Oxidized aporphine and biguanide derivative mixed rhodium complex as well as synthesis method and application thereof

Abstract

The invention discloses two rhodium complexes mixed by oxoaporphine and biguanide derivatives as well as a synthesis method and application of the rhodium complexes, and belongs to the technical field of medicines. The synthesis method of the rhodium complex comprises the following steps: placing aporphine oxide, rhodium chloride hydrate and metformin hydrochloride or phenformin hydrochloride which is excessive relative to the aporphine oxide into a reaction container, adding a mixed solvent, and reacting under a heating condition to obtain a complex Rh-Met or Rh-Phen; wherein the mixed solvent is a composition composed of acetonitrile and methanol according to a volume ratio of (1-2): (1-3). Test results of the applicant show that the compound has a good inhibition effect on various tumor cell strains, has dual effects of inhibiting tumor growth and reducing blood sugar level in a mouse experiment of a type 2 diabetes mellitus combined tumor model, and is expected to be used as a therapeutic drug for diabetes mellitus combined tumor patients.

Description

Technical Field

[0001] The invention relates to a rhodium complex of aporpheniramine oxide and a biguanide derivative, a synthesis method and application thereof, and belongs to the technical field of medicine. Background Art

[0002] There is a correlation between diabetes and cancer. A growing number of studies indicate that the incidence of cancer coexisting with diabetes (particularly type 2 diabetes mellitus (T2DM)) is significantly increased. Currently, there is no standard clinical treatment for patients with cancer and diabetes. Metformin and phenformin are first-line drugs for the clinical treatment of diabetes. In recent years, they have also attracted considerable attention in anti-cancer research. Studies have shown that metformin can alter cancer cell metabolism, thereby exerting anti-tumor activity. The mechanism of metformin's cancer treatment relies on activation of 5'-adenosine monophosphate-activated protein kinase (AMPK), thereby inhibiting the glycolytic activity of tumor cells. In the context of cancer treatment, metformin exhibits anti-cancer effects only at high doses (e.g., the optimal dose for humans is generally 1000-2000 mg / day), which can lead to severe toxic side effects. Therefore, researchers are attempting to enhance the anti-cancer efficacy of metformin through other approaches, including conjugation with other active pharmacophores or combination therapies.

[0003] Metformin contains two cis-imino groups, which can coordinate with metal ions in a bidentate chelation mode to form stable metal complexes. In recent years, metformin complexes have been reported to possess promising antitumor activities. For example, cyclometallated Au(III) prodrugs exert anti-triple-negative breast cancer activity by interfering with energy production and mitochondrial function; and platinum-metformin conjugates achieve tumor immunotherapy through AMPK-mediated lysosomal degradation of PD-L1. However, research on these complexes has been limited to their anticancer activities; metal complexes with both antitumor and hypoglycemic activities have not yet been investigated.

[0004] Aporphine oxide, as an active ingredient in traditional Chinese medicine, has certain coordination abilities and anti-cancer activity. Currently, there are no reports on rhodium (III) complexes obtained by mixing aporphine oxide with biguanide derivatives and their use in hypoglycemic and anti-cancer treatments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide two rhodium complexes of mixed aporpheniramine oxide and biguanide derivatives with novel structures and both hypoglycemic and anti-tumor effects, as well as their synthesis methods and applications.

[0006] In the first aspect, the present invention provides a mixed rhodium complex formed by mixing rhodium (III) metal ions with aporpheniramine oxide and a biguanide derivative, which are the complex Rh-Met and the complex Rh-Phen, and their structures are shown below:

[0007]

[0008] In a second aspect, the present invention provides a method for synthesizing the above-mentioned rhodium complex, comprising: placing aporphenanthrene oxide, rhodium trichloride hydrate, and an excess of metformin hydrochloride or phenformin hydrochloride relative to aporphenanthrene oxide in a reaction vessel, adding a mixed solvent, and reacting under heating conditions to obtain a complex Rh-Met or a complex Rh-Phen; wherein the mixed solvent is a composition composed of acetonitrile and methanol in a volume ratio of 1 to 2:1 to 3.

[0009] The applicant discovered in the experiment that the amount of metformin hydrochloride or phenformin hydrochloride relative to aporpheniramine oxide must be in excess to obtain the target product complex Rh-Met or complex Rh-Phen of the present application. Otherwise, the complex Rh1 with the following structure is obtained:

[0010]

[0011] Furthermore, the excess metformin hydrochloride or phenformin hydrochloride generally means that the amount of metformin hydrochloride or phenformin hydrochloride is 1.5 times or more of the molar amount of aporpheniramine oxide, preferably 2 to 3 times the molar amount of aporpheniramine oxide.

[0012] Furthermore, the reaction is carried out at a temperature of ≥50°C, preferably at 80-120°C, and the reaction time is suitably controlled at 24-72h.

[0013] Furthermore, the reaction container is a high-pressure reactor.

[0014] The applicants also discovered that, under certain conditions, the ratio of acetonitrile to methanol in the mixed solvent affects the properties of the resulting Rh-Met complex. When the volume ratio of acetonitrile to methanol is 1:2, the resulting Rh-Met complex is crystalline (reddish-brown), while other ratios yield a powdery complex. Regarding the amount of mixed solvent used, it is ideal to control the amount of mixed solvent used for all raw materials involved in the reaction to 5-12 mL, based on 0.1 mmol of aporpheniramine oxide.

[0015] In a third aspect, the present invention provides a crystalline form of a complex Rh-Met, which belongs to the triclinic system, the P-1 space group, and the unit cell parameters are: α=111.124(2)°,β=94.915(2)°,γ=99.112(2)°. The single crystal structure shows that the complex Rh-Met is an ionic complex containing a pair of anions and cations. The Rh(III) center of the cation is coordinated with an oxygen atom and a nitrogen atom from aporpheniramine oxide, two nitrogen ions from metformin and two free chloride ions to form a hexacoordinated octahedral geometry ([Rh(L 1 )(Met)Cl2] + ), the Rh(III) center of the anion is chelated with an oxygen ion and a nitrogen atom from aporphyrin oxide and four free chloride ions to form a hexacoordinated octahedral geometry ([Rh(L 1 )Cl4] - ). The complex is generally electrically neutral.

[0016] In a fourth aspect, the present invention provides a method for synthesizing the Rh-Met crystal form of the above-mentioned complex, comprising: placing aporphenanthrene oxide, rhodium trichloride hydrate, and metformin hydrochloride in an excess amount relative to aporphenanthrene oxide in a reaction vessel, adding acetonitrile and methanol, reacting under heating conditions, cooling the reactants, precipitating crystals, and collecting the crystals to obtain the target product; wherein the volume ratio of acetonitrile to methanol is 1:2.

[0017] The selection of the excess amount of metformin hydrochloride relative to aporpheniramine oxide, reaction temperature and time, reaction vessel, etc. involved in the synthesis method of the above-mentioned crystal form is the same as that of the above-mentioned rhodium complex synthesis method.

[0018] The oxidized aporphines involved in the synthesis method of the rhodium complex or the synthesis method of the crystal form described in the present invention can be synthesized with reference to the previous literature reports of the inventor team of this application, using commercial 2-bromobenzaldehyde derivatives (or 2-boronic acid benzaldehyde derivatives) as starting materials, and a series of oxidized aporphines derivatives are synthesized by palladium-catalyzed Suzuki coupling-cross dehydrogenation coupling with 8-boronic acid isoquinoline derivatives (or 8-bromoisoquinoline derivatives) (Liao LS, Tan LJ, Chen Y, Yang QY, Choudhary MI, Pan YM, Tang HT, Su GF, Liang H, Chen ZF. One-pot synthesis of oxoaporphines as potent antitumor agents and investigation of their mechanisms of actions [J]. European Journal of Medicinal Chemistry, 2022, 231: 114141-144159.).

[0019] The applicant discovered through in vitro cell growth inhibition tests that the above-mentioned rhodium complex or the crystalline form of the complex Rh-Met has a very significant inhibitory effect on cell proliferation of various tumor cells under high sugar conditions (cell culture medium is 4.5g / L glucose), and the inhibitory effect is stronger than that under low sugar conditions (cell culture medium is 1g / L glucose). In a mouse experiment with a type 2 diabetes mellitus combined with tumor model, the complex Rh-Met can effectively reduce the blood sugar level of the mice while inhibiting the growth of MB-49 tumors. Therefore, the present invention also includes the use of the above-mentioned rhodium complex or the crystalline form of the complex Rh-Met mixed with aporphenanthrene oxide and a biguanide derivative in the preparation of hypoglycemic drugs or anti-tumor drugs. Furthermore, it also includes the use of the complex Rh-Met or the crystalline form of the complex Rh-Met in the preparation of drugs for treating patients with diabetes combined with tumors.

