According to WHO, the acquired secondary form of hematopoietic-depressive states increases the risk of death in people with cancer, infectious, and hormonal diseases. The choice of drugs that stimulate the proliferative activity of bone marrow cells is limited. The stimulus to the search for hematopoietic-stimulating compounds among pyridine derivatives was the manifestation of the activity of the cerebroprotective drug Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate) to restore the granulocytes and B-lymphocytes homeostasis in clinical practice. The hematopoietic-stimulating activity of the newly synthesized compound Complex of 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo [4,3-c]pyridine complex with β-cyclodextrin (BIV) was studied on a model of cyclophosphamide-induced myelodepression in C57BL6/J mouse line. The BIV compound demonstrated a stimulating effect on the process of restoring the level of Ly-6G+ Ly-6C+ granulocytes, monocytes/macrophages, CD19+ B-lymphocytes in the bone marrow, exceeding the activity of the reference drug Methyluracil. The BIV compound did not affect on the proliferative activity of c-kit (CD117+)-expressing cells, CD3e+ pre-T-lymphocytic cells and Ter-119+ erythroid cells in the bone marrow. The total number of bone marrow myelokaryocytes in the experimental groups was significantly higher than in the intact group. The pronounced hematopoietic-stimulating effect of the BIV compound gives in the future the prospect of development as a drug used for the treatment of B-lymphocytic and granulocytic-macrophage hematopoietic-depressive states.
Research Article
Improvement of myelopoiesis in a cyclophosphamide-induced myelodepression model by administration of a Newly Synthesized Pyrazolo- Piperidine β-Cyclodextrine complex compound
https://doi.org/10.21203/rs.3.rs-1433939/v1
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5-benzyl-7-(o-fluorobenzylidene)-2
3-bis(o-fluorophenyl)-3
3a
4
5
6
7-hexahydro-2H-pyrazolo[4
3-c]pyridine complex with β-cyclodextrin · hematopoietic-stimulating activity · sodium cyclophosphamide
According to WHO, the acquired form of hematopoietic-depressive states increases the risk of death in people with cancer, infectious, and hormonal diseases. The secondary cytopenic syndrome occurs for a number of reasons: work at mining enterprises, living in ecologically unfavorable areas, the use of herbicides, pesticides and insecticides in agriculture, the use of equipment with high radiation in diagnostic and therapeutic practice, the use of cytostatic drugs in therapeutic practice, trauma, extensive surgical operations, abuse of food additives marked with the letter E (Aziz et al. 2010; Makheja et al. 2013). Also, secondary cytopenic syndrome develops secondary to the underlying disease (diabetes, kidney disease, malignant neoplasms). Cytopenic syndrome is expressed by erythropenia, thrombocytopenia and leucopenia. Clinical manifestations of leucopenia are expressed in the form of frequently recurrent or chronic infectious and inflammatory processes of the respiratory, genitourinary, gastrointestinal, and cardiovascular systems, eyes, skin and soft tissues, sometimes caused by opportunistic microorganisms. In 63% of the reasons of hematopoietic-depressive syndromes are disorders of proliferative activity of the bone marrow (Fu et al. 2016; Setdikova et al. 2006; Luss et al. 2013). Timely treatment of secondary cytopenic syndrome prolongs the life of patients. Therefore, there is a growing interest in drugs that stimulate hematopoiesis. But the range of drugs used is limited to hormonal, peptide, cytokine preparations and vitamin-mineral complexes.
Drugs that stimulate leukopoiesis are divided into nucleic acid derivatives, peptide and cytokine drugs. Preparations based on sodium deoxyribonucleate: Sodium nucleate, Sodium nucleinate, Sodium nucleospermate, Pentoxyl (4-Methyl-5-oxymethyluracil)). These were the first drugs approved for medical use. Based on these drugs, a number of synthetic drugs have been developed: Poludan (Polyadenilic acid + Polyuridilic acid), Inosine pranobex (inosine acedoben dimepranol), Methyluracil (2,4-dihydroxy-6-methylpyrimidine) and Inosine. They contribute the activation of proliferative activity of bone marrow cells, but also contribute to the intensive division of bacterial microflora, cancer cells in metastasized foci of the body, so they are prohibited for use in many countries (Klinman et al. 1996).
