CN115368345B

CN115368345B - Small molecular compound targeting tumor cell mitochondria and application and preparation method thereof

Info

Publication number: CN115368345B
Application number: CN202210879310.XA
Authority: CN
Inventors: 周在刚; 沈建良; 谢聪颖
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2022-07-25
Filing date: 2022-07-25
Publication date: 2023-08-11
Anticipated expiration: 2042-07-25
Also published as: CN115368345A9; CN115368345A

Abstract

The application relates to a small molecular compound targeting tumor cell mitochondria, application and a preparation method, wherein the application designs a micro-environment with the tumor mitochondria targeting small molecular compound IR-LND through a rapid, economical and efficient synthetic route, and can reverse oxygen deficiency and immunosuppression in tumors, wherein the IR-LND simultaneously enhances photodynamic therapy and immunotherapy, and has wide application prospect in the treatment of solid tumors, metastasis and hematoma.

Description

Small molecular compound targeting tumor cell mitochondria and application and preparation method thereof

Technical Field

The application relates to the field of compound compounds, in particular to synthesis, application and preparation methods of a small molecular compound targeting tumor cell mitochondria.

Background

Photodynamic therapy (PDT) has been successfully incorporated into the treatment of a variety of tumors since the last 70 th century. PDT requires the co-participation of a photosensitizer, a laser of a specific wavelength, and oxygen, which converts oxygen in cells into Reactive Oxygen Species (ROS) having cytotoxicity under irradiation of the laser of the specific wavelength, thereby inducing apoptosis. In addition, PDT can also promote antigen presentation to cytotoxic T lymphocytes to reverse the immunosuppressive microenvironment to initiate anti-tumor immunity. PDT has advantages of small trauma, small side effects and being able to treat locally, compared to conventional chemotherapy, radiotherapy and cell therapy, and thus PDT is expected to develop into one of the effective schemes for treating cancer clinically.

Three generations of photosensitizers have been developed so far, including heptamethylcyanates, porphyrin analogues and phenothiazine derivatives, although these emerging photosensitizers can solve to some extent the internal drawbacks of traditional photosensitizers, such as low ROS yield, short half-life, no tumor targeting etc. Among them, the seven-methyl-cyanamide dyes such as IR-780 and MHI-148 are the most promising photosensitizer molecules. Because the molecules can be specifically accumulated at a tumor part and can be excited by near infrared light of 780-800nm, the tissue penetration depth can reach about 8mm-1cm, and PDT can be implemented on cancerous tissues deep in the organism. However, tumor hypoxia severely limits ROS production, resulting in poor PDT effects, and PDT can further promote PD-L1 expression at the tumor site by increasing IFN-gamma secretion, exacerbating the immunosuppressive microenvironment. However, few photosensitizer molecules have been able to solve the problems of tumor hypoxia and high expression of PD-L1 at the same time. Therefore, there is an urgent need to develop a photosensitizer molecule with other functions to solve these two challenges faced by PDT.

It is well known that tumors are primarily energized by "Warberg effect", but that many malignant cell lines still exhibit mitochondrial-related oxidative phosphorylation (OXPHOS) enhancement during tumorigenesis, progression and metastasis, consuming more endogenous O ₂ Causing tumor hypoxia. Currently, there are many methods for nanocarriers to increase oxygenation of tumors by oxygen loading or oxygen generation by redox reactions occurring in cells, however, related computational models indicate that reducing intrinsic oxygen consumption is more effective than increasing oxygen supply in alleviating the hypoxic state of tumor cells. Thus, studies have shown that drugs that inhibit mitochondrial OXPHOS, such as metformin, tamoxifen, lonidamine, etc., can reduce oxygen consumption to some extent to alleviate intra-tumor hypoxia. However, current drugs that inhibit mitochondrial OXPHOS are generally less potent, and the institute of wisconsin, especially teaches that the team improves the potency by a factor of 100 relative to before the modification by modifying lonidamine to a Mito-LND that targets mitochondria. It is another object of the present application to find more Mito-LNDs that have high efficacy in inhibiting mitochondrial OXPHOS.

