Compound useful in detecting ion and method of preparing it

A novel compound is disclosed having the structure ##STR1## in which R is lower alkyl and X is halogen or pseudohalogen. Also disclosed in a novel process for preparing the compound which comprises the steps of combining a compound having the structure ##STR2## in which X is a halogen or pseudohalogen, and a compound having the structure ##STR3## in which R is lower alkyl and A is lower alkylidene to produce a first reaction mixture; adjusting the pH of the first reaction mixture to at least about 8 to produce a second reaction mixture; and recovering the compound or its salt from the second reaction mixture.

INTRODUCTION 
The present invention relates to a novel compound useful in the measurement 
of ions, in particular ions in aqueous solution, and to a method for its 
preparation. The invention makes possible a quick, facile way of assaying 
ions whereby results are available to the assayist momentarily after 
merely contacting a test sample solution with a test means or device 
containing the compound. There is no need for cumbersome, expensive 
electronic equipment such as ion-specified electrodes, flame photometers, 
atomic absorption spectrophotometers or the like. Nor is it necessary to 
resort to time-consuming wet chemistry techniques such as titration and 
other laboratory procedures. The compound of the present invention enables 
the analyst to merely contact the test sample with a strip device or 
similar test means configuration, and observe any color change. 
The determination of aqueous ion concentration has application in numerous 
technologies. In the water purification art, calcium concentration must be 
carefully monitored to assess the degree of saturation of an ion exchange 
resin deionizer. Measurement of sodium and other ions in seawater is 
important in the preparation of drinking water aboard a ship at sea. 
Measurement of the potassium level in blood aids the physician in 
diagnosis of conditions leading to muscle irritability and excitatory 
changes in myocardial function. Such conditions include oliguria, auria, 
urinary obstruction and renal failure due to shock. 
Needless to say, a quick, facile method for determining ion concentration 
would greatly enhance the state of these technologies, as well as any 
others where such rapid, accurate determinations would be beneficial. 
Thus, for example, if a medical laboratory technician could accurately 
measure the potassium or calcium level of a serum or whole blood sample in 
a matter of seconds or minutes, not only would such rapid results aid the 
physician in diagnosis, but also laboratory efficiency would increase 
manyfold. The present compound is the linchpin of such a test, being a 
reporter substance which, when present in the composition containing an 
ionophore for the ion to be detected, produces a detectable response to 
the presence of the ion. 
BACKGROUND OF THE INVENTION 
Prior to the present invention, phenolic imine compounds were prepared by 
the so-called Gibbs Reaction. H. D. Gibbs, Chem. Review 13, 291-319 
(1927). See also D. Svobodova, et al., Mikrochimica Acta, pp. 251-264 
(1978). These references, the contents of which are incorporated herein by 
reference, describe the coupling of phenols with imines in accordance with 
##STR4## 
Such reactions are useful in a test for determining the presence phenols. 
The novel compound of the present invention is not only useful as a 
reporter substance for detecting ions in a test sample, but provide 
stability during storage and is relatively free from interfering side 
reactions in a sample. 
DEFINITIONS 
Certain terms used in the present discussion should at this point be 
mentioned to assure that the reader is of the same mind as the author as 
to their respective meanings. Thus the following definitions are provided 
to clarify the scope of the present invention, and to enable its 
formulation and use. 
3.1 The term "ionophore" includes molecules capable of forming a complex 
with a particular ion, in some instances to the substantial exclusion of 
others. For example the cyclic polypeptide, valinomycin, binds selectively 
to potassium ions in solution to form a cationic complex. Also included in 
the term are coronands, cryptands and podands. 
3.2 The term "nonporous" is intended to mean substantially impervious to 
the flow of water. Thus a nonporous carrier matrix is one which 
substantially precludes the passage of water through it, one side to the 
other. For example, a polyvinyl chloride film would be considered for the 
purposes herein as being nonporous. 
3.3 A "reporter substance" is a compound which is capable of interacting 
with an ionophore/ion complex to produce a color change or other 
detectable response. Thus, a reporter substance can be one which is 
relatively colorless in the non-ionized state, but which colors when in 
the presence of a complex of an ionophore and an ion. The compound of the 
present invention is such a substance, i.e., it produces color or change 
in light reflectance in the presence of such a complex. 
