JP6892103B2 - Dinuclear ligand or dinuclear metal complex - Google Patents

Description

The present invention relates to a dinuclear ligand or a dinuclear metal complex having the dinuclear ligand thereof.

Cisplatin is a metal complex that is clinically used as a chemotherapeutic agent for cancer. Cisplatin exhibits anticancer activity by directly binding to the DNA of cancer cells and distorting the three-dimensional structure of the DNA. However, cisplatin may show side effects such as vomiting and nephrotoxicity, and cisplatin-resistant cancer has also been reported in recent years. Therefore, a chemotherapeutic drug to replace cisplatin is required.

For example, a zinc complex binds to the phosphate portion of DNA, activates water molecules to promote hydrolysis of the phosphate ester of DNA, and this action mildly cleaves DNA to exhibit anticancer activity (non-cancerous). Patent Document 1). However, the rate of hydrolysis is slow, and the hydrolyzed DNA is easily regenerated by repair enzymes and does not cause apoptosis of cancer cells.

In addition, for example, bleomycin binds to iron in cancer cells to activate oxygen, thereby cleaving a DNA strand and suppressing the growth of cancer cells (Non-Patent Document 2). Bleomycin is widely used in clinical medicine as an excellent chemotherapeutic agent for squamous epithelial cancers and malignant lymphomas of human skin, head and neck, cervix and the like. However, bleomycin is a water-soluble glycopeptide antibiotic obtained from the actinomycete Streptomyces verticillus, and a chemotherapeutic agent obtained by a simple synthesis method independent of microorganisms is required.

S. Anbu, M. Kandaswamy, S. Kamalraj, J. Muthumarry, B. Varghese, “Phosphatase-like activity, DNA binding, DNA polymerase, anticancer and lactate dehydrogenase inhibition activity promoting by a new bis-phenanthroline dicopper (II) complex ”, Dalton Trans. (2011) 7310-7318. H. Umezawa, K. Maeda, T. Takeuchi, Y. Okami, J. Antibiot., 19 A, 200 (1966)

The present invention has been made in view of the above problems, and a dinuclear metal complex having a dinuclearization ligand or a dinuclearization ligand thereof, which can be easily synthesized and has an accurate anticancer effect. The purpose is to provide.

The dinuclear ligand according to the present invention is represented by the following chemical formula (I).

Here, (i) R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently hydrogen atoms or linear or branched alkyl groups having 1 to 6 carbon atoms, and (i) ii) X is a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, an alkoxy group, an alkoxyalkyl group, an ester group, an ester alkyl group, or a phenyl group, a pyridyl group, an amino group, a hydroxyl group, It is a thiol group, a fluorine atom, and a chlorine atom.

The binuclear metal complex thereof according to the present invention is represented by the following chemical formula (III).

Here, M is Cu, Fe, Zn, Co, Mn, or Ce.

According to the present invention, it is possible to easily obtain a dinuclearized ligand or a dinuclear metal complex that has little effect on normal cells and has an accurate nucleic acid cleaving action on cancer cells.

It is a figure which shows ^{the 1} H NMR spectrum of the binuclearization ligand which concerns on this invention. It is a figure which shows the ESI-MS of the dinuclear metal complex which concerns on this invention. It is a figure which shows the X-ray crystal structure analysis using the single crystal obtained by the recrystallization of the dinuclear metal complex which concerns on this invention. It is a figure which shows the hydrogen peroxide concentration dependence of DNA oxidative cleavage in the dinuclear metal complex which concerns on this invention. It is a figure which shows the DNA oxidative cleavage activity catalyzed by the dinuclear metal complex which concerns on this invention.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiment, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

As a result of diligent research, the present inventor has found as a new finding that the dinuclear ligand according to the following formula has a high nucleic acid cleaving action, and completed the present invention based on such fact.

Here, (i) R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are independently hydrogen atoms or linear or branched alkyl groups having 1 to 6 carbon atoms, and (i) ii) X is a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, an alkoxy group, an alkoxyalkyl group, an ester group, an ester alkyl group, or a phenyl group, a pyridyl group, an amino group, a hydroxyl group, It is a thiol group, a fluorine atom, and a chlorine atom. Here, the amino group is 'can described as radical, R ₇ and R _7' NR ₇ R ₇ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted Alternatively, it represents an unsubstituted aryl group.