[0020] In addition, the present invention also provides a pharmaceutical composition, which contains the rhodium complex or the crystal form of the complex Rh-Met mixed with the above-mentioned aporpheniramine oxide and a biguanide derivative.

[0021] Compared with the prior art, the present invention provides two novel rhodium complexes of mixed aporpheniramine oxide and biguanide derivatives and their synthesis methods; the applicant's experimental results show that the compounds described in the present invention exhibit good inhibitory effects on various tumor cell lines, and in experiments on mice with a type 2 diabetes mellitus combined with tumor models, they exhibited the dual effects of inhibiting tumor growth and lowering blood sugar levels, and are expected to be used as therapeutic drugs for patients with diabetes and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the crystal structure diagram of the complex Rh1 (ellipsoidal ratio 50%).

[0023] Figure 2 Figure 3 is the crystal structure of the complex Rh-Met (ellipsoidal ratio 50%).

[0024] Figure 3 The UV stability test of Rh-Met was conducted in physiological saline, PBS containing 10% serum, PBS at pH 5.0 and 8.0.

[0025] Figure 4 The UV stability test of Rh-Phen was conducted in physiological saline, PBS containing 10% serum, PBS with pH=5.0 and PBS with pH=8.0.

[0026] Figure 5 The high performance liquid chromatograms of Rh1, Rh-Met and Rh-Phen in methanol solution.

[0027] Figure 6The graph shows the effect of Rh-Met and Met treatment on T-24 cell apoptosis under high glucose and low glucose conditions for 24 hours.

[0028] Figure 7 The figure shows the effect of Rh-Phen and DDP on T-24 cell apoptosis after 24 hours of treatment under high glucose and low glucose conditions.

[0029] Figure 8 This is a diagram showing the distribution of Rh1, Rh-Met and Rh-Phen (6 μM) in T-24 cells under high sugar and low sugar conditions detected by ICP-MS.

[0030] Figure 9 Graph showing the effects of Rh-Met and Rh-Phen (6, 12, 18, and 24 μM) on the cell cycle of T-24 cells under high glucose (A) and low glucose (B) conditions.

[0031] Figure 10 Mitochondrial stress test experiments were conducted to examine the effects of Rh-Met and Rh-Phen on mitochondrial function in T-24 cells after 24 h of treatment under high glucose (A) and low glucose (B) conditions.

[0032] Figure 11 Glycolysis stress experiments were performed to detect the effects of Rh-Met and Rh-Phen on the glycolytic function of T-24 cells after treatment with high glucose (A) and low glucose (B) conditions for 24 hours.

[0033] Figure 12 This figure shows the changes in blood glucose levels in diabetic mice after 15 days of continuous treatment.

[0034] Figure 13 In vivo evaluation of the antitumor effect of Rh-Met in diabetic mice with tumors. (A) Images of tumors in each group after 15 days of treatment; (B) Average tumor weight and tumor inhibition rate in each group; (C) Changes in tumor volume in each group; (D) Changes in average body weight of mice in each group.

[0035] Figure 14 Figure 3. Changes in blood glucose (A) and the area under the curve (B) of mice in each group after oral administration of glucose in the OGTT experiment from 0 to 120 min.

[0036] Figure 15 It is the percentage change of blood glucose in each group of mice from 0 to 120 minutes after intraperitoneal injection of insulin in the ITT experiment.

[0037] Figure 16 This is a diagram showing the distribution of Rh complexes in various organs and tumors of mice detected by ICP-MS.

[0038] Figure 17These are histological morphology images (400×) of the heart, liver, spleen, lung, and kidney sections of mice in each group after 15 days of continuous treatment. DETAILED DESCRIPTION

[0039] In order to better explain the technical solution of the present invention, the present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0040] Example 1: Preparation of complex Rh-Met

[0041] 23.1 mg (0.1 mmol) of aporphenanthrene oxide, 26.7 mg (0.1 mmol) of rhodium trichloride hydrate, and 34.2 mg of metformin hydrochloride were weighed into a high-temperature reactor. 3 mL of acetonitrile and 6 mL of methanol (the volume ratio of acetonitrile to methanol was 1:2) were added. The reactor was then placed in an 80°C oven for 72 hours. After the reaction, the temperature was slowly lowered to room temperature. Reddish-brown blocky crystals were observed in the reactor. These crystals were collected and dried. The yield was 31.21% (15.7 mg, calculated based on aporphenanthrene oxide).

[0042] Single crystals of suitable size and shape were selected and their structures were determined using single crystal X-ray diffraction analysis and other methods. The specific characterization data are as follows:

[0043] (1) H NMR spectrum: 1 H NMR(600MHz,DMSO-d6)δ9.77(d,J=6.1Hz,1H),9.57–9.50(m,1H),9.19(t,J=7.0Hz,1H),9.11(d,J=7.5Hz,1 H),8.88(dt,J=14.8,7.7Hz,3H),8.79(d,J=8.0Hz,1H),8.66(d,J=6.8Hz,1H),8.58–8.53(m,2H),8.30(td,J =7.9,3.2Hz,2H),8.23(t,J=7.9Hz,1H),8.19(d,J=8.0Hz,1H),8.12(t,J=7.0Hz,1H),7.90(dt,J=27.8,7.6H z,2H),7.41(s,1H),6.96(s,1H),6.67(d,J=2.2Hz,1H),6.40(s,1H),6.04(s,1H),3.20(s,3H),2.89(s,3H).

[0044] (2) NMR carbon spectrum: 13C NMR(151MHz,DMSO)δ193.49,193.31,155.05,152.61,152.17,151.06,149.46, 149.04,145.38,143.62,137.40,137.31,137.05,135.35,134.74,134.59,133. 65,133.07,130.80,130.29,130.10,129.77,129.62,127.93,127.48,127.30,126.98,126.12,125.69,124.49,124.39,123.46,123.16,123.05,36.82,33.65.

[0045] (3) High-resolution mass spectrometry: (ESI) calculation for C 20 H 20 Cl2N6ORh[Rh(L 1 )(Met)Cl2] + (m / z):533.0131;Found,533.0150,C 20 H 20 Cl2N6ORh[Rh(L 1 )(Met)Cl1-H] + (m / z):497.0364;Found,497.0382,C 16 H9Cl4NORh[Rh(L 1 )Cl4] - (m / z):473.8493;Found,473.8485,C 17 H 12 Cl4NO2Rh[Rh(L 1 )Cl4+CH3OH+Na-H] - (m / z):527.3153; Found,527.3164,C 17 H 11 Cl3NO2Rh[Rh(L 1 )Cl3+CH3OH+Na-2H] - (m / z):491.3365; Found,491.3398.

[0046] (4) Elemental analysis (C 36 H 29 Cl6N7O2Rh2):

[0047] Calculated values ​​(%): C, 42.80; H, 2.89; N, 9.71;

[0048] Found (%): C, 42.85; H, 2.91; N, 9.60.

[0049] (5) Single crystal X-ray diffraction analysis:

[0050] A reddish-brown block crystal of moderate size was selected and placed on an Agilent SuperNova X-ray single crystal diffractometer. α The initial crystal structures of the products obtained in this example were solved by the SHELXS-97 direct method. The diffraction data were corrected by LP factors and empirical absorption. All non-hydrogen atomic coordinates and their anisotropic thermal parameters were refined by full matrix least squares method. All hydrogen atomic coordinates were determined by theoretical calculations. The obtained crystallographic and structural refinement data are shown in Table 1 below. The obtained crystal structure is shown in Figure 2 As shown, the planar structure is shown below for Rh-Met. The single crystal structure shows that the complex Rh-Met is an ionic complex containing a pair of anions and cations. The Rh(III) center of the cation is coordinated with an oxygen atom and a nitrogen atom from aporpheniramine oxide, two nitrogen ions from metformin, and two free chloride ions to form a hexacoordinated octahedral geometry ([Rh(L 1 )(Met)Cl2] + ), the Rh(III) center of the anion is chelated with an oxygen ion and a nitrogen atom from aporphyrin oxide and four free chloride ions to form a hexacoordinated octahedral geometry ([Rh(L 1 )Cl4] - ). The complex is generally electrically neutral.