Peptide preparations (Filgrastim (N-L-Methionyl genetically engineered human colony-stimulating factor), Molgramostim (recombinant granulocytic-macrophage colony-stimulating factor), Lenograstim (human glycosylated granulocyte colony stimulating factor), Pegfilgrastim (recombinant human granulocyte colony-stimulating factor), Lipegfilgrastim (conjugate of genetically engineered human colony-stimulating factor (filgrastim) with monomethoxypolyethylene glycol), Empegfilgrastim (1-(N-{4-[omega-methoxypoli(oxyethylene)]butyl}-L-methionine)) human granulocyte colony-stimulating factor), based on the content of colony-stimulating factors, are able to stimulate and regulate bone marrow hematopoiesis, regulate immune responses and angiogenesis. But they cause uncontrolled pyrogenic reactions of the body, "bone" pains, dose selection is carried out and used strictly in clinical practice under the supervision of a doctor. It is quite obvious that cytokines have a huge potential and can claim to be the most effective medicines. But cytokines can cause cascading uncontrolled immune reactions(Liu et al. 2010: Lundqvist et al. 2013). Cytokines can lead to septic shock, decreased contractility of the myocardium and vascular smooth muscle cells, increased permeability of the endothelium, impaired microcirculation in organs, hypotension and hypoglycemia (Aiello et al. 2007; Azuma 1992). Vitamin and mineral preparations should be classified as dietary supplements. Some synthetic drugs have activity that stimulate leucopoiesis, but also have a wide range of side effects and high toxicity (Marcova, 2019).
Currently, there is an interest in synthetic compounds with a heterocyclic core. Heterocyclic nuclei form the basis for the construction of numerous homological series containing hydrocarbon residues in the form of side chains, as well as various functional groups. The most important are pyrrol, pyrazole, imidazole, pyridine, pyrimidine, piperidine, furan, thiophene, indole, quinoline, purine, benzimidazole, etc (Dambaeva et al. 2003; Eryanti et al. 2015; Holt et al. 2005; Purygin et al. 2004). Heterocyclic compounds include alkaloids - nitrogen-containing physiologically active substances of plants. Among them there are strong poisons (strychnine, nicotine) and important medicines (Quinine, Reserpine). Of the registered medicines, 6% are compounds with a pyridine ring and its derivatives. These are anti-tuberculosis, antihypertensive, antihistamines, cardiovascular drugs, antidotes of toxic organophosphorus compounds, neuroleptics and other drugs. The prerequisite for the synthesis of a new compound was the manifestation of leucopoiesis-stimulating activity with the drug Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate). Mexidol is a nootropic, cerebroprotective, stress-protective, antihypoxic, antioxidant, anticonvulsant, anxiolytic and membrane-protective drug. When used in postoperative practice, an increase in the proportion of examined patients with vegetative equilibrium was revealed (from 62.86–88.57%; p < 0.05), normalization of vegetative reactivity (from 8.57–34.29%; p < 0.05), blood leukogram analysis showed effective positive dynamics in restoring granulocytes and B-lymphocytes, an increase in IgM indicators (from 1.89 g/l to 2.12 g/l; p < 0.05) with a simultaneous tendency to increase IgA and IgG (Kabdualiev et al. 2007; Tolkacheva et al. 2021).
The aim of this work was to study the hematopoietic-stimulating activity of the newly synthesized compound Complex of 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo [4,3-c]pyridine complex with β-cyclodextrin (BIV) on a model of cyclophosphamide-induced myelodepression in C57BL6/J mouse line.
