 NH ¹ Asn).

e) Hexamethylenediamide ditrifluoroacetate bis-D-asparaginyl-L-asparagine), (CF ₃ COOH*HD-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

A suspension of 1.60 g (2.07 mmol) (Boc-D-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of a mixture of DCM and TFA (1:1) was stirred for 2.5 h, then the reaction mass was evaporated, reevaporated with DCM (2 × 15 ml), the residue was triturated under dry diethyl ether with decantation (2 × 20 ml) and left under diethyl ether (20 ml) for 2 hours to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . 1.51 g (90%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.06 (A), R _ƒ 0 (B), R _ƒ 0.20 (E); m.p. 148-154°C (with decomposition); [α] _D ²² -5.4° (c = 1, DMSO), [α] _D ²⁴ -10.0° (c = 1, MeOH), [α] _D ²⁴ -25.8° (c = 1, H ₃ O). ¹ Н-ЯМР (DMSO-d ₆ ): 1.21 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.36 (м, 4Н, -2 С ² Н ₂ спейсер), 2.34 - 2.69 (м, 8Н, 4 С ^β Н ₂ ² Asn), 3.01 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.11 (м, 2Н, 2 C ^α H ² Asn), 4.53 (м, 2Н, 2 С ^α Н ¹ Asn), 6.87 и 7.35, 7.25 и 7.67 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.89 (т, ³ J=5.6 Гц, 2Н, - NH(CH ₂ ) ₆ NH-), 8.11 (уш с, 6Н, 2 N ⁺ H ₃ ² Asn), 8.62 (д, 2Н, ³ J=7.9 Гц, 2 NH ¹ Asn).

g) Hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine), (HOOC-(CH ₂ ) ₂ -CO-D-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ , TTC-301DL

Received similarly to example 1 item g.

To a solution of 0.70 g (0.87 mmol) of (CF ₃ COOH*HL-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of DMF was added 0.32 ml (1.83 mmol) of DIEA, then 0.20 g (1.91 mmol) of succinic anhydride was sprinkled with stirring. The reaction mass was stirred at room temperature for 4 h, then left overnight without stirring. DMF was evaporated (53°C), reevaporated with water (3 × 20 ml), 40 ml of acetone was added to the residue and left for 2 h under stirring, the formed precipitate was filtered off, washed with hot MeOH (3 × 10 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . Received 0.57 g (85%) chromatographically homogeneous product in the form of a white powder with a slight creamy tint. R _ƒ 0.39 (A), R _ƒ 0.14 (B), R _ƒ 0.59 (E); m.p. 193-222°C (from ethanol, with decomposition); [α] _D ²³⁵ -1.68° (c = 1, DMSO). Found, m/z: 773.34 [M+H] ⁺ ; m/z: 771.33 [M-N] ^- . C ₃₀ H ₄₈ N ₁₀ O ₁₄ . Calculated, m/z: 772.77. ¹ H-NMR (DMSO-d ₆ ): 1.19 (s, 4H, 2 C ³ H ₂ spacer), 1.36 (m, 4H, 2 C ² H ₂ spacer), 2.34-2.56 (m, 16H, 4 C ^β H ₂ ¹ Asn and ² Asn; 2-CH ₂ CH ₂ -COOH Suc), 3.00 (m, 4 H, 2 C ¹ H ₂ spacer), 4.38-4.47 (m, 4H, 4 C ^α H ¹ Asn and ² Asn), 6.89 and 7.30; 6.96 and 7.41 (four s, 8Н, 4 -C(О)NH ₂ ¹ Asn and ² Asn), 7.63 (t, ³ J=5.4 Hz, 2Н, -NH(CH ₂ ) ₆ NH-), 8.15 (d, 2Н, ³ J=8.1 Hz, 2 NH ² Asn), 8.30 (d, 2Н, ³ J= 7.0 Hz, 2 NH ¹ Asn), 12.14 (br s, 2 H, 2 COOH Sue). ¹³ С -ЯМР (DMSO-d ₆ ): 174.41 (с, 2 СО, СООН Suc), 172.47 (с, 2 CONH ₂ ¹ Asn), 172.31 (с, 2 CONH ₂ ² Asn), 172.10 (с, 2 CONH Suc), 171.28 (с, 2 CONH ¹ Asn), 170.82 (с, 2 CONH ² Asn), 50.75 (с, 4С ^{α 1} Asn и ² Asn), 39.23 (s, 2 С ¹ спейсер), 37.10 (с, 2 С ^{β 1} Asn), 36.92 (с, 2 С ^{β 2} Asn), 30.40 (с, 2 С ² Sue), 29.50 (с, 2 С Sue), 29.31 (с, 2 С ² спейсер), 26.42 (с, 2 С ³ спейсер).

EXAMPLE 4 Preparation of bis-(N-monosuccinyl-D-asparaginyl-D-asparagine) hexamethylenediamide (TTC-301DD)

a) N-tert-butyloxycarbonyl-D-asparagine, Boc-D-Asn-OH

See example 2 point a.

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-D-asparagine, Boc-D-Asn-OPfp

See example 2 point b.

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-D-asparagine), (Boc-D-Asn-NH-) ₂ (CH ₂ ) ₆

See example 2 point c.

d) Hexamethylenediamide ditrifluoroacetate bis-(D-asparagine), (CF ₃ COOH*HD-Asn-NH-) ₂ (CH ₂ ) ₆

See example 2 point d.

e) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-D-asparaginyl-D-asparagine), (Boc-D-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

To a solution of 1.24 g (2.17 mmol) of (CF ₃ COOH*HD-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml of DMF was added 0.84 ml (4.80 mmol) of DIEA, and 1.90 g (4.77 mol) of Boc-D-Asn-OPfp was sprinkled with stirring. The reaction mixture was stirred for 4 h at room temperature, left overnight without stirring. niya. DMF was evaporated (53°С), reevaporated with water (3×20 ml), 50 ml of distilled water (45°С) was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), hexane (20 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.54 g (92%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.75 (A), R _ƒ 0.47 (B), R _ƒ 0.87 (E); m.p. 224-227°C (with decomposition); [α] _D ¹⁹ +13.2° (с = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.19 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.38 (м, 22Н, 2 С ² Н ₂ спейсер, 2 -ОС(СН ₃ ) ₃ Boc), 2.34-2.54 (м, 8Н, 4 С ^β Н ₂ ¹ Asn и ² Asn), 3.00 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.14-4.20 и 4.38-4.44 (два м, 4Н, 2 C ^α H ¹ Asn и ² Asn), 6.89 и 7.34, 6.97 и 7.40 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.62 (т, ³ J=5.5 Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 7.02 и 8.13 (два д, 4Н, ⁵ J=7.3 и ³ J=1.9 Гц, 4 NH ¹ Asn и ² Asn).

f) Hexamethylenediamide ditrifluoroacetate bis-(N-asparaginyl-D-asparagine), (CF ₃ COOH*HD-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item e.