[0051] Therefore, it can be confirmed that the obtained reddish-brown block crystals are a rhodium complex of aporpheniramine oxide and metformin, namely, the complex Rh-Met.

[0052]

[0053] Table 1 Crystallographic and structural revision data of Rh1 and Rh-Met

[0054]

[0055]

[0056] Comparative Example 1-1: Preparation of complex Rh1

[0057] Example 1 was repeated, except that metformin hydrochloride was not added.

[0058] After the reaction was completed, the temperature was gradually lowered to room temperature. Red block crystals were observed to form in the reactor. The crystals were collected and dried. The yield was 25.26% (12.1 mg, calculated based on the ligand oxidized aporpheniramine).

[0059] Single crystals of suitable size and shape were selected and their structures were determined using single crystal X-ray diffraction analysis and other methods. The specific characterization data are as follows:

[0060] (1) H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ9.72(d,J=6.3Hz,1H),9.24(d,J=7.4Hz,1H),9.04(s,1H),8.88(d,J=8.1Hz ,1H),8.65(t,J=9.2Hz,2H),8.34(t,J=7.9Hz,1H),8.22(s,1H),7.92(t,J=7.6Hz,1H),2.97(s,3H).

[0061] (2) High-resolution mass spectrometry: HR-MS (ESI) calculation for C 20 H 20 Cl3N2O3RhS[Rh(L 1 )(CH3CN)Cl3+H2O+DMSO+H] + (m / z):576.9394

[0062] (3) Elemental analysis (C 18 H 12 Cl3N2ORh):

[0063] Calculated value (%): C, 44.90; H, 2.51; N, 5.82;

[0064] Found (%): C, 44.82; H, 2.72; N, 5.86.

[0065] (4) Single crystal X-ray diffraction analysis:

[0066] A red block crystal of appropriate size was selected and placed on an Agilent SuperNova X-ray single crystal diffractometer. α The initial crystal structures of the products obtained in this example were solved by the SHELXS-97 direct method. The diffraction data were corrected by LP factors and empirical absorption. All non-hydrogen atomic coordinates and their anisotropic thermal parameters were refined by full matrix least squares method. All hydrogen atomic coordinates were determined by theoretical calculations. The obtained crystallographic and structural refinement data are shown in Table 1 above. The obtained crystal structure is shown in Figure 1As shown, the planar structure is shown below for Rh1. Rh1 has a six-coordinated octahedral geometry, with the central Rh(III) coordinated by one nitrogen and one oxygen from aporpheniramine oxide, one nitrogen atom from an acetonitrile molecule, and three free chloride ions.

[0067] Comparative Example 1-2:

[0068] Example 1 was repeated, except that the amount of metformin hydrochloride was halved, that is, 17.1 mg of metformin hydrochloride was added.

[0069] As a result, red block crystals were obtained with a yield of 20.13% (10.12 mg, calculated based on the ligand aporpheniramine oxide).

[0070] High-resolution mass spectrometry and single-crystal X-ray diffraction analysis of the resulting red block crystals confirmed that it was the complex Rh1 that did not contain metformin. The applicant speculated that this might be due to the poor coordination ability of metformin.

[0071] Comparative Examples 1-3:

[0072] Example 1 was repeated except that chloroform was used instead of acetonitrile.

[0073] After stopping the reaction, the temperature was slowly lowered to room temperature. The kettle contained a clear liquid with no crystals or precipitates at the bottom. The liquid in the kettle was further concentrated under reduced pressure, but no crystals or precipitates were observed.

[0074] Example 2: Preparation of complex Rh-Met

[0075] Example 1 was repeated, except that 2 mL of acetonitrile and 6 mL of methanol (the volume ratio of acetonitrile to methanol was 1:3) were added.

[0076] The resulting reddish brown powder had a yield of 28.37% (14.27 mg, calculated based on the ligand oxidized aporpheniramine).

[0077] The obtained reddish-brown powder was characterized by H-NMR, C-NMR and high-resolution mass spectrometry, and it was confirmed that the obtained powder was the complex Rh-Met.

[0078] Example 3: Preparation of complex Rh-Met

[0079] Example 1 was repeated, except that 3 mL of acetonitrile and 3.9 mL of methanol (the volume ratio of acetonitrile to methanol was 1:1.3) were added, the reaction temperature was changed to 100° C., and the reaction time was changed to 48 hours.

[0080] The resulting reddish brown powder had a yield of 26.25% (13.20 mg, calculated based on the ligand aporpheniramine oxide).

[0081] The obtained reddish-brown powder was characterized by H-NMR, C-NMR and high-resolution mass spectrometry, and it was confirmed that the obtained powder was the complex Rh-Met.

[0082] Example 4: Preparation of complex Rh-Met

[0083] Example 1 was repeated, except that 6 mL of acetonitrile and 3 mL of methanol were added (the volume ratio of acetonitrile to methanol was 2:1).

[0084] The resulting reddish brown powder had a yield of 30.31% (15.24 mg, calculated based on the ligand aporpheniramine oxide).

[0085] The obtained reddish-brown powder was characterized by H-NMR, C-NMR and high-resolution mass spectrometry, and it was confirmed that the obtained powder was the complex Rh-Met.

[0086] Example 5: Preparation of complex Rh-Phen

[0087] 23.1 mg (0.1 mmol) of the ligand aporphenanthrene oxide, 26.7 mg (0.1 mmol) of rhodium trichloride hydrate, and 48 mg of phenformin hydrochloride were weighed into a high-temperature reactor. 3 mL of acetonitrile and 6 mL of methanol (the volume ratio of acetonitrile to methanol was 1:2) were added. The reactor was then placed in an 80°C oven for 72 hours. After the reaction, the temperature was slowly lowered to room temperature. A reddish-brown powder was observed in the reactor. The powder was collected and dried. The yield was 39.37% (21.3 mg, calculated based on the ligand aporphenanthrene oxide).

[0088] Its structure was determined by H / C NMR, high-resolution mass spectrometry, elemental analysis and other methods. The specific characterization methods are as follows:

[0089] (1) H NMR spectrum: 1 H NMR(600MHz,DMSO-d6)δ9.80(dd,J=6.1,1.5Hz,1H),9.30(s,1H),9.19(s,1H),9.16–8.9 8(m,2H),8.96–8.84(m,3H),8.82–8.73(m,1H),8.66–8.55(m,3H),8.34–8.11(m,4H),7. 98–7.86(m,2H),7.55(t,J=5.5Hz,1H),7.48–7.39(m,3H),7.33(dt,J=5.8,3.0Hz,1H),7 .26–7.14(m,1H),6.68(s,2H),6.49–6.31(m,2H),3.81–3.65(m,2H),3.03–2.95(m,2H).

[0090] (2) NMR carbon spectrum:13 C NMR (151MHz, DMSO) δ194.62,194.42,153.36,152.30,150.51,149.58,146.41,138.99,138. 59,138.17,136.43,136.06,135.81,135.62,134.93,134.76,134.17,131.97,131.35,131. 22,131.13,130.98,130.85,130.70,129.45,129.02,128.96,128.39,128.31,128.02,127.13,127.01,126.72,126.35,126.17,125.56,125.41,124.52,124.26,43.57,40.40,34.92.

[0091] (3) High-resolution mass spectrometry: HR-MS (ESI) calculation for C 26 H 24 Cl2N6ORh[Rh(L 1 )(Phen)Cl2] + (m / z):609.0444;Found,609.0418,C 28 H 26 ClN7ORh[Rh(L 1 )(Phen)Cl-H] + (m / z):573.0677;Found,573.0652,C 18 H 15 Cl4NO2RhS[Rh(L 1 )Cl4+DMSO] - (m / z):551.3033;Found,551.3069,C 17 H 12 Cl4NO2Rh[Rh(L 1 )Cl4+CH3OH+Na-H] - (m / z):527.3153; Found,527.3170,C 17 H 11 Cl3NO2Rh[Rh(L 1 )Cl3+CH3OH+Na-2H] - (m / z):491.3365; Found,491.3404.

[0092] (4) Elemental analysis (C 42 H 33 Cl6N7O2Rh2):

[0093] Calculated value (%): C, 46.44; H, 3.06; N, 9.03;

[0094] Found (%): C, 46.51; H, 3.14; N, 9.01.