3.4 By "interacting" is meant any coaction between a reporter substance and 
an ionophore/ion complex which leads to a detectable response. An example 
of the reporter substance interacting with the complex is in the case 
where the reporter is changed by the complex from a colorless to a colored 
state, such as in the case of 
2-methyl-4-(3',5'-dichlorophen-4'-one)-indonaphth-1-ol. 
3.5 The term "detectable response" is meant herein as a change in or 
occurrence of a parameter in a test means system which is capable of being 
perceived, either by direct observation or instrumentally, and which is a 
function of the presence of a specific ion in an aqueous test sample. 
3.6 The term "lower alkyl", as used in the present disclosure includes an 
alkyl moiety, substituted or unsubstituted, containing about 1-4 carbon 
atoms. Included in the meaning of lower alkyl are methyl, ethyl, n-propyl, 
isopropyl, n-butyl, sec-butyl and tert-butyl. These may be unsubstituted, 
or they may be substituted provided any such substituents not interfere 
with the operation or functioning of the presently claimed test means or 
device in its capability to detect ions. "Lower alkylidene" is used in the 
same context as "lower alkyl", but designates an alkylene group (i.e., a 
divalent alkyl) having 1-4 carbon atoms. Thus, lower alkylidene includes 
methylene, ethylidene, n-propylidene, isopropylidene, n-butylidene, 
sec-butylidene and tert-butylidene. 
3.7 By "pseudohalogen" is meant atoms or groups of atoms which, when 
attached to an unsaturated or aromatic ring system, affect the 
electrophilicity or nucleophilicity of the ring system, and/or have an 
ability to distribute an electrical charge through delocalization or 
resonance, in a fashion similar to the halogens. Thus, whereas halogen 
signifies Group VII atoms such as F, Cl, and I, pseudohalogens embrace 
such moieties as --CN, --SCN, --OCN, --N.sub.3, --COR, --COOR, --CONHR, 
--CF.sub.3, --CCl.sub.3, --NO.sub.2, --SO.sub.2 CF.sub.3, --SO.sub.2 
CH.sub.3, and --SO.sub.2 C.sub.6 H.sub.4 CH.sub.3, in which R is alkyl or 
aryl. 
SUMMARY OF THE INVENTION 
The present invention resides in the discovery of a novel compound which 
has been found useful as a reporter substance, or indicator, in a test for 
the presence of a specific ion or group of ions in an aqueous test sample. 
The compound is one having the structure 
##STR5## 
in which R is lower alkyl and X is a halogen such as F, Cl, Br or I, or X 
is a pseudohalogen. in preferred embodiments, R is methyl and/or X is Cl. 
In addition to the novel compound, the present invention also comprises a 
process for preparing it. Basically, the process involves the reaction 
between a 2,6-dihaloquinone-4-haloimide and N-[2'-(lower 
alkyl)-1'-naphthyl]-aminoalkanol. The former compound has the structure 
##STR6## 
in which X is as defined above. The N-naphthylaminoalkanol has the 
structure 
##STR7## 
in which R is lower alkyl and A is lower alkylidene. Upon combining these 
precursor compounds to form a first reaction mixture, the pH is adjusted 
to at least about 8 to produce a second reaction mixture. Compound I is 
then isolated from the second reaction mixture. In a preferred embodiment, 
the isolation step includes acidifying the second reaction mixture to a pH 
in the range of about 2-4. Preferred reactants are 
2,6-dichloroquinone-4-chloroimide and 
N-[1'-(2'-methyl)naphthyl]aminoethanol. These latter compounds produce the 
specific version of compound (I) having the name 
2-methyl-4-(3',5'-dichlorophen-4'-one)indonaphth-1-ol. 
PREATION OF THE COMPOUND OF THE PRESENT INVENTION 
Because of the relative instability of compound (II) it is preferable to 
use a nonaqueous solution in combining it with (III). Acetone has been 
found particularly suited for this step, but of course other solvents such 
as other ketones and alcohols might be equally compatible. Such a 
determination is easily within the ability of the routineer, given the 
present disclosure. 
Generally it is preferred to combine stoichiometrically equal amounts of 
(II) and (III) as solutions to form a first reaction mixture. The pH of 
the first mixture is then adjusted to at least about 8 using a suitable 
base, thereby forming a second reaction mixture. Preferably an aqueous 
buffer (pH in the range of about 8 to 11) is utilized. It has been found 
that good results are obtained with a 100 mM aqueous solution of 
3-(cyclohexylamino)propanesulfonic acid which has been adjusted to pH 10 
with LiOH. 
