A photoactive ruthenium complex and its preparation method and application

Abstract

The present invention belongs to the field of organic synthesis technology and specifically provides a photoactive ruthenium complex and a preparation method thereof. The photoactive ruthenium complex provided by the present invention not only decomposes and releases isoniazid under light but also generates singlet oxygen under light excitation, exhibiting dual antibacterial effects, including significant antibacterial effects against Mycobacterium smegmatis, and can be used to treat tuberculosis infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to a photoactive ruthenium complex and a preparation method and application thereof. Background Art

[0002] Tuberculosis (TB) is a serious disease that primarily affects the lungs. The TB pathogen is Mycobacterium tuberculosis, and the death toll from TB remains high each year. Treatment for TB primarily relies on antibiotics, with isoniazid and rifampicin commonly used as first-line treatment. However, inappropriate use of antibiotics can easily lead to drug resistance. Approximately half of isoniazid-resistant TB strains isolated and cultured harbor deletions or mutations in the KatG enzyme, which activates isoniazid to form INH-nicotinamide adenine dicyclobutyrate (INH-NAD). This enzyme inhibits the InhA enzyme involved in the synthesis of mycophenolic acid, a key lipid component of the mycobacterial cell wall. The emergence of drug-resistant strains requires longer treatment, incurs higher costs, and presents more toxic side effects for patients. Therefore, the development of new drugs to combat M. tuberculosis infection is urgently needed.

[0003] In new drug development, the use of metal complexes has emerged as a promising candidate for promising biological effects. IQG-67 can directly activate isoniazid without requiring an enzymatic reaction, thus avoiding its inactivation in drug-resistant bacteria by replacing the enzyme activation reaction. There are also reports of octahedral ruthenium(II) polypyridine complexes combined with isoniazid as a means of combating mycobacteria, demonstrating promising results in antibacterial experiments. Furthermore, the INH-Fe(II) complex exhibits in vitro and in vivo inhibitory activity against the InhA enzyme and mycobacteria, including against drug-resistant strains.

[0004] Photoactive ruthenium complexes have also been used as precursors of antibacterial drugs, which can achieve the purpose of light-controlled drug release. Currently, relevant literature has reported ruthenium complexes containing isoniazid and have shown potential antimycobacterial activity. [Ru(bpy)2(INH)2] 2+ It can release INH under blue light irradiation and has anti-Aspergillus fumigatus Mycobacterium. [Ru(tyy)(LL)(INH)] 2+ A type of photoactive ruthenium(II) complex can inhibit the growth of Mycobacterium kansasii at low concentrations under visible light stimulation, and high concentrations also have a certain antibacterial effect in the dark. However, the antibacterial mechanism of these complexes relies solely on the release of isoniazid. Continuous drug treatment may still cause drug resistance, and it does not address the core issue of isoniazid inactivation in drug-resistant bacteria. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem of isoniazid inactivation in drug-resistant bacteria in the prior art.

[0006] To this end, the present invention provides a photoactive ruthenium complex, the structure of which is shown below:

[0007]

[0008] The present invention also provides a method for preparing a photoactive ruthenium complex, comprising the following steps:

[0009] S1, synthesis of Ru(tpy)Cl3;

[0010] S2, mixing Ru(tpy)Cl3 and 2,2'-biquinoline, adding a reducing agent, reacting under an inert gas atmosphere, and washing the product with HCl to obtain [Ru(tpy)(biq)(Cl)2];

[0011] S3, cis-[Ru(tpy)(biq)(Cl)2] and isoniazid are added to water, reacted under inert gas, filtered, NH4PF6 is added to the filtrate, the precipitate is collected and washed to obtain a photoactive ruthenium complex (Ru3-INH).

[0012] Specifically, the above step S1 is specifically as follows: dissolving RuCl3·3H2O and 2,2:6',2"-terpyridine in methanol, heating and reacting under an inert gas atmosphere to obtain Ru(tpy)Cl3.

[0013] Specifically, in the above step S2, the mass ratio of Ru(tpy)Cl3 to 2,2'-biquinoline is 172:100.