[0095] Thus, it can be confirmed that the obtained reddish-brown powder is a rhodium complex of aporphyrin oxide and phenformin, namely, the complex Rh-Phen, the structure of which is shown below:

[0096]

[0097] Comparative Example 5-1:

[0098] Example 5 was repeated, except that the amount of phenformin hydrochloride was halved, i.e., 24 mg of phenformin hydrochloride was added.

[0099] The reddish-brown powder obtained was the control compound Rh1 without phenformin, which may be due to the poor coordination ability of phenformin.

[0100] Comparative Example 5-2:

[0101] Example 5 was repeated except that chloroform was used instead of acetonitrile.

[0102] After stopping the reaction, the temperature was slowly lowered to room temperature. The kettle contained a clear liquid with no crystals or precipitates at the bottom. The liquid in the kettle was further concentrated under reduced pressure, but no crystals or precipitates were observed.

[0103] Example 6: Preparation of complex Rh-Phen

[0104] Example 1 was repeated, except that 5 mL of acetonitrile and 5 mL of methanol (the volume ratio of acetonitrile to methanol was 1:1) were added, the reaction temperature was changed to 120° C., and the reaction time was changed to 48 hours.

[0105] The resulting reddish brown powder had a yield of 35.96% (19.45 mg, calculated based on the ligand aporpheniramine oxide).

[0106] The obtained reddish-brown powder was characterized by H-NMR, C-NMR and high-resolution mass spectrometry, and it was confirmed that the obtained powder was the complex Rh-Phen.

[0107] Example 7: Preparation of complex Rh-Phen

[0108] Example 1 was repeated, except that 3 mL of acetonitrile and 7 mL of methanol (the volume ratio of acetonitrile to methanol was 1:3) were added.

[0109] The resulting reddish brown powder had a yield of 32.18% (17.41 mg, calculated based on the ligand aporpheniramine oxide).

[0110] The obtained reddish-brown powder was characterized by H-NMR, C-NMR and high-resolution mass spectrometry, and it was confirmed that the obtained powder was the complex Rh-Phen.

[0111] Experimental Example 1: Stability test of the rhodium complex of the present invention

[0112] 1.1 Determination of compound stability by UV-visible spectroscopy

[0113] The applicant used UV-visible spectroscopy to detect the stability of the synthesized compound in PBS solution containing 10% serum, physiological saline, and PBS solution with pH = 5.0 and 8.0, providing an experimental basis for subsequent cell level experiments. -5 The complex was placed in 10% serum PBS solution, physiological saline, PBS solution with pH=5.0 and 8.0 at room temperature for 0, 24 and 48 hours, and the absorbance was measured once using a UV spectrometer. Figure 3 and Figure 4 As shown, the complexes Rh-Met and Rh-Phen exhibited poor stability in physiological saline, with their absorbance decreasing slightly after 24 and 48 hours. However, the complexes exhibited excellent stability in PBS containing 10% serum, with no new absorption peaks appearing and no significant change in absorbance over 48 hours. UV spectroscopy of the complexes in PBS solutions within a near-physiological pH range demonstrated that the complexes were more stable in weakly acidic environments than in weakly alkaline environments.

[0114] 1.2 Determination of compound stability and purity by high performance liquid chromatography (HPLC)

[0115] A Waters series HPLC system was used, with a C-18 column (Agilent Eclipse XDB-C18 5μm, 4.6mm×150mm, USA). The mobile phase was methanol to water at a ratio of 10:1. 1 mg of freshly prepared complex crystals or powder was weighed and dissolved in 1 mL of methanol solution. The elution time of the sample at 0, 24, and 48 hours was measured by HPLC. Figure 5 As shown, the purity of the tested compounds exceeded 95%, and the retention times of the complexes at 0, 24, and 48 h were almost the same, with no new peaks appearing, indicating that the complexes remained stable in methanol solution for at least 48 h.

[0116] Experimental Example 2: Study on the anti-tumor activity of the rhodium complex of the present invention in vitro

[0117] 2.1 Cell culture

[0118] The basal media used for cell culture were DMEM high-glucose medium (LOT P2959819, glucose content 4.5 g / L) and DMEM low-glucose medium (LOT P2613093, glucose content 1 g / L), both purchased from Shanghai Titan Technology Co., Ltd., and supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin. Before use, the cells were equilibrated in a 37°C incubator for 30 minutes. The experimental cells were serially subcultured in the corresponding glucose concentration medium for ≥3 generations before use in subsequent experiments.

[0119] Table 2 Chinese and English names of cell lines used in the experiment

[0120]

[0121] 2.2 Determination of IC of the complex by MTT assay 50 value

[0122] The MTT assay is a commonly used cell viability assay. Its main principle is that exogenous MTT (also known as thiazolyl blue) reduces succinate dehydrogenase, present only in the mitochondria of living cells, to blue-purple crystalline formazan. Crystalline formazan can be dissolved in DMSO, and the absorbance of the solution is measured using a microplate reader to reflect cell viability. The more viable cells there are, the darker the solution color and the greater the absorbance. Within a certain range of cell counts, the amount of blue-purple crystalline formazan formed is positively correlated with the number of viable cells.

[0123] Well-growing cells were seeded into 96-well cell culture plates and incubated in a 37°C incubator with 5% carbon dioxide for 24 hours, until the cells reached 80-90% growth. 20 μL of the test compound was then added according to a concentration gradient, with final concentrations ranging from 2.5 to 40 μM (2.5, 5.0, 10.0, 20.0, and 40.0 μM). The plates were then incubated in an incubator for 48 hours. Subsequently, 10 μL of MTT was added to each well and incubated for another 4-6 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were shaken on a shaker for 10 minutes to completely dissolve the precipitated formazan crystals. The absorbance difference at 570 / 630 nm was measured using a microplate reader, and cytotoxicity was assessed based on the percentage of cells surviving compared to the negative control. The final inhibitory concentration (IC) was calculated using the Bliss method (n=5). All tests were repeated in at least three independent experiments.

[0124] 2.3 Results and Discussion

[0125] We used MTT assay to detect the IC values ​​of the synthesized mixed complexes on four human tumor cells and one human normal cell line under high glucose and low glucose conditions.50 The activity of the control compound Rh1 and the positive control drugs cisplatin (DDP), metformin (Met), and phenformin (Phen) were also tested. To explore the impact of the formation of mixed complexes on their antitumor activity, the antitumor activity of the Rh1+Met group and the Rh1+Phen group, composed of the same proportions of the mixed compounds, was also studied.

[0126] IC values ​​of each compound under high glucose conditions 50 The values ​​are shown in Table 3. The results show that the mixed complexes Rh-Met, Rh-Phen and the control compound Rh1 all exhibited good antitumor activity under high glucose conditions, IC 50 The values ​​showed the following trend: Rh-Met<Rh1<Rh-Phen<Rh1+Met<Rh1+Phen. This result shows that the formation of metformin mixed complexes enhances its anti-tumor activity. Among them, Rh-Met has the best anti-proliferative activity against T-24 cells under high glucose conditions, IC 50 The value was 5.87±0.08μM. Metformin did not show significant antitumor activity under high glucose conditions, which is consistent with the literature reports. The IC values ​​of each compound under low glucose conditions were 50 The values ​​are shown in Table 4. Compared with high glucose conditions, the cytotoxicity of Rh1, Rh-Met and Rh-Phen to these tumor cells under high glucose conditions was better than that under low glucose conditions. There was no significant difference in the cytotoxicity of DDP under high glucose and low glucose conditions. The IC 50 The values ​​were lower than those under high glucose conditions. This result indicates that the formation of the mixed rhodium complex of oxidized aporpheniramine and metformin significantly enhances its antitumor activity under high glucose conditions. Because Rh-Met exhibits good inhibitory activity and selectivity against T-24 cells, we further investigated the biological effects of these Rh complexes on T-24 cells under high and low glucose conditions.

[0127] Table 3 IC values ​​of the complexes against various cell lines under high glucose (4.5 g / L glucose) conditions 50 Value (μM).

[0128]

[0129] Table 4 IC values ​​of the complexes against various cell lines under low glucose (1 g / L glucose) conditions 50 Value (μM).

