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US5185450.Pdf |||||||||||| USOO518545OA United States Patent (19) 11 Patent Number: 5,185,450 Owen 45 Date of Patent: Feb. 9, 1993 (54) TETRAZOLIUM COMPOUNDS FORCELL 52 U.S.C. .................................... 548/194; 548/250; VIABILITY ASSAYS 548/252, 548/193; 548/112 75) Inventor: Terence C. Owen, Tampa, Fla. 58) Field of Search ............... 548/252, 204, 73) Assignee: versity of South Florida, Tampa, Primary Examiner-David B. Springer as Attorney, Agent, or Firm-Reising, Ethington, Barnard, (21) Appl. No.: 899,155 Perry & Milton (22) Filed: Jun. 12, 1992 57 ABSTRACT Novel tetrazolium salts, which are biologically reduc Related U.S. Application Data ible to blue water-soluble formazan compounds by liv 63 Situation of Ser. No. 657,462, Feb. 19, 1991, aban- ing cells, are utilized in cell culture viability assays. Oe 51 Int. Cl.................... CO7D 417/04; CO7D 251/01 8 Claims, No Drawings 5,185,450 1. 2 TETRAZOLIUM COMPOUNDS FOR CELL (5). An assay utilizing this compound capitalizes on the VABILITY ASSAYS water solubility of the formazan disulfonic acid pro duced upon reduction of the tetrazolium zwitterion-salt. This is a continuation of copending application Ser. 5 The dye diffuses out of the cells in which it is produced No. 07/657,462 filed on Feb. 19, 1991, now abandoned. and into the culture medium. Optical density is read TECHNICAL FIELD directly. The steps involving removal of the medium The present invention generally relates to the use of and extraction with solvent are obviated and the proce tetrazolium salts in assays to distinguish living cells 10 dure becomes amenable to full automation. from dead cells. More particularly, the invention pro There are problems with XTT however. The synthe vides novel tetrazolium compounds for cell viability sis for XTT is problematic and the XTT formazan is assays and their method of use. yellow-orange in color (in water), leading to difficulties BACKGROUND OF THE INVENTION 15 in the differentiation of the formazan from a generally The biological reduction of a colorless tetrazolium yellow cell culture medium background. Problems also salt 1 to a colored formazan 2 is widely used to indicate arise in certain cell types where XTT forms precipi and measure cell viability in cell cultures (1,2). tates. To be most useful in spectrophotometric assays, a formazan should be purple, blue or violet. XTT shows that there is an advantage in utilizing a tetrazolium compound which reduces to a water soluble formazan which is capable of diffusing out of the cells into the 25 culture medium where it could be measured by standard Numerous laboratories have consequently used tetrazo spectrophotometers, and also the utility of sulfonate lium salts for many years in assays to distinguish living groups in achieving such water solubility in the forma cells from dead ones. There are several characteristics zans. The present invention provides these advantages which suit tetrazolium salts to this purpose. Tetrazolium salts are reduced to the intensely colored 30 while further providing structural flexibility allowing formazans by the functioning electron transport systems chemical modification to overcome many of the prob of viable cells, either directly or through the intermedi lens associated with XXT. acy of electron carriers such as phenazine methosulfate or menadione. The direction of the color change, from SUMMARY OF THE INVENTION the colorless oxidized form to a colored reduced form, 35 is practically unique among dye-stuffs. Viable cells de In accordance with the present invention, there is velop color in direct proportion to their metabolic ac provided a tetrazolium compound having attached to tivity. The colorless tetrazolium reagent is an ionized the tetrazolium ring either (a) at least one side chain salt, soluble in water and capable of passing into cells. capable of having at least two acidic groups thereon for The colored reduction product, the formazan, is not rendering the compound water soluble: or (b) at least ionic, is insoluble in water, and is deposited within via ble cells in a manner well suited to histochemical evalu two side chains each capable of having one or more ation. A number of different structural groups can be acidic groups thereon for the same purpose of rendering attached to the tetrazolium core to influence color, the compound water soluble. redox potential, solubility, and histochemical character 45 The present invention further provides a cell viability istics of the tetrazolium - formazan couples. assay including the steps of exposing cells to the above Over a thousand different tetrazolium compounds have been synthesized of which about a dozen are in described tetrazolium compound, the viable cells reduc common use for cell viability assays. Some of the proce ing the compound to a blue or purple