[0014] Specifically, the reducing agent in the above step S2 includes LiCl and triethylamine.

[0015] Specifically, the inert gas in the above step S2 includes argon.

[0016] Specifically, in the above step S3, the mass ratio of [Ru(tpy)(biq)(Cl)2], isoniazid, and NH4PF6 is 132:68:163.

[0017] Specifically, [Ru(tpy)(biq)(Cl)2] and isoniazid in the above step S3 are added to water and reacted under nitrogen at 80°C for 6 hours.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The photoactive ruthenium complex provided by the present invention can not only decompose and release isoniazid under light, but also generate singlet oxygen under light excitation, and has a dual antibacterial effect, has a significant antibacterial effect on Mycobacterium smegmatis, and can be used to treat tuberculosis infection.

[0020] The present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The diagram is a synthetic route diagram of the photoactive ruthenium complex provided by the present invention.

[0022] Figure 2 1 is a synthetic route diagram of Ru(tpy)Cl3 provided in an embodiment of the present invention.

[0023] Figure 3 This is a pathway diagram for synthesizing Ru3 from Ru(tpy)Cl3 provided in an embodiment of the present invention.

[0024] Figure 4 This is a pathway diagram for synthesizing Ru3-INH from Ru3 provided in an embodiment of the present invention.

[0025] Figure 5 : This is a graph showing the results of ROS detection using DPBF in Example 2 of the present invention; wherein, a is the fluorescence spectrum of DPBF after green light irradiation of Ru3-INH for different time periods; b is the fluorescence spectrum of DPBF after green light irradiation of Ru1-INH for different time periods; c is the ratio of the fluorescence intensity of DPBF at different time periods to the initial fluorescence intensity after green light irradiation of Ru3-INH and Ru1-INH for different time periods; d is the comparison of the fluorescence intensities of the control group, Ru1-INH, and Ru3-INH after 5 minutes of irradiation.

[0026] Figure 6 : This is a graph showing the results of ROS detection using DCFH in Example 2 of the present invention; wherein, a is the fluorescence spectrum of DCFH after green light irradiation of Ru3-INH and Ru1-INH for different times; b is the ratio of the fluorescence intensity of DCFH at different times to the initial fluorescence intensity after green light irradiation of Ru3-INH and Ru1-INH for different times; c is the comparison of the fluorescence intensities of the control group, Ru1-INH, and Ru3-INH 5 minutes after irradiation.

[0027] Figure 7 This is the result of fluorescence microscopy detection after mixing Ru3-INH and M. Smegmatis with green light irradiation in Example 2 of the present invention.

[0028] Figure 8 3 is a graph showing the results of light-controlled drug release detection in Example 3 of the present invention; wherein, a is a graph showing the change in the ultraviolet spectrum of Ru3-INH in PBS under green light irradiation for different times (dark room), [Ru3-INH] = 61.5 μM; b is a graph showing the change in the ultraviolet spectrum of Ru1-INH in PBS under green light irradiation for different times (dark room), [Ru1-INH] = 61.5 μM.

[0029] Figure 9 This is a schematic diagram of the isoniazid release reaction of the light-controlled drug in Example 3 of the present invention.

[0030] Figure 10 These are the bacterial survival rate results in Example 4 of the present invention; wherein, a is the bacterial survival rate detection after 40 minutes of green light irradiation after mixing different concentrations of Ru3-INH and E. coli; b is the bacterial survival rate detection after 40 minutes of green light irradiation after mixing different concentrations of Ru3-INH and B. subtilis.

[0031] Figure 11 The green light (LED 520nm, 16mw / cm2) is obtained by mixing different concentrations of Ru3-INH, different concentrations of Ru1-INH and M. Smegmatis in Example 4 of the present invention. 2 ) Bacterial survival rate test results after 20 minutes of irradiation or in a dark environment.

[0032] Figure 12 The green light (LED 520nm, power density 16mw / cm2) produced by mixing Ru3-INH and M. Smegmatis in Example 4 of the present invention 2 ) Flat plate test results after 20 minutes of irradiation.