[0130]

[0131] Experimental Example 3: Study on the anti-tumor mechanism of the rhodium complex of the present invention in vitro

[0132] 3.1 Cellular uptake and distribution experiments

[0133] T-24 cells in good growth condition were digested in a 100mm culture dish and incubated for 24 hours. After the cells covered 80-90% of the culture dish surface, the supernatant was aspirated and 10mL of new DMEM medium (high sugar or low sugar) was added. The cells were then treated with Rh1, Rh-Met and Rh-Phen (6μM) for 24 hours. Subsequently, the supernatant was discarded, and the cells were gently scraped and collected with a cell scraper and washed 2-3 times with PBS. The organelles were then extracted according to the instructions of the kit, and the nucleus, cytoplasm, mitochondria and nucleus were extracted separately. 1mL of nitric acid was added for digestion overnight, 9mL of distilled water was added for dilution, and the membrane was passed. The Rh element content in each sample was then determined using a high-performance liquid chromatography inductively coupled mass spectrometer (ICP-MS).

[0134] 3.2 Cell apoptosis assay

[0135] Apoptosis is an active, highly regulated process of programmed cell death. It is typically detected by flow cytometry using Annexin V-FITC / PI double staining. During early apoptosis, the phosphatidylserine (PS) on the inner membrane flips outward, allowing Annexin V to bind to this flipped phosphatidylserine. During late apoptosis, the cell membrane is severely damaged, allowing propidium iodide (PI) to enter the cell and stain the nucleus. Double staining with Annexin V and PI can distinguish the two stages of apoptosis.

[0136] Main experimental steps:

[0137] (1) T-24 cells in good growth condition were digested into six-well plates and then placed in a cell culture incubator for 24 h. After the cells grew to 80-90% density, the supernatant was removed and 2 mL of new DMEM medium (high glucose or low glucose) was added. The cells were then treated with different concentrations of Rh-Met and Rh-Phen (6, 12, and 18 μM) for 24 h.

[0138] (2) The cells were then digested with trypsin and collected, centrifuged at 1500 rpm for 5–10 min, the supernatant removed, and washed 2–3 times with PBS;

[0139] (3) Add 5 μL of Annexin V-FITC to each sample in 500 μL of 1× Binding Buffer and incubate in the dark for 5 minutes. Then add 5 μL of PI and continue incubation for 15 minutes.

[0140] (4) After incubation, the cells were passed through a membrane and then detected using an ultrasonic NxT acoustic focusing flow cytometer.

[0141] 3.3 Cell cycle assay

[0142] There is a close interaction between the cell cycle and energy metabolism. Cell proliferation requires a continuous supply of energy and biosynthetic raw materials, and the cell cycle process is also dynamically regulated by the state of energy metabolism. Flow cytometry detection of the cell cycle is an experimental method that determines the different stages of the cell cycle (G0 / G1 phase, S phase, G2 / M phase) by analyzing the distribution of cellular DNA content. Its core principle is based on the changes in DNA content during the cell cycle, combined with fluorescent dye labeling and the high-throughput detection technology of flow cytometry.

[0143] The main experimental steps are as follows:

[0144] (1) T-24 cells in good growth condition were digested into a culture dish and placed in a cell culture incubator for 24 h. After the cells grew to 80-90% density, the supernatant was removed and 4 mL of new DMEM medium (high glucose or low glucose) was added. The cells were then treated with different concentrations of Rh-Met and Rh-Phen (6, 12, 18, and 24 μM) for 24 h.

[0145] (2) Collect the cells and centrifuge them, wash them 2-3 times with PBS, resuspend them in ice-cold ethanol solution, and place them in a -20℃ refrigerator for 24 hours.

[0146] (3) Centrifuge at 2000 rpm to remove the supernatant, add 500 μL PI / RNase staining buffer (Cat. No. 550825, BD Pharmaceuticals TM ) and incubated at 37°C in the dark for 30 min.

[0147] (4) The samples were filtered through a double membrane and then analyzed using an NxT acoustic focusing flow cytometer.

[0148] 3.4 Mitochondrial stress test

[0149] The Seahorse Mitostress Assay is a test used to assess mitochondrial function in cells, typically performed using the Seahorse XF Analyzer. This assay measures the oxygen consumption rate (OCR) of cells to assess mitochondrial respiratory function, thereby understanding the cell's energy metabolism. Specific drugs are sequentially injected to disrupt the mitochondrial electron transport chain (ETC), allowing the oxygen consumption rate of cells to be measured under different conditions.

[0150] Main experimental steps:

[0151] (1) T-24 cells were seeded in a Seahorse XF cell culture plate, with an appropriate number of cells per well. DMEM high-glucose medium or DMEM low-glucose medium was added. After the cells grew to an appropriate density, the original medium was removed and new medium was added. Then, different concentrations of Rh complexes were added to make the final concentrations of 6, 12, 18, and 24 μM, and the cells were incubated for 12 hours.

[0152] (2) Prepare drugs: Oligomycin, FCCP, and Rotenone / Antimycin A, dilute to appropriate concentrations according to the experimental design, and calibrate the Seahorse XF analyzer;

[0153] (3) Place the cell culture plate into the Seahorse XF Analyzer. Set up the experimental protocol, including the sequence and timing of baseline measurements and drug infusions. The experiment is then run, first measuring the baseline oxygen consumption rate (OCR) of the cells, followed by the gradual addition of oligomycin, FCCP, and rotenone / antimycin A, and data collection.

[0154] (4) The data were analyzed using Seahorse XF analysis software to calculate various parameters, such as basal respiration, ATP-related respiration, maximum respiratory capacity, and spare respiratory capacity.

[0155] 3.5 Glycolysis Stress Test

[0156] The Glycolysis Stress Test Assay is an experimental method used to evaluate the glycolytic function of cells, usually performed using a Seahorse XF analyzer. This experiment evaluates the glycolytic activity of cells by measuring the extracellular acidification rate (ECAR) of cells, thereby understanding the state of the glycolytic pathway in the cell's energy metabolism. Glycolysis is the main way for cells to produce energy under anaerobic or hypoxic conditions, and its end product, lactate, causes acidification of the extracellular environment. The glycolysis stress test experiment interferes with the glycolytic pathway by sequentially injecting specific drugs to measure the ECAR value of cells under different conditions.

[0157] Main experimental steps:

[0158] (1) T-24 cells were seeded in a Seahorse XF cell culture plate, with an appropriate number of cells seeded into each well. DMEM high-glucose medium or DMEM low-glucose medium was added. After the cells grew to an appropriate density, the original medium was removed and new medium was added. Then, different concentrations of rhodium complex were added to make the final concentrations of 6, 12, 18, and 24 μM, and the cells were incubated for 12 hours.

[0159] (2) Prepare drugs: glucose, oligomycin, and 2-D, dilute to appropriate concentrations according to the experimental design, and calibrate the Seahorse XF analyzer;

[0160] (3) Place the cell culture plate into the Seahorse XF analyzer. Set up the experimental protocol, including the order and timing of baseline measurements and drug infusions. The experiment is then run, first measuring the cells' baseline ECAR (reflecting basal glycolytic activity), followed by the gradual addition of glucose, oligomycin, and 2-DG, and data collection.

[0161] (4) Use Seahorse XF analysis software to analyze the data and calculate various parameters, such as basal glycolysis, glycolytic capacity, and glycolytic reserve capacity.

[0162] 3.6 Results and Discussion

[0163] 3.6.1 Effect of flow cytometry on apoptosis

[0164] To verify the results of the MTT assay, we first used flow cytometry to detect the apoptosis of T-24 cells induced by these Rh complexes under high and low glucose conditions. Figure 6 and Figure 7 As shown, metformin mixed with rhodium complex Rh-Met exhibited a stronger apoptosis-inducing effect on T-24 cells under high glucose conditions than under low glucose conditions. Under high glucose conditions, treatment with 6, 12, and 18 μM Rh-Met for 24 hours resulted in 16.7%, 28.4%, and 47.6% apoptosis in T-24 cells, respectively. This was compared to 14.1%, 30.7%, and 34.2% apoptosis in T-24 cells treated with the same concentrations under low glucose conditions. Phenformin mixed with rhodium complex Rh-Phen exhibited a similar trend, with apoptosis rates of 13.3%, 20.6%, and 34.1% under high glucose conditions and 8.8%, 12.1%, and 21.3% under low glucose conditions. This result suggests that metformin or phenformin mixed complexes have a more pronounced apoptosis-inducing effect under high glucose conditions, consistent with the results of the MTT assay.