The dinuclear ligand represented by the above formula is a compound having a novel structure having an amide bond in the compound structure. As will be described later, the dinuclear ligand and the dinuclear metal complex according to the present invention can cleave the nucleic acid only by the reaction between the compound and hydrogen peroxide, but the amide-bonded portion is oxidized by hydrogen peroxide. Since it is not decomposed, the dinuclear ligand and the dinuclear metal complex according to the present invention can stably cleave nucleic acids.

In the present invention, the dinuclearization ligand represented by the following chemical formula (II) is preferable.

In addition, the present inventor has found as a new finding that the dinuclear metal complex according to the following formula (III) has a high nucleic acid cleaving action. Here, M is Cu, Fe, Zn, Co, Mn, or Ce.

In the present invention, a dinuclear metal complex represented by the following chemical formula (IV) is preferable.

In the above, the nucleic acid to be cleaved is DNA or RNA.

Further, since the dinuclear ligand and the dinuclear metal complex according to the present invention have a high nucleic acid cleavage action, they can be used, for example, as a tool for analyzing the gene structure. Further, the dinuclear ligand and the dinuclear metal complex according to the present invention can be used as an anticancer agent because they can cleave the nucleic acid of cancer cells. Normal cells have scavenging enzymes such as catalase, so hydrogen peroxide can be decomposed into water and oxygen, but cancer cells have almost no enzymes such as catalase, so hydrogen peroxide can be decomposed like normal cells. It cannot be degraded and therefore has a higher hydrogen peroxide concentration in cancer cells than in normal cells. The dinuclear ligand and the dinuclear metal complex according to the present invention can cleave nucleic acids only by the reaction between the compound and hydrogen peroxide, and can specifically cleave the nucleic acids of cancer cells. Therefore, according to the present invention, the effect on normal cells is small. Further, since the binuclearization ligand and the dinuclear metal complex according to the present invention bind hydrogen peroxide with two metal ions (for example, two copper ions), they have high hydrogen peroxide affinity. Therefore, even when used in vivo, it reacts with a small amount of hydrogen peroxide and exhibits high nucleic acid cleavage activity.

In the dinuclearization ligand represented by the above chemical formula (I), X is preferably a functional group attracted to the surface of cancer cells. Compared to normal cells, the surface of cancer cells contains more anionic compounds such as sialic acid and heparan sulfate and is negatively charged. Therefore, for example, X can be a functional group attracted to a negative charge on the surface of a cancer cell.

The dinuclearized ligand and the dinuclear metal complex according to the present invention can be used for various cancers and are not particularly limited, but for example, colorectal cancer, gastric cancer, esophageal cancer, and the like. Colon cancer, liver cancer, pancreatic cancer, breast cancer, lung cancer, bile sac cancer, bile duct cancer, biliary tract cancer, rectal cancer, ovarian cancer, uterine cancer, renal cancer, bladder cancer, prostate It can be used for the treatment of cancer, osteosarcoma, brain tumor, leukemia, myoma, skin cancer, malignant melanoma, malignant lymphoma, tongue cancer, myeloma, thyroid cancer, skin metastatic cancer, skin melanoma, etc. ..

The administration form of the anticancer agent having the dinuclear ligand and the dinuclear metal complex according to the present invention is not particularly limited, and may be either an oral or parenteral administration form. In addition, it can be made into an appropriate dosage form according to the administration form, for example, oral preparations such as injections, capsules, tablets, granules, powders, pills, fine granules, rectal administrations, oily suppositories. , Can be prepared into various formulations such as aqueous suppositories.

Various preparations can be prepared by appropriately adding pharmacologically acceptable additives such as excipients, binders, lubricants, disintegrants, surfactants, and fluidity promoters. As excipients, lactose, fructose, glucose, corn starch, sorbit, etc., as binders, methyl cellulose, ethyl cellulose, arabic rubber, gelatin, hydroxypropyl cellulose, polyvinylpyrrolidone, etc., as lubricants, talc, magnesium stearate, polyethylene glycol, etc. Etc., as a disintegrant, starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, synthetic magnesium silicate, etc., and as a surfactant, sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80 As the fluidity accelerator, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like can be used.