[0033] Figure 13 These are the results of cell activity after culture of Ru3-INH (a), Ru1-INH (b) and MCF10A in Example 5 of the present invention (p≤0.05=*, p≤0.02=**).

[0034] Figure 14 This is the TEM result of ginger vesicles in Example 6 of the present invention.

[0035] Figure 15 This is the ginger vesicle particle size result in Example 6 of the present invention.

[0036] Figure 16 This is the result of drug release measurement over time of Ru3-INH-loaded ginger vesicles in Example 6 of the present invention.

[0037] Figure 17 This is a graph showing the changes in the ultraviolet spectrum of Ru3-INH ginger vesicles in PBS under green light irradiation at different times in Example 6 of the present invention (dark room).

[0038] Figure 18 This is the UV characterization of Ru3-INH before drug loading and the UV characterization of Ru3-INH-loaded ginger vesicles in Example 6 of the present invention.

[0039] Figure 193 is a diagram of wound status at different times in Example 7 of the present invention; wherein a is the Ru3-INH+green light group; b is the Ru3-INH+darkness group; c is the PBS+green light group; d is the PBS+darkness group.

[0040] Figure 20 This is the wound area detection result in Example 7 of the present invention.

[0041] Figure 21 This is the weight detection result in Example 7 of the present invention. DETAILED DESCRIPTION

[0042] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0043] The present invention provides a photoactive ruthenium complex, the structure of which is shown below:

[0044]

[0045] The present invention also provides a method for preparing a photoactive ruthenium complex, comprising the following steps:

[0046] S1. Synthesis of Ru(tpy)Cl3

[0047] RuCl3·3H2O and 2,2:6',2"-terpyridine were dissolved in methanol and heated to react under an inert gas atmosphere to prepare Ru(tpy)Cl3.

[0048] Specifically, refer to Figure 2 RuCl3·3H2O and 2,2:6',2"-terpyridine were dissolved in methanol (distilled over Mg). The mixture was heated to reflux under N2. The reaction solution was cooled to room temperature and then cooled in an ice bath. The brown solid was collected by vacuum filtration, washed with cold methanol until the filtrate was colorless, then washed with ether and air-dried.

[0049] S2, synthesis of Ru3

[0050] Ru(tpy)Cl3 and 2,2'-biquinoline are mixed, a reducing agent is added, and the mixture is reacted under an inert gas atmosphere. The product is washed with HCI to obtain [Ru(tpy)(biq)(Cl)2], i.e., Ru3.

[0051] Specifically, refer to Figure 3 Ru(tpy)Cl3 and 2,2'-biquinoline were mixed in 75% ethanol. LiCl and triethylamine were added as reducing agents. The mixture was purged with argon and then heated under argon reflux. The mixture was filtered while hot and the product was collected by evaporation under reduced pressure. The reaction mixture was then frozen in a refrigerator. The solid was collected on a frit, partially washed with HCl, then washed with acetone and anhydrous ether, and air-dried.

[0052] The mass ratio of Ru(tpy)Cl3 to 2,2'-biquinoline is preferably 172:100.

[0053] S3. Synthesis of Ru3-INH

[0054] Cis-[Ru(tpy)(biq)(Cl)2] and isoniazid are added to water, reacted under inert gas, filtered, NH4PF6 is added to the filtrate, and the precipitate is collected and washed to obtain a photoactive ruthenium complex, namely Ru3-INH.

[0055] Specifically, refer to Figure 4 cis-[Ru(tpy)(biq)(Cl)2] and isoniazid were added to water and reacted under nitrogen at 80°C for 6 hours. The solution was allowed to cool to room temperature and filtered to remove any unreacted starting material. NH4PF6 was then added to form a precipitate. The precipitate was collected by filtration and washed with cold water and diethyl ether.

[0056] The photoactive ruthenium complex of the present invention, its preparation method and application effects are studied below through specific examples.

[0057] Example 1:

[0058] Reference Figure 1 , the present application provides a photoactive ruthenium complex, which is prepared using the following steps.