Disclosure of Invention

Aiming at the limited effectiveness of Mito-LND for inhibiting mitochondria OXPHOS and limited compound types at present, the application aims to provide an intermediate of a small molecular compound for targeting mitochondria of tumor cells, wherein the intermediate is a novel oxidative phosphorylation inhibitor, and the structural formula is as follows:

the application also provides a preparation method of the oxidative phosphorylation inhibitor, which comprises the following steps:

the lonidamine, 1- (Boc-amino) -4-aminobutane and 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride are dissolved in an organic solvent and stirred at room temperature for reaction for 24 hours, then trifluoroacetic acid and triethylamine are added to remove the Boc group, and the oxidative phosphorylation inhibitor is obtained after purification.

In the technical scheme, the mol ratio of the lonidamine to the 1- (Boc-amino) -4-aminobutane and the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride is 1:1.2:1.2.

In the above technical scheme, the organic solvent is methanol and dichloromethane.

In the above technical solution, the purification step: the product was further purified by high performance preparative liquid chromatography techniques and freeze dried.

The application also provides a small molecule compound of targeted tumor cell mitochondria, which is a photosensitizer and has the structural formula:

the application also provides a preparation method of the small molecule compound targeting the mitochondria of tumor cells, which comprises the following steps:

sodium acetate, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] aniline hydrochloride, 1-N-butyl-2, 3-trimethyl-3H-indole iodide and 1- (5-carboxyhexyl) -2, 3-trimethyl-3H-indole iodide are dissolved in absolute ethyl alcohol, reflux is carried out for 3H at a constant temperature of 85 ℃, the reacted liquid is decompressed and distilled in a rotary way, and precipitation, filtration and purification are carried out by using a precipitator, thus obtaining the IR-68 compound.

In the above technical scheme, the precipitating agent is diethyl ether.

Aiming at the defects of the existing photodynamic therapy, the application aims to provide a small molecular compound targeting tumor cell mitochondria with an oxidative phosphorylation inhibitor as a raw material, which has the structural formula as follows:

dissolving the IR-68 compound and the oxidative phosphorylation inhibitor in an organic solvent, adding a dehydration condensing agent, reacting for 24-48 hours at room temperature, precipitating with a precipitating agent, filtering, purifying to obtain the small molecular compound of the targeted tumor cell mitochondria of claim 2,

the structural formula of the compound IR-68 is as follows:

in the above technical scheme, the molar ratio of the IR-68 compound to the oxidative phosphorylation inhibitor is 1:1.

In the above technical scheme, the dehydration condensing agent is DMTMM.

In the above technical scheme, the precipitating agent is diethyl ether.

The present application also provides a composition comprising: small molecule compounds targeting the mitochondria of tumor cells as described above and pharmaceutically acceptable carriers.

The application also provides application of the small molecular compound of the targeted tumor cell mitochondria in preparation of antitumor therapeutic drugs.

In the above technical scheme, the tumor is solid tumor, metastasis tumor and hematological tumor, wherein the solid tumor comprises pancreatic tumor, liver tumor, lung tumor, stomach tumor, intestinal tumor, breast tumor or cervical tumor.

In the technical scheme, the anti-tumor photodynamic therapy drug is combined with 808nm wavelength laser.

The application also provides application of the small molecular compound of the targeted tumor cell mitochondria in preparing an anti-tumor photodynamic therapy drug or a tumor immunotherapy drug.

The application also provides application of the small molecular compound targeting the mitochondria of tumor cells in preparation of a tumor living body fluorescence imaging reagent.

A synthesis method and application of photosensitizer molecules with tumor mitochondria targeting function for regulating tumor oxygen and PD-L1 expression are provided.

The small molecular compound of the application which targets the mitochondria of tumor cells is named as IR-LND, and the oxidative phosphorylation inhibitor prepared by the application is named as LND-3.

The application has the following advantages: IR-68, a near infrared two-region photosensitizer with tumor mitochondrial selectivity, brings the antineoplastic drug Lonidamine (LND) acting on mitochondria to accumulate more efficiently in mitochondria. Meanwhile, the IR-LND can improve tumor hypoxia and the immunosuppressive microenvironment of high expression of PD-L1, and the efficacy of the IR-LND is 100 times that of the LND. The IR-LND not only solves the bottleneck of photodynamic therapy, but also starts the multimode anti-tumor therapy of photodynamic therapy and immunotherapy.