[0165] 3.6.2 ICP-MS Detection of Cellular Uptake and Distribution of Rh Complexes

[0166] Studies have shown that the ability of cells to absorb drugs can affect the effects of drugs, thereby directly affecting the therapeutic effect. 50 To determine whether the difference in values ​​was related to the uptake capacity of the complex by T-24 cells, we first used ICP-MS to detect the uptake capacity of the complex in T-24 cells cultured in high glucose and low glucose conditions. Figure 8 As shown, the accumulation levels of Rh1 and Rh-Met in T-24 cells were similar under high- and low-glucose conditions, while the accumulation of Rh-Phen was slightly higher under high-glucose conditions than under low-glucose conditions. This indicates that Rh complexes can effectively accumulate in T-24 tumor cells, with no significant difference in accumulation between high- and low-glucose conditions. The distribution of the complexes in various organelles revealed that Rh complexes were primarily concentrated in mitochondria, similar to the cellular accumulation profile of most mitochondrial metallodrugs.

[0167] 3.6.3 Flow cytometry to detect the effect of Rh complexes on cell cycle

[0168] Studies have shown that the cell cycle is closely related to changes in cellular metabolism. For example, the metabolic regulatory enzyme pyruvate kinase M2 (PKM2) in cells plays a key role in regulating the progression of the G1 phase of cells and is highly expressed in tumor cells. It promotes the synthesis of biological macromolecules (such as nucleotides and lipids) by regulating glycolytic flux (such as reducing enzyme activity to accumulate upstream metabolic intermediates), thereby indirectly promoting the G1 / S phase transition. The energy provided by glycolysis and oxidative phosphorylation (OXPHOS) is essential for DNA replication in the S phase of cells.

[0169] We further investigated the effects of Rh-Met and Rh-Phen on the cell cycle of T-24 tumor cells by flow cytometry. Figure 9As shown, Rh-Met and Rh-Phen effectively induce cell cycle arrest under both high and low glucose conditions. Under high glucose conditions, Rh-Met and Rh-Phen arrested T-24 cells in the G1 phase, and the percentage of cells arrested in the G1 phase increased with increasing dose concentration. At Rh-Met concentrations of 6, 12, 18, and 24 μM, the percentages of cells arrested in the G1 phase were 54.97%, 57.39%, 67.80%, and 100%, respectively. At the same Rh-Phen concentrations, the percentages of cells arrested in the G1 phase were 51.58%, 61.32%, 66.69%, and 73.50%, respectively. Under low glucose conditions, Rh-Met and Rh-Phen primarily arrested T-24 cells in the S phase, also in a concentration-dependent manner. When Rh-Met was administered at concentrations of 6, 12, 18, and 24 μM, the percentages of cells arrested in the S phase were 52.63%, 60.69%, 63.44%, and 73.86%, respectively. In contrast, when treated with Rh-Phen at the same concentrations, the percentages of cells arrested in the S phase were 40.42%, 45.75%, 42.25%, and 43.26%, respectively. This result indicates that the effects of Rh-Met and Rh-Phen on the cell cycle of T-24 cells are related to extracellular glucose concentration, with high glucose conditions primarily arresting T-24 cells in the G1 phase and low glucose conditions primarily arresting them in the S phase.

[0170] 3.6.4 Effects of Rh Complexes on T-24 Cell Metabolism

[0171] Research has shown that the PI3K / AKT / mTOR signaling pathway plays a crucial role in reprogramming tumor energy metabolism. Tumor cell growth and proliferation require substantial amounts of energy and other substances. The PI3K / AKT / mTOR signaling pathway enhances metabolic pathways such as glycolysis and lipid synthesis during this process, enabling tumor cells to adapt and survive even in the harsh environment of hypoxia. Inhibition of the PI3K / AKT / mTOR signaling pathway may lead to mitochondrial respiration suppression and energy metabolism defects.

[0172] We used the Seahorse Cell Energy Metabolism Analyzer to further analyze the effects of Rh-Met and Rh-Phen treatment on the mitochondrial oxidative phosphorylation system of T-24 cells under high glucose and low glucose conditions for 24 hours. Figure 10As shown, Rh-Met significantly inhibited mitochondrial bioenergetics parameters in T-24 cells under high glucose conditions, and this inhibitory effect became more pronounced with increasing concentration. Under low glucose conditions, Rh-Met's inhibitory effect on mitochondrial respiration in T-24 cells was even more pronounced, exhibiting a strong inhibitory effect at only a 6 μM concentration. Rh-Phen also inhibited mitochondrial respiration in T-24 cells at 18 μM, and this was observed under both high and low glucose conditions.

[0173] Effects of 3.6.5 Rh Complexes on Glycolysis in T-24 Cells

[0174] Glucose metabolic reprogramming (GMR) is the process by which tumor cells reshape their glucose metabolism to adapt to the pressures of rapid proliferation and survival. Its core characteristic is that, even in the presence of ample oxygen, tumor cells preferentially obtain energy and metabolic intermediates through aerobic glycolysis (the Warburg effect), rather than relying on efficient mitochondrial oxidative phosphorylation. This shift in metabolic pattern not only provides energy for tumor cells but also supports their biosynthesis and antioxidant needs.

[0175] We used glycolysis stress test to further study the effects of different concentrations of Rh-Met and Rh-Phen on the glycolytic function of T-24 cells after 24 hours. Figure 11 As shown, under high glucose conditions, glycolysis in T-24 cells was significantly inhibited after 24 hours of treatment with different concentrations of Rh-Met (6, 12, 18, and 24 μM) and Rh-Phen (18 μM). We also compared the effects of Rh-Met and Rh-Phen on glycolysis in T-24 cells under low glucose conditions. Under low glucose conditions, the ECAR values ​​of the control group were essentially the same as those under high glucose conditions, indicating that, in the absence of drugs, glycolysis in T-24 cells is unaffected by external glucose levels. However, after treatment with Rh-Met, glycolysis in T-24 cells was significantly inhibited, and this inhibitory effect was more pronounced than under high glucose conditions. The results of the glycolysis stress experiment showed that Rh-Met and Rh-Phen effectively inhibited glycolysis in T-24 cells under both high and low glucose conditions.

[0176] Experimental Example 4: Study on the anti-tumor and hypoglycemic activities of the rhodium complex of the present invention in a type 2 diabetes mellitus combined with tumor mouse model

[0177] 4.1 Construction of a Type 2 Diabetes Mellitus Combined with Subcutaneous MB-49 Tumor Model

[0178] The BKS-DB mouse strain is often used as a model for type 2 diabetes and obesity. BKS-DB mice carrying a spontaneous diabetes mutation (Leprdb) exhibit morbid obesity, persistent hyperglycemia, and reduced insulin production by pancreatic beta cells, which further leads to decreased insulin levels. Obesity symptoms begin to appear 3-4 weeks after birth, plasma insulin gradually increases from 10-14 days after birth, and blood glucose levels rise from 4-8 weeks after birth. BKS-DB mice also show symptoms such as increased food intake, increased water intake, and increased urine output.

[0179] To evaluate the hypoglycemic and tumor-suppressing effects of Rh-Met in a type 2 diabetes mellitus (T2DM)-tumor model, we established a T2DM-MB-49 subcutaneous tumor model using BKS-DB mice. The specific experimental steps are as follows:

[0180] (1) Animal husbandry: Mice for animal experiments were purchased from Hangzhou Resource Laboratory Animal Co., Ltd. (Hangzhou, China, approval number: SCXK(ZHE)2024-0004). All animal experimental procedures were performed in accordance with protocols approved by the Laboratory Animal Care and Animal Ethics Committee of Guangxi Normal University. The experiments were conducted at the Animal Culture Center on the sixth floor of the State Key Laboratory of Medicinal Resource Chemistry and Drug Molecular Engineering, Guangxi Normal University. The animal room was maintained at a constant temperature of 23–25°C. Cages, feed, and drinking water required for animal husbandry were sterilized by ultraviolet or high temperature and replaced every 2 days.

[0181] (2) Preparation of MB-49 cell suspension: MB-49 mouse bladder cancer cells with good growth status were digested and centrifuged, the supernatant was removed, and the cells were resuspended in a small amount of serum-free medium. The cells were then counted and the cell number was adjusted to 2×10 per ml. 6 Add biological matrix gel at a ratio of 1:1 and pipette the cells evenly to obtain a cell suspension. Prepare enough cell suspension according to the number of mice to be inoculated, then place on ice and complete the inoculation within 1-2 hours.