water soluble dures using tetrazolium salts currently in vogue have 50 formazan and detecting the blue or purple color of the problems in that they are cumbersome, labor intensive, formazan in the media as an indication of cell viability. and ill-suited to volume throughput or automation. Typically, in such prior art procedures the formazan DETALED DESCRIPTION OF THE dye is deposited as an insoluble precipitate within cells INVENTION of the culture which, in the standard procedure, must be 55 spun down, mounted on a microscope slide, and evalu The present invention is comprised of the use of ated (rather subjectively) by a skilled microscopist. novel tetrazolium salts that will reduce to blue or purple Recently procedures have been described in which the water soluble formazans. cells are lysed with solvents such as DMF, DMSO, and 2-propanol, and the dye thereby brought into solution The first general design of the present invention ob to be measured spectrophotometrically (3,4). These tains new tetrazolium compounds having attached to procedures give less subjective quantitation, but are no the tetrazolium ring a naphthalenedisulfonic acid moi more convenient nor less labor intensive than the earlier ety. The naphthalenedisulfonic moiety provides a side methods. chain having two acidic groups thereon for rendering One tetrazolium compound used recently for cell 65 viability assays is the tetrazolium disulfonic acid XTT, a the compound water soluble. For this purpose, tetrazo tetrazolium disulfonic acid wherein R1 = carbanilido, lium compounds of the formula 3 have been synthesized and R2 sR3 = 2-methoxy-4-nitrobenzene5-sulfonic acid 5,185,450 3 4. then oxidized to yield the desired tetrazolium com R3 SO3- po und 3. 5 N S. N CO 5 NR V - SO3Na R-c1 H NH-1SR(R- Ase20R3) X N1 I Y O R 1 4. O R2 7 H - NS-R30R2) in which the R3 group is a naphthalene with two sulfo- R- N=NRR) >r- n nate groups attached. Points of attachment of sulfonate -NR (R3) N1,(R3) groups and of tetrazolium nitrogen to the naphthalene is 2K3 ring may be varied. Two sulfonate groups are necessary 6 8 for water solubility since tetrazolium zwitterions, like diti h li described most large-molecule zwitterions, tend to be poorly solu- In ad to the St. ium compounds descri l ble in water. However, unlike XTT which utilizes one R te St. of the genera acidic group (sulfonate group) on each of the two rings 20 El 9 s d Set cell viability ty in a attachedring, the presentto nitrogen invention atoms provides of the central a single tetrazolium side chain aSOlar as S12Part of thee presenttetrazolium E. compounds. bis-tetrazolium In partic having two acidic groups thereon for rendering the coPonds S. the formula g would be synthesized by compoundwater soluble. The other side chains this are "ethods l ogous to those leading to the tetrazolium available for further modification to render the com- "P" ' N' R3 R3Y 9 N1A /SN N-R-N s / \ a R1 N N R R R1 10 X X N N 1 \ / N N NV + N Y Y O3S O O SO3 SO3Na SO3Na Those variants of the tetrazolium compound 3 and 50 the bis-tetrazolium compound 10 which are useful in the pound more useful. present invention include those variants in which X can The present invention provides means necessary for be a hydrogen, a nitro group, or a methoxy group; Y water solubility of the compound while also allowing can be a hydrogen, a nitro group or a methoxy group; for two other side chains which can be modified for R can be a pyridyl group or a methoxy-diiodophenyl providing a blue or purple color to the compound when 55 group; R2 can be a dimethylthiazolyl group or a benzo reduced to the corresponding formazan. Moreover, the thiazolylphenyl group: and R3 is the previously men large conjugation of the naphthalene moiety contributes tioned naphthalene disulfonate group. to the blue color of the formazans produced by reduc In the tetrazolium compounds 3 and 10, the naphtha tion of the above mentioned compounds. Thus, the lene ring not only provides a carrier structure for the present invention provides means for gaining the water 60 sulfonate groups and contributes to the ease of purifica solubility of prior art compounds such as XTT but also tion of the compounds, but it also enhances the color of renders the present invention more structurally flexible the corresponding formazan, as in the case of known to overcome the problems of the XTT compound. tetrazolium violet compounds. Virtually any desired Tetrazolium compounds of the formula 3 were syn color of formazan may be had by manipulating groups thesized in the following manner. An aldehyde 4 was 65 R1 and R2, since R3 is capable of carrying both acidic reacted with a hydrazine 5 to give the hydrazone 6. The groups. If both R1 and R2 are simple benzene rings, the hydrazone 6 was then reacted with the diazonium salt 7 formazan is crimson.
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