Drawings

FIG. 1A is a synthetic route for IR-68; 1B is the synthetic route for IR-LND.

FIG. 2A is in vivo fluorescence imaging of MB49 tumor model at various time points following rat tail intravenous injection of 100. Mu.L of IR-LND (2 mg/kg);

FIG. 2B shows the distribution of IR-LND after 48 hours with heart, liver, spleen, lung, kidney, tumor, muscle, small intestine removed;

FIG. 2C is the average fluorescence intensity statistic of FIG. 2B;

FIG. 2D is a graph showing the IR-LND fluorescence profile of the main organ and tumor 24h or 48h after 100. Mu.L of IR-LND (2 mg/kg) or IR-LND (5 mg/kg) was intravenously injected into the rat tail of the CT26 tumor model;

FIG. 2E is a fluorescence image of the major viscera of 100. Mu.L of IR-LND (2 mg/kg) after 24 hours in normal mice and pulmonary transfer mice by tail vein injection;

FIG. 2F shows the average fluorescence intensity statistics of FIG. 2E.

FIG. 3A is a schematic illustration of the treatment process of example 6;

FIG. 3B is the change in tumor volume for the different treatment regimens of example 6;

FIG. 3C is a photograph of the tumor sizes of each group after the end of the treatment of example 6;

FIG. 3D is the tumor weights of the groups after the end of the treatment of example 6;

FIG. 3E is a graph of tumor volume change in the Vehicle group of example 6; FIG. 3F is a graph of tumor volume change in the Vehicle+Laser group of example 6; FIG. 3G is a graph of the change in tumor volume for the LND group of example 6; FIG. 3H is a plot of the tumor volume change for the IR-68 group of example 6; FIG. 3I is a plot of tumor volume change in the IR-68+ laser group of example 6; FIG. 3J is a plot of tumor volume change for the IR-LND group of example 6; FIG. 3K is a plot of tumor volume change for the IR-LND+laser set of example 6;

FIG. 3L is a graph of tumor volume change for each group of example 6.

FIG. 4A is a schematic illustration of the treatment process of example 9;

FIG. 4B is a graph showing the change in tumor volume for the different treatment regimens of example 9;

FIG. 4C is a photograph of tumor sizes of each group after the end of the treatment of example 9;

FIG. 4D is the tumor weights of the groups after the end of the treatment of example 9;

FIG. 4E is a graph of the tumor volume change in the Vehicle group of example 9; FIG. 4F is a graph of LND group tumor volume change over example 9; FIG. 4G is a plot of the volume change of the IR-68 group tumor of example 9; FIG. 4H is a plot of PD-L set tumor volume change for example 9; FIG. 4I is a plot of tumor volume change in the IR-LND group of example 9;

fig. 4K is a graph showing the change in body weight of mice under the different treatment regimens of example 9.

FIG. 5A is a fluorescent image of IR-68 and IR-LND taken preferentially by cancer cells (Hela, MB 49) over normal cells (L929, LO-2); FIG. 5B is a photograph of the red fluorescence of IR-68 and IR-LNDs highly co-localized with the green fluorescence of mitochondrial dye.

FIG. 6A is an inhibition curve of IR-LND and LND against mitochondrial complex I;

FIG. 6B is an IR-LND and LND inhibition curve for mitochondrial complex II;

FIG. 6C is the effect of different concentrations of IR-LND and LND on mitochondrial complex IV.

Fig. 7A: is a WesternBlot band of different concentrations of IR-LND for activating AMPK to induce PD-L1 low expression by disrupting mitochondrial oxidative phosphorylation;

fig. 7B: gray scale statistics of p-AMPK in fig. 7A;

fig. 7C: gray statistics of PD-L1 in fig. 7A.

Detailed Description

The present application will be further described in detail with reference to examples and effect examples, without limiting the scope of the present application.