Chemical methods
The course of the reaction and the individuality of the compounds were controlled by TLC on aluminum oxide of the III degree of activity with the appearance of spots with iodine vapor. IF (FT-IR) spectra were registered on a «Nicolet 5700 FT-IR» spectrometer. The 1H and 13C NMR spectra were obtained using a JNM-ECA Jeol 400 spectrometer (frequency is equal to 399.78 and 100.53 MHz, respectively) using CDCl3 solvent. Chemical shifts were measured relative to the signals of residual protons or carbon atoms of deuterated chloroform (δН 7.26, δС 77 m.d. (t)).
Biology methods
Animals
Mice C57BL6/J, acquired in the Charles River Laboratory (USA) and propagated at the Kazakh Scientific Center for Quarantine and Zoonotic Infections named after Aikimbayev. Female mice with body weight of 18–23 g and 5–7 weeks of age were selected for research. The animals were bred and maintained in a pathogen free barrier facility.
Experimental groups
Intact group, BIV group, Control group, and Methyluracil group were used in the experiments. Each group consisted of six mice. All animals were kept at a temperature of 21–23ºC, in standard polypropylene cages with free access to standard food and water.
The cytostatic drug sodium cyclophosphamide (Baxter Oncology GmbH) was dissolved in 0.9% saline solution and injected intramuscularly to three treated groups of mice (BIV group, Methyluracil group, and Control group) in a dose of 100 mg/kg, in a volume of 0.2–0.24 ml (1% solution), at 9:00 am, every other day, three injections (1st, 3rd, 5th day of the experiment). Then, on the 8th, 10th and 12th day of the experiment, at 9:00 am: BIV group animals were injected with the BIV compound (BIV compound was dissolved in 0.9% saline solution and administered intramuscularly at a dose of 10 mg/kg, in a volume of 0.2–0.24 ml (0.1% solution)), Methyluracil group animals were injected with Methyluracil as a comparative compound (Methyluracil was dissolved in 0.9% saline solution and administered intramuscularly at a dose of 10 mg/kg, in a volume of 0.2–0.24 ml (0.1% solution)), and Control group animals were administered with similar volumes (0.2–0.24 ml) of 0.9% saline, Intact group was consisted of intact animals (Fig. 1). After 7 days, the hind limb bones was surgically removed from anesthetized animals sacrificed by decapitation. The studies were performed in accordance with the Order of the Minister of Healthcare of the Republic of Kazakhstan No. KR DSM-181/2020 of November 4, 2020 and by the protocol of the Local Ethics Commission of the Al-Farabi Kazakh National University No. 20136/10 dated June 12, 2020. The validity period is 3 years.
Preparation of phosphate-buffer saline (PBS) and column buffer
PBS was used to wash the cells and prepare the column buffer solution. To prepare the PBS solution, NaH2PO4 and NaCl were dissolved in purified water, and NaOH was used to achieve pH 7.4. To prepare the column buffer, 0.5% FBS and 0.002 M EDTA were added to the PBS solution. All obtained solutions were passed through a sterile 0.22 µm membrane filter and stored in the refrigerator at 4–8ºC until use.
Bone marrow cellularity assessment
The bones of the hind limb were surgically removed from the muscles, the bone marrow was washed with a PBS solution using a 10-milliliter syringe. Obtained cell suspension was filtered through a 30 µm membrane and washed twice by centrifugation for 10 minutes. Lysing solution with pH 7.2–7.4 was used for lysis of erythrocytes. Cells were counted and their viability was determined using trypan blue.
Flow cytometry
The following mouse monoclonal antibodies (mAbs) were used for surface staining: Per CP-labeled anti-Ly6G, PE-labeled anti-Ly6C, Per CP-labeled anti-CD19, PE-labeled anti-CD3e, Per CP-labeled anti-Ter119, APC-labeled anti-CD117. Briefly, 106 cells were incubated with mAbs specific for surface markers according to the manufacturer’s protocols and then fixed with Fixation solution (cat. No 554722, BD Biosciences) for 20 min in dark at room temperature. Afterward, the cells were washed with PBS, re-suspended in flow solution, and immediately analyzed by flow cytometry on a FACSCalibur using CellQuest Pro software (BD Biosciences). Unstained cells, single fluorochrome stained cells, and cells stained as fluorescence minus-one controls were used to set-up the flow cytometer. Multiparameter data were analyzed as described previously (Carleton and Nicholson, 2000).