[0182] (3) Construction of Type 2 Diabetes + MB-49 Subcutaneous Tumor Model: The prepared MB-49 cell suspension was inoculated into the right thigh of 7-8 week old BKS-DB mice. The inoculation site was then disinfected to prevent infection. After injection, the cage was replaced with a new one, and the tumor growth status was continuously observed.

[0183] (4) Experimental grouping: When the tumor grows to 80-100 mm 3 At the same time, tumor-bearing mice with good growth status and uniform size were selected and randomly divided into 4 groups, with 5 mice in each group. The groups were: control group (5 mice), cisplatin group (5 mice), metformin group (5 mice), and Rh-Met group (5 mice).

[0184] (5) Drug administration: At the beginning of the experiment, mice were treated according to their grouping. Drugs were administered intraperitoneally, and the dosage was calculated based on the mouse body weight. The control group received a daily injection of saline containing 1.5% DMSO, the metformin group and the Rh-Met group received daily administration at a dosage of 20 mg / kg, and the cisplatin group received administration every other day at a dosage of 2 mg / kg.

[0185] (6) Data Collection: Changes in body weight, tumor volume, fasting blood glucose levels, and other indicators of the mice were monitored. After 14 days of continuous administration, the mice were anesthetized and sacrificed on the 15th day. The heart, liver, spleen, lung, kidney, blood, and tumor of the mice were removed for subsequent testing of various indicators.

[0186] Fasting blood glucose level detection: The fasting blood glucose concentration of mice was measured every two days. The measurement time was fixed, 4-6 hours after administration. During this period, the mice were given normal water but no food was provided. Blood glucose levels were measured three times using a Roche blood glucose meter. Blood was collected from the tip of the mouse's tail, and fresh blood was used each time.

[0187] Tumor volume measurement: Use a vernier caliper to measure the tumor volume of the mouse, record the length and width of the tumor, and then calculate the tumor volume. The calculation formula of tumor volume (TV): TV = lw 2 / 2 (l and w represent the long and short diameters of the tumor, respectively).

[0188] Tumor Weight and Tumor Inhibition Rate: After 15 days of dosing, tumors were harvested and weighed, and the average tumor weight for each group was recorded. The tumor inhibition rate was calculated using the formula: (TGI%) = (1-m / m0), where m0 is the average tumor weight of the control group and m is the average tumor weight of the treatment group.

[0189] 4.2 Oral glucose tolerance test in mice

[0190] The Oral Glucose Tolerance Test (OGTT) in mice is an experimental method commonly used to evaluate the glucose metabolism ability and insulin sensitivity of mice. It helps to understand whether the glucose metabolism function of mice is normal by monitoring the changes in blood glucose levels after the mice ingest glucose. It is often used in the study of diabetes, obesity and related metabolic diseases. The blood glucose of normal mice will return to baseline levels within 1-2 hours. If the blood glucose level of mice rises rapidly after ingesting glucose and recovers slowly, it indicates that insulin resistance may exist (such as the type 2 diabetes model).

[0191] The main experimental steps are as follows:

[0192] (1) Mice were fasted for 12 hours overnight to eliminate the effect of eating on blood glucose. Water was provided normally during this period.

[0193] (2) Blood glucose levels of mice in each group were measured by taking blood from the tip of the tail, which was the baseline blood glucose at 0 min;

[0194] (3) Prepare a glucose solution of a certain concentration in advance, calculate the amount of glucose solution according to the weight of each mouse, and administer the glucose solution by gavage at a dose of 1 g / kg;

[0195] (4) Continuously monitor the blood glucose changes of mice by collecting blood samples at specific time points (15, 30, 60, 90, and 120 minutes) after glucose ingestion to measure blood glucose concentration;

[0196] (5) Draw a curve of blood glucose changes over time and calculate the area under the curve (AUC).

[0197] 4.3 Intraperitoneal insulin tolerance test in mice

[0198] The intraperitoneal insulin tolerance test (ITT) is an experimental method to directly assess the insulin sensitivity of mice by intraperitoneal injection of insulin and monitoring blood glucose changes. [8] It is primarily used to study the responsiveness of mice to exogenous insulin. A slow or small decrease in blood sugar levels suggests insulin resistance (common in models of type 2 diabetes and obesity).

[0199] The main experimental steps are as follows:

[0200] (1) Mice were fasted for 4–6 h (to avoid stress caused by overnight fasting) to stabilize basal blood glucose levels, and water was supplied normally during this period;

[0201] (2) baseline blood glucose measurement, fasting blood glucose was measured after fasting (0 min);

[0202] (3) Intraperitoneal injection of a certain dose of insulin. The injection volume is calculated based on the weight of the mouse, and the injection volume for each mouse is 1.0 U / kg.

[0203] (4) Continuously monitor the blood glucose changes of mice by collecting blood samples at specific time points (15, 30, 60, 90, and 120 minutes) after glucose ingestion to measure blood glucose concentration;

[0204] (5) Draw a curve of blood glucose changes over time and calculate the area under the curve (AUC).

[0205] 4.4 ICP-MS Detection of Drug Distribution in Mouse Organs and Tumors

[0206] We used high-performance liquid chromatography-mass spectrometry (ICP-MS) to examine the distribution of Rh-Met in various organs and tumors in mice after 15 days of continuous administration. The main experimental steps are as follows:

[0207] (1) Remove mouse heart, liver, spleen, lung, kidney, and tumor tissues, accurately cut 0.1 g, add 500 μL of RIPA lysis buffer containing 1% PMSF, and grind thoroughly to lyse tissue cells;

[0208] (2) Collect the cleavage solution and add 1.5 mL of concentrated nitric acid for digestion overnight;

[0209] (3) Add distilled water to dilute the sample 10 times and then pass it through the membrane;

[0210] (4) The Rh content in the samples was analyzed using a high performance liquid chromatography-mass spectrometer.

[0211] 4.5 Hematoxylin-eosin staining experiment

[0212] Hematoxylin-eosin staining (H&E staining) is a fundamental staining technique in histopathology, used to visualize the morphology of the cell nucleus and cytoplasm, aiding in the observation of histological features, pathological changes, and cellular localization. Hematoxylin primarily binds to DNA (negatively charged phosphate groups) within the cell nucleus, forming complexes with metal mordants (such as aluminum ions), staining the cell nucleus blue-purple. Hematoxylin also binds to basic proteins in the cytoplasm (such as collagen and cytoplasmic matrix), staining the cytoplasm and extracellular matrix pink or red.

[0213] Main experimental steps:

[0214] (1) Remove the tissue sample, dehydrate it, embed it in paraffin, cut it into 4-6 μm slices, and attach it to a slide;

[0215] (2) Dissolve paraffin by soaking in xylene for 10-20 minutes;

[0216] (3) Rehydration in a series of ethanol solutions with a concentration gradient of 100% → 95% → 80% → 70%, with each immersion lasting 3–5 minutes;

[0217] (4) Stain with hematoxylin for 3-10 minutes, then rinse with running water to remove excess dye;

[0218] (5) Soak in alcohol containing 1% hydrochloric acid for several seconds to 10 seconds, then rinse with running water for 15 to 30 minutes until the cell nucleus returns to blue;

[0219] (6) Stain with eosin solution for 1 to 3 minutes and continue rinsing with running water;

[0220] (7) Dehydration was performed using a series of ethanol solutions with a concentration gradient of 70% → 80% → 90% → 95% → 100% alcohol, each for 1 to 2 minutes, for gradual dehydration;

[0221] (8) Encapsulate the slices with neutral gum;

[0222] (9) Analyze the pathological sections of the heart, liver, kidney and other tissues of the mice after administration under a microscope.