Example 1: preparation method of photosensitizer IR-68

Sodium acetate (975 mg,11.9 mM), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] aniline hydrochloride (4.7 g,13.0 mM), N-N-butyl-2, 3-trimethylindole iodide (4.05 g,11.8 mmol) and 1- (5-carboxyhexyl) -2, 3-trimethylindole iodide (4.75 g,11.8 mmol) were dissolved in 150mL of absolute ethanol and refluxed at 85℃for 3 hours in an oil bath. The reacted liquid was distilled under reduced pressure to 40-50mL, and then 100mL of diethyl ether was added. The dark green crystals were collected in aqueous ethanol and the product was further purified using high performance preparative liquid chromatography followed by 3 days in a lyophilizer to give 3.52g of dark green powder (IR-68) having the formula shown in FIG. 1 and chemical name 1- (5-carboxypentyl) -2- [ (1E) -2- { 2-chloro-3- [ (1E) -2- [ (2E) -1-butyl-3, 3-dimethyl-2, 3-dihydro-1H-indol-2-ylidene ] ethylidene ] cyclohex-1-enyl } vinyl ] -3, 3-dimethyl-3H-indol-1-positive ion iodide.

Example 2: preparation method of oxidative phosphorylation inhibitor LND-3

Lonidamine (3.21 g,10 mM), 1- (Boc-amino) -4-aminobutane (2.26 g,12 mM), and 4- (4, 6-dimethoxy triazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM, 2.26g,12 mmol) are dissolved in 75mL of methanol and 75mL of dichloromethane, reacted at room temperature with stirring for 24 hours, and then trifluoroacetic acid (TFA) and Triethylamine (TEA) are added to remove Boc group (t-butoxycarbonyl) to obtain compound LND-3, and the product is further purified by high performance liquid chromatography technique and freeze-dried to obtain 1.68g of compound LND-3 with a yield of 43.1% and a purity of 95%.

Example 3: preparation method of small molecular compound IR-LND (infrared-low-density nucleic acid) targeting tumor cell mitochondria

The compound LND-3 (780 mg,2 mM), IR-68 (1.50 g,2 mM) and DMTMM (0.38 g,2 mM) were taken and dissolved in 50mL of methanol and 50mL of methylene chloride, and reacted at room temperature with stirring for 24 hours, followed by spin-evaporation under reduced pressure to 15-20mL, and further addition of 50mL of diethyl ether, and the dark green crystals were collected in an aqueous ethanol solution and the product was further purified by a high performance preparative liquid chromatography technique, followed by being placed in a freeze-dryer for 3 days to give 1.08g of a dark green powder (IR-LND) having the chemical name of 2- [ (1E) -2- { 2-chloro-3- [ (1E) -2- [ (2E) -1-butyl-3, 3-dihydro-1H-indol-2-ylidene ] cyclohex-1-enyl } vinyl ] -1- (1- {1- [ (2, 4-dichlorophenyl) methyl ] indazol-3-yl } -1, 8-dioxy-2, 7-diaza-3-dimethyl-3-iodo-1-ion of the formula shown in FIG. 1.

Example 4: evaluation of IR-68 and IR-LND targeting tumor cells and tumor cell mitochondria effect

The evaluation steps are as follows:

1) Hela, MB49, L929, LO-2 cells were cultured in confocal dishes, incubated for 4h with 2. Mu. MIR-68 and IR-LND, respectively, and then photographed under laser confocal conditions to give FIG. 5A, demonstrating the selectivity of IR-68 and IR-LND for tumor cells.

2) MB49 cells were cultured in confocal dishes, followed by incubation with 2. Mu.M IR-68 and IR-LND for 4h, followed by addition of mitochondrial Green fluorescent probes (Mito tracker Green) for 20min, washing three times with PBS, and photographing under laser confocal to give FIG. 5B, indicating that IR-68 and IR-LND accumulate mainly in mitochondria after entering the cells.

Example 5: evaluation of in vivo tumor targeting effect of IR-LND

The evaluation steps are as follows:

1) Preparation of animal models

C57BL/6 and BALB/C mice (age 6-8 weeks, weight 20-22g, animal culture center of Etsuzhou university of medical science laboratory animal) were subcutaneously injected with 50 ten thousand MB49 cells or CT26 cells in the armpit of the right forelimb until tumor volume reached 150-200mm ³ Can be used for in vivo fluorescence imaging.