The dose of the anticancer agent having the dinuclear ligand and the dinuclear metal complex according to the present invention is appropriately determined in consideration of the usage, the age, sex, degree of symptom, etc. of the patient. Adults 10-800 mg per day, preferably 100-200 mg, which can be administered once or in several divided doses per day.

(1) Synthesis of dinuclear ligand
(1-1) N, N, N-tri (toluene-4-sulfonyl) diethylenetriamine
The rotor was placed in a 500 mL eggplant flask, a 200 mL isobaric dropping funnel, a three-way cock, and a balloon were attached and vacuum dried. Add 45.6 g (0.239 mol) of p-toluene sulfonyl chloride dissolved in 250 mL Et ₂ O to the reaction vessel, and dissolve 8.26 g (0.0801 mol) and 9.6 g (0.24 mol) of NaOH in 90 mL distilled water, etc. When dropped over 2 hours with a pressure dropping funnel, a white solid was precipitated. When stirring was continued for 2 hours after the dropping, a white solid having a high viscosity was obtained. This was washed and filtered with distilled water and Et ₂ O, vacuum dried, and then recrystallized with MeOH to obtain a white solid (Yield 29.299 g, 65%).

(1-2) N, O, O-tri (toluene-4-sulfonyl) diethanolamine
The rotor was placed in a 300 mL three-mouth reaction vessel, a 200 mL isobaric dropping funnel, a three-way cock, and a balloon were attached and vacuum dried. _{CH 2} Cl ₂ 70 mL and p-toluenesulfonyl chloride 57.25 g (0.300 mol) were added to the reaction vessel, and the mixture was stirred while being immersed in an ice bath. Next, a 75 mL aqueous solution of 12.0 g of NaOH was added to a mixture of 10.52 g (0.100 mol) of diethanolamine and 9.11 g of benzyl-triethylammonium chloride, which was placed in an isobaric dropping funnel and slowly added dropwise. After the dropping, the mixture was stirred for a while, then the ice bath was removed and stirring was continued for 1 hour. The organic phase was taken in a liquid separation flask and washed with distilled water three times. The organic phase was taken _{, dehydrated with anhydrous Na 2} SO ₄ , concentrated with an evaporator, and further vacuum dried to obtain an oily substance. When MeOH was added to this and mixed well, a white solid was precipitated, which was filtered to obtain a white solid (Yield 36.571 g, 64%).

(1-3) 1,4,7,10-tetrakis (toluene-4-sulfonyl) -1,4,7,10-tetraazacyclododecane
Put the rotor in a 300 mL three-mouth reaction vessel, attach a 200 mL isobaric dropping funnel, a three-way cock, and a balloon, shield the reaction vessel with aluminum foil, soak it in an ice bath, vacuum dry it, and then put it in a nitrogen-substituted reaction vessel. 50 mL was added using a syringe with nitrogen flow. Furthermore, add these to the reaction vessel while flowing nitrogen in the order of NaH 1.69 g, N, N, N-tri (toluene-4-sulfonyl) diethylenetriamine 12.0 g (0.0212 mol), _{and stir until H 2} gas is no longer generated. did. The reaction vessel was transferred to an oil bath, the temperature was slowly raised to 70 ° C., and the mixture was stirred at this temperature for 1 hour. Then, the temperature of the oil bath was slowly raised to 90 ° C., and the mixture was stirred at this temperature for 1 hour. A solution of 12.0 g (0.0211 mol) of N, O, O-tri (tluene-4- sulfonyl) diethanolamine in 50 mL of dry DMF was placed in an isobaric dropping funnel, slowly added dropwise, and then stirring was continued for 1 day. DMF was removed by vacuum distillation, distilled water was added to the remaining solid, and the solid was cooled in the refrigerator. This was washed and filtered through distilled water, Et ₂ O and Et OH, and hot filtered through MeOH to give a white solid (Yield 9.2402 g, 55%).