[0223] 4.6 Experimental Results and Discussion

[0224] 4.6.1 Study on the Hypoglycemic Activity of Rh-Met in a Diabetes and Tumor Model

[0225] Studies have shown that tumor cells exposed to high glucose levels exhibit faster growth and stronger invasiveness compared to tumor cells under normal conditions. To evaluate the anti-tumor activity of the oxidized aporpheniramine-metformin mixed rhodium complex under high glucose conditions, we established a type 2 diabetes mellitus combined with MB-49 subcutaneous tumor mouse model. MB-49 single cell suspension was injected subcutaneously into the right buttock of each mouse. After the tumor grew to 80-100 mm 3 Mice were then randomly divided into four groups (n=5): a control group, a metformin (20 mg / kg) group, a DDP (2.0 mg / kg) group, and a Rh-Met (20 mg / kg) group. The control group received daily injections of 1.5% DMSO-containing saline. The Rh-Met (20 mg / kg) and metformin (20 mg / kg) groups were treated with Rh-Met or metformin dissolved in 1.5% DMSO-containing saline and administered daily according to the mice's body weight for 14 consecutive days, for a total of 14 doses. The DDP (2.0 mg / kg) group was treated with DDP dissolved in saline and administered every two days according to the mice's body weight for 14 consecutive days, for a total of 7 doses. The mice's body weight, fasting blood glucose level, and tumor volume were measured every two days.

[0226] We first studied the changes in blood glucose in diabetic mice during continuous treatment. Figure 12As shown, after treatment with Rh-Met (20 mg / kg), the fasting blood glucose levels of diabetic mice decreased significantly. On the ninth day of continuous treatment, the average fasting blood glucose level of mice in the Rh-Met (20 mg / kg) group stabilized at around 8 mmol / L. On the 15th day, the average fasting blood glucose concentration in the Rh-Met (20 mg / kg) group was 7.1 mmol / L, significantly lower than the 24.6 mmol / L in the control group. The average fasting blood glucose concentrations in the metformin (20 mg / kg) and DDP (2.0 mg / kg) groups were 18.0 mmol / L and 18.1 mmol / L, respectively. This result indicates that the oxidized aporpheniramine-metformin mixed rhodium complex can effectively lower the blood glucose levels of diabetic mice at a dose of 20 mg / kg, and is superior to 20 mg / kg of metformin alone.

[0227] 4.6.2 Study on the Antitumor Activity of Rh-Met in a Diabetes-Tumor Model

[0228] We also analyzed the tumor-suppressing effect of Rh-Met (20 mg / kg) in a diabetic mouse model with tumors. Figure 13 As shown, after 15 days of continuous treatment, tumors in all groups of mice were suppressed to varying degrees. After treatment with Rh-Met (20 mg / kg), the average tumor weight in mice decreased from 0.864 g in the control group to 0.404 g, while it decreased to 0.468 g in the cisplatin (2.0 mg / kg) group and 0.688 g in the metformin (20 mg / kg) group. The tumor inhibition rate in the Rh-Met group was 53.24%, higher than the 44.68% in the DDP group and 18.68% in the Met group. After treatment with Rh-Met (20 mg / kg), the body weight of diabetic mice decreased slightly, but the average weight was higher than that in the cisplatin group. These results demonstrate that Rh-Met, at a dose of 20 mg / kg, effectively inhibits tumor growth in mice with type 2 diabetes and MB-49 subcutaneous tumors, and outperforms the positive control drug, cisplatin.

[0229] 4.6.3 Effects of Rh-Met Treatment on Diabetes-Related Parameters in Mice

[0230] To evaluate the effect of Rh-Met treatment on the alleviation of diabetes in mice, we performed an oral glucose tolerance test (OGTT) on the treated mice. Figure 14As shown, after oral administration of 1 g / kg glucose based on body weight, the blood glucose levels of diabetic mice in the Rh-Met-treated group increased minimally, and blood glucose levels essentially returned to normal by 90 minutes. However, the blood glucose levels in the DDP and metformin groups remained similar to those in the control group. Following oral administration of 1 g / kg glucose based on body weight, blood glucose levels increased significantly and then decreased more slowly, demonstrating significant glucose intolerance. We also calculated the area under the curve (AUC) for each group in the OGTT experiment. The results showed that the AUC for the Rh-Met group was significantly lower from 0 to 120 minutes than those in the control, metformin, and DDP groups. This result suggests that Rh-Met significantly improves glucose tolerance in diabetic mice, significantly alleviating diabetic symptoms.

[0231] We further verified the insulin resistance of diabetic mice after Rh-Met treatment by intraperitoneal insulin tolerance test (ITT). Figure 15 As shown, after intraperitoneal injection of 1.0 U / kg insulin based on body weight, the blood glucose level of mice in the Rh-Met treatment group decreased significantly, reaching 42.36±3.2% at 30 minutes, which was significantly better than the 63.65±5.4% in the untreated group, 57.87±5% in the Met group, and 63.83±4.9% in the DDP group. This result indicates that after continuous Rh-Met treatment, the responsiveness of diabetic mice to insulin is enhanced and insulin resistance is improved.

[0232] 4.6.4 ICP-MS Detection of Rh-Met Accumulation in Various Organs and Tumors in Mice

[0233] We used ICP-MS to detect the accumulation of drugs in major organs and tumor tissues of mice. Figure 16 As shown, in the control group, Rh accumulation in various organs of mice was very low. However, after Rh-Met treatment, Rh levels in various organs of the mice increased significantly. Rh accumulated to varying degrees in the heart, liver, spleen, lungs, kidneys, and pelvis of the mice. Rhodium accumulated to a high level in the liver, with an Rh content of 310.2 μg / g tissue, and also in tumor tissue, with a high content of 92.8 μg / g tissue. These results indicate that after Rh-Met treatment, Rh complexes in diabetic mice with tumors are primarily concentrated in the liver and tumor tissues.

[0234] 4.6.5 H&E staining to detect the effects of Rh complexes on various organs of mice

[0235] We used hematoxylin-eosin staining (H&E) to examine the histomorphological characteristics of the heart, liver, spleen, lung, and kidney of diabetic mice after 15 days of treatment to study the effects of drug treatment on various organs of the mice. Figure 17 As shown in the results, after Rh-Met treatment, the histological morphology of the heart, liver, spleen, lung, and kidney of the mice was basically the same as that of the control group, with no obvious lesions or damage. This result indicates that Rh-Met is safe for BSK-DB type 2 diabetic mice with tumors at a dose of 20 mg / kg.

Claims

1. The rhodium complexes of aporpheniramine oxide and biguanide derivatives are the complexes Rh-Met and Rh-Phen, and their structures are shown below:

2. The synthetic method of the rhodium complex according to claim 1, wherein Aporpheniramine oxide, rhodium trichloride hydrate, and an excess of metformin hydrochloride or phenformin hydrochloride relative to aporpheniramine oxide are placed in a reaction container, a mixed solvent is added, and the mixture is reacted under heating conditions to obtain a complex Rh-Met or a complex Rh-Phen; wherein the mixed solvent is a composition composed of acetonitrile and methanol in a volume ratio of 1-2:1-3.

3. A crystal form of a complex Rh-Met, which belongs to the triclinic system, P-1 space group, and has unit cell parameters: α=111.124(2) o , β=94.915(2) o ,γ=99.112(2) o .

4. The method for synthesizing the Rh-Met crystal form of the complex according to claim 3, characterized in that: Aporpheniramine oxide, rhodium trichloride hydrate and metformin hydrochloride in excess relative to aporpheniramine oxide are placed in a reaction vessel, acetonitrile and methanol are added, and the reaction is carried out under heating conditions. The reactants are cooled, crystals are precipitated, and the crystals are collected to obtain the target product; wherein the volume ratio of acetonitrile to methanol is 1:

2.

5. The preparation method according to claim 2, characterized in that: The excess metformin hydrochloride or phenformin hydrochloride means that the amount of metformin hydrochloride or phenformin hydrochloride is 1.5 times or more of the molar amount of aporpheniramine oxide.

6. The preparation method according to claim 2 or 4, characterized in that: The reaction was carried out at ≥50°C.

7. The preparation method according to claim 2 or 4, characterized in that: The reaction container is a high-pressure reactor.

8. Use of the rhodium complex of aporpheniramine oxide and a biguanide derivative according to claim 1 or the crystalline form of the complex Rh-Met according to claim 3 in the preparation of hypoglycemic drugs or anti-tumor drugs.

9. Use of the Rh-Met complex according to claim 1 or the crystal form of the Rh-Met complex according to claim 3 in the preparation of a medicament for treating diabetes mellitus combined with tumors in patients.

10. A pharmaceutical composition comprising the rhodium complex of aporphenanthrene oxide and a biguanide derivative according to claim 1 or the crystalline form of the complex Rh-Met according to claim 3.

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