Graphics methods
The ChemDraw software solution from Perkin Elmer Informatics for chemists and scientists was used to reproduce the unique chemical structures of compounds. The GraphPad Software Prism 9.2.0 software solution was used to reproduce graphs.
Statistical analysis
The obtained data were processed by mathematical statistics methods using Microsoft Excel and "Statistica 6.0" software. The data are shown as mean value (M) ± standard deviation (SD) (n = 6 mice/group) from three independent experiments. Student's t-test was used to determine statistically significant difference and p < 0.05 was considered significant.
Chemical study results
1-(Benzyl)-3,5-bis(o-fluorobenzylidene)piperidone-4 (II). 4.2 g (0.105 mol) of NaOH was placed in 40 ml of water and 40 ml of ethanol in a flask equipped with a reflux condenser, a thermometer, a stirrer and a dropping funnel. Half of the freshly prepared mixture was poured 4.0 g (0.021 mol) of 1-(benzyl)-4-oxopiperidine (I) and 5.3 g (0.043 mol) of o-fluorobenzaldehyde. After 15 minutes, the second half of the mixture was added. The reaction was carried out at a temperature of 20–25 ° C. The reaction mixture was stirred for 4 hours. The precipitate was filtered, washed with water to a neutral state, dried and recrystallized from hexane. 1-(benzyl)-3,5-bis(o-fluorobenzylidene)piperidone-4(II) was obtained with a yield of 8.0 g (95%), Rf 0.43 (eluent: benzene: dioxane = 7:1), which was a crystalline product at a temperature of 150-151oC.
Calculated for C26H21NOF2, %: C 77.79; H 5.27; N 3.49. Found,%: C 77.80; H 5.23; N 3.21.
IR spectrum, cm− 1: 1670.0 (C = O); 1073.1 (C-N); 1582 (C = C) 1147.4 (C-F).
13С NMR spectrum, δ, ppm (CDCI3): 53.91 (С2,6); 140.21 (С3,5); 186.06 (С4); 140.21 (C3(5)=С); 161.34-164.23 (C-F); 123.99; 115.47-129.92 (СqC4H4CF); 61.52 (CH2); 137.21; 127.23-128.85 (CqC5H5).
5-Benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3а,4,5,6,7-hexahydro-2Н-pyrazolo[4,3-c]pyridine (III). 0.49 g (0.003 mol) of o-fluorophenylhydrazine hydrochloric acid was added to a suspension of 1.2 g (0.003 mol) of 1-(benzyl)-3,5-bis(o-fluorobenzylidene) piperidone-4(II) in 15 ml of MeOH. The mixture was stirred for 4 hours at a temperature of 70 ° C. The solvents were evaporated, the residue was recrystallized from methanol. Hydrochloride 5-(benzyl)-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo [4,3-c]pyridine (III.HCl) was obtained with a yield of 0.92 g (56%), m.p. 145-146oC. Treatment with a solution of potash of an aqueous hydrochloride solution by followed by chloroform extraction yields 5-(benzyl)-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine (III), Rf 0.49 (eluent: benzene: dioxane = 10:1).
Calculated for C32H26N3F3,%: C 75.43; H 5.14; N 8.25.
Found,%: C 75.39; Н 5.35; N 8.29.
IR spectrum, cm− 1: 1604.7 (C = N); 1019.5 (С-N); 1499,4 (С=С); 1161,6 (С-F).
13С NMR spectrum, δ, ppm (CDCl3): 56.67 (С3); 58.04 (С4); 54,04 (С6); 128.78 (C7); 155,04 (C8); 53.22 (C9); 132,05 (C10); 63,01 (CН2); A: 138.53; 115,11–125,08 (СqC5H5); B: 161,3; 163,89 (C-F); 123,63; 115,41–129,58 (СqC4H4CF); C: 160,62; 164,01 (C-F); 105,22; 115,61–129,70 (СqC4H4CF); D: 154.72; 156.06 (C-F); 130.49; 115,61–122,70 (СqC4H4CF).