2) Preparation of IR-LND solution

10mg of IR-LND was weighed, 0.5mL of absolute ethanol and 0.5mL of castor oil were added, and the mixture was sonicated for 2min to give an IR-LND solution (10 mg/mL). Then diluted to 0.4mg/mL (2 mg/kg) with PBS solution. 3) Fluorescence distribution of IR-LND in MB49 tumor mice and CT26 tumor mouse animal models

The MB49 tumor mice of step 1) were injected with 100uL of IR-LND solution (0.4 mg/mL) intravenously, and the distribution of IR-LND in vivo was observed with a multimode optical in vivo imaging system at 1h,6h,12h,24h, and 48h after injection. After 48h the mice were dissected and hearts, livers, spleens, kidneys, tumors, muscles, and small intestine were removed and the fluorescence distribution of the IR-LND was observed with a multimode optical biopsy system.

The mice were dissected after 100uL,24h or 48h of intravenous IR-LND solution (0.4 mg/mL or 1 mg/mL) for the CT26 tumor mice of step 1), and the heart, liver, spleen, kidney, tumor were removed, and the fluorescence distribution of IR-LND was observed.

The results in FIG. 2 show that IR-LND is metabolized by the liver and accumulates primarily at tumor sites. Indicating that the IR-LND can be used as a near infrared fluorescent imaging agent for indicating tumor sites.

Example 6: evaluation of tumor growth inhibition effect by combination of IR-LND and photodynamic therapy

The evaluation steps are as follows:

1) BALB/c mice (year)Age 6-8 weeks, weight 20-22g, winzhou university of medical science laboratory animal culture center) 50 ten thousand CT26 cells are subcutaneously injected into the armpit of the right forelimb until the tumor volume reaches about 120mm ³ At this time, the mice were randomly divided into 7 groups, vehicle, vehicle + Laser, LND, IR-68, IR-68+Laser, IR-LND, IR-LND+Laser, respectively.

2) 100. Mu.L of different drugs were intravenously administered to mice in groups at days 0,4,8, wherein Vehicle consisted of control solvents of absolute ethanol and castor oil, at a dose of 1mg/mL (5 mg/kg), wherein + Laser group, was administered 24h after dosing with 808nm Laser (1W/cm ² ) The irradiation was performed for 10 cycles, 10s each, at intervals of 30s.

3) During the treatment period, the body weight of the mice and the tumor volume were recorded every two days.

4) After the treatment, the mice were euthanized, the subcutaneous tumors were removed, photographed and the tumors were weighed.

The results in FIG. 3 show that the tumor growth inhibition after IR-LND treatment was 76.3%, the tumor growth inhibition after combination photodynamic treatment increased to 88.9%, there was a significant difference from the Vehicle, vehicle + Laser, LND, IR-68, IR-68+Laser groups, and that no significant fluctuations in the mouse body weight profile occurred during the treatment period. Thus, it is demonstrated that IR-LND is a relatively safe antitumor drug that works better as an antitumor photodynamic therapy drug.

Example 7: evaluation of IR-LND effect of inhibiting mitochondrial oxidative phosphorylation of tumor cells

MB49 cells were cultured in 6cm cell culture dishes, a series of concentration gradient LND and IR-LND solutions were prepared, then LND and IR-LND of different concentrations were added for 24 hours, and the activity of the mitochondrial complex was evaluated by continuously detecting absorbance changes for 15 consecutive minutes by means of a microplate reader, according to the mitochondrial complex detection kit instructions. Mitochondrial Complex I Activity Assay, mitochondrial Complex II Activity Assay and Mitochondrial Complex IV Activity Assay are purchased from Cayman. The results in FIGS. 6A-6C show that the LND and the IR-LND inhibit mainly mitochondrial complexes I and II, and that the IR-LND has a much greater inhibitory capacity than the LND.

Example 8: effect on AMPK and PD-L1 expression in IR-LND mediated immunotherapy

MB49 cells were cultured in 6cm cell culture dishes, then IR-LND was added at various concentrations for 24 hours, and then proteins in the cells were extracted, and expression of p-AMPK, AMPK, PD-L1, and beta-actin in the cells was evaluated by Western Blot experiments. FIGS. 7A-7C show that IR-LND induced high p-AMPK expression and low PD-L1 expression. From this, it was demonstrated that IR-LND, after inhibiting mitochondrial complex, would disrupt mitochondrial oxidative phosphorylation, thereby activating AMPK pathway to regulate PD-L1 expression.