A suspension of 1.12 g (14.49 mmol) (Boc-D-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml of a mixture of DCM and TFA (1:1) was stirred for 2.5 h, then the reaction mass was evaporated, reevaporated with DCM (2 × 15 ml), the residue was triturated under dry diethyl ether with decantation (2 × 20 ml) and left under diethyl ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.70 g (90%) of the product was obtained, which was then dissolved in 25 ml of deionized water and lyophilized. R _ƒ 0.25 (A), R _ƒ 0.03 (B), R _ƒ 0.28 (E); m.p. not measured (lyophilisate); [α] _D ²⁰ +13.3° (c = 0.92, H ₂ O). ¹ Н-ЯМР (DMSO-d ₆ ): 1.20 (м, 4Н, 2 С ³ Н ₂ спейсер), 1.36 (м, 4Н, -2 С ² Н ₂ спейсер), 2.41 и 2.52 (два дд, ² J=16.0 Гц, ³ J=8.0 Гц, ³ J=5.5 Гц, 4Н, 2 C ^β H ₂ ² Asn), 2.61 и 2.74 (два дд, ² J=17 Гц, ³ J=7.8 Гц, ³ J=5.2 Гц, 4Н, 2 C ^β H ₂ ¹ Asn), 3.00 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.05 (м, 2Н, 2 С ^α Н ² Asn), 4.50-4.57 (м, 2Н, 2 С ^α Н ¹ Asn), 6.93 и 7.37; 7.31 and 7.75 (four s, 8Н, 4 -C(О)NH ₂ ¹ Asn and ² Asn), 7.86 (t, ³ J=5.4 Hz, 2Н, -NH(CH ₂ ) ₆ NH-), 8.12 (br s, 6Н, 2 N ⁺ H ₃ ² Asn), 8.61 (d, 2Н, ³ J=1.5 Hz, 2 NH ¹ Asn).

g) Hexamethylenediamide bis-(N-monosuccinyl-D-asparaginyl-D-asparagine), (HOOC-(CH ₂ ) ₂ -CO-D-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ , TTC-301DD

Received similarly to example 1 item g.

To a solution of 0.50 g (0.62 mmol) (CF ₃ COOH*HD-Asn-D-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml DMF was added 0.23 ml (1.30 mmol) DIEA, then 0.20 g (1.99 mmol) succinic anhydride was sprinkled with stirring. The reaction mass was stirred at room temperature for 4 h, then left overnight without stirring. DMF was evaporated (53°C), reevaporated with water (3 × 20 ml), 40 ml of acetone was added to the residue and left for 2 h under stirring, the formed precipitate was filtered off, washed with hot MeOH (3 × 10 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . Received 0.30 g (63%) chromatographically homogeneous product in the form of a white powder. R _ƒ 0.24 (A), R _ƒ 0.08 (B), R _ƒ 0.42 (E); m.p. 207-218°C (from methanol, with decomposition); [α] _D ²⁰ +23.5° (с = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.19 (с, 4Н, 2 С ³ Н ₂ спейсер), 1.36 (м, 4Н, 2 С ² Н ₂ спейсер), 2.37-2.60 (м, 16Н, 4 С ^β Н ₂ ¹ Asn и ² Asn; 2 -СН ₂ СН ₂ СООН Suc), 3.00 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.38-4.47 (м, 4Н, 4 C ^α H ¹ Asn и ² Asn), 6.89 и 7.30, 7.00 и 7.46 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.66 (т, ³ J=5A Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 8.15 (д, 2Н, ³ J=8.0 Гц, 2 NH ² Asn), 8.25 (д, 2Н, ³ J=13 Гц, 2 NH ¹ Asn), 12.12 (уш с, 2 Н, 2 СООН Suc).

¹³C-NMR (DMSO-d₆): 174.43 (s, 2 CO, COOH Suc), 172.31 (s, 4 CONH₂ ¹Asn and²Asn), 172.01 (s, 2 CONH Suc), 171.13 (s, 2 CONH¹Asn), 170.91 (s, 2 CONH²Asn), 50.51 (s, 4C^{α 1}Asn and²Asn), 39.18 (s, 2 C spacer), 37.48 (s, 2 C^{β 2}Asn), 37.09 (s, 2 C^{β 1}Asn), 30.31 (s, 2 C² Sue), 29.52 (s, 2 С Suc), 29.32 (s, 2 С² spacer), 26.40 (s, 2 C³ spacer).

EXAMPLE 5. Preparation of hexamethylenediamide bis-(N-γ-hydroxybutyryl-L-glutamyl-L-asparagine), (GHB-L-Glu-L-Asn-NH-) ₂ (CH ₂ ) ₆ , GTS-302

a) N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OH

See example 1 point a.

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OPfp

See example 1 point b.

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparagine), (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₆

See example 1 point c.

d) Hexamethylenediamide ditrifluoroacetate bis-(L-asparagine), (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆

See example 1 paragraph d.

e) Hexamethylenediamide bis-(N ^α -benzyloxycarbonyl-γ-tert-butyl-glutamyl-asparagine), (ZL-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆

To a solution of 1.16 g (2.03 mmol) (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml DMF was added 0.74 ml (4.26 mmol) DIEA, and 1.94 g (4.46 mmol) ZL-Glu(tBu)-OSu was added with stirring. The reaction mixture was stirred for 4 h at room temperature, then left overnight without stirring. DMF was evaporated in a rotary evaporator vacuum at a temperature of 53°C, then reevaporated with water (3 × 20 ml), 50 ml of distilled water, preliminarily heated to 45°C, was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water to neutral pH, then with acetone (20 ml), petroleum ether (20 ml), diethyl ether (2 × 20 ml) and dried in air to obtain 1.10 g (55%) chromatographically homogeneous (ZL-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆ , in the form of a white crystalline substance with a creamy tinge . R _ƒ 0.94 (A), R _ƒ 0.83 (B); m.p. 211-218°С with decomposition; [α] ²⁵ _D -5.2° (c = 1, DMSO).

¹H-NMR (DMSO-d₆): 1.19 (m, 4H, -NH-(CH₂)₂(CH₂)₂(CH₂)₂NH-), 1.38 (br.s, 22Н, -NHCH₂-CH₂-(CH₂)₂CH₂CH₂NH-, 2-OC(CH₃)₃ tBu), 1.72 and 1.85 (two m, 4H, 2C^βH₂ Glu), 2.24 (t,³J=7.6Hz, 4H, 2C^γH₂ Glu), 2.46-2.50 (m, 4H, 2C^βH₂ Asn), 2.99 (m, 4H, -NHCH₂(CH₂)₄CH₂NH), 3.95-4.01 (m, 2Н, 2 С^αH Clu), 4.41-4.48 (m, 2H, 2C^αH Asn), 5.00 and 5.06 (two d,²J=12.7 Hz, 4H, 2-OCH₂- Z), 6.90 and 7.34 (two s, 4Н, 2 -C(О)NH₂ Asn), 7.36 (m, 10H, 2 -C₆H₅ Z), 7.56 (d,³J 7.36 Hz, 2 H, -NH-Glu), 7.57 (br. t, 2 H, -NH-(CH₂)₆-NH-), 8.13 (d,³J 7.9 Hz, 2Н, NH Asn).

f) Hexamethylenediamide bis-(γ-tert-butyl-glutamyl-asparagine), (HL-Glu(tBu)-L-Asn-NH-)2(CH ₂ ) ₆

A suspension of (ZL-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆ (5.0 g, 5.08 mmol) in 180 mL of methanol was subjected to hydrogenolysis in the presence of 1 g of 10% Pd/C, 50% humidity at room temperature for 7 h; _CO2 was evacuated every 2 h. The catalyst was filtered off, washed with 100 ml of methanol. The solvent was removed in vacuo, 30 ml of benzene was added to the residue and distilled off again in vacuo, the residue was crystallized in petroleum ether as an oil. The precipitate in the form of a white powder was dried under vacuum in a desiccator over CaCl ₂ to obtain 3.06 g (84.5%) of chromatographically homogeneous (HL-Glu(tBu)-L-Asn-NH-) ₂ -(CH ₂ ) ₆ as an amorphous white powder. R _ƒ 0.81 (A), R _ƒ 0.48 (B); mp 174-187°C with decomposition; [α] _D ²⁵ -5.7° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.20 (м, 4Н, -NH(CH ₂ ) ₂ (CH ₂ )2(CH ₂ ) ₂ -NH-), 1.38 (уш.с, 22Н, -NHCH ₂ CH ₂ (CH ₂ ) ₂ CH ₂ CH ₂ NH-, 2 -ОС(СН ₃ ) ₃ tBu), 1.56 и 1.79 (два м, 4Н, 2 С ^β Н ₂ Glu), 1.91 (ш.с, 4Н, 2 NH ₂ Glu), 2.24 (т, ³ J=7.6 Гц, 4Н, 2 С ^γ H ₂ Glu), 2.37-2.50 (м, 4Н, 2 С ^β Н ₂ Asn), 3.00 (м, 4 Н, -NHCH ₂ (CH ₂ ) ₄ CH ₂ NH), 3.16 (м, 2Н, 2 С ^α Н Clu), 4.45 (уш.с, 2Н, 2 С ^α Н Asn), 6.86 и 7.38 (два с, 4Н, 2 -C(О)NH ₂ Asn), 7.74 (уш.с, 2 Н, -NH(CH ₂ ) ₆ NH-), 8.21 (уш. с, 2 Н, NH Asn).