[0059] S1, synthesis of Ru(tpy)Cl3;

[0060] 0.553 g (2.50 mmol) of RuCl3·3H2O and 0.583 g (2.50 mmol) of 2,2:6',2"-terpyridine were dissolved in 50 mL of methanol (distilled over magnesium). The mixture was heated at reflux at 70°C under N2 for 3 h. The reaction solution was cooled to room temperature and then cooled in an ice bath for 0.5 h. The brown solid was collected by vacuum filtration, washed with cold methanol until the filtrate was colorless, then washed four times with diethyl ether and air-dried.

[0061] S2, synthesis of Ru3

[0062] Ru(tpy)Cl3 (172 mg, 0.39 mmol) and 2,2'-biquinoline (biq, 100 mg, 0.39 mmol) were mixed in 75% ethanol (20 mL). 0.093 g of LiCl and 0.086 mL of triethylamine were added as reducing agents. The mixture was purged with argon for 7 minutes and then heated under argon reflux for 4 hours. The mixture was then filtered while hot. The product was collected by evaporation under reduced pressure, and the reaction mixture was then frozen in a refrigerator for 24 hours. The solid was collected on a frit and washed with 2 mL portions of chilled 3M HCl. The solid was then washed with 3 mL of acetone and 15 mL of anhydrous ether and air-dried.

[0063] S3. Synthesis of Ru3-INH

[0064] cis[Ru(tpy)(biq)(Cl)2] (132 mg, 0.2 mmol) and isoniazid (INH) (68 mg, 0.5 mmol) were reacted in 15 mL of water at 80°C under nitrogen for 6 hours. The solution was allowed to cool to room temperature and filtered to remove any unreacted starting material. NH4PF6 (163 mg, 1 mmol) was then added to form a precipitate. The precipitate was collected by filtration and washed with cold water and diethyl ether. Ru(bpy)(biq)(INH)2 was dissolved in DMSO.

[0065] Example 2:

[0066] This example studies the photocontrolled release of isoniazid and reactive oxygen species production by Ru3-INH and Ru1-INH synthesized in Example 1. Ru1-INH was prepared using a method described in the literature, and PBS was used as a control group. The structure of Ru1-INH is shown below:

[0067]

[0068] 1,3-Diphenylisobenzofuran (DPBF) is a highly specific fluorescent probe for singlet oxygen. It reacts with singlet oxygen to form an endoperoxide, which then decomposes into 1,2-dibenzoylbenzene, a compound that can be used to detect the production of reactive oxygen species (ROS) in vivo.

[0069] 2',7'-dichlorodihydrofluorescein (DCFH) is a fluorescent probe for measuring reactive oxygen species. When oxidized by ROS to 2',7'-dichlorofluorescein (DCF), it emits intense fluorescence. DCFH has improved lipid solubility and membrane permeability, making it widely used for detecting intracellular ROS.

[0070] In this example, the ROS fluorescent probes DPBF and DCFH were used to detect the ROS at different time periods under green light (520 nm, 16 mW / cm 2 )Ru3-INH and Ru1-INH fluorescence results, such as Figure 5-6 As shown in Figure 2, the absorbance of Ru3-INH NPs on DPBF gradually increased before and after light irradiation. In contrast, the absorbance of DPBF without Ru3-INH NPs did not change significantly ( Figure 5 By comparing the absorbance of DCFH before and after light irradiation, the DCFH without Ru3-INH NPs showed no significant change, and only the group using Ru3-INH-NPs showed a significant decrease ( Figure 6 These results indicate that Ru3-INH can produce 1 O2.

[0071] 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as an indicator again. 50 μM Ru3-INH was mixed with M. Smegmatis and the mixture emitted green light (LED 520 nm, power density 16 mW / cm 2 ) After 20 min of irradiation, fluorescence microscopy was performed. Ru1-INH and PBS were used as controls. Figure 7 As shown. After entering the bacteria, the non-luminescent DCFH-DA reacts with the ROS produced by Ru3-INH and is converted into highly fluorescent 2',7'-dichlorofluorescein (DCF). Under green LED light, bacteria incubated with Ru3-INH showed obvious green fluorescence, while bacteria in the PBS group and Ru1-INH group did not show fluorescence. At the same time, in the absence of green LED light illumination, none of the test groups glowed. The results further prove that Ru3-INH has the ability to produce ROS in bacteria.