Example 9: evaluation of the tumor growth inhibition effect of IR-LND mediated immunotherapy

The evaluation steps are as follows:

1) BALB/c mice (age 6-8 weeks, weight 20-22 g) were subcutaneously injected with 50 ten thousand CT26 cells in the armpit of the right forelimb, until tumor volume reached about 120mm ³ At this time, the mice were randomly divided into 5 groups, vehicle, LND, IR-68, PD-L1, IR-LND, respectively.

2) 100 μl of each of the different drugs was intravenously injected into the tail of the mice on days 0,3,6,9 in groups at a dose of 1mg/mL (5 mg/kg).

4) After the treatment, the mice were euthanized, the subcutaneous tumors were removed, photographed and the tumor seeds were weighed.

The results in FIG. 4 show that the tumor growth inhibition after PD-L1 treatment was 63.7% and that the tumor growth inhibition after IR-LND treatment was increased to 79.1% and that no significant fluctuations in the body weight curve of the mice occurred during the treatment period.

Finally, what is necessary here is: the above embodiments are only for further detailed description of the technical solutions of the present application, and should not be construed as limiting the scope of the present application, and some insubstantial modifications and adjustments made by those skilled in the art from the above description of the present application are all within the scope of the present application.

Claims

1. A small molecule compound targeting a tumor cell mitochondria, characterized in that: the structure is that

2. A composition comprising: the small molecule compound that targets a tumor cell mitochondria of claim 1 and a pharmaceutically acceptable carrier.

3. Use of a small molecule compound targeting a tumor cell mitochondria according to claim 1 or 2 for the preparation of an antitumor drug.

4. The use according to claim 3, wherein the tumor is a solid tumor, a metastasis and a hematological tumor, wherein the solid tumor is selected from pancreatic tumor, liver tumor, lung tumor, stomach tumor, intestinal tumor, breast tumor or cervical tumor.

5. The use of a small molecule compound targeting a tumor cell mitochondria according to claim 4 for the preparation of a medicament for tumor immunotherapy.

6. The use of a small molecule compound targeting a tumor cell mitochondria according to claim 1 for the preparation of a tumor in vivo fluorescence imaging agent.

7. An intermediate compound of the small molecule compound targeting a tumor cell mitochondria according to claim 1, characterized in that: the intermediate compound is an oxidative phosphorylation inhibitor, and has a structural formula of

8. A process for the synthesis of an intermediate compound according to claim 7, wherein: the lonidamine, 1- (Boc-amino) -4-aminobutane and 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride are dissolved in an organic solvent and stirred at room temperature for reaction for 24 hours, then trifluoroacetic acid and triethylamine are added to remove the Boc group, and the oxidative phosphorylation inhibitor is obtained after purification.

9. The method for synthesizing a small molecule compound targeting mitochondria of tumor cells according to claim 1, wherein the method comprises the following steps: dissolving an IR-68 compound and the oxidative phosphorylation inhibitor of claim 7 in an organic solvent, adding a dehydration condensing agent, reacting for 24-48 hours at room temperature, precipitating with a precipitating agent, filtering, purifying to obtain the small molecular compound of the targeted tumor cell mitochondria of claim 2,

the structural formula of the compound IR-68 is as follows:

10. the method of synthesizing a small molecule compound targeting a tumor cell mitochondria according to claim 9, wherein: the preparation method of the IR-68 compound comprises the following steps: sodium acetate, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene ] aniline hydrochloride, 1-N-butyl-2, 3-trimethyl-3H-indole iodide and 1- (5-carboxyhexyl) -2, 3-trimethyl-3H-indole iodide are dissolved in absolute ethyl alcohol, reflux is carried out for 3H at a constant temperature of 85 ℃, the reacted liquid is decompressed and distilled in a rotary way, and precipitation, filtration and purification are carried out by using a precipitator, thus obtaining the IR-68 compound.