Complex 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3а,4,5,6,7-hexahydro-2Н-pyrazolo[4,3-c]pyridine with β-cyclodextrin (IV). To obtain the complex, solutions of 0.30 g (0.0005 mol) 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine (III) in 30 ml of ethanol and 0.68 g (0.0005 mol) β-cyclodextrin in 30 ml of distilled water were used. The mixture was placed in the oven, ethanol and water were evaporated at a temperature of 50–55°C. A complex of 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine with β-cyclodextrin (IV) was obtained with a yield of 0.98 g a in the form of a white powder melting at decomposition above 240°C.
Calculated for C74H96O35N3F3, %: C 54.02; H 5.84; N 2.55. Found,%: C 54.04; H 5.85; N 2.56.
The complex of 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3а,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine with β-cyclodextrin (IV, BIV) was obtained by coupling 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3а,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c] pyridine with equimolar amount of β-cyclodextrin.
Biological study results
Analysis of the bone marrow cellularity
The average number of bone marrow myelokaryocytes in the Intact group was (23.3 ± 1.0) ×106 cells and was 3.2 times lower (p < 0.01) than in the BIV group (75.8 ± 5.1) ×106 cells and 2.6 times lower (p < 0.01) than in the Methyluracil group (62.1 ± 2.8) ×106 cells (Table 1). The increased number of bone marrow myelokaryocytes in the Control group, the BIV group, and the Methyluracil group can be explained by stimulated and compensatory proliferation of bone marrow cell pools after cyclophosphamide-induced myelodepression.
|
Average number of myelokaryocytes, ×106 cells |
|
|---|---|
|
Intact group |
23.3 ± 1.0 |
|
Control group |
73.9 ± 4.2 |
|
Group BIV |
75.8 ± 5.1 |
|
Methyluracil group |
62.1 ± 2.8 |
Analysis of the hemostimulating activity of the BIV compound on the c-kit (CD117+)-, Ly6G+-, Ly6C+-, Ter119+-, CD19+-, CD3e+-expressing bone marrow cells after cyclophosphamide-induced myelosuppression
C-kit (CD117+) is a type III receptor tyrosine kinase and is expressed on the surface of hematopoietic stem cells. The analysis showed that the BIV compound and the comparison drug Methyluracil did not statistically affect on the volume of CD117+ cells. The average level of c-kit (CD117+) cells in the BIV group was 4.3 ± 0.1% and was similar to the average level of c-kit (CD117+) cells in the Control group 4.5 ± 0.6% and in the Methyluracil group 5.4 ± 0.4%. The average level of c-kit (CD117+) cells in the placebo, Methyluracil and BIV groups were lower than in the Intact group 7.6 ± 1.6% by 1.7–1.4 times (p < 0.05) (Fig. 2a).
Subsequently, the committed bone marrow cells follow various pathways of cellular differentiation and, in particular, granulocyte-macrophage differentiation. The expression of Ly6G+ molecules on myeloblastic cells directly correlates with the level of differentiation and maturation of granulocytic leukocytes. Ly6C+ molecules expression is recorded on monoblastic cells with different levels of differentiation and maturation of monocyte/macrophage cells. The compound BIV significantly stimulated an increase in the level of granulocytes and monocytes/macrophags in the bone marrow. The average volume of Ly-6G+ Ly-6C+ granulocytes and monocytes/macrophages in the group BIV was 47.5 ± 1.6% and this index was at the level of Methyluracil group 45.5 ± 1.0%. The level of Ly-6G+ Ly-6C+ cells of the BIV group significantly exceeded the level of the Control group 26.7 ± 1.6% by 1.7 times (p < 0.05) and even exceeded the level of the Intact group 36.2 ± 1.0% by 1.3 times (Fig. 2b). Ter119+ is an erythroid-specific antigen expressed on early pro-erythroblasts to mature erythrocytes, but not on erythroid colony-forming cells. The average level of Ter119+ erythroid cells in the BIV group was 11.7 ± 1.6%. Exceeded the indicator of the Control group 5.3 ± 0.8% by 2.2 times (p < 0.01). It was inferior in activity to the comparison drug Methyluracil with an index of 17.8 ± 1.6% in 1.5 times (p < 0.05) and did not reach the values of the Intact group (Fig. 2d).