[0072] Example 3:

[0073] In order to investigate the effect of light-controlled drug release of Ru3-INH and Ru1-INH in PBS, the green light (520 nm, V≈2.7 v, 20 mW / cm 2 The UV spectra of Ru3-INH and Ru1-INH in PBS were studied under irradiation. Figure 8-9 After exposing Ru3-INH solution (61.5uM) to green light for 5 minutes at room temperature, the UV-visible spectrum changed significantly ( Figure 8a), the main absorption band shifted, and the peak intensity at 560nm increased. In less than 2 minutes, the release reaction reached a steady state. The peak at 560nm increased with time in a single exponential relationship, which also indicated that the time required to reach equilibrium was about 2 minutes. In Ru1-INH solution (61.5uM), a similar photorelease reaction was observed under green light irradiation. After 5 minutes of light irradiation, the UV-visible spectrum changed ( Figure 8 b) The main absorption band shifts, and the peak intensity at 460 nm increases. The release reaction reaches a plateau within 3 minutes. The peak at 560 nm increases monoexponentially with time, indicating that approximately 3 minutes are required to reach equilibrium.

[0074] Both Ru3-INH and Ru1-INH can release isoniazid under green light excitation. Ru3-INH reaches equilibrium two minutes after irradiation, which is faster than Ru1-INH.

[0075] Example 4:

[0076] This example investigates the bactericidal effect of Ru3-INH.

[0077] Different concentrations of Ru3-INH were mixed with E. coli and B. subtilis respectively, and then green light (LED 520nm, 16mw / cm 2 ) irradiated for 40 minutes, and the bacterial survival rate was detected. The results were as follows Figure 10 For representatives of Gram-negative and Gram-positive bacteria, Escherichia coli and Staphylococcus aureus, high concentrations of Ru3-INH drugs can kill both cells under light excitation, presumably due to the production of a large amount of reactive oxygen species.

[0078] In order to further investigate the bactericidal effect of Ru3-INH and Ru1-INH, different concentrations of Ru3-INH and Ru1-INH were mixed with M. Smegmatis (Mycobacterium smegmatis) and then illuminated with green light (LED 520nm, 16mw / cm 2 ) After 20 minutes of irradiation, the bacterial survival rate was tested. Figure 11 The results of the Ru3-INH and M. Smegmatis plate experiments are shown in Figure 12 shown.

[0079] For Mycobacterium smegmatis, low concentrations of Ru3-INH and Ru1-INH both have bactericidal effects after green light excitation, but Ru3-INH has a better bactericidal effect, indicating that the superposition of isoniazid and photodynamic therapy has a better antibacterial effect.

[0080] Example 5:

[0081] This example investigates the cytotoxicity of Ru3-INH and Ru1-INH, and the specific steps are as follows:

[0082] 1. Place normal human breast MCF-10A cells into two 96-well plates (5000 cells per well, total volume 100 μl) and culture overnight until the cells adhere.

[0083] 2. Dissolve Ru3-INH and Ru1-INH in different culture media and prepare different concentrations (5-100uM), then replace the original culture medium in the well with 100ul of drug-containing culture medium.

[0084] 3. After replacing all the culture medium with drug-containing culture medium, the two 96-well plates were incubated in a dark incubator for 1 hour. Subsequently, one of the 96-well plates was irradiated with excitation light for 20 minutes, while the other plate was kept in the dark. The two 96-well plates were then incubated in a dark incubator for another 24 hours.

[0085] 4. Use the CCK8 method to detect cell survival rate.

[0086] The results are as follows Figure 13 In the dark, both drugs were non-toxic to cells. After light excitation, Ru1-INH remained non-cytotoxic, but Ru3-INH exhibited a significant cell-killing effect. This was due to the generation of reactive oxygen species, which are detrimental to cell survival. This was confirmed in fluorescence experiments, where green light excitation revealed the presence of reactive oxygen species in both bacteria and cells.

[0087] Example 6:

[0088] Ginger vesicles are an ideal drug carrier with good biocompatibility and high biosafety. Some plant vesicles have therapeutic effects and can be used for cancer or antibacterial treatment. Therefore, ginger vesicles were selected as the carrier of Ru3-INH in this example. Ginger exosomes were extracted according to the reported differential centrifugation method and characterized accordingly: TEM and particle size. The results are as follows Figure 14-15 shown.

[0089] Ru3-INH was loaded into ginger vesicles by high-speed centrifugation. The drug release results of Ru3-INH loaded ginger vesicles over time were as follows: Figure 16 Green light (520nm, V≈2.7v, 20mW / cm 2 ) irradiation download Ru3-INH ginger vesicles in PBS UV spectrum changes were studied, the results are as follows Figure 17 UV characterization of Ru3-INH before loading and UV characterization of Ru3-INH loaded ginger vesicles as shown. Figure 18 shown.

[0090] The drug-loaded ginger vesicles can be released in vitro, and the ultraviolet absorption also changes before and after drug loading, indicating that the drug has been successfully loaded; in the ultraviolet photolysis release experiment, it can be seen that the Ru3-INH ginger vesicles can achieve drug release under green light excitation.

[0091] Example 7:

[0092] After creating wounds of the same size on the same part of healthy experimental mice, 10μl of Mycobacterium smegmatis liquid was inoculated and the mice were randomly divided into four groups for treatment. Group a was treated with Ru3-INH and green light (Ru3-INH+light), group b was treated with Ru3-INH and placed in a dark room (Ru3-INH+dark), group c was treated with PBS and green light (PBS+light), and group d was treated with PBS and placed in a dark room (PBS+dark). The experimental period was 8 days, and the wound conditions of the mice in each group were observed every day. The results are shown in the figure below. Figure 19 The percentage of wound area and body weight changes over the days are shown in Figure 20-21 The wound healing effect of mice in the Ru3-INH+light group was the fastest. The weight of mice in all groups decreased in the early stage and gradually increased in the later stage, indicating that the drug had no effect on the survival of mice.

[0093] The above examples are merely illustrative of the present invention and do not limit the scope of protection of the present invention. Any design that is identical or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. A photoactive ruthenium complex, characterized in that The structure of the photoactive ruthenium complex is shown below: 。 2. A method for preparing a photoactive ruthenium complex, characterized in that: The following steps are involved: S1, synthesis of Ru(tpy)Cl3; S2, mixing Ru(tpy)Cl3 and 2,2'-biquinoline, adding a reducing agent, reacting under an inert gas atmosphere, and washing the product with HCl to obtain [Ru(tpy)(biq)(Cl)2]; S3, cis-[Ru(tpy)(biq)(Cl)2] and isoniazid are added to water, reacted under inert gas, filtered, NH4PF6 is added to the filtrate, the precipitate is collected, and washed to obtain a photoactive ruthenium complex.

3. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: The step S1 specifically comprises: dissolving RuCl3·3H2O and 2,2:6',2"-terpyridine in methanol, and heating the mixture under an inert gas atmosphere to react to obtain Ru(tpy)Cl3.

4. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: The mass ratio of Ru(tpy)Cl3 to 2,2'-biquinoline in step S2 is 172:

100.

5. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: In step S2, the reducing agent consists of LiCl and triethylamine.

6. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: The inert gas in step S2 is argon.

7. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: In step S3, the mass ratio of [Ru(tpy)(biq)(Cl)2], isoniazid, and NH4PF6 is 132:68:

163.

8. The method for preparing the photoactive ruthenium complex according to claim 2, wherein: In step S3, [Ru(tpy)(biq)(Cl)2] and isoniazid are added to water and reacted under nitrogen at 80°C for 6 hours.

9. Use of the photoactive ruthenium complex as claimed in claim 1 in the preparation of a medicament for treating tuberculosis.

10. The use according to claim 9, characterized in that: The drug for treating tuberculosis is a drug that inhibits the growth of Mycobacterium tuberculosis.

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