Following the formation of the second reaction mixture, the product (I) is 
recovered by any suitable means. Acidification of the second reaction 
mixture is one such means of recovery, in that the basic form of (I), 
i.e., the deprotonated form which is soluble in water, is rendered 
insoluble through addition of of acidic hydrogen ions. Thus addition of 1N 
HCl with rapid stirring causes precipitation of (I), and the precipitate 
is easily recovered via centrifugation. Ideally the second reaction 
mixture is acidified to a pH in the range of about 2-4. 
Further purification can be effected by dissolving the precipitate in a 
suitable solvent, such as acetone, and passing this solution through a 
purification procedure, such as recrystallization, column chromotography 
or thin layer chromatography. 
USE OF COMPOUND (I) 
The compounds of the present invention find use as reporter substances, or 
indicators, in a system for measuring specific ions or groups of ions. 
Such a system comprises, in addition to (I), an ionophore and a carrier 
matrix. The carrier matrix has incorporated within it the ionophore, 
compound (I) either being incorporated with the matrix or added separately 
to the test sample being analyzed. If the ion to be assayed is present in 
the test sample, it can complex with the ionophore, and the formation of 
such complex causes (I) to change color. Typically a blue color is formed. 
In a preferred use, the ionophore and (I) are incorporated with the carrier 
matrix in such a way as to be substantially isolated from the aqueous test 
sample. For example, the ionophore and (I) can be taken up in a solution 
of vinyl chloride/vinylidene chloride copolymer in a suitable solvent and 
cast as a film on a polyester substrate film. Despite the hydrophobicity 
of such a film, the ion under analysis can penetrate it by complexing with 
the ionophore. Such penetration is to the exclusion of other test sample 
components. The formation of the ionophore/ion complex evokes the 
appearance of color in the film due to interacting of the complex with 
compound (I). 
Although the mechanism of the color formation is not known, it may well be 
due to the formation of an ion having a resonant structure capable of 
absorbing light at certain wavelengths. It is probable that Compound (I) 
exhibits tautomerism, in accordance with 
##STR8## 
In the presence of a charged ion/ionophore complex, through a mechanism 
not thoroughly understood, it is theorized that the tautomer could lose a 
hydroxyl proton to become a resonating ion in accordance with 
##STR9## 
such a resonant structure theory is a plausible explanation for the 
generation of blue color observed when a film containing valinomycin and 
(I) is contacted with aqueous potassium.

EXAMPLES 
A series of experiments was conducted whereby a unique synthesis procedure 
was utilized in preparing the novel compound (I). Following its 
preparation, (I) was then tested as to its utility as a reporter substance 
in a test means and device for measuring ions in solution. The preparative 
procedures utilized, as well as the evaluation are described in the 
following examples. 
Preparation of 2-Methyl-4-(3',5'-dichlorophen-4'-one)indonaphth-1-ol 
The captioned compound (hereafter, MEDPIN) was prepared in accordance with 
the following procedure. 
Equimolar amounts of 2,6-dichloroquinone-4-chloroimide (DQCI) and 
N-[1'-(2-methyl)naphthyl]aminoethanol (MeNAE) were mixed in acetone to a 
concentration of 100 mM of each solute. A brown solution resulted. 
To a one mL (milliliter) portion of this solution, was added 6 mL of 100 mM 
CAPS buffer (pH=10), CAPS buffer is an aqueous solution of 
3-(cyclohexylamino)propanesulfonic acid titrated to pH 10 with LiOH. The 
resulting solution was red in color. 
1N HCl was added dropwise to the red solution with efficient mixing until 
the pH dropped to about 2.6. The solution became turbid rapidly, a 
brick-red precipitate forming. Care was taken not to allow the pH below 
about 1.9 to avoid product decomposition. 
The mixture was then centrifuged, the precipitate dried at RT under 
nitrogen, and redissolved in 3 mL acetone. After standing in a 
refrigerator at 4.degree. C. for 30 minutes, the liquid was passed onto a 
silica gel column and eluted with a 1:4 mixture of ethyl acetate and 
toluene. A reddish brown band formed in the column. 
Thin layer chromatography of the product on a silica gel plate eluted by a 
1:4 solution of ethyl acetate and toluene gave a single spot at Rf 0.76. 