(1-4) 1,4,7,10-tetraazacyclododecane (cyclen)
A rotator was placed 200 mL eggplant flask, cyclen · 4Ts 9.24 g (0.01171 mol) and H ₂ SO ₄ to 6 mL was added, and subjected to vacuum drying and nitrogen substitution fitted with a reflux condenser fitted with a three-way cock and a balloon. The reaction vessel was immersed in an oil bath, the temperature was slowly raised to 160 ° C., and the mixture was stirred at this temperature for 30 minutes. Then, the oil bath was removed, the mixture was cooled to room temperature, the reflux tube was removed, a 200 mL isobaric dropping funnel was attached, and 50 mL of EtOH was slowly added dropwise. Next, when _{80 mL of Et 2} O was added dropwise a little earlier, a dark brown solid was precipitated, which was washed and filtered with _{Et OH and Et 2 O.} This solid was dissolved in distilled water, filtered through Celite, and the filtrate was concentrated with an evaporator to obtain a dark brown oily substance. When HCl and EtOH were added to this, a solid was precipitated. When this was washed and filtered with EtOH, a light brown solid of the desired hydrochloride was obtained (Yield 1.564 g, 42%). 1.500 g (4.777 mmol) of this cyclen ・ 4 HCl was applied to an ion exchange column, and fractions showing basicity were collected and concentrated to obtain a white solid. When CHCl ₃ was added to this, impurities were precipitated and removed, and when concentrated again, a white solid was obtained (Yield 0.4649 g, 57%).

(1-5) 1,4,7,10-tetraazacyclododecane-1,4,7-carboxylate (tri (Boc) -cyclen)
The rotor was placed in a 50 mL eggplant flask, cyclen 0.4649 g (2.703 mmol) _{was dissolved in CHCl 3} 13.2 mL, Et ₃ N 1.16 mmol was added, and the mixture was immersed in an ice bath at 0 ° C. A solution prepared by dissolving 1.780 g (8.213 mmol) of (Boc) _{2 O in 10.1 mL of CHCl 3} was added dropwise at 0 ° C. over 2 hours. After the dropping, the ice bath was removed and the mixture was stirred at room temperature for 18 hours. After this, the solution was _{washed 3 times with H 2} O in a separatory funnel, and the organic phase was _{dehydrated and concentrated with Na 2} SO ₄ to obtain an oily substance, which was vacuum dried to obtain a white solid. The purpose is to dissolve this solid in the minimum amount of CHCl ₃ and purify it by open column chromatography (filler: silica gel, developing solvent: CHCl ₃ : MeOH = 20: 1) to recover and concentrate the fraction containing the desired product. White solid was obtained (Yield 0.6133 g, 48%).

(1-6) 2,6-diformyl-p-cresol
The rotor was placed in a 200 mL eggplant flask, and 3.84 g (27.4 mmol) of hexamethylenetetramine and 1.48 mL (13.7 mmol) of p-cresol were dissolved in 24 mL of TFA, _{replaced with N 2,} and then refluxed at 130 ° C. for 24 hours. After 24 hours, the reaction solution was returned to room temperature, 80 mL of 4 M HCl was added, and the mixture was stirred under aerobic conditions for 30 minutes. The solution was then transferred to a separatory funnel and extracted 3 times with _{CH 2} Cl _{2 60 mL.} The extracted organic phase is washed twice with 80 mL of 4 M HCl, once with 80 mL of H ₂ O, and once with 50 mL of saturated saline, and the organic phase is _{dehydrated with Na 2} SO ₄ and concentrated to obtain the desired yellow color. Obtained a solid. This was subjected to silica gel column chromatography (developing solvent: hexane: CHCl ₃ = 1: 1), and the fraction containing the desired product was recovered and concentrated to obtain 0.953 g of the desired pale yellow solid (5.81 mmol, Yield 42%). ).