Many researchers have noted that sodium cyclophosphamide has the most pronounced suppressive effect on T-lymphocytes. They observed that the complete restoration of T-lymphopoiesis in the bone marrow begins only by the 60th day after the first administration of sodium cyclophosphamide. The CD3e+ marker was taken; it is registered at all stages of proliferation, maturation, differentiation of T-lymphocytes. The BIV compound did not have a stimulating effect on restoring the volume of CD3e+ cells of the pre-T-lymphocytic series. The average level of CD3e+T-lymphocytes in the BIV group was 15.7 ± 1.5%, in the Control group was 13.4 ± 2.7% and was at the same level. The average volume of cells in the Methyluracil group was 35.6 ± 1.3% and exceeded the indicator of the BIV group by 2.39 times (р<0.01) (Fig. 2e).
The huge role of the bone marrow is that it has a lymphoid pathway of proliferation and differentiation of lymphocytic cells. CD19+ is a marker of B-lymphocytes; it represents the activation of the tyrosine kinas cascade. The BIV compound significantly stimulated the process of increasing the level of CD19+ pro-, pre-B-lymphocytic cells in the bone marrow. The average volume of CD19+ B-lymphocytic cells in the Group BIV was 15.9 ± 4.0% and this level was slightly higher than the level of the Intact group 14.0 ± 4.5%. The average volume of CD19+ cells in the BIV group exceeded the average volume of CD19+ cells in the Methyluracil group 9.6 ± 1.5% and in the Control group 7.6 ± 0.4% by 1.6 and 2.0 times (p < 0.01), respectively (Fig. 3).
Chemical study discussion
The simplicity of the synthesis of 3,5-dienone derivatives of piperidine under the conditions of aldol-crotonic condensation and the availability of starting reagents attracted our attention in the search for new potential biologically active substances. The synthesis of α,β-unsaturated piperidin-4-ones is carried out by the Claisen-Schmidt reaction by condensation of N-substituted piperidone-4 with an aromatic aldehyde. A new target synthetic compound was obtained by the interaction of 1-benzylpiperidin-4-one (I) and o-fluorobenzaldehyde in a ratio of 1:2 in the presence of an alkaline agent:
1-Benzyl-3,5-bis(o-fluorobenzylidene)piperidine-4-one (II) (m.p. 150–151оС) was formed in 95% yield using 2 mol o-fluorobenzaldehyde per mol 1-benzylpiperidone-4 (I) in the presence of 15% potassium hydroxide in ethanol.
In the IR spectrum of compound II, the bands at 1670.0 cm− 1 are attributed to the stretching vibrations of the carbonyl group, and the C = C bonds conjugated with the carbonyl had absorption bands in the region of 1582 cm− 1. The intensity of the bands of the multiple carbon-carbon bond is higher than that of the conjugated carbonyl group. The absorption bands at 1073.1 cm− 1 are attributed to the stretching vibrations of the C-N bond, and 1147.4 cm− 1- C-F. The most informative was the 13C NMR spectrum, in which most of the carbon atoms appeared as signals of double intensity due to the molecule symmetry, with the exception of C4 of the piperidine ring and carbons of the benzyl radical.