The fractions containing the product were pooled, and the solvent removed 
under vacuum in a rotary evaporator. The purified product can be stored as 
a dried powder or in acetone at 0.degree. C. 
Characterization of the Product of 7.1 
A series of experiments was undertaken to characterize the compound 
isolated in example 7.1, and to elicit its structure. 
Mass spectrum analysis yielded a strong 3-line pattern at 331, 333 and 335. 
Such a pattern is indicative of the presence of two chlorine atoms. 
Elemental analysis gave evidence of an empirical formula of C.sub.17 
H.sub.11 NO.sub.2 Cl.sub.2 in accordance with the following data: 
______________________________________ 
% C % H % N % O % Cl 
______________________________________ 
Found 61.79 3.76 3.98 9.50 20.69 
Calculated 
61.45 3.31 4.22 9.64 21.39 
______________________________________ 
A molecular weight of 332 was deduced from mass spectroscopy. 
Proton nuclear magnetic resonance (nmr) spectra showed the presence of 
seven groups of protons in addition to those of the solvent. The most 
upfield signal of the spectrum is due to methyl protons split by the 
neighboring lone proton. The next signal is due to two equivalent single 
protons on a benzene ring. The third signal, a quarter, is attributable to 
a proton at the 3-position of a naphthalene ring, split by three methyl 
protons. The remaining signals further downfield are due to four protons 
on the 5-, 6-, 7- and 8-positions of a naphthalene ring. Interactions 
among these signals give rise to this complex ABCD-type spectrum. 
The results of these experiments are, in their sum total, strong evidence 
that the product of example 7.1 has the structure of compound (I) in which 
R is methyl and X is Cl. 
Use of Compound (I) in Detecting Potassium 
A solution was prepared containing 6.7 mg/mL valinomycin and 1.67 mg/mL 
MEDPIN in o-nitrophenyloctyl ether. A buffered gelatin solution was 
prepared using 3.13 g Type I gelatin (Sigma Chemical Co.) which had been 
dialyzed at 10.degree. C. to remove ionic impurities, and 20.8 g of 
dionized water. To this was added 0.25 mL of a buffer prepared by 
adjusting 1M Trizma base (Sigma Chemical Co.) to pH 8 with HCL (Baker) and 
then to pH 5 with acetic acid (Baker). 
The oil and gelatin solutions were mixed and placed in a 12-37 mL mini 
sample container for a Waring Blender (Fisher Scientific) and blended for 
2 minutes at high speed. 
After allowing 15-30 minutes at 45.degree. C. for bubbles to rise, the 
emulsion was spread onto a polyester film support which had been 
pretreated to accept gelatin (40 GAB 2S, 3M Co.). The film was spread to a 
thickness of 6.75.times.10.sup.-3 inches, (#75 Mayer Rod, RDS Co., Webster 
N.Y.) The film was air dried, then 0.2.times.0.4 inch pieces were mounted 
onto polystyrene film support handles using double-faced adhesive tape 
(Double Stick, 3M Co.) to form test devices. 
Test samples were prepared containing 0, 0.2, 0.6, 0.6, 0.8 and 1.1 mM KCl, 
100 mM tris-Cl pH 8.5. These concentrations correspond to those found in 
serum diluted ninefold. A 30 .mu.L (microliter) sample drop was placed on 
the reagent portion of a test device and incubated at 37.degree. C. in a 
Seralyzer.RTM. (Ames Division, Miles Laboratories, Inc.) reflectance 
spectrophotometer for 2.5 minutes, at which time the reflectance at 640 nm 
(nanometers) were measured. The reflectance data is tabulated below. 
______________________________________ 
K.sup.+ (mM) 
(K/S).sup.2 
______________________________________ 
0 0.2048 
0.2 1.4945 
0.6 5.3038 
1.1 8.4158 
______________________________________ 
The data shows a linear correlation between K.sup.+ concentration and 
(K/S).sup.2. 
(K/S).sup.2 is defined as 
##EQU1## 
in which R is the fraction of reflectance from the test device, K is a 
constant, and S is the light scattering coefficient of the particular 
reflecting medium. The above equation is a simplified form of the 
well-known Kubelka-Munk equation (See Gustav Kortum, "Reflectance 
Spectroscopy", pp 106-111, Springer Verlag, New York (1969). 
The above data shows that potassium concentration corresponds linearly to 
(K/S).sup.2. Moreover, the data shows that various concentrations can be 
accurately measured.