(1-7) p-cresol 2,6-dicarboxylic acid
A rotator was placed in a 50 mL eggplant flask, dissolved NaOH 1.56 g in H ₂ O 13.7 mL, heated to 55-60 ° C. added _{Ag 2 O 1.78 g (7.80 mmol} ). To this, 0.63 g (3.90 mmol) of 2,6-diformyl-p-cresol was added, _{and stirring was continued until Ag 2} O was reduced to Ag. After that, the mixture was stirred for another 10 minutes, the solution was filtered through hot water, and HCl was added to the filtrate to lower the pH to 1, and a pale yellow solid was precipitated. This was collected by filtration to give 0.56 g of the compound of interest (2.86 mmol, 82%).

(1-8) p-cresol 2,6-dicarboxylic acid dichloride
Place the rotor in a 20 mL eggplant flask, add ₂ 3 mL of SOCl, add 0.020 g (0.10 mmol) of p-cresol 2,6-dicarboxylic acid, raise the temperature to 50-60 ° C, and stir for 4 hours. Continued. After 4 hours, SOCL ₂ was distilled off from the solution to give 0.019 g of a pale yellow oil (0.081 mmol, 82%).

(1-9) 2,6-di (1,4,7,10-tetraazacyclododecane-1-carboxyamide-1,4,7-triBoc-amide) -4-cresol
The rotor was placed in a 50 mL eggplant flask, triboc cyclen 0.088 g (0.187 mmol) was dissolved in _{10 mL dry CH 2} Cl _{2 , and K 2} CO ₃ 0.103 g (0.747 mmol) was added. Gradually add 0.0202 g (0.085 mmol) of p-cresol 2,6-dicarboxylic acid dichloride dissolved in 10 mL of dry CH ₂ Cl _{2 , replace the container with N 2,} and then stir the solution overnight. Continued. The reaction solution was filtered, the filtrate was separated with water, the organic phase was _{dehydrated with Na 2} SO ₄ , and then concentrated with an evaporator to obtain a white solid. This was subjected to silica gel column chromatography (developing solvent: CHCl ₃ : AcOEt = 1: 1), and the fraction containing the desired product was recovered and concentrated to obtain 0.0702 g of the desired white solid (0.0635 mmol, Yield 75%).

(1-10) 2,6-di (1,4,7,10-tetraazacyclododecane-1-carboxyamide) -4-cresol? 8HCl (Hbcamide? 8HCl)
The rotor was placed in a 50 mL eggplant flask to prepare a solution of EtOH: HCl = 3: 1, to which Hbcamide (Boc) ₆ 0.0702 g (0.0635 mmol) was added and stirring was continued overnight. After the reaction, the solution was filtered to obtain 0.0480 g of the desired white solid (0.0603 mmol, Yield 95%). ^{The 1} H NMR spectrum is shown in FIG.

(2) Synthesis of dinuclear metal complex [Cu ₂ (μ-OH ₂ ) bcamide] (ClO ₄ ) ₃ (4)
Hbcamide ・ 8HCl 0.0480 g (0.0603 mmol) was dissolved in 5 mL MilliQ water in a 10 mL eggplant flask, and a 1 M NaOH aqueous solution was added thereto to adjust the pH to 7. After filtration This was dissolved in methanol there Cu because _{(ClO 4) 2 · 6H 2} O 0.0589 g (0.159 mmol) Concentration of the solution was added a green solid precipitated, gas-liquid filtrate with Et ₂ O By recrystallization by diffusion, the desired green complex [Cu ₂ (μ-OH ₂ ) bcamide] (ClO ₄ ) ₃ (hereinafter, this complex may be referred to as complex 4) 0.034 g (0.0359 g) was obtained. mmol, Yield 60%). The ESI-MS is shown in FIG.

In addition, X-ray crystal structure analysis was performed using a single crystal obtained by recrystallization of _{[Cu 2} (μ-OH ₂ ) bcamide] (ClO ₄ ) _{3 (complex 4).} FIG. 3 is an ORTEP (Oak Ridge Thermal Ellipsoid Plot) diagram of the crystal structure.
(3) Hydrogen peroxide concentration-dependent complex 4 of the oxidative cleavage reaction of the dinuclear metal complex [Cu ₂ (μ-OH ₂ ) bcamide] (ClO ₄ ) _{3 (4) is peroxidized as shown below.} Oxidative cleavage of DNA with hydrogen peroxide was performed. For measurement of hydrogen peroxide concentration dependence, [complex] = 50 μM, [pUC19 DNA] = 0.05 mM bp, [buffer (pH 6.0)] = 10 mM (pH 5.5, 6.0 (MES), [NaCl]] The solution was prepared and measured so that = 10 mM, [H ₂ O ₂ ] = 125, 250, 375, 500 μM.