g) Hexamethylenediamide bis-(N-γ-hydroxybutyryl-γ-tert-butyl-glutamyl-asparagine), (GHB-L-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆

A suspension of 2.5 g (3.49 mmol) of (HL-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 20 ml of γ-butyrolactone was kept at a temperature of 60–70°C for 4 h. Then it was stirred at room temperature for 12 h, the precipitate that formed was thoroughly triturated under diethyl ether (4 × 15 ml), decanting the solvent. The residue was dried in vacuum (15 mm Hg) over CaCl ₂ , crystallized from hot acetone. 2.07 g (67%) of chromatographically homogeneous (GHB-L-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆ was obtained as a white crystalline powder, R _ƒ 0.79 (A), R _ƒ 0.64 (B); m.p. 162-173°C; [α] _D ²⁵ -4.8° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.21 (м, 4Н, -NH-(CH ₂ ) ₂ (CH ₂ ) ₂ (CH ₂ ) ₂ NH-), 1.39 (уш.с, 22Н, -NHCH ₂ CH ₂ (CH ₂ ) ₂ CH ₂ CH ₂ -NH-, 2 -ОС(СН ₃ ) ₃ But), 1.65 (м, 4Н, 2 С ² Н ₂ GHB), 1.72 и 1.85 (два м, 4Н, 2 -С ^β Н ₂ Glu), 2.19 (т, 4Н, ³ J=7.8 Гц -С ³ Н ₂ GHB), 2,24 (т, 4Н, ³ J=7.8 Гц, 2СН ₂ Glu), 2.40-2.50 (м, 4Н, 2 С ^β Н ₂ Asn), 3.00 (м, 4 Н, -NHCH ₂ (CH ₂ ) ₄ CH ₂ NH), 3.38 (т, ³ J=6.4 Гц, 2 С ¹ Н ₂ GHB), 4.08-4.15 (м, 2Н, 2 С ^α Н Clu), 4.38-4.41 (м, 2Н, 2 С ^α Н Asn), 6.90 и 7.39 (два с, 4Н, 2 -C(О)-NH ₂ Asn), 7.53 (уш. с, 2 Н, -NH(CH ₂ ) ₆ NH-), 8.16-8.18 (м, 4 Н, 2 NH Asn и 2 NH Glu).

h) Hexamethylenediamide bis-(N-γ-hydroxybutyryl-glutamyl-asparagine), (GHB-L-Glu-L-Asn-NH-) ₂ (CH ₂ ) ₆ , GTS-302

To a suspension of 1.50 g (1.69 mmol) (GHB-L-Glu(tBu)-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 45 ml of CH ₂ Cl ₂ at room temperature was added 15 ml of TFA, stirred for 2 h, then the reaction mass was evaporated, reevaporated with methylene chloride (2 × 15 ml), the residue was triturated under dry diethyl ether with decane cation (2×20 ml) and left under diethyl ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuo over CaCl ₂ (15 mm Hg). GTS-302 was purified by preparative RP HPLC (τ=10.99 min), then lyophilized. 1.0 g (76%) of the target GTS-302 peptide was obtained in the form of a white hygroscopic solid. R _ƒ 0.75 (A), R _ƒ 0.25 (B); [α] _D ²⁵ -4.3°(c = 1, DMSO).

¹H-NMR (DMSO-d₆): 1.20 (m, 4H, -NH(CH₂)₂(CH₂)₂(CH₂)₂NH-), 1.35 (m, 4Н, -NHCH₂CH₂(CH₂)₂CH₂CH₂-NH-), 1.64 (m, 4H, 2C²H₂ GHB), 1.73 and 1.88 (two m, 4H, 2 C^βH₂Glu), 2.19 (t,³J=7.7 Hz, 2 C³H₂ GHB), 2.26 (t,³J=7.7Hz, 4H, 2C^γH₂ Glu), 2.42-2.50 (m, 4H, 2C^βH₂ Asn), 3.00 (m, 4 H, -NHCH₂(CH₂)₄CH₂NH), 3.38 (t,³J=6.3Hz, 4H, 2C¹H₂ GHB), 4.08-4.15 (m, 2H, 2C^αH Clu), 4.38-4.44 (m, 2H, 2C^αH Asn), 6.90 and 7.34 (two s, 4H, 2-C(O)-NH₂Asn), 7.51 (t,³J=5.5 Hz, 2 H, -NH(CH₂)₆NH-), 8.08 (d,³J=8.0 Hz, 2Н, 2 NH Asn), 8.12 (d,³J=7.0 Hz, 2 H, 2-NH Glu), 12.07 (br.s 2H, 2 COOH Glu).

¹³C-NMR (DMSO-d₆): 174.44 (s, 2C, 2CO 2COOH), 173.71 (s, 2C, 2CO GHB), 172.31 (s, 2C, 2CO Asn side), 171.51 (s, 2C, 2CO, Glu), 170.81 (s, 2C, 2CO (Asn) spacer), 60.80 (s, 2C, 2C³GHB), 53.04 (s, 2С, 2С^α Glu), 50.26 (s, 2С, 2С^α Asn), 39.11 (s, 2С, 2С¹ spacer), July 37 (s, 2C, 2 C^βAsn), 32.39 (s, 2С, 2С¹ GHB), 30.57 (s, 2С, 2С^γGlu), 29.30 (s, 2С, 2С² spacer), 28.87 (s, 2С, 2С² GHB), 27.25 (s, 2C, 2C^βGlu), 26.38 (s, 2С, 2С³ spacer).

EXAMPLE 6. Obtaining bis-(N-monoglutaryl-L-asparaginyl-L-asparagne) hexamethylenediamide (GTS-304)

a) N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OH

See example 1 point a.

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OPfp

See example 1 point b.

c) Hexamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparagine), (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₆

See example 1 point c.

d) Ditrifluoroacetate of hexamethylenediamide bme-(L-asparagine), (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₆

See example 1 paragraph d.

e) Hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine), (Glt-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ , GTS-304

To a solution of 0.90 g (1.12 mmol) (CF ₃ COOH*HL-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆ in 30 ml DMF at room temperature was added 0.43 ml (2.46 mmol) DIEA, then 0.34 g (2.46 mmol) glutaric anhydride was added with stirring. The reaction mixture was stirred for 7 hours at room temperature. DMF was evaporated with the addition of water (3×20 ml), then acetone (20 ml). The paraffin-like residue was washed with hot acetonitrile with filtration on a nozzle for hygroscopic substances (3 × 10 ml), 15 ml of diethyl ether was added and left for 30 min with stirring on a magnetic stirrer, the ether was filtered off on a nozzle, the precipitate was dried in a vacuum of a water jet pump, and finally dried in a vacuum (15 mm Hg) over CaCl ₂ . 0.74 g (83%) of a chromatographically homogeneous product was obtained in the form of a white crystalline powder with a slight cream tint. R _ƒ 0.3S (A), R _ƒ 0.22 (B); m.p. 198-224°С (with decomposition); [α] _D ²³ -17.9° (c=1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.19 (с, 4Н, 2 С ³ Н ₂ спейсер), 1.36 (м, 4Н, 2 С ² Н ₂ спейсер), 1,70 (м, 4Н, 2 C ^p H ₂ Glt), 2,15-2,20 (м, 8Н, 2 С ^α Н ₂ и 2 C ^Y H ₂ Git), 2.37-2.59 (м, 16Н, 4 С ^β Н ₂ ¹ Asn и ² Asn), 3.01 (м, 4 Н, 2 С ¹ Н ₂ спейсер), 4.42 (м, 4Н, 4 С ^α Н ¹ Asn и ² Asn), 6.88 и 7.32 (два с, 4Н, 2 -C(О)NH ₂ ¹ Asn), 6.99 и 7.45, 6.95 и 7.40 (четыре с, 4Н, 2 -C(О)NH ₂ ² Asn), 7.70 (т, ³ J=5.5 Гц, 2Н, -NH(CH ₂ ) ₆ NH-), 8.13 и 8.23 (два м, 4Н, 2 NH ² Asn, 2 NH ¹ Asn).

EXAMPLE 7. Obtaining heptamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-306)

a) N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OH

See example 1 point a.

b) Pentafluorophenyl ester of N-tert-butyloxycarbonyl-L-asparagine, Boc-L-Asn-OPfp

See example 1 point b.

c) Heptamethylenediamide bis-(N-tert-butyloxycarbonyl-L-asparagine), (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₇

Received similarly to example 1 point in.

To a solution of 2.67 g (6.7 mmol) of Boc-L-Asn-OPfp in 20 ml of DMF, a solution of 0.48 ml of 95% heptamethylenediamine in 15 ml of DMF was added with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (40°С), 200 ml of distilled water (43–45°С) was added to the residue and left at room temperature until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), petroleum ether (2×40 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.56 g (92%) chromatographically homogeneous product in the form of a white crystalline powder. R _ƒ 0.87 (A), R _ƒ 0.76 (B), R _ƒ 0.9 (E); m.p. 196-207°C (with decomposition); [α] _D ²⁷ -1.0° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.22 и 1.37 (два уш.с, 28Н, -NHCH ₂ (CH ₂ )sCH ₂ NH-, 2 -ОС(СН ₃ ) ₃ ), 2.29-2.43 (м, 4Н, 2 С ^β Н ₂ Asn), 3.02 (м, 4Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 4.13-4.20 (м, 2Н, С ^α Н Asn), 6.80 (д, 2H, ³ J=7.92 Гц, NH Asn) 6.85 и 7.25 (два с, 4Н, 2 -C(О)NH ₂ Asn), 7.64 (уш.с, 2Н, -NH(CH ₂ ) ₇ NH-).

d) Heptamethylenediamide ditrifluoroacetate bis(H-L-asparagine), 2CF ₃ COOH x (HL-Asn-NH-) ₂ (CH ₂ ) ₇

Received similarly to example 1 item d.

A solution of 1.40 g (2.5 mmol) (Boc-L-Asn-NH-) ₂ (CH ₂ ) ₇ in 30 ml of a mixture of DCM and TFA (1:1) was stirred at room temperature for 2 h, then evaporated, reevaporated with DCM (2×15 ml), the residue was triturated under dry diethyl ether with decantation (2×20 ml) and left under diethyl ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.32 g (90%) chromatographically homogeneous product in the form of a white crystalline substance. R _ƒ 0.18 (A), R _ƒ 0.0 (B), R _ƒ 0.35 (E). ¹ Н-ЯМР (DMSO-d ₆ ): 1.25 и 1.40 (два уш.с, 10Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 2.58 и 2.66 (два дд, 4Н, ² J=17.0 Гц, ³ J=5.12 и ³ J=7.92 Гц, 2 С ^β Н ₂ Asn), 2.99-3.16 (м, 4 Н, -NHCH ₂ (CH ₂ ) ₄ CH ₂ NH-), 3.99 (уш.с, 2Н, 2 С ^α Н Asn), 7.22 и 7.67 (два с, 4Н, 2 -C(О)NH ₂ Asn), 8.10 (уш.с, 6Н, N ⁺ H ₃ Asn), 8.33 (т, 2Н, ^J J=5.5 Гц, NH(CH ₂ ) ₇ NH-).

e) Heptamethylenediamide bis-(N-tert-butyloxycarbonyl L-asparaginyl-L-asparagine), (Boc-Z,-Asn-i-Asn-NH-) ₂ (CH ₂ ) ₇

Received similarly to example 1 item e.

To a solution of 1.32 g (2.17 mmol) of (CF ₃ COOH*HL-Asn-NH-) ₂ (CH ₂ ) ₇ in 20 ml of DMF was added 1.12 ml (6.45 mmol) of DIEA, and 2.20 g (4.77 mmol) of Boc-L-Asn-OPfp was sprinkled with stirring. The reaction mass was stirred for 4 h at room temperature and left overnight without stirring. DMF was evaporated (53°С), reevaporated with water (3×20 ml), 50 ml of distilled water (45°С) was added to the residue and left until a precipitate formed. The precipitate was filtered off, washed with water, acetone (20 ml), hexane (20 ml), dried in vacuum (15 mm Hg) over CaCl ₂ . Received 1.51 g (85%) chromatographically homogeneous product in the form of a white crystalline substance. R _ƒ 0.73 (A), R _ƒ 0.42 (B), R _ƒ 0.88 (E); m.p. 195-200°C (with decomposition). [α] _D ²⁴ -10.2° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.21 и 1.38 (два уш. с.м, 28Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-, 2 -ОС(СН ₃ ) ₃ Boc), 2.34-2.54 (м, 8Н, 4 С ^β Н ₂ ¹ Asn и ² Asn), 3.00 (м, 4Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 4.13-4.20 и 4.38-4.44 (два м, 4Н, С ^α Н ¹ Asn и ² Asn), 6.87 и 7.32, 6.95 и 7.37 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.60 (уш.с, 2Н, -NH(CH ₂ ) ₇ NH-), 7.00 и 8.11 (два д, 4Н, ^J J=7.0 и ³ J=7.82 Гц, 4 NH ¹ Asn и ² Asn).

f) Heptamethylenediamide ditrifluoroacetate bis-(L-asparaginyl-L-asparagine), 2CF ₃ COOH x (HD)-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₇

Received similarly to example 1 item e.

A suspension of (Boc-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₇ (0.504 g, 0.64 mmol) in 30 ml of a mixture of DCM and TFA (1:1) was stirred for 2.5 h; ether (20 ml) for 2 h to form a precipitate. The precipitate was filtered off and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.46 g (88%) of a chromatographically homogeneous product was obtained in the form of a white powder. R _ƒ OAO (A), R _ƒ 0.23 (E); m.p. 165-172°C decomp.; [α] _D ^{24 5} -5.3° (c = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.22 и 1.37 (два уш.с, 4Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 2.42 и 2.52 (два дд, 4Н, ² J=16.0 Гц, ³ J=5.0 и ³ J=7.9 Гц, 4 C ^β H ₂ ² Asn), 2.62 и 2.74 (два дд, ² J=17.0 Гц, ³ J=5.0 и ⁵ J=7.6 Гц, 4 С ^β Н ₂ ¹ Asn), 2.93-3.12 (м, 4 Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 4.05-4.07 (уш. с, 2Н, 2 -C ^a H ¹ Asn), 4.50-4.57 (м, 2Н, 2 С ^α Н ² Asn), 6.90 и 7.36, 7.28 и 7.74 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.83 (т, ³ J=5.6 Гц, 2Н, -NH(CH ₂ ) ₇ NH-), 8.14 (уш.с, 6Н, 2N ⁺ H ₃ , ¹ Asn), 8.60 (д, 2Н, ³ J=7.7 Гц, 2 NH ² Asn).

g) Hexamethylenediamide bis-(N-nioniosuccinyl-L-asparaginyl-L-asparagine), (Suc-L-Asn-L-Asn-NH-) ₂ (CH ₂ ) ₆

Received similarly to example 1 item g.

To a solution of 0.46 g (0.56 mmol) of (CF ₃ COOH*HL-Asn-L-Asn-NH-)2(CH ₂ ) ₇ in 20 ml of DMF was added 0.23 ml (1.30 mmol) of DIEA, then 0.15 g (1.30 mmol) of succinic anhydride was sprinkled with stirring. The reaction mass was stirred at room temperature for 4 h, then left overnight without stirring. DMF was evaporated (53°C), reevaporated with water (3 × 20 ml), 40 ml of acetone was added to the residue and left for 2 h under stirring, the formed precipitate was filtered off, washed with hot MeOH (3 × 10 ml), and dried in vacuum (15 mm Hg) over CaCl ₂ . 0.345 g (78%) of a chromatographically homogeneous product was obtained as a white powder. R _ƒ 0.37 (A), R _ƒ 0.62 (E), R _ƒ 0.07 (B); m.p. 186-194°C (from methanol, with decomposition); [α] _D ²² +3.4° (с = 1, DMSO). ¹ Н-ЯМР (DMSO-d ₆ ): 1.21 и 1.37 (два уш.с, 10Н, -NHCH(CH ₂ ) ₅ CH ₂ NH-), 2.32-2.59 (м, 16Н, 4 С ^β Н ₂ ¹ Asn и ² Asn; 2 -СН ₂ -СН ₂ -СООН Sue), 2.93-3.07 (м, 4 Н, -NHCH ₂ (CH ₂ ) ₅ CH ₂ NH-), 4.36-4.45 (м, 4Н, 4C ^α H ¹ Asn и ² Asn), 6.85 и 7.34, 6.96 и 7.41 (четыре с, 8Н, 4 -C(О)NH ₂ ¹ Asn и ² Asn), 7.57 (т, ³ J=5.5 Гц, 2Н, -NH(CH ₂ ) ₇ NH-), 8.17 и 8.27 (два д, 4Н, ³ J=8.0, ³ J=7.0 Гц, 4 NH ¹ Asn и ² Asn), 12.20 (уш.с, 2 Н, 2 СООН Suc).

Pharmacological activity of dimeric dipeptide mimetics of the 4th loop of neurotrophin-3 (NT3) in experiments in vitro

EXAMPLE 8. Neuroprotective activity of NT3 loop 4 mimetics

The neuroprotective activity of mimetics has been studied in three models:

Fig. 1. A model of oxidative stress on a culture of immortalized NT-22 mouse hippocampal neurons in the presence of hydrogen peroxide.

2. Model of glatamate toxicity on HT-22 cells.

3. Cellular model of Parkinson's disease on a culture of human neuroblastoma cell line SH-SY5Y using the neurotoxin 6-hydroxydopamine (6-OHDA)

The model of oxidative stress in the culture of hippocampal neurons of the NT-22 line was carried out in accordance with the description of Jackson GR et al. [Jackson GR, Werrbach-Perer K, Ezell EL, Post JF, Perez-Polo JR Brain Res. 1992; V. 592 (1): 239-248]. Cells were incubated in the presence of H ₂ O ₂ at a final concentration of 1.5 mm in an atmosphere of 5% CO ₂ for 30 min at 37°C in DMEM containing 5% FBS and 2 mm L-glutamine. Next, the culture medium containing H2O2 was replaced with a normal one, and cell viability was determined 4 h later using the MTT assay.

The model of glutamate toxicity in the culture of NT-22 hippocampal neurons was performed according to Tan S. et al [Tan S, Wood M, Maher P. Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells // Neurochem. 1998; V.71: 95-105]. Glutamate toxicity was modeled by introducing into the culture medium glutamic acid at a final concentration of 5 mM in an atmosphere of 5% CO ₂ for 30 min at 37°C in a DMEM medium containing 5% FBS and 2 mM L-glutamine. The incubation time with glutamic acid was 24 h, after which the medium was changed to normal and cell viability was determined after 24 h using the MTT assay.

A cell model of Parkinson's disease using the neurotoxin 6-hydroxydopamine (6-ONDA) was performed according to Riveles K. et al. [Riveles K., Huang LZ, Quik M. Cigarette smoke, nicotine and cotinine protect against 6-hydroxydopamine-induced toxicity in SH-SY5Y cells // Neurotoxicology. 2008. V.29(3): 421-427]. To induce 6-hydroxydopamine toxicity, 6-OHDA at a final concentration of 100 μM was added to the SH-SY5Y cell culture and incubated for 24 h at 37°C in 5% CO ₂ . after which the medium was changed to normal and cell viability was determined after 24 h using the MTT assay.

Peptides were dissolved in distilled water when heated to 70°C for 1-3 h and introduced 24 h and immediately after cell damage at final concentrations from 10 ^-5 to 10 ^-13 M. NT3 (100 ng/ml) was used as a positive control [Huang EJ, Wilkinson G. A, Farinas I, Backus C, Zang K, Wong S. L, Reichardt L F. Expression of T rk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT3 activation of TrkA and TrkB in addition to TrkC // Development. 1999; V. 126(10): 2191-2203].

Statistical data processing was performed using one-way analysis of variance ANOVA and the Kruskal-Wallis test, followed by Dunn's test. Activity was calculated by the formula А(%) = (D _exp - D _Н2О2 ) / (D _counter - D _Н2О2 ) × 100%, where _Dexp - optical absorption of the solution in the experiment, D _Н2О2 - optical absorption of the active control solution (with damage), D _counter - optical absorption; passive control (no damage).

Dipeptide GTS-301 on the model of oxidative stress significantly increased cell viability in the concentration range of 10 ^-5 -10 ^-12 M when applied 24 hours before peroxide and at concentrations of 10 ^-5 -10 ^-9 M when applied after peroxide (table 2). The activity of GTS-301 was 20-30%, the activity of NT3 was 40-50%. On the model of glutamate toxicity, GTS-301 protected cells from death when applied 24 before glutamic acid in the concentration range of 10 ^-5 -10 ^-8 M and when applied after glutamic acid in the concentration range of 10 ^-5 -10 ^-9 M (Table 2).

At the same time, the activity of the dipeptide when added after glutamic acid at a concentration of 10 ^-6 M (74%) did not differ from the activity of NT3. In the model of cellular Parkinson's disease (6-OHDA-induced toxicity), GTS-301 was also active when administered both 24 hours before the toxin (at concentrations of 10 ^-5 -10 ^-6 M) and when administered after the toxin (at concentrations of 10 ^-5 -10 ^-8 M) (Table 2). The activity of the dipeptide in this model was comparable to that of NT3, and at concentrations of 10 ^-5 and 10 ^-6 M it exceeded it.

The study of the neuroprotective activity of LD, DL and DD diastereomers of the GTS-301 compound (Table 3) showed that only GTS-301 (LD) protects cells from death. The dipeptide was active in the concentration range of 10 ^-5 -10 ^-7 M when it was added 24 hours before the peroxide and at concentrations of 10 ^-6 -10 ^-7 M when it was added after the peroxide. Thus, in vitro experiments revealed the stereospecificity of the neuroprotective effect of GTS-301: when the N-terminal L-Asn was replaced by D-Asn, the activity disappeared.

Compound GTS-302 showed neuroprotective activity in models of oxidative stress and glutamate toxicity only when applied after the toxin, at concentrations of 10 ^-6 -10 ^-10 M and 10 ^-6 -10 ^-7 M, respectively (Table 4). In the model of oxidative stress, GTS-302 showed relatively weak activity (8-14%), and in the model of glutamate toxicity, its activity (81%) did not differ from that of NT3.

In the model of 6-OHDA- ^induced toxicity, GTS- ³⁰² was active both when applied 24 hours before the toxin (at concentrations of ^{10–6–10–7} M) and when applied after the toxin (at concentrations of ^{10–5–10–7} M). The activity of the dipeptide at a concentration of 10 ^-6 did not differ from the activity of NT3 in both schemes of the experiment (table 4).

The neuroprotective activity of the dipeptide GTS-304 was revealed in the model of oxidative stress (Table 5). The compound protected cells from death at concentrations of 10 ^-6 -10 ^-8 M when applied 24 hours before peroxide and at concentrations of 10 ^-5 -10 ^-8 M when applied after peroxide.

Compound GTS-306 showed neuroprotective activity in the model of oxidative stress when applied only 24 hours before peroxide in the concentration range of 10 ^-6 -10 ^-9 M (table 6).

Thus, the dipeptide mimetics of the 4th loop of neurotrophin NT3 in vitro experiments revealed neuroprotective activity in the concentration range of 10 ^-5 -10 ^-10 M.

The compound GTS-301 was found to have a stereospecific neuroprotective effect.

Pharmacological activity of dimeric dipeptide mimetics of the 4th loop of neurotrophin-3 (NT3) in experiments in vivo

EXAMPLE 9 Antidepressant-like activity

The study was carried out on male mice of the BALB/c line weighing 20-22 g obtained at the Stolbovaya Branch of the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency". The animals were kept under a natural change in the light regime with free access to standard food and water. Experiments with animals were carried out in accordance with the Decision of the Council of the Eurasian Economic Commission dated November 3, 2016 No. 81 "On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the field of circulation of medicines." The experiments were approved by the Commission on Biomedical Ethics of the Federal State Budgetary Scientific Institution “Research Institute of Pharmacology named after N.N. V.V. Zakusova.

NT3 mimetics were administered intraperitoneally (ip) as a suspension in a 1% aqueous solution of Tween 80. Control animals received a 1% aqueous solution of Tween 80. The classic tricyclic antidepressant amitriptyline was used as a reference drug. Amitriptyline was administered i.p. in saline at a dose of 10 mg/kg. The dose of amitriptyline was chosen according to the literature [Abdelhamid, R.E, Kovacs, K.J, Nunez, M.G. & Larson, A.A. Depressive behavior in the forced swim test can be induced by TRPV1 receptor activity and is dependent on NMDA receptors. Pharmacol. Res. 2014; V79:21-27]. The volume of administration was 10 ml per 1 kg of mice body weight.

Antidepressant-like activity was studied in the forced swimming test. The test setup consisted of 5 transparent plastic cylinders with a diameter of 10 cm and a height of 30 cm, between which black plastic opaque partitions were installed to prevent visual contact between the animals during the study. Behind the cylinders, a black plastic panel is installed as a background. The cylinders were filled with water at a temperature of 22-23°C to 2/3 of the height - the level at which the animals are not able to rest on the bottom of the cylinder with their paws or tail. Animals were placed in cylinders filled with water for 10 min. After 24 hours, a test landing was carried out - the animals were repeatedly placed under the same conditions for 5 minutes. The experiment was recorded on a video camera, the resulting video materials were processed in the program of Open Science Research and Production Company LLC (Moscow, Russia) with the calculation of the total immobility time (refusal of active-defensive and exploratory behavior).

Experiment design. Animals were randomly divided into groups of 8-10 mice. NT3 mimetics were administered according to two schemes - acutely or subchronically (7 days). In the first case, the mimetics were administered 1 hour after the first landing in cylinders with water and 24 hours before the second landing (amitriptyline was administered 1 hour before the second landing). In the case of subchronic administration, the first landing in the cylinders was carried out 24 hours after the last administration of mimetics or amitriptyline.

Statistical data analysis was performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). The data were checked for normal distribution using the Shapiro-Wilk test. To identify intergroup differences, one-way analysis of variance (one-way ANOVA) was used, followed by pairwise intergroup comparisons using Dunn's test. Differences were considered statistically significant at p<0.05. Data are presented as means and standard errors of the mean.

9.1. Antidepressant-like activity of GTS-301 and its LD, DL, DD diastereomers

The NT3 mimetic GTS-301 showed no activity in the acute forced swimming test in the dose range of 1-20 mg/kg (Table 7).

With subchronic administration, GTS-301 (Table 7) significantly reduced the immobility time of mice at doses of 10, 20 and 40 mg/kg by 22, 26 and 26%, respectively, indicating its antidepressant-like activity. The activity of GTS-301 is comparable to that of amitriptyline (10 mg/kg), which reduced the immobility time by 27%.

GTS-301 (LD) showed antidepressant-like activity both at acute administration at doses of 10 and 20 mg/kg and at subchronic administration at doses of 20 mg/kg, significantly reducing the immobility time of mice with effects (24% in both cases) comparable to those of amitriptyline (22%) (Table 7).

GTS-301 (DL) and GTS-301 (DD) showed no antidepressant-like effects in the acute forced swimming test in the dose range of 1-20 mg/kg (Table 7). GTS-301 (DL) showed no activity even after subchronic administration at a dose of 20 mg/kg.

Thus, GTS-301 exhibited antidepressant-like activity in the forced swimming test in BALB/c mice.

As in the case of neuroprotective activity in vitro, the stereospecificity of the antidepressant-like activity of GTS-301 was shown: when switching from the LL isomer (GTS-301) to the DL or DD isomers, the activity disappeared.

9.2. Antidepressant-like activity of the GTS-302 compound

The GTS-302 dipeptide exhibited antidepressant-like effects upon acute administration at doses of 1, 5, and 10 mg/kg (ip), statistically significantly reducing the immobility time in BALB/c mice in the forced swimming test by 19, 17, and 22%, respectively (Table 8).

9.3. Antidepressant-like activity of GTS-304 compound

The GTS-304 dipeptide exhibited antidepressant-like effects upon acute administration at doses of 5 and 10 mg/kg, reducing the immobility time in BALB/c mice in the forced swimming test by 27% and 39%, respectively (Table 9).

Thus, dipeptide mimetics of the 4th loop of NT3, compounds GTS-301, GTS-302, and GTS-304, showed antidepressant-like activity when administered intraperitoneally in the forced swimming test in mice. The dipeptide GTS-301, when administered subchronically at doses of 10-40 mg/kg, had effects comparable to those of the classical antidepressant Amitriptyline.

EXAMPLE 10. Antidiabetic activity of the compound GTS-301

The studies were performed on male mice of the C57 B1/6 line (n=60) with an initial body weight of 16-20 g obtained from the Federal State Budgetary Scientific Institution "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency" (Stolbovaya branch). The animals were kept under standard vivarium conditions with free access to food (with the exception of 16 hours prior to the administration of streptozotocin) and water. Experiments with animals were carried out in accordance with the Decision of the Council of the Eurasian Economic Commission dated November 3, 2016 No. 81 "On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the field of circulation of medicines." The experiments were approved by the Commission on Biomedical Ethics of the V.V. Zakusov.

Experiment design. Streptozotocin diabetes is a widely accepted experimental model for diabetes mellitus. Experimental diabetes in mice was induced by a single intraperitoneal (ip) administration of streptozotocin (STZ) at a dose of 110 mg/kg after 16 h of fasting. The STZ solution was prepared in citrate buffer (pH 4.5).

GTS-301 at doses of 0.5 and 5.0 mg/kg was administered intravenously as a suspension in a 1% aqueous solution of Tween 80 for 14 days before and 17 days after induction of diabetes. Control animals received a 1% aqueous solution of Tween 80.

Mice were randomly divided into the following groups:

(1) "Control" (mice were injected with a 1% solution of Tween 80 for 31 days);

(2) "Diabetes" (mice were injected with 1% Tween 80 solution for 14 days, STZ was administered on the 15th day, and then continued with 1% Tween 80 solution for 17 days);

(3) "Diabetes + GTS-301 (0.5 mg/kg)" (mice were injected with GTS-301 at a dose of 0.5 mg/kg for 14 days, STZ was administered on the 15th day, and then GTS-301 for 17 days);

(4) "Diabetes + GTS-301 (5 mg/kg)" (mice were injected with GTS-301 at a dose of 5 mg/kg for 14 days, STZ was administered on the 15th day, and then GTS-301 for 17 days);

(5) and (6) - groups "GTS-301, 0.5" and "GTS-301, 5 mg/kg" mg/kg, respectively. Mice were injected intravenously with GTS-301 at a dose of 0.5 or 5.0 mg/kg for 31 days.

The level of glucose in the blood taken from the tail vein of mice was measured using a One Touch Select Plus Flex glucometer (Switzerland) before and on the 5th day after the administration of STZ. The body weight of mice was measured every 3-4 days, and the intake of food and water - daily.

Statistical data processing was carried out using the Statistica 10 program. The Shapiro-Wilk test was used to assess the normality of the distribution. Because the mouse blood glucose data were not normally distributed, a nonparametric Mann-Whitney test with Bonferroni correction was used to detect between-group differences. Fisher's exact test was used to assess differences between groups in the number of sick animals and in the amount of water and feed consumed. A p<0.05 probability value was considered statistically significant.

Introduction GTS-301 for 14 days and 20 days at doses of 0.5 and 5.0 mg/kg did not cause changes in blood glucose levels in healthy animals (table 10). The introduction of STZ at a dose of 110 mg/kg caused a pronounced increase in glucose levels. Animals were considered diabetic when reaching a threshold glucose level of 13 mmol/l [Watcho P, Stavniichuk R, Tane P. et al. Evaluation of PMI-5011, an ethanolic extract of Artemisia dracunculus L, on peripheral neuropathy in streptozotocindiabetic mice // Int J Mol Med. 2011. V. 27(3): 299-307]. In the group of diabetic mice that were injected with GTS-301 at a dose of 0.5 mg/kg on the 5th day after the administration of STZ, there was a significant decrease in hyperglycemia compared with the group of untreated animals (Table 10). Fisher's exact test was used to assess the significance of differences in the number of animals with diabetes mellitus (blood glucose ≥13 mmol/l). It was shown that in the group "Diabetes + GTS-301 (0.5 mg/kg)" the number of sick animals was statistically significantly lower (4 out of 11 mice) than in the group "Diabetes" (12 out of 13 mice). GTS-301 at a dose of 5.0 mg/kg did not reduce hyperglycemia.

Continued administration of GTS-301 at doses of 0.5 and 5 mg/kg (groups 5 and 6) for 20 days had no effect on blood glucose levels in healthy mice.

The introduction of GTS-301 in both doses studied had no effect on the body weight of the animals before the administration of STZ, as well as on the 5th day after the administration of STZ.

One of the important indicators of diabetes is polydipsia. In the animals of the "Diabetes" group, the daily water intake increased 3 times compared to the water intake before the induction of diabetes (Table 11), and also compared to the control group (Fisher's exact test).

In diabetic animals treated with GTS-301 at a dose of 0.5 mg/kg, daily water intake increased by 2 times compared with water intake before diabetes induction; however, it was statistically significantly different from the group of untreated diabetic animals and did not differ from the control group.

In diabetic animals treated with GTS-301 at a dose of 5.0 mg/kg, daily water intake increased by almost 6 times compared to water intake before diabetes induction, and was statistically significantly different both from the group of diabetic untreated animals and from the group of healthy animals.

There were no differences in food intake on the 5th day after diabetes induction between the groups.

The totality of the data obtained indicates that the NT3 mimetic GTS-301, when administered chronically intraperitoneally at doses of 0.5 and 5.0 mg/kg, does not affect the blood glucose level in healthy animals. At the same time, at a dose of 0.5 mg/kg, GTS-301 exhibits hypoglycemic activity, reduces the number of sick animals (with blood glucose levels ≥13 mmol/l) and normalizes water consumption in mice with CT3-induced diabetes.

Thus, the compound GTS-301, when administered chronically at a dose of 0.5 mg/kg, exhibited antidiabetic effects in a mouse model of streptozotocin diabetes, exerting an antihyperglycemic effect and normalizing increased water intake.

EXAMPLE 11 Compounds GTS-302 and GTS-304 do not affect the pain response threshold upon stimulation of nociceptors

The experiments were performed on inbred male mice of the C57 B1/6 line weighing 18–22 g (n=76), obtained from the Federal State Budgetary Scientific Institution “Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency” (Stolbovaya branch). Animals were kept 15 individuals in a T/3 standard cage in a vivarium under natural light with an air temperature of 21–23°C and a relative humidity of 40–60%, free access to water and briquetted food for 10 days prior to testing. The keeping of animals complied with the rules of good laboratory practice when conducting preclinical studies in the Russian Federation in accordance with the Decision of the Council of the Eurasian Economic Commission dated November 3, 2016 N 81, GOST 33215-2014 and GOST 33216-2014. The experiments were approved by the Commission on Biomedical Ethics of the V.V. Zakusova.

GTS-302 and GTS-304 were injected i.p. as a suspension in a 1% aqueous solution of Tween 80 at doses of 1-10 mg/kg once at a rate of 0.1 ml/10 g of animal weight 30 min before testing. Animals of the control groups received water for injection with Tween-80.

The hot plate test was used to assess the nociceptive response. Using an analgesimeter "Ugo Basile" (Italy), the latent reaction time (licking the hind paw or jumping) was recorded. Animals were selected 2 hours before the start of the experiment on the basis of basic reactivity under the conditions of the experimental model, excluding mice that remained on the plate heated to 55±0.5°C for more than 10 s. A latent period of 20 s (maximum exposure time) was regarded as 100% analgesia. The time of appearance of the reaction in mice was recorded 30, 60, 90, and 120 min after a single injection of the studied compounds. The results obtained were expressed as the maximum possible effect (MPE) in %. MRE = (reaction latency after drug administration minus background reaction latency)/(maximum exposure time minus background reaction latency) X 100%.

Statistical processing of the obtained results was carried out using one-way analysis of variance (ANOVA) and subsequent pairwise intergroup comparisons using Duncan's test. Data are presented as means and standard error of means Mean ± SEM. Critical significance level p=0.05.

The average baseline level of nociceptive response for C67B 1/6 mice in the "hot plate" test was 6.5±0.5 s. Animals with a latent reaction period of more than 10 s were not included in the experiment.

When modeling acute somatic pain, it was found that after 30, 60, 90 and 120 minutes after the administration of the compounds, the pain reaction thresholds in the experimental groups did not differ from the control ones (Table 12). The data obtained indicate that GTS-302 and GTS-304 in the studied dose range with systemic administration do not change the threshold of pain response to thermal stimulation of nociceptors, which confirms the absence of hyperalgesia with the introduction of low molecular weight neurotrophin-3 mimetics, as in full-sized neurotrophin-3 [X-Q Shu, A Llinas, L M Mendell Effects of trkB and trkC neurotrophin receptor agonists on thermal nociception: a behavioral and electrophysiological study // Pain. 1999; V 80(3): 463-470].

It is known that one of the main side effects of neurotrophins is hyperalgesia [Pezet S, McMahon SB. Neurotrophins: mediators and modulators of pain. Annu Rev Neurosci. 2006;29:507-538]. In our in vivo experiments, we have proved the absence of nociceptive and antinociceptive properties of neurotrophin-3 loop 4 mimetics GTS-302 and GTS-304 in modeling acute somatic pain at the supraspinal level. The results obtained indicate that the studied compounds do not cause hyperalgesia, which is characteristic of full-sized neurotrophins.

EXAMPLE 12. Anxiolytic activity of the compound GTS-302

Experiments were performed on inbred male BALB/c mice (n=40) with a genetically determined passive response to emotional stress [Carola V, D'Olimpio F, Brunamonti E, Mangia F, Renzi P. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice // Behav Brain Res. 2002; V.134(1-2): 49-57] with a body weight of 18-22 g (FSBSI "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency", branch "Stolbovaya"). The animals were kept in T/3 standard cages under vivarium conditions (temperature 21–23°C, relative air humidity 40–60%) with natural light and free access to water and briquetted food for 10 days prior to testing. The content of animals corresponded to GOST 33215-2014 and GOST 33216-2014. The experiments were approved by the Commission on Biomedical Ethics of the V.V. Zakusov.

Emotional stress was modeled in the elevated plus maze test (EPM). PKL devices for mice (NPK Open Science, Moscow, Russia) are made of gray plastic and have opaque side walls painted in the color of the floor. The labyrinths were raised to a height of 38 cm. Each animal was placed in the center of the maze for 300 s. Registered the following indicators: the time spent in the open sleeves; number of entries into open arms; number of entries into closed arms. The total motor activity was calculated as the sum of visits to the open and closed arms of the maze. The anxiolytic effect was assessed by the increase in the duration of stay in the open arms and the number of animal entries into the open arms of the labyrinth. The indicators “time in open arms” and “number of entries into open arms” were compared in addition to absolute values in relative units (in %), which were calculated by the formula:

relative time in open arms, % = (time in open arms in sec/300 sec) × 100%; relative number of entries into open arms, % = (number of entries into open arms/total number of entries into open and closed arms) × 100%.

Statistical processing of the results was performed using one-way analysis of variance (ANOVA) followed by Duncan's test. Differences were considered statistically significant at p<0.05. Data are presented as means and standard error of the means (mean ± SEM).

GTS-302 was administered i.p. as a suspension in a 1% aqueous solution of Tween 80 at doses of 1.0, 5.0 and 10.0 mg/kg, i.p. 30 min before testing in PCL at the rate of 0.1 ml/10 g of animal weight. Animals of the control groups received water for injection with Tween-80.

In the PCL test, the compound GTS-302, when administered acutely, prevented the development of novelty anxiety in BALB/c mice in a dose-dependent manner. The most pronounced effect of GTS-302 was demonstrated at the minimum studied dose of 1.0 mg/kg, statistically significantly increasing the residence time by 1.9 times and the number of exits into the open arms of the maze by 2.7 times compared with the control group, with no effect on the number of entries into closed arms and general motor activity (Table 13). GTS-302 at doses of 5.0 and 10.0 mg/kg did not affect the time spent in the open arms, but increased by 1.9 and 2.1 times, respectively, the number of exits into the open arms of the maze in BALB/c mice, which indicates the formation of a stable anxiolytic effect upon administration of the neurotrophin-3 mimetic.

Thus, it has been established in vivo experiments that GTS-302, a mimetic of the 4th loop of neurotrophin-3, has an anxiolytic effect in highly emotional BALB/c mice after a single injection.

EXAMPLE 13. Antiaddictive effects of the compound GTS-302

The experiments were performed on outbred white male rats weighing 230-250 g (n=40) (FSBSI "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency", branch "Stolbovaya"). Animals were kept 6 individuals per cage in a vivarium (temperature 21-23°C, relative humidity 40-60%) with natural light and free access to water and briquetted food for 10 days prior to testing. The content of animals corresponded to GOST 33215-2014 and GOST 33216-2014. The experiments were approved by the Commission on Biomedical Ethics of the V.V. Zakusov.

Morphine hydrochloride (Minmedbioprom Association "Chimkentbiopharm", substance) was dissolved in water for injection and administered intraperitoneally (ip) at the rate of 0.1 ml/100 g of rat body weight. GTS-302 was administered i.p. as a suspension in a 1% aqueous solution of Tween 80 at doses of 0.1-10.0 mg/kg at the rate of 0.1 ml/100 g of animal weight.

The formation of opiate dependence and the assessment of somatic manifestations of morphine withdrawal syndrome in rats were carried out in accordance with the scheme described earlier [Kolik L.G, Konstantinopolsky M.A. Comparative assessment of the effectiveness of noncompetitive NMDA receptor antagonists amantadine and hemantane in morphine withdrawal syndrome model // Bulletin of Experimental Biology and Medicine. 2019; T. 166(6): 739-743].

To obtain animals dependent on morphine, the drug was administered to animals in increasing doses (10-20 mg/kg) 2 times a day with an interval of 8 hours for 5 days:

1st day in total - 10 and 10 mg / kg,

2nd day - 10 and 20 mg/kg,

3rd day - 20 and 20 mg / kg,

4th day - 20 and 20 mg/kg,

5th day - 20 mg/kg.

On the 5th day of the experiment, 5 hours after the last injection of morphine and 60 minutes before testing, GTS-302 was administered at doses of 0.1 (n=10); 1.0 (n=10) and 10.0 (n=10) mg/kg ip, and animals from the active control group (n=10) - water for injection in an equivalent volume. Animals were tested for specific signs of morphine withdrawal syndrome (SD) for 5 min in an “open field” (illuminated round arena with a diameter of 80 cm) 15 min after administration of the opiate receptor antagonist naloxone (Du Pont De Nemours Int. S.A, Swiss) at a dose of 1.0 mg/kg to provoke SO. For all groups, specific signs of morphine SD were recorded (17 indicators). Discrete signs of abstinence (diarrhea - in points, shaking and grinding of teeth - by the number of acts) were evaluated quantitatively and alternatively, the rest - in an alternative form according to the principle "yes" - "no". The total index (SI) of OS severity for each animal and the average values for the experimental and control groups were calculated on the basis of alternative signs with the maximum possible SI value equal to 17 points. The mean value of OS severity in the morphine group (active control) was taken as 100%.

Statistical processing of the obtained results was carried out using one-way analysis of variance (ANOVA) followed by Dunnett's test. Differences were considered statistically significant at p<0.05. Data are presented as means and standard error of the means (mean±SEM).

The results show that GTS-302 has a significant dose-dependent effect on specific behavioral measures of withdrawal in morphine-treated rats (Table 14).

In comparison with the active control group, the decrease in the total SD index of morphine for the "GTS-302 0.1 mg/kg" group was 26.9%, for the "GTS-302 1.0 mg/kg" group - 41.4% and for the "GTS-302 10.0 mg/kg" group - 36.5%. The effect of GTS-302 was most pronounced at a dose of 1.0 mg/kg. Assessing the correction of individual manifestations of morphine SR, it should be noted a decrease in the severity or complete elimination of such indicators as posture disorder (p<0.001), shaking off the “wet dog” (p<0.05), vocalization (p<0.05) and head shaking (p<0.01). Despite the absence of statistically significant differences, one can note a tendency to weaken withdrawal symptoms such as diarrhea, ptosis, dyspnea and convulsions with an increase in the dose of GTS-302 (Table 14).

There were no significant changes in locomotor activity during 5 min in the OP, vertical activity ("racks"), the number of acts of grooming and defecation, as well as the effect on such signs of morphine withdrawal as rhinorrhea, piloerection, flight, stereotypy and writhing in animals treated with GTS-302, in comparison with the control group.

Thus, the ability of a low-molecular analogue of neurotrophin-3 to dose-dependently reduce the value of the total index of morphine withdrawal syndrome without affecting the overall motor activity has been experimentally proven.

Claims (10)

1. Compounds of the general formula (R-CO-L-AK-X-Asn-NH-) ₂ (CH ₂ ) ₆ , where R = HOOS-(CH ₂ ) ₂ -, or HOOS-(CH ₂ ) ₃ -, or HO-(CH ₂ ) ₃ -, AK = Glu or Asn, X = L - or D - configuration. 2. The compound according to claim 1, characterized in that R = HOOC-(CH ₂ ) ₂ -, AK = Asn, X = L. 3. The compound according to claim 1, characterized in that R = HOOC-(CH ₂ ) ₂ -, AK = Asn, X = D. 4. The compound according to claim 1, characterized in that R = HOOC-(CH ₂ ) ₃ -, AK = Asn, X = L. 5. The compound according to claim 1, characterized in that R = HO-(CH ₂ ) ₃ -, AK = Glu, X = L. 6. A method for the treatment of acute and chronic neurodegenerative diseases such as stroke, cerebral ischemia, Alzheimer's disease, Parkinson's disease, including the introduction of an effective amount of the compound according to any one of paragraphs. 2-5. 7. A method for the treatment of depression, including the introduction of an effective amount of a compound according to any one of paragraphs. 2-5.