Next, heterocyclization of 3,5-diarylidene-substituted piperidone-4 II was carried out into a polycyclic system containing a “pharmacophoric” piperidine ring in combination with a pyrazole one - the interaction of 1-benzyl-3,5-bis(o-fluorobenzylidene)piperidine-4-one (II) with hydrochloric acid o-fluorophenylhydrazine led to the formation of the corresponding hydrochloride (III.HCl), isolated in crystalline form (56%), m.p. 145-146oC. Treatment of III.HCl with an aqueous solution of potash followed by extraction with chloroform of the product led to III:
The formation of pyrazo-piperidine was confirmed by IR absorption bands at 1604.7 (C = N); 1019.5 (С-N); 1499.4 (C = C); 1161.6 (C-F) cm-1. In the 13С NMR spectrum of compound III, there was no signal of the carbon atom of the carbonyl group ( at 186.06 ppm), and a broad singlet signal in the region of 155.04 ppm, a decrease in the intensity of the signals of carbon atoms at the double bond also indicates heterocyclization. The symmetry breaking of the molecule also supports this conclusion.
The pharmacological activity of III under the BIV font was evaluated as a complex with β-cyclodextrin, obtained by mixing aqueous-alcoholic solutions of β-cyclodextrin and pyrazolo-piperidine, followed by stirring the mixture at the temperature of 50–55°C.
Biological study discussion
Analysis of the bone marrow cellularity
The average level of bone marrow myelokaryocytes in the BIV group was significantly higher than in the Intact group, which is associated with compensatory proliferation of bone marrow cell populations after cyclophosphamide-induced myelodepression and stimulating activity of BIV compound. The effect of the BIV compound was reflected in high indicators of the average level of bone marrow myelokaryocytes and exceeded the average levels of bone marrow myelokaryocytes in the Methyluracil group. The stimulating activity of BIV compound was more higher than that of Methyluracil.
Analysis of the hemostimulating activity of the BIV compound on the c-kit (CD117+)-, Ly6G+-, Ly6C+-, Ter119+-, CD19+-, CD3e+-expressing bone marrow cells after cyclophosphamide-induced myelosuppression
The cyclophosphamide model of pancytopenia was shosen because cyclophosphamide is widely used in the treatment of cancer, autoimmune diseases as a leading chemotherapeutic drug. The alkylating cytostatic cyclophosphane causes a cytostatic effect in the pool of cancer cells, but also suppresses the mitotic activity of the myeloid and lymphoid pool of the bone marrow. According to the research of Grinko et al., on the 10th day of the study, after a single administration of sodium cyclophosphamide at a dose of 125 mg/kg body weight of C57BL/6 mouse line, the researchers recorded a threefold decrease in the number of thymocytes in the thymus, thymus atrophy, depletion of the morphology of thymus lobules with myelometaplastic changes in subcapsular and corticomedullary zones, an increase in the number of apoptotic cells with fragmented DNA (Grinko et al. 2020). The content of cells decreased in the tissues of the spleen and other secondary lymphomyeloid organs. Also, many researchers have noted that the complete restoration of hematopoiesis and thymopoiesis is observed only by the 60th day after the first administration of sodium cyclophosphamide (Grinko et al. 2020; Setdikova et al. 2006). Persistent deficiency of peripheral T-lymphocytes led to increased homeostatic proliferation and conversion of the Tnaiv phenotype and accumulation of Th1 and CTL1. There was on accumulation of autoaggressive clones. Many researchers have noted the need for timely restoration of mitotic activity of bone marrow pools with timely colonization of the thymus and reduction in the threat of accumulation of autoaggressive CTL1.
The compound BIV did not stimulate an increase in the level of c-kit (CD117+) hematopoietic stem cells of the bone marrow after cytotoxic emptying induced by cyclophosphamide. But the non-responsiveness of hematopoietic stem cells to the BIV compound is a positive result, since bone marrow stem cells at the stage of primary differentiation should not react to physical and chemical factors from the outside in order to exclude the possibility of cancer mutations, malignant transformation as a result of unlimited cell division. Mutations of the c-kit proto-oncogene play an important role in the formation of various types of tumors of gastrointestinal stromal tumors, acute myeloid leukemia, melanoma, testicular cancer.