The results of the hydrogen peroxide concentration dependence of the oxidative cleavage reaction of complex 4 are shown. Samples are collected every 0, 10, 20, 40, 60 minutes for hydrogen peroxide concentrations of 500, 375 μM and every 0, 30, 60, 90, 120 minutes for 250, 125 μM. Then, the cleavage status of pUC19 DNA at each time was measured by agarose gel electrophoresis. A diagram plotting the cutting rate from Form I to Form II at this time is shown in FIG. It was found that when the hydrogen peroxide concentration was 500, 375 μM, Form II became 90% or more in 1 hour, and a saturation curve was drawn. The rate of Form II formation decreased as the concentration of hydrogen peroxide decreased. At a hydrogen peroxide concentration of 250 μM, the cleavage rate was almost 100% after 2 hours, and at 125 μM, the cleavage rate was about 80% after 2 hours.

When the DNA cleaving activity was examined using the complex 4 in the presence of hydrogen peroxide, it showed a very high DNA cleaving activity. Specifically, a cleaving experiment of pUC19 DNA was carried out using 50 μM complex 4 under the condition of 500 μM hydrogen peroxide (10 equivalents with respect to the complex). [H ₂ O ₂ ] = 500 μM, [4] = 50, 37.5, 25, 12.5 μM, [pUC19 DNA] = 0.05 mM bp, [buffer] = 10 mM (pH 5.5, 6.0 (MES), pH 7.4 (Tris) -HCl)), [NaCl] = 10 mM at 37 ° C. As a result, Form I disappeared at pH 7.0 in 1 hour, and 30% of Form II was converted to Form III after 5 hours (Fig. 5). Therefore, this oxidative cleavage of DNA by hydrogen peroxide is a reaction that specifically accelerates to complex 4. The reason for this is considered to be that the complex 4 exists stably under these reaction conditions. That is, the complex 4 is not oxidatively decomposed by hydrogen peroxide because p-cresol and cyclen are connected by an amide bond.

Since the complex 4 has a binuclear structure, two copper ions combine with two oxygen atoms of hydrogen peroxide to easily form a dinuclear copper peroxo complex even with a low concentration of hydrogen peroxide. Therefore, it is considered that the dinuclear copper complex can easily activate hydrogen peroxide. Bleomycin catalyzes oxidative cleavage of DNA. Bleomycin is an antibiotic (anticancer antibiotic) that exhibits high anticancer activity. This is because bleomycin oxidatively cleaves the DNA of cancer cells, causing them to die. Here, bleomycin is an iron complex, and in the iron (III) state, it reacts with hydrogen peroxide to rapidly produce an active form of bleomycin called active bleomycin. It is believed to be a hydroperoxo complex of iron (III). Furthermore, it is presumed that cleavage of the O-O bond of hydroperoxo produces oxidatively active species. Thus, complex 4 is considered to be useful as a new anticancer agent in place of bleomycin, which acts as an oxidizing agent using hydrogen peroxide.

It can be used as an anticancer drug.

Claims (7)

Binuclear ligand you characterized by being represented by the following formula (II).

(II) A nucleic acid cleavage agent comprising the dinuclearization ligand according to claim 1. An anticancer agent comprising the dinuclear ligand according to claim 1. Following chemical formula (III) binuclear metal complex thereof, characterized in that represented by (in the following formula, M is Cu, Fe, Zn, Co, Mn or Ce.).

(III) Binuclear metal complex of claim 4, characterized in that represented by the following formula (IV).

(IV) Nucleic acid cleaving agent, characterized in that it comprises a binuclear metal complex of claim 4 or 5, wherein. Anticancer agent characterized in that it has a binuclear metal complex of claim 4 or 5, wherein.

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