The BIV compound did not stimulate the process of increasing the level of CD3e+ pre-T lymphocytes in the bone marrow, which also characterizes the compound on the positive side. Subsequently, a high level of CD3e+ pre-T lymphocytes can enhance the production of elevated levels of effectors (T-killer and other) and regulatory (T-helper, T-regulatory, including T-suppressors) cells. An increased amount of one of the Th1 and Th2 subpopulations can lead to a violation of the balance of humoral and cellular immunity, to various hyperactive immune reactions. Excessive amounts of T-suppressors can lead to rapid suppression and intermittent immune response and can even cause immunological tolerance. High levels of T-suppressors and high functional activity can lead to the development of severe infectious, autoimmune and oncological diseases. A high level of cytotoxic T-lymphocytes can lead to the development of septic shock.
The BIV compound showed pronounced stimulating activity in relation to the process of increasing the level of Ly-6G+ Ly-6C+ granulocytes, monocytes/macrophages, exceeding the activity of the comparison drug Methyluracil. The BIV compound stimulated the process of restoring the level of Ly-6G+ Ly-6C+ granulocytes, monocytes/macrophages, which play an important role in innate immunity and in all types of adaptive immunity. Timely restoration of the level of granulocyte and monocyte-macrophage pools of cells contributes to the restoration of the functional activity of humoral and cellular immunity. It is known that monocytopenia is accompanied by non-tuberculosis mycobacterial infections, fungal infections, and viral diseases with a subsequent risk of progression to secondary cancers; there is a risk of developing various hematological disorders. With neutropenia, the risk of developing opportunistic infections, fungal diseases with damage to extensive areas of mucous tissues, etc. increases.
The BIV compound had weak stimulating activity in relation to the process of increasing the level of Ter119+ erythroid cells and was inferior in activity to the comparison drug Methyluracil.
The BIV compound showed pronounced stimulating activity in relation of the process of increasing the level of CD19+ B-lymphocytes, exceeding the activity of the comparison drug Methyluracil. The BIV compound effectively influenced the process of restoring the population of B lymphocytes in the bone marrow, which will further contribute to an increase in the volume of Transitional B Cells, which plays an important role for further differentiation and formation of a sufficient number of Mature B1 Cells and Mature B2 Cells, where Mature B2 Cells make up the bulk of the functional population of B lymphocytes.
On the model of cyclophosphamide-induced hematopoietic-depression of the bone marrow of C57BL6/J mouse line the newly compound Complex 5-benzyl-7-(o-fluorobenzylidene)-2,3-bis(o-fluorophenyl)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine complex with β-cyclodextrin showed high stimulating activity in the process of restoring the level of Ly-6G+ Ly-6C+ granulocytes, monocytes/macrophages, CD19+ B-lymphocytes.
Acknowledgments
The authors thank anyone who contributed to the article.
Funding
Funding for this research was financially supported by the Grant from Committee of Science of the Ministry of Education and Science of the Republic of Kazakhstan [Chief of Grant Yu V.K. AP 08856051]
Data Availability
All data in this study are available from the corresponding author.
Ethics approval
The studies were performed in accordance with the Order of the Minister of Healthcare of the Republic of Kazakhstan No. KR DSM-181/2020 of November 4, 2020 and by the protocol of the Local Ethics Commission of the Al-Farabi Kazakh National University No. 20136/10 dated June 12, 2020. The validity period is 3 years.
Consent for publication
All authors have read the paper and agree that it can be published.
Competing interest
The authors declare no ethical, scientific, commercial or financial conflict of interest.
Permission to reproduce material from other sources
Data from other resources were not used.
Authors’ contributions
All authors contributed to the study conception and design. Conceptualization by L. Baktybayeva, V.Yu. Material preparation, data collection and analysis were performed by L. Baktybayeva,V.Yu, G.Daulet, N.Belyaev, A.Zazybin, Ye.Ostapchuk, Yu.Perfilyeva, A.Kali, N.Abdolla, R.Tleulieva, A.Ten and Zh.Koshetova. The first draft of the manuscript was written by L.Baktybayeva, G.Daulet and V.Yu and all authors commented on previous of the manuscript. All authors read and approved the final manuscript.
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